David J Hartshorne

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• Mizutani, H., Okamoto, R., Moriki, N., Konishi, K., Taniguchi, M., Fujita, S., Dohi, K., Onishi, K., Suzuki, N., Satoh, S., Makino, N., Itoh, T., Hartshorne, D. J., & Ito, M. (2010). Overexpression of myosin phosphatase reduces Ca²⁺ sensitivity of contraction and impairs cardiac function. Circulation Journal, 74(1), 120-128.
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  PMID: 19966500;Abstract: Background: Phosphorylation of the regulatory light chain of myosin (MLC) has roles in cardiac function. In vitro, myosin phosphatase target subunit 2 (MYPT2) is a strongly suspected regulatory subunit of cardiac myosin phosphatase (MP), but there is no in-vivo evidence regarding the functions of MYPT2 in the heart. Methods and Results: Transgenic mice (Tg) overexpressing MYPT2 were generated using the α-MHC promoter. Tg hearts showed an increased expression of MYPT2 and concomitant increase of the endogenous catalytic subunit of type 1 phosphatase (PP1cδ), resulting in an increase of the MP holoenzyme. The level of phosphorylation of ventricular MLC was reduced. The pCa-tension relationship, using β-escin permeabilized fibers, revealed decreased Ca2+ sensitization of contraction in the Tg heart. LV enlargement with associated impairment of function was observed in the Tg heart and ultrastructural examination showed cardiomyocyte degeneration. Conclusions: Overexpression of MYPT2 and the increase in PP1cδ resulted in an increase of the MP holoenzyme and a decrease in the level of MLC phosphorylation. The latter induced Ca2+ desensitization of contraction and decreased LV contractility, resulting in LV enlargement. Thus, MYPT2 is truly the regulatory subunit of cardiac MP in-vivo and plays a significant role in modulating cardiac function.
• Funabara, D., Osawa, R., Ueda, M., Kanoh, S., Hartshorne, D. J., & Watabe, S. (2009). Myosin loop 2 is involved in the formation of a trimeric complex of twitchin, actin, and myosin. Journal of Biological Chemistry, 284(27), 18015-18020.
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  PMID: 19439402;PMCID: PMC2709379;Abstract: Molluscan smooth muscles exhibit a low energy cost contraction called catch. Catch is regulated by twitchin phosphorylation and dephosphorylation. Recently, we found that the D2 fragment of twitchin containing the D2 site (Ser-4316) and flanking immunoglobulin motifs (TWD2-S) formed a heterotrimeric complex with myosin and with actin in the region that interacts with myosin loop 2 (Funabara, D., Hamamoto, C., Yamamoto, K., Inoue, A., Ueda, M., Osawa, R., Kanoh, S., Hartshorne, D. J., Suzuki, S., and Watabe, S. (2007) J. Exp. Biol. 210, 4399-4410). Here, we show that TWD2-S interacts directly with myosin loop 2 in a phosphorylation-sensitive manner. A synthesized peptide, CAQNKEAETTGTHKKRKSSA, based on the myosin loop 2 sequence (loop 2 peptide), competitively inhibited the formation of the trimeric complex. Isothermal titration calorimetry showed that TWD2-S binds to the loop 2 peptide with a Ka of (2.44 ± 0.09) × 105 M-1 with two binding sites. The twitchin-binding peptide of actin, AGFAGDDAP, which also inhibited formation of the trimeric complex, bound to TWD2-S with a Ka of (5.83 ± 0.05) × 104 M-1 with two binding sites. The affinity of TWD2-S to actin and myosin was slightly decreased with an increase of pH, but this effect could not account for the marked pH dependence of catch in permeabilized fibers. The complex formation also showed a moderate Ca2+ sensitivity in that in the presence of Ca2+ complex formation was reduced. © 2009 by The American Society for Biochemistry and Molecular Biology, Inc.
• Kusaka, M., Ikeda, D., Funabara, D., Hartshorne, D. J., & Watabe, S. (2008). The occurrence of tissue-specific twitchin isoforms in the mussel Mytilus galloprovincialis. Fisheries Science, 74(3), 677-686.
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  Abstract: The catch state in Mytilus anterior byssus retractor muscle is regulated by phosphorylation and dephosphorylation of twitchin, a member of the titin/connectin superfamily, and involves two serine residues, Ser-1075 (D1) and Ser-4316 (D2). This study was undertaken to examine whether isoforms of twitchin were expressed in various muscles of the mussel Mytilus galloprovincialis by reverse transcription-polymerase chain reaction. Mussel tissues, including both catch and non-catch muscles, contained various twitchin isoforms that all contained the D2 site and the kinase domain. However, sequence alterations were detected around the D1 site, notably a potential deletion of the D1 site. All isoforms from catch muscles contained both the D1 and D2 sites, whereas those from non-catch muscles also expressed the D2 site, but some of them lacked the D1 site. This suggests that the D1 site of twitchin is essential to the mechanism of catch. Genomic DNA analysis revealed that twitchin isoforms are produced by alternative splicing. © 2008 Japanese Society of Fisheries Science.
• Matsumura, F., & Hartshorne, D. J. (2008). Myosin phosphatase target subunit: Many roles in cell function. Biochemical and Biophysical Research Communications, 369(1), 149-156.
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  PMID: 18155661;PMCID: PMC2367208;Abstract: Phosphorylation of myosin II is important in many aspects of cell function and involves a myosin kinase, e.g. myosin light chain kinase, and a myosin phosphatase (MP). MP is regulated by the myosin phosphatase target subunit (MYPT1). The domain structure, properties, and genetic analyses of MYPT1 and its isoforms are outlined. MYPT1 binds the catalytic subunit of type 1 phosphatase, δ isoform, and also acts as an interactive platform for many other proteins. A key reaction for MP is with phosphorylated myosin II and the first process shown to be regulated by MP was contractile activity of smooth muscle. In cell division and cell migration myosin II phosphorylation also plays a critical role and these are discussed. However, based on the wide range of partners for MYPT1 it is likely that MP is implicated with substrates other than myosin II. Open questions are whether the diverse functions of MP reflect different cellular locations and/or specific roles for the MYPT1 isoforms. © 2007 Elsevier Inc. All rights reserved.
• Yamashiro, S., Yamakita, Y., Totsukawa, G., Goto, H., Kaibuchi, K., Ito, M., Hartshorne, D. J., & Matsumura, F. (2008). Myosin Phosphatase-Targeting Subunit 1 Regulates Mitosis by Antagonizing Polo-like Kinase 1. Developmental Cell, 14(5), 787-797.
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  PMID: 18477460;PMCID: PMC2680213;Abstract: Myosin phosphatase-targeting subunit 1 (MYPT1) binds to the catalytic subunit of protein phosphatase 1 (PP1C). This binding is believed to target PP1C to specific substrates including myosin II, thus controlling cellular contractility. Surprisingly, we found that during mitosis, mammalian MYPT1 binds to polo-like kinase 1 (PLK1). MYPT1 is phosphorylated during mitosis by proline-directed kinases including cdc2, which generates the binding motif for the polo box domain of PLK1. Depletion of PLK1 by small interfering RNAs is known to result in loss of γ-tubulin recruitment to the centrosomes, blocking centrosome maturation and leading to mitotic arrest. We found that codepletion of MYPT1 and PLK1 reinstates γ-tubulin at the centrosomes, rescuing the mitotic arrest. MYPT1 depletion increases phosphorylation of PLK1 at its activating site (Thr210) in vivo, explaining, at least in part, the rescue phenotype by codepletion. Taken together, our results identify a previously unrecognized role for MYPT1 in regulating mitosis by antagonizing PLK1. © 2008 Elsevier Inc. All rights reserved.
• Funabara, D., Hamamoto, C., Yamamoto, K., Inoue, A., Ueda, M., Osawa, R., Kanoh, S., Hartshorne, D. J., Suzuki, S., & Watabe, S. (2007). Unphosphorylated twitchin forms a complex with actin and myosin that may contribute to tension maintenance in catch. Journal of Experimental Biology, 210(24), 4399-4410.
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  PMID: 18055628;PMCID: PMC2219545;Abstract: Molluscan smooth muscle can maintain tension over extended periods with little energy expenditure, a process termed catch. Catch is thought to be regulated by phosphorylation of a thick filament protein, twitchin, and involves two phosphorylation sites, D1 and D2, close to the N and C termini, respectively. This study was initiated to investigate the role of the D2 site and its phosphorylation in the catch mechanism. A peptide was constructed containing the D2 site and flanking immunoglobulin (Ig) motifs. It was shown that the dephosphorylated peptide, but not the phosphorylated form, bound to both actin and myosin. The binding site on actin was within the sequence L10 to P29. This region also binds to loop 2 of the myosin head. The dephosphorylated peptide linked myosin and F-actin and formed a trimeric complex. Electron microscopy revealed that twitchin is distributed on the surface of the thick filament with an axial periodicity of 36.25 nm and it is suggested that the D2 site aligns with the myosin heads. It is proposed that the complex formed with the dephosphorylated D2 site of twitchin, F-actin and myosin represents a component of the mechanical linkage in catch.
• Lubomirov, L. T., Reimann, K., Metzler, D., Hasse, V., Stehle, R., Ito, M., Hartshorne, D. J., Gagov, H., Pfitzer, G., & Schubert, R. (2006). Urocortin-induced decrease in Ca²⁺ sensitivity of contraction in mouse tail arteries is attributable to cAMP-dependent dephosphorylation of MYPT1 and activation of myosin light chain phosphatase. Circulation Research, 98(9), 1159-1167.
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  PMID: 16574904;Abstract: Urocortin, a vasodilatory peptide related to corticotropin-releasing factor, may be an endogenous regulator of blood pressure. In vitro, rat tail arteries are relaxed by urocortin by a cAMP-mediated decrease in myofilament Ca sensitivity through a still unclear mechanism. Here we show that contraction of intact mouse tail arteries induced with 42 mmol/L KCl or 0.5 μmol/L noradrenaline was associated with a ≈2-fold increase in the phosphorylation of the regulatory subunit of myosin phosphatase (SMPP-1M), MYPT1, at Thr696, which was reversed in arteries relaxed with urocortin. Submaximally (pCa 6.1) contracted mouse tail arteries permeabilized with α-toxin were relaxed with urocortin by 39±3% at constant [Ca], which was associated with a decrease in myosin light chain (MLC20), MYPT1, and MYPT1 phosphorylation by 60%, 28%, and 52%, respectively. The Rho-associated kinase (ROK) inhibitor Y-27632 decreased MYPT1 phosphorylation by a similar extent. Inhibition of PP-2A with 3 nmol/L okadaic acid had no effect on MYPT1 phosphorylation, whereas inhibition of PP-1 with 3 μmol/L okadaic acid prevented dephosphorylation. Urocortin increased the rate of dephosphorylation of MLC20 ≈2.2-fold but had no effect on the rate of contraction under conditions of, respectively, inhibited kinase and phosphatase activities. The effect of urocortin on MLC20 and MYPT1 phosphorylation was blocked by Rp-8-CPT-cAMPS and mimicked by Sp-5,6-DCl-cBIMPS. In summary, these results provide evidence that Ca-independent relaxation by urocortin can be attributed to a cAMP-mediated increased activity of SMPP-1M which at least in part is attributable to a decrease in the inhibitory phosphorylation of MYPT1. © 2006 American Heart Association, Inc.
• Okamoto, R., Kato, T., Mizoguchi, A., Takahashi, N., Nakakuki, T., Mizutani, H., Isaka, N., Imanaka-Yoshida, K., Kaibuchi, K., Zhaojiang, L. u., Mabuchi, K., Tao, T., Hartshorne, D. J., Nakano, T., & Ito, M. (2006). Characterization and function of MYPT2, a target subunit of myosin phosphatase in heart. Cellular Signalling, 18(9), 1408-1416.
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  PMID: 16431080;Abstract: Characterization of cardiac MYPT2 (an isoform of the smooth muscle phosphatase [MP] target subunit, MYPT1) is described. Several features of MYPT2 and MYPT1 were similar, including: a specific interaction with the catalytic subunit of type 1 phosphatase, δ isoform (PP1cδ); interaction of MYPT2 with the small heart-specific MP subunit; interaction of the C-terminal region of MYPT2 with the active form of RhoA; phosphorylation by Rho-kinase at an inhibitory site, Thr646 and thiophosphorylation at Thr646 inhibited activity of the MYPT2-PP1cδ complex. MYPT2 activated PP1cδ activity, using light chains from smooth and cardiac muscle, by reducing Km and increasing kcat. The extent of activation (kcat) was greater than for MYPT1 and could reflect distinct N-terminal sequences in the two MYPT isoforms. Adenovirus-mediated gene transfer of MYPT2 and PP1cδ reduced the phosphorylation level of cardiac light chains following stimulation with A23187. Overexpression of MYPT2 and PP1cδ blocked the angiotensin II-induced sarcomere organization in cultured cardiomyocytes. Electron microscopy indicated locations of MYPTs, at, or close to, the Z-line, the A band and mitochondria. Similarity of the two MYPT isoforms suggests common enzymatic mechanisms and regulation. Cardiac myosin is a substrate for the MYPT2 holoenzyme, but the Z-line location raises the possibility of other substrates. © 2005 Elsevier Inc. All rights reserved.
• Funabara, D., Kanoh, S., Siegman, M. J., Butler, T. M., Hartshorne, D. J., & Watabe, S. (2005). Twitchin as a regulator of catch contraction in molluscan smooth muscle. Journal of Muscle Research and Cell Motility, 26(6-8), 455-460.
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  PMID: 16453161;PMCID: PMC1483069;Abstract: Molluscan catch muscle can maintain tension for a long time with little energy consumption. This unique phenomenon is regulated by phosphorylation and dephosphorylation of twitchin, a member of the titin/connectin family. The catch state is induced by a decrease of intracellular Ca2+ after the active contraction and is terminated by the phosphorylation of twitchin by the cAMP-dependent protein kinase (PKA). Twitchin, from the well-known catch muscle, the anterior byssus retractor muscle (ABRM) of the mollusc Mytilus, incorporates three phosphates into two major sites D1 and D2, and some minor sites. Dephosphorylation is required for re-entering the catch state. Myosin, actin and twitchin are essential players in the mechanism responsible for catch during which force is maintained while myosin cross-bridge cycling is very slow. Dephosphorylation of twitchin allows it to bind to F-actin, whereas phosphorylation decreases the affinity of the two proteins. Twitchin has been also been shown to be a thick filament-binding protein. These findings raise the possibility that twitchin regulates the myosin cross-bridge cycle and force output by interacting with both actin and myosin resulting in a structure that connects thick and thin filaments in a phosphorylation-dependent manner. © Springer 2006.
• Lontay, B., Kiss, A., Gergely, P., Hartshorne, D. J., & Erdodi, F. (2005). Okadaic acid induces phosphorylation and translocation of myosin phosphatase target subunit 1 influencing myosin phosphorylation, stress fiber assembly and cell migration in HepG2 cells. Cellular Signalling, 17(10), 1265-1275.
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  PMID: 16038801;Abstract: It was determined that the myosin phosphatase (MP) activity and content of myosin phosphatase target subunit 1 (MYPT1) were correlated in subcellular fractions of human hepatocarcinoma (HepG2) cells. In control cells MYPT1 was localized in the cytoplasm and in the nucleus, as determined by confocal microscopy. Treatment of HepG2 cells with 50 nM okadaic acid (OA), a cell-permeable phosphatase inhibitor, induced several changes: 1) a marked redistribution of MYPT1 to the plasma membrane associated with an increased level of phosphorylation of MYPT1 at Thr695. Both effects showed only a slight influence with the Rho-kinase inhibitor, Y-27632; 2) an increase in phosphorylation of MYPT1 at Thr850 associated with its accumulation in the perinuclear region and nucleus. These effects were markedly reduced by Y-27632; 3) an increased phosphorylation of the 20 kDa myosin II light chain at Ser19 associated with an increased location of myosin II at the cell center. These effects were partially counteracted by Y-27632; 4) an increase in stress fiber formation and a decrease in cell migration, both OA-induced effects were blocked by Y-27632. In HepG2 lysates, OA (5-100 nM) did not affect MP activity but inhibited PP2A activity. These results indicate that OA induces differential phosphorylation and translocation of MYPT1, dependent on PP2A and, to varying extents, on ROK. These changes are associated with an increased level of myosin II phosphorylation and attenuation of hepatic cell migration. © 2005 Elsevier Inc. All rights reserved.
• Murányi, A., Derkach, D., Erdodi, F., Kiss, A., Ito, M., & Hartshorne, D. J. (2005). Phosphorylation of Thr695 and Thr850 on the myosin phosphatase target subunit: Inhibitory effects and occurrence in A7r5 cells. FEBS Letters, 579(29), 6611-6615.
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  PMID: 16297917;Abstract: Major sites for Rho-kinase on the myosin phosphatase target subunit (MYPT1) are Thr695 and Thr850. Phosphorylation of Thr695 inhibits phosphatase activity but the role of phosphorylation at Thr850 is not clear and is evaluated here. Phosphorylation of both Thr695 and Thr850 by Rho-kinase inhibited activity of the type 1 phosphatase catalytic subunit. Rates of phosphorylation of the two sites were similar and efficacy of inhibition following phosphorylation was equivalent for each site. Phosphorylation of each site on MYPT1 was detected in A7r5 cells, but Thr850 was preferred by Rho-kinase and Thr695 was phosphorylated by an unidentified kinase(s). © 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
• Okamoto, R., Ito, M., Suzuki, N., Kongo, M., Moriki, N., Saito, H., Tsumura, H., Imanaka-Yoshida, K., Kimura, K., Mizoguchi, A., Hartshorne, D. J., & Nakano, T. (2005). The targeted disruption of the MYPT1 gene results in embryonic lethality. Transgenic Research, 14(3), 337-340.
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  PMID: 16145842;Abstract: Myosin phosphatase (MP) is a major phosphatase responsible for the dephosphorylation of the regulatory light chain of myosin II. MYPT1, a target subunit of smooth and nonmuscle MP, is responsible for activation and regulation of MP. To identity the physiological roles of MP, we have generated MYPT1-deficient mice by gene targeting. The heterozygous mice showed no changes in expression levels of MYPT1 and no distinct phenotype compared to wild-type mice was observed. None of the F2 mice were homozygous for the MYPT1 deletion, indicating that the targeted disruption of the MYPT1 gene resulted in embryonic lethality. The point of embryonic lethality is before 7.5 dpc. These findings indicate that MYPT1 is essential for mouse embryogenesis. © Springer 2005.
• Yue, W. u., Murányi, A., Erdodi, F., & Hartshorne, D. J. (2005). Localization of myosin phosphatase target subunit and its mutants. Journal of Muscle Research and Cell Motility, 26(2), 123-134.
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  PMID: 15999227;Abstract: Transient transfection of NIH3T3 cells with various constructs of myosin phosphatase target subunit (MYPT1) and GFP showed distinct cellular localizations. Constructs containing the N-terminal nuclear localization signals (NLS), i.e. full-length MYPT1 and N-terminal MYPT1 fragments, were concentrated in the nucleus. Full-length chicken and human MYPT1-GFP showed discrete nuclear foci. Deletion of the N-terminal NLS or use of central or C-terminal MYPT1 fragments did not show unique nuclear distributions (C-terminal NLS are present). Transient transfection of NIH3T3 cells (in the presence of serum) with full-length MYPT1-GFP caused a marked decrease in number of attached cells, an apparent block in the cell cycle prior to M phase and signs of increased apoptosis. Under conditions of serum starvation the unique nuclear localization of MYPT1-GFP was not found and there was no marked decrease in the number of attached cells (after 48 h). Stable transfection of HEK 293 cells with GFP-MYPT1 was obtained. MYPT1 and its N-terminal mutants bound to retinoblastoma protein (Rb), raising the possibility that Rb is implicated in the effects caused by overexpression of MYPT1. © Springer 2005.
• Hartshorne, D. J., Ito, M., & Erdödi, F. (2004). Role of protein phosphatase type 1 in contractile functions: Myosin phosphatase. Journal of Biological Chemistry, 279(36), 37211-37214.
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  PMID: 15136561;
• Ito, M., Nakano, T., Erdodi, F., & Hartshorne, D. J. (2004). Myosin phosphatase: Structure, regulation and function. Molecular and Cellular Biochemistry, 259(1-2), 197-209.
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  PMID: 15124925;Abstract: Phosphorylation of myosin II plays an important role in many cell functions, including smooth muscle contraction. The level of myosin II phosphorylation is determined by activities of myosin light chain kinase and myosin phosphatase (MP). MP is composed of 3 subunits: a catalytic subunit of type 1 phosphatase, PP1c; a targeting subunit, termed myosin phosphatase target subunit, MYPT; and a smaller subunit, M20, of unknown function. Most of the properties of MP are due to MYPT and include binding of PP1c and substrate. Other interactions are discussed. A recent discovery is the existence of an MYPT family and members include, MYPT1, MYPT2, MBS85, MYPT3 and TIMAP. Characteristics of each are outlined. An important discovery was that the activity of MP could be regulated and both activation and inhibition were reported. Activation occurs in response to elevated cyclic nucleotide levels and various mechanisms are presented. Inhibition of MP is a major component of Ca2+-sensitization in smooth muscle and various molecular mechanisms are discussed. Two mechanisms are cited frequently: (1) Phosphorylation of an inhibitory site on MYPT1, Thr696 (human isoform) and resulting inhibition of PP1c activity. Several kinases can phosphorylate Thr696, including Rho-kinase that serves an important role in smooth muscle function; and (2) Inhibition of MP by the protein kinase C-potentiated inhibitor protein of 17 kDa (CPI-17). Examples where these mechanisms are implicated in smooth muscle function are presented. The critical role of RhoA/Rho-kinase signaling in various systems is discussed, in particular those vascular smooth muscle disorders involving hypercontractility. © 2004 Kluwer Academic Publishers.
• Lontay, B., Serfozo, Z., Gergely, P., Ito, M., Hartshorne, D. J., & Erdodi, F. (2004). Localization of myosin phosphatase target subunit 1 in rat brain and in primary cultures of neuronal cells. Journal of Comparative Neurology, 478(1), 72-87.
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  PMID: 15334650;Abstract: Myosin phosphatase (PP1M) is composed of the δ isoform of the PP1 catalytic subunit (PP1cδ), the myosin phosphatase target subunit (MYPT), and a 20 kDa subunit. Western blots detected higher amounts of the MYPT1 isoform compared to MYPT2 in whole brain extracts. The localization of MYPT1 was studied in rat brain and in primary cell cultures of neurons using specific antibodies. Analysis of lysates of brain regions for MYPT1 and PP1M by Western blots using anti-MYPT1 antibodies and by phosphatase assays with myosin as substrate suggested a ubiquitous distribution. Immunohistochemistry of tissue sections revealed that MYPT1 was distributed in all areas of the brain, with staining observed in many different cell types. Depending on the method used for fixation, the MYPT1 appeared with varying intensity in nuclei, in nucleoli, and in the cytoplasm. In primary hippocampal cultures, MYPT1 was identified by confocal microscopy in the cytoplasm and in the nucleus, whereas a predominantly cytoplasmic localization was found in cochlear nucleus cells. In cultured cells, MYPT1 and PP1cδ colocalized with synaptophysin. PP1M activity was high in synaptosomes isolated from the cerebral cortex, but was relatively low in the postsynaptic densities. The interaction of MYPT1 with synaptophysin and with known partners (Rho-kinase, PP1cδ) in brain extracts was shown by immunoprecipitation with anti-MYPT1. Pull-down assays from synaptosomes, using GST-MYPT1, also confirmed these interactions. In conclusion, the widespread cellular and subcellular localization of MYPT1 implies that PP1M may play an important role in the dephosphorylation of key regulatory proteins in neuronal cells. © 2004 Wiley-Liss, Inc.
• Totsukawa, G., Yue, W. u., Sasaki, Y., Hartshorne, D. J., Yamakita, Y., Yamashiro, S., & Matsumura, F. (2004). Distinct roles of MLCK and ROCK in the regulation of membrane protrusions and focal adhesion dynamics during cell migration of fibroblasts. Journal of Cell Biology, 164(3), 427-439.
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  PMID: 14757754;PMCID: PMC2172229;Abstract: We examined the role of regulatory myosin light chain (MLC) phosphorylation of myosin II in cell migration of fibroblasts. Myosin light chain kinase (MLCK) inhibition blocked MLC phosphorylation at the cell periphery, but not in the center. MLCK-inhibited cells did not assemble zyxin-containing adhesions at the periphery, but maintained focal adhesions in the center. They generated membrane protrusions all around the cell, turned more frequently, and migrated less effectively. In contrast, Rho-associated kinase (ROCK) inhibition blocked MLC phosphorylation in the center, but not at the periphery. ROCK-inhibited cells assembled zyxin-containing adhesions at the periphery, but not focal adhesions in the center. They moved faster and more straight. On the other hand, inhibition of myosin phosphatase increased MLC phosphorylation and blocked peripheral membrane ruffling, as well as turnover of focal adhesions and cell migration. Our results suggest that myosin II activated by MLCK at the cell periphery controls membrane ruffling, and that the spatial regulation of MLC phosphorylation plays critical roles in controlling cell migration of fibroblasts.
• Wooldridge, A. A., MacDonald, J. A., Erdodi, F., Chaoyu, M. a., Borman, M. A., Hartshorne, D. J., & A., T. (2004). Smooth muscle phosphatase is regulated in Vivo by exclusion of phosphorylation of threonine 696 of MYPT1 by phosphorylation of serine 695 in response to cyclic nucleotides. Journal of Biological Chemistry, 279(33), 34496-34504.
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  PMID: 15194681;Abstract: Regulation of smooth muscle myosin phosphatase (SMPP-1M) is thought to be a primary mechanism for explaining Ca2+ sensitization/desensitization in smooth muscle. Ca2+ sensitization induced by activation of G protein-coupled receptors acting through RhoA involves phosphorylation of Thr-696 (of the human isoform) of the myosin targeting subunit (MYPT1) of SMPP-1M inhibiting activity. In contrast, agonists that elevate intracellular cGMP and cAMP promote Ca2+ desensitization in smooth muscle through apparent activation of SMPP-1M. We show that cGMP-dependent protein kinase (PKG)/cAMP-dependent protein kinase (PKA) efficiently phosphorylates MYPT1 in vitro at Ser-692, Ser-695, and Ser-852 (numbering for human isoform). Although phosphorylation of MYPT1 by PKA/PKG has no direct effect on SMPP-1M activity, a primary site of phosphorylation is Ser-695, which is immediately adjacent to the inactivating Thr-696. In vitro, phosphorylation of Ser-695 by PKA/PKG appeared to prevent phosphorylation of Thr-696 by MYPT1K. In ileum smooth muscle, Ser-695 showed a 3-fold increase in phosphorylation in response to 8-bromo-cGMP. Addition of constitutively active recombinant MYPT1K to permeabilized smooth muscles caused phosphorylation of Thr-696 and Ca2+ sensitization; however, this phosphorylation was blocked by preincubation with 8-bromo-cGMP. These findings suggest a mechanism of Ca2+ desensitization in smooth muscle that involves mutual exclusion of phosphorylation, whereby phosphorylation of Ser-695 prevents phosphorylation of Thr-696 and therefore inhibition of SMPP-1M.
• Erdodi, F., Kiss, E., Walsh, M. P., Stefansson, B., Deng, J. T., Eto, M., Brautigan, D. L., & Hartshorne, D. J. (2003). Phosphorylation of protein phosphatase type-1 inhibitory proteins by integrin-linked kinase and cyclic nucleotide-dependent protein kinases. Biochemical and Biophysical Research Communications, 306(2), 382-387.
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  PMID: 12804574;Abstract: Protein phosphatases play key roles in cellular regulation and are subjected to control by protein inhibitors whose activity is in turn regulated by phosphorylation. Here we investigated the possible regulation of phosphorylation-dependent type-1 protein phosphatase (PP1) inhibitors, CPI-17, PHI-1, and KEPI, by various kinases. Protein kinases A (PKA) and G (PKG) phosphorylated CPI-17 at the inhibitory site (T38), but not PHI-1 (T57). Phosphorylated CPI-17 inhibited the activity of both the PP1 catalytic subunit (PP1c) and the myosin phosphatase holoenzyme (MPH) with IC50 values of 1-8nM. PKA predominantly phosphorylated a site distinct from the inhibitory T73 in KEPI, whereas PKG was ineffective. Integrin-linked kinase phosphorylated KEPI (T73) and this dramatically increased inhibition of PP1c (IC50=0.1nM) and MPH (IC50=8nM). These results suggest that the regulatory phosphorylation of CPI-17 and KEPI may involve distinct kinases and signaling pathways. © 2003 Elsevier Science (USA). All rights reserved.
• Funabara, D., Watabe, S., Mooers, S. U., Narayan, S., Dudas, C., Hartshorne, D. J., Siegman, M. J., & Butler, T. M. (2003). Twitchin from molluscan catch muscle. Primary structure and relationship between site-specific phosphorylation and mechanical function. Journal of Biological Chemistry, 278(31), 29308-29316.
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  PMID: 12756258;Abstract: The phosphorylation state of the myosin thick filament-associated mini-titin, twitchin, regulates catch force maintenance in molluscan smooth muscle. The full-length cDNA for twitchin from the anterior byssus retractor muscle of the mussel Mytilus was obtained using PCR and 5′ rapid amplification of cDNA ends, and its derived amino acid sequence showed a large molecule (∼530 kDa) with a motif arrangement as follows: (Ig)11(IgFn2)2Ig(Fn)3 Ig(Fn)2Ig(Fn)3(Ig)2(Fn)2 (Ig)2 FnKinase(Ig)4. Other regions of note include a 79-residue sequence between 0Ig domains 6 and 7 (from the N terminus) in which more than 60% of the residues are Pro, Glu, Val, or Lys and between the 7th and 8th Ig domains, a DFRXXL motif similar to that thought to be necessary for high affinity binding of myosin light chain kinase to F-actin. Two major phosphorylation sites, i.e. D1 and D2, were located in linker regions between Ig domains 7 and 8 and Ig domains 21 and 22, respectively. Correlation of the phosphorylation state of twitchin, using antibodies specific to D1 and D2, with mechanical properties suggested that phosphorylation of both D1 and D2 is required for relaxation from the catch state.
• Seko, T., Ito, M., Kureishi, Y., Okamoto, R., Moriki, N., Onishi, K., Isaka, N., Hartshorne, D. J., & Nakano, T. (2003). Activation of RhoA and inhibition of myosin phosphatase as important components in hypertension in vascular smooth muscle. Circulation Research, 92(4), 411-418.
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  PMID: 12600888;Abstract: Two mechanisms are proposed to account for the inhibition of myosin phosphatase (MP) involved in Ca2+ sensitization of vascular muscle, ie, phosphorylation of either MYPT1, a target subunit of MP or CPI-17, an inhibitory phosphoprotein. In cultured vascular aorta smooth muscle cells (VSMCs), stimulation with angiotensin II activated RhoA, and this was blocked by pretreatment with 8-bromo-cGMP. VSMCs stimulated by angiotensin II, endothelin-1, or U-46619 significantly increased the phosphorylation levels of both MYPT1 (at Thr696) and CPI-17 (at Thr38). The angiotensin II-induced phosphorylation of MYPT1 was completely blocked by 8-bromo-cGMP or Y-27632 (a Rho-kinase inhibitor), but not by GF109203X (a PKC inhibitor). In contrast, phosphorylation of CPI-17 was inhibited only by GF 109203X. Y-27632 dramatically corrected the hypertension in Nω-nitro-L-arginine methyl ester (L-NAME)-treated rats, and this hypertension also was sensitive to isosorbide mononitrate. The level of the active form of RhoA was significantly higher in aortas from L-NAME-treated rats. Expression of RhoA, Rho-kinase, MYPT1, CPI-17, and myosin light chain kinase were not significantly different in aortas from L-NAME-treated and control rats. Activation of RhoA without changes in levels of other signaling molecules were observed in three other rat models of hypertension, ie, stroke-prone spontaneously hypertensive rats, renal hypertensive rats, and DOCA-salt rats. These results suggest that independent of the cause of hypertension, a common point in downstream signaling and a critical component of hypertension is activation of RhoA and subsequent activation of Rho-kinase.
• Yue, W. U., Erdodi, F., Murányi, A., Nullmeyer, K. D., Lynch, R. M., & Hartshorne, D. J. (2003). Myosin phosphatase and myosin phosphorylation in differentiating C2C12 cells. Journal of Muscle Research and Cell Motility, 24(8), 499-511.
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  PMID: 14870965;Abstract: C2C12 cells offer a useful model to study the differentiation of non-muscle cells to skeletal muscle cells. Myosin phosphorylation and changes in related enzymes, with an emphasis on myosin phosphatase (MP) were analyzed over the first 6 days of C2C12 differentiation. There was a transition from myosin phosphatase target subunit 1 (MYPT1), predominant in the non-muscle cells to increased expression of MYPT2. Levels of MYPT1/2 were estimated, and both isoforms were higher in non- or partially differentiated cells compared to the concentrations in the differentiated isolated myotubes from day 6. A similar profile of expression was estimated for the type 1 protein phosphatase catalytic subunit, δ isoform (PPlcδ). Phosphatase activities, using phosphorylated smooth and skeletal muscle myosins, were estimated for total cell lysates and isolated myotubes. In general, smooth muscle myosin was the preferred substrate. Although the expression of MYPT1/2 and PPlcδ was considerably reduced in isolated myotubes the phosphatase activities were not reduced to corresponding levels. Most of the MP activity was due to PPlc, as indicated by okadaic acid. In spite of relatively high expression of MYPT1/2 and PPlcδ, marked phosphorylation of non-muscle myosin (over 50% of total myosin) was observed at day 2 (onset of expression of muscle-specific proteins) and both mono- and diphosphorylated light chains were observed. Partial inhibition of MLCK by 1-(5-chloronaphthalene-1-sulphonyl)-1H-hexahydro-1,4- diazepine HCl (ML-9) or by a construct designed from the autoinhibitory domain of MLCK, resulted in an increase in small myotubes (3-5 nuclei) after 3 days of differentiation and a decrease in larger myotubes (compared to control). The effect of ML-9 was not due to a reduction in intracellular Ca2+ levels. These results suggest that phosphorylation of non-muscle myosin is important in growth of myotubes, either in the fusion process to form larger myotubes or indirectly, by its role in sarcomere organization.
• Borman, M. A., MacDonald, J. A., Murányi, A., Hartshorne, D. J., A., T., Pharmacology, D. O., Biology, C., Group, M. B., & Arizona, U. O. (2002). Smooth muscle myosin phosphatase-associated kinase induces Ca²⁺ sensitization via myosin phosphatase inhibition. Journal of Biological Chemistry, 277(26), 23441-23446.
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  PMID: 11976330;Abstract: Smooth muscle calcium sensitization reflects an inhibition of myosin light chain phosphatase (SMPP-1M) activity; however, the underlying mechanisms are not well understood. SMPP-1M activity can be modulated through phosphorylation of the myosin targeting subunit (MYPT1) by the endogenous myosin phosphatase-associated kinase, MYPT1 kinase (MacDonald, J. A., Borman, M. A., Muranyi, A., Somlyo, A. V., Hartshorne, D. J., and Haystead, T. A. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 2419-2424). Recombinant chicken gizzard MYPT1 (M130) was phosphorylated in vitro by a recombinant MYPT1 kinase, and the sites of phosphorylation were identified as Thr654, Ser808, and Thr675. Introduction of recombinant MYPT1 kinase elicited a calcium-independent contraction in β-escin-permeabilized rabbit ileal smooth muscle. Using an antibody that specifically recognizes MYPT1 phosphorylated at Thr654 (M130 numbering), we determined that this calcium-independent contraction was correlated with an increase in MYPT1 phosphorylation. These results indicate that SMPP-1M phosphorylation by MYPT1 kinase is a mechanism of smooth muscle calcium sensitization.
• Kiss, E., Murányi, A., Csortos, C., Gergely, P., Ito, M., Hartshorne, D. J., & Erdodi, F. (2002). Integrin-linked kinase phosphorylates the myosin phosphatase target subunit at the inhibitory site in platelet cytoskeleton. Biochemical Journal, 365(1), 79-87.
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  PMID: 11931630;PMCID: PMC1222641;Abstract: The myosin phosphatase (MP) composed of the catalytic subunit of type 1 protein phosphatase and myosin phosphatase target subunit isoform 1 (MYPT1) was identified as the major serine/threonine phosphatase component in the platelet-cytoskeleton fraction. MYPT1 was phosphorylated by cytoskeletal kinase(s), but the identity of the kinase(s) and the effect of phosphorylation were not established. Incubation of platelet-cytoskeletal fraction with MgATP or MgATP[S] (magnesium adenosine 5′-[γ-thio]triphosphate) caused a decrease in the 20 kDa light-chain of smooth-muscle myosin (MLC20) phosphatase and phosphorylase phosphatase activities. MYPT1 contains a phosphorylation site, Thr-695, involved in the inhibition of MP in a RhoA/Rho kinase-dependent manner. The cytoskeletal kinase(s) phosphorylated Thr-695 of glutathione S-transferase (GST)-MYPT1, as determined with an antibody specific for phosphorylated Thr-695. The level of Rho kinase was low in the cytoskeletal fraction and was detected primarily in the membrane and cytosolic fractions. The phosphorylation of Thr-695 by the cytoskeletal kinase(s) was nof affected by Rho kinase inhibitor, Y-27632, suggesting that kinase(s) other than Rho kinase were involved. In-gel kinase assay identified a kinase at 54-59 kDa that phosphorylated the C-terminal fragment of MYPT1 (GST-MYPT1667-1004). Western blots detected both zipper-interacting protein kinase (ZIPK) and integrin-linked kinase (ILK) at 54-59 kDa in the cytoskeleton and membrane fractions. Cytoskeletal ZIPK and ILK were separated and partially purified by chromatography on SP-Sepharose and on MonoQ. ZIPK preferentially phosphorylated MLC20 and had low activity on MYPT1. ILK phosphorylated both MLC20 and MYPT1 and phosphorylation of MYPT1 occured on Thr-695. The above results raise the potential for regulation of MP activity in platelet cytoskeleton by ILK and suggest an alternative to the Rho-linked pathway.
• Murányi, A., MacDonald, J. A., Deng, J. T., Wilson, D. P., A., T., Walsh, M. P., Erdödi, F., Kiss, E., Yue, W. u., & Hartshorne, D. J. (2002). Phosphorylation of the myosin phosphatase target subunit by integrin-linked kinase. Biochemical Journal, 366(1), 211-216.
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  PMID: 12030846;PMCID: PMC1222775;Abstract: A mechanism proposed for regulation of myosin phosphatase (MP) activity is phosphorylation of the myosin phosphatase target subunit (MYPT1). Integrin-linked kinase (ILK) is associated with the contractile machinery and can phosphorylate myosin at the myosin light-chain kinase sites. The possibility that ILK may also phosphorylate and regulate MP was investigated. ILK was associated with the MP holoenzyme, shown by Western blots and in-gel kinase assays. MYPT1 was phosphorylated by ILK and phosphorylation sites in the N- and C-terminal fragments of MYPT1 were detected. From sequence analyses, three sites were identified: a primary site at Thr709, and two other sites at Thr695 and Thr495. One of the sites for cAMP-dependent protein kinase (PKA) was Ser694. Assays with the catalytic subunit of type 1 phosphatase indicated that only the C-terminal fragment of MYPT1 phosphorylated by zipper-interacting protein kinase, and ILK inhibited activity. The phosphorylated N-terminal fragment activated phosphatase activity and phosphorylation by PKA was without effect. Using full-length MYPT1 constructs phosphorylated by various kinases it was shown that Rho kinase gave marked inhibition; ILK produced an intermediate level of inhibition, which was considerably reduced for the Thr695 → Ala mutant; and PKA had no effect. In summary, phosphorylation of the various sites indicated that Thr695 was the major inhibitory site, Thr709 had only a slight inhibitory effect and Ser694 had no effect. The findings that ILK phosphorylated both MYPT1 and myosin and the association of ILK with MP suggest that ILK may influence cytoskeletal structure or function.
• Shin, H., Je, H., Gallant, C., Tao, T. C., Hartshorne, D. J., Ito, M., & Morgan, K. G. (2002). Differential association and localization of myosin phosphatase subunits during agonist-induced signal transduction in smooth muscle. Circulation Research, 90(5), 546-553.
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  PMID: 11909818;Abstract: It has been known for some time that agonist-induced contractions of vascular smooth muscle are often associated with a sensitization of the contractile apparatus to intracellular Ca2+. One mechanism that has been suggested to explain Ca2+ sensitization is inhibition of myosin phosphatase activity. In the present study, we tested the hypothesis that differential localization of the phosphatase might be associated with its inhibition. Quantitative confocal microscopy of freshly dissociated, fully contractile smooth muscle cells was used in parallel with measurements of myosin light chain and myosin phosphatase phosphorylation. The results indicate that, in the smooth muscle cells, the catalytic and targeting subunits of the phosphatase are dissociated from each other in an agonist-specific manner and that the dissociation is accompanied by a slower rate of myosin phosphorylation. Targeting of myosin phosphatase to the cell membrane precedes the dissociation of subunits and is associated with phosphorylation of the targeting subunit at a Rho-associated kinase (ROK) phosphorylation site. The phosphorylation and membrane translocation of the targeting subunit are inhibited by a ROK inhibitor. This dissociation of subunits may provide a mechanism for the decreased phosphatase activity of phosphorylated myosin phosphatase.
• Araki, S., Ito, M., Kureishi, Y., Feng, J., Machida, H., Isaka, N., Amano, M., Kaibuchi, K., Hartshorne, D. J., & Nakano, T. (2001). Arachidonic acid-induced Ca²⁺ sensitization of smooth muscle contraction through activation of Rho-kinase. Pflugers Archiv European Journal of Physiology, 441(5), 596-603.
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  PMID: 11294240;Abstract: Arachidonic acid activates isolated Rho-kinase and contracts permeabilized smooth muscle fibres. Various assays were carried out to examine the mechanism of this activation. Native Rho-kinase was activated 5-6 times by arachidonic acid but an N-terminal, constitutively-active fragment of Rho-kinase, expressed as a glutathione-S-transferase (GST) fusion protein and including the catalytic subunit (GST-Rho-kinase-CAT), was not. GST-Rho-kinase-CAT was inhibited by a C-terminal fragment of Rho-kinase and arachidonic acid removed this inhibition. These results suggest that the C-terminal part of Rho-kinase, containing the RhoA binding site and the pleckstrin homology domain, acts as an autoinhibitor. It is suggested further that activation by arachidonic acid is due to its binding to the autoinhibitory region and subsequent release from the catalytic site. Arachidonic acid, at concentrations greater than 30 μM, increases force in α-toxin-permeabilized femoral artery but not in Triton X-100-skinned fibres. The content of Rho-kinase in the latter was lower than in α-toxin-treated or intact fibres. The arachidonic acid-induced contraction was not observed at a pCa above 8.0 and was inhibited by Y-27632 and wortmannin, inhibitors of Rho-kinase and myosin light-chain kinase (MLCK), respectively. The activation of Rho-kinase and subsequent phosphorylation of the myosin phosphatase target sub-unit inhibits myosin phosphatase and increases myosin phosphorylation.
• Butler, T. M., Narayan, S. R., Mooers, S. U., Hartshorne, D. J., & Siegman, M. J. (2001). The myosin cross-bridge cycle and its control by twitchin phosphorylation in catch muscle. Biophysical Journal, 80(1), 415-426.
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  PMID: 11159412;PMCID: PMC1301243;Abstract: The anterior byssus retractor muscle of Mytilus edulis was used to characterize the myosin cross-bridge during catch, a state of tonic force maintenance with a very low rate of energy utilization. Addition of MgATP to permeabilized muscles in high force rigor at pCa > 8 results in a rapid loss of some force followed by a very slow rate of relaxation that is characteristic of catch. The fast component is slowed 3-4-fold in the presence of 1 mM MgADP, but the distribution between the fast and slow (catch) components is not dependent on [MgADP]. Phosphorylation of twitchin results in loss of the catch component. Fewer than 4% of the myosin heads have ADP bound in rigor, and the time course (0.2-10 s) of ADP formation following release of ATP from caged ATP is similar whether or not twitchin is phosphorylated. This suggests that MgATP binding to the cross-bridge and subsequent splitting are independent of twitchin phosphorylation, but detachment occurs only if twitchin is phosphorylated. A similar dependence of detachment on twitchin phosphorylation is seen with AMP-PNP and ATPγS. Single turnover experiments on bound ADP suggest an increase in the rate of release of ADP from the cross-bridge when catch is released by phosphorylation of twitchin. Low [Ca2+] and unphosphorylated twitchin appear to cause catch by 1) markedly slowing ADP release from attached cross-bridges and 2) preventing detachment following ATP binding to the rigor cross-bridge.
• Funabara, D., Kinoshita, S., Watabe, S., Siegman, M. J., Butler, T. M., & Hartshorne, D. J. (2001). Phosphorylation of molluscan twitchin by the cAMP-dependent protein kinase. Biochemistry, 40(7), 2087-2095.
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  PMID: 11329277;Abstract: Catch in certain molluscan muscles is released by an increase in cAMP, and it was suggested that the target of cAMP-dependent protein kinase (PKA) is the high molecular weight protein twitchin [Siegman, M. J., Funabara, J., Kinoshita, S., Watabe, S., Hartshorne, D. J., and Butler, T. M. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 5384-5388]. This study was carried out to investigate the phosphorylation of twitchin by PKA. Twitchin was isolated from Mytilus catch muscles and was phosphorylated by PKA to a stoichiometry of about 3 mol of P/mol of twitchin. There was no evidence of twitchin autophosphorylation. Two phosphorylated peptides were isolated and sequenced, termed D1 and D2. Additional cDNA sequence for twitchin was obtained, and the D2 site was located at the C-terminal side of the putative kinase domain in a linker region between two immunoglobulin C2 repeats. Excess PKA substrates, e.g., D1 and D2, blocked the reduction in force on addition of cAMP, confirming the role for PKA in regulating catch. Papain proteolysis of 32P-labeled twitchin from permeabilized muscles showed that the D1 site represented about 50% of the 32P labeling. Proteolysis of in-situ twitchin with thermolysin suggested that the D1 and D2 sites were at the N- and C-terminal ends of the molecule, respectively. Thermolysin proteolysis also indicated that D1 and D2 were major sites of phosphorylation by PKA. The direct phosphorylation of twitchin by PKA is consistent with a regulatory role for twitchin in the catch mechanism and probably involves phosphorylation at the D1 and D2 sites.
• MacDonald, J. A., Borman, M. A., Murányi, A., Somlyo, A. V., Hartshorne, D. J., & A., T. (2001). Identification of the endogenous smooth muscle myosin phosphatase-associated kinase. Proceedings of the National Academy of Sciences of the United States of America, 98(5), 2419-2424.
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  PMID: 11226254;PMCID: PMC30153;Abstract: Ca2+ sensitization of smooth muscle contraction involves inhibition of myosin light chain phosphatase (SMPP-1M) and enhanced myosin light chain phosphorylation. Inhibition of SMPP-1M is modulated through phosphorylation of the myosin targeting subunit (MYPT1) by either Rho-associated kinase (ROK) or an unknown SMPP-1M-associated kinase. Activated ROK is predominantly membrane-associated and its putative substrate, SMPP-1M, is mainly myofibrillar-associated. This raises a conundrum about the mechanism of interaction between these enzymes. We present ZIP-like kinase, identified by "mixed-peptide" Edman sequencing after affinity purification, as the previously unidentified SMPP-1M-associated kinase. ZIP-like kinase was shown to associate with MYPT1 and phosphorylate the inhibitory site in intact smooth muscle. Phosphorylation of ZIP-like kinase was associated with an increase in kinase activity during carbachol stimulation, suggesting that the enzyme may be a terminal member of a Ca2+ sensitizing kinase cascade.
• Machida, H., Ito, M., Okamoto, R., Shiraki, K., Isaka, N., Hartshorne, D. J., & Nakano, T. (2001). Molecular cloning and analysis of the 5′-flanking region of the human MYPT1 gene. Biochimica et Biophysica Acta - Gene Structure and Expression, 1517(3), 424-429.
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  PMID: 11342221;Abstract: We have cloned and sequenced a 5 kb genomic fragment in the 5′-flanking region of the human myosin phosphatase target subunit 1. The transcription initiation site (+1) was 268 bp upstream from the translation start site. In this promoter there are no canonical TATA or CAAT box elements but there is a high GC-rich sequence. Basal promoter activity was due to the GC-rich region that contained one Sp1 transcription factor binding site, thus demonstrating that the MYPT1 gene is a housekeeping gene. Luciferase reporter assays showed the presence of two regions for positive elements and one for a negative element. © 2001 Elsevier Science B.V.
• Murányi, A., Zhang, R., Liu, F., Hirano, K., Ito, M., Epstein, H. F., & Hartshorne, D. J. (2001). Myotonic dystrophy protein kinase phosphorylates the myosin phosphatase targeting subunit and inhibits myosin phosphatase activity. FEBS Letters, 493(2-3), 80-84.
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  PMID: 11287000;Abstract: Myotonic dystrophy protein kinase (DMPK) and Rho-kinase are related. An important function of Rho-kinase is to phosphorylate the myosin-binding subunit of myosin phosphatase (MYPT1) and inhibit phosphatase activity. Experiments were carried out to determine if DMPK could function similarly. MYPT1 was phosphorylated by DMPK. The phosphorylation site(s) was in the C-terminal part of the molecule. DMPK was not inhibited by the Rho-kinase inhibitors, Y-27632 and HA-1077. Several approaches were taken to determine that a major site of phosphorylation was T654. Phosphorylation at T654 inhibited phosphatase activity. Thus both DMPK and Rho-kinase may regulate myosin II phosphorylation. © 2001 Federation of European Biochemical Societies.
• Yamawaki, K., Ito, M., Machida, H., Moriki, N., Okamoto, R., Isaka, N., Shimpo, H., Kohda, A., Okumura, K., Hartshorne, D. J., & Nakano, T. (2001). Identification of human CPI-17, an inhibitory phosphoprotein for myosin phosphatase. Biochemical and Biophysical Research Communications, 285(4), 1040-1045.
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  PMID: 11467857;Abstract: CPI-17 is a phosphorylation-dependent inhibitor of myosin phosphatase. cDNA clones encoding CPI-17 were isolated from a human aorta library. Overlapping clones indicated two isoforms: CPI-17α was 147 residues and mass of 16.7 kDa; CPI-17β (120 residues, mass 13.5 kDa) resulted from a deletion in the α-isoform of 27 residues, sequence 68-94. N-terminal 67 residues of all CPI-17 isoforms (human, porcine, rat and mouse) were highly conserved (for the human and porcine isoforms the identity was 91%). The presence of the two human isoforms was detected from cDNA sequences amplified by RT-PCR and by Western blots on human aorta. The cloned human CPI-17 gene indicated 4 coding exons and CPI-17β was an alternative splice variant due to deletion of the second exon. FISH analysis located the human CPI-17 gene on chromosome 19q13.1. © 2001 Academic Press.
• Hamaguchi, T., Ito, M., Feng, J., Seko, T., Koyama, M., Machida, H., Takase, K., Amano, M., Kaibuchi, K., Hartshorne, D. J., & Nakano, T. (2000). Phosphorylation of CPI-17, an inhibitor of myosin phosphatase, by protein kinase N. Biochemical and Biophysical Research Communications, 274(3), 825-830.
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  PMID: 10924361;Abstract: CPI-17 Is a phosphorylation-dependent inhibitory protein for smooth muscle myosin phosphate. Phosphorylation at Thr38, in vitro, by protein kinase C or Rho-kinase enhances the inhibitory potency toward myosin phosphatase. Phosphorylation of CPI-17 by protein kinase N (PKN), a fatty acid- and Rho- activated serine/threonine kinase, and its effect on smooth muscle myosin phosphatase activity were investigated. CPI-17 was phosphorylated by GST-PKN-CAT, a constitutively active GST-fusion fragment of PKN, to 1.46 mol of P/mol of CPI-17, in vitro. The K(m) value of CPI-17 for PKN was 0.96 μM. Phosphorylation of PKN dramatically increased the inhibitory effect of CPI-17 on myosin phosphatase activity. The major and inhibitory phosphorylation site was identified as Thr38 using a point mutant of CPI-17 and a phosphorylation - state specific antibody. Thus, CPI-17 is a substrate of PKN and might be involved in the Ca2+ sensitization of smooth muscle contraction as a downstream effector of Rho and/or arachidonic acid. (C) 2000 Academic Press.
• Koyama, M., Ito, M., Feng, J., Seko, T., Shiraki, K., Takase, K., Hartshorne, D. J., & Nakano, T. (2000). Phosphorylation of CPI-17, an inhibitory phosphoprotein of smooth muscle myosin phosphatase, by Rho-kinase. FEBS Letters, 475(3), 197-200.
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  PMID: 10869555;Abstract: Phosphorylation of CPI-17 by Rho-associated kinase (Rho-kinase) and its effect on myosin phosphatase (MP) activity were investigated. CPI-17 was phosphorylated by Rho-kinase to 0.92 mol of P/mol of CPI-17 in vitro. The inhibitory phosphorylation site was Thr38 (as reported previously) and was identified using a point mutant of CPI-17 and a phosphorylation state- specific antibody. Phosphorylation by Rho-kinase dramatically increased the inhibitory effect of CPI-17 on MP activity. Thus, CPI-17 as a substrate of Rho-kinase could be involved in the Ca2+ sensitization of smooth muscle contraction as a downstream effector of Rho-kinase. (C) 2000 Federation of European Biochemical Societies.
• Swärd, K., Dreja, K., Susnjar, M., Hellstrand, P., Hartshorne, D. J., & Walsh, M. P. (2000). Inhibition of Rho-associated kinase blocks agonist-induced Ca²⁺ sensitization of myosin phosphorylation and force in guinea-pig ileum. Journal of Physiology, 522(1), 33-49.
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  PMID: 10618150;PMCID: PMC2269742;Abstract: 1. Ca2+ sensitization of smooth muscle contraction involves the small GTPase RhoA, inhibition of myosin light chain phosphatase (MLCP) and enhanced myosin regulatory light chain (LC20) phosphorylation. A potential effector of RhoA is Rho-associated kinase (ROK). 2. The role of ROK in Ca2+ sensitization was investigated in guinea-pig ileum. 3. Contraction of permeabilized muscle strips induced by GTPγS at pCa 6.5 was inhibited by the kinase inhibitors Y-27632, HA1077 and H-7 with IC50 values that correlated with the known K1 values for inhibition of ROK. GTPγS also increased LC20 phosphorylation and this was prevented by HA1077. Contraction and LC20 phosphorylation elicited at pCa 5.75 were, however, unaffected by HA1077. 4. Pre-treatment of intact tissue strips with HA1077 abolished the tonic component of carbachol-induced contraction and the sustained elevation of LC20 phosphorylation, but had no effect on the transient or sustained increase in [Ca2+]1 induced by carbachol. 5. LC20 phosphorylation and contraction dynamics suggest that the ROK-mediated increase in LC20 phosphorylation is due to MLCP inhibition, not myosin light chain kinase activation. 6. In the absence of Ca2+, GTPγS stimulated 35S incorporation from [35S]ATPγS into the myosin targeting subunit of MLCP (MYPT). The enhanced thiophosphorylation was inhibited by HA1077. No thiophosphorylation of LC20 was detected. 7. These results indicate that ROK mediates agonist-induced increases in myosin phosphorylation and force by inhibiting MLCP activity through phosphorylation of MYPT. Under Ca2+-free conditions, ROK does not appear to phosphorylate LC20 in situ, in contrast to its ability to phosphorylate myosin in vitro. In particular, ROK activation is essential for the tonic phase of agonist-induced contraction.
• Totsukawa, G., Yamakita, Y., Yamashiro, S., Hartshorne, D. J., Sasaki, Y., & Matsumura, F. (2000). Distinct roles of ROCK (Rho-kinase) and MLCK in spatial regulation of MLC phosphorylation for assembly of stress fibers and focal adhesions in 3T3 fibroblasts. Journal of Cell Biology, 150(4), 797-806.
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  PMID: 10953004;PMCID: PMC2175273;Abstract: ROCK (Rho-kinase), an effector molecule of RhoA, phosphorylates the myosin binding subunit (MBS) of myosin phosphatase and inhibits the phosphatase activity. This inhibition increases phosphorylation of myosin light chain (MLC) of myosin II, which is suggested to induce RhoA-mediated assembly of stress fibers and focal adhesions. ROCK is also known to directly phosphorylate MLC in vitro; however, the physiological significance of this MLC kinase activity is unknown. It is also not clear whether MLC phosphorylation alone is sufficient for the assembly of stress fibers and focal adhesions. We have developed two reagents with opposing effects on myosin phosphatase. One is an antibody against MBS that is able to inhibit myosin phosphatase activity. The other is a truncation mutant of MBS that constitutively activates myosin phosphatase. Through microinjection of these two reagents followed by immunofluorescence with a specific antibody against phosphorylated MLC, we have found that MLC phosphorylation is both necessary and sufficient for the assembly of stress fibers and focal adhesions in 3T3 fibroblasts. The assembly of stress fibers in the center of cells requires ROCK activity in addition to the inhibition of myosin phosphatase, suggesting that ROCK not only inhibits myosin phosphatase but also phosphorylates MLC directly in the center of cells. At the cell periphery, on the other hand, MLCK but not ROCK appears to be the kinase responsible for phosphorylating MLC. These results suggest that ROCK and MLCK play distinct roles in spatial regulation of MLC phosphorylation.
• Tóth, A., Kiss, E., Gergely, P., Walsh, M. P., Hartshorne, D. J., & Erdödi, F. (2000). Phosphorylation of MYPT1 by protein kinase C attenuates interaction with PP1 catalytic subunit and the 20 kDa light chain of myosin. FEBS Letters, 484(2), 113-117.
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  PMID: 11068043;Abstract: The effect of phosphorylation in the N-terminal region of myosin phosphatase target subunit 1 (MYPT1) on the interactions with protein phosphatase 1 catalytic subunit (PP1c) and with phosphorylated 20 kDa myosin light chain (P-MLC20) was studied. Protein kinase C (PKC) phosphorylated threonine-34 (1 mol/mol), the residue preceding the consensus PP1c-binding motif (35KVKF38) in MYPT11-38, but this did not affect binding of the peptide to PP1c. PKC incorporated 2 mol P(i) into MYPT11-296 suggesting a second site of phosphorylation within the ankyrin repeats (residues 40-296). This phosphorylation diminished the stimulatory effect of MYPT11-296 on the P-MLC20 phosphatase activity of PP1c. Binding of PP1c or P-MLC20 to phosphorylated MYPT11-296 was also attenuated. It is concluded that phosphorylation of MYPT1 by PKC may therefore result in altered dephosphorylation of myosin. Copyright (C) 2000 Federation of European Biochemical Societies.
• Tóth, A., Kiss, E., Herberg, F. W., Gergely, P., Hartshorne, D. J., & Erdödi, F. (2000). Study of the subunit interactions in myosin phosphatase by surface plasmon resonance. European Journal of Biochemistry, 267(6), 1687-1697.
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  PMID: 10712600;Abstract: The interactions of the catalytic subunit of type 1 protein phosphatase (PP1c) and the N-terminal half (residues 1-511) of myosin phosphatase target subunit 1 (MYPT1) were studied. Biotinylated MYPT1 derivatives were immobilized on streptavidin-biosensor chips, and binding parameters with PP1c were determined by surface plasmon resonance (SPR). The affinity of binding of PP1c was: MYPT11-296 > MYPT11-38 > MYPT123-38. No binding was detected with MYPT11-34, suggesting a critical role for residues 35- 38, i.e. the PP1c binding motif. Binding of residues 1-22 was inferred from: a higher affinity binding to PP1c for MYPT11-38 compared to MYPT123-38, as deduced from SPR kinetic data and ligand competition assays; and an activation of the myosin light chain phosphatase activity of PP1c by MYPT11-38, but not by MYPT123-38. Residues 40-296 (ankyrin repeats) in MYPT11-296 inhibited the phosphorylase phosphatase activity of PP1c (IC50 = 0.2 nM), whereas MYPT11-38, MYPT123-38 or MYPT11-34 were without effect. MYPT140-511, which alone did not bind to PP1c, showed facilitated binding to the complexes of PP1c-MYPT11-38 and PP1c- MYPT123-38. The inhibitory effect of MYPT140-511 on the phosphorylase phosphatase activity of PP1c also was increased in the presence of MYPT11-38. The binding of MYPT1304-511 to complexes of PP1c and MYPT11-38, or MYPT11-296, was detected by SPR. These results suggest that within the N-terminal half of MYPT1 there are at least four binding sites for PP1c. The essential interaction is with the PP1c-binding motif and the other interactions are facilitated in an ordered and cooperative manner.
• Feng, J., Ito, M., Ichikawa, K., Isaka, N., Nishikawa, M., Hartshorne, D. J., & Nakano, T. (1999). Inhibitory phosphorylation site for Rho-associated kinase on smooth muscle myosin phosphatase. Journal of Biological Chemistry, 274(52), 37385-37390.
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  PMID: 10601309;Abstract: It is clear from several studies that myosin phosphatase (MP) can be inhibited via a pathway that involves RhoA. However, the mechanism of inhibition is not established. These studies were carried out to test the hypothesis that Rho-kinase (Rho-associated kinase) via phosphorylation of the myosin phosphatase target subunit 1 (MYPT1) inhibited MP activity and to identify relevant sites of phosphorylation. Phosphorylation by Rho-kinase inhibited MP activity and this reflected a decrease in V(max). Activity of MP with different substrates also was inhibited by phosphorylation. Two major sites of phosphorylation on MYPT1 were Thr695 and Thr850. Various point mutations were designed for these phosphorylation sites. Following thiophosphorylation by Rho-kinase and assays of phosphatase activity it was determined that Thr695 was responsible for inhibition. A site- and phosphorylation-specific antibody was developed for the sequence flanking Thr895 and this recognized only phosphorylated Thr695 in both native and recombinant MYPT1. Using this antibody it was shown that stimulation of serum-starved Swiss 3T3 cells by lysophosphatidic acid, thought to activate RhoA pathways, induced an increase in Thr695 phosphorylation on MYPT1 and this effect was blocked by a Rho-kinase inhibitor, Y-27632. In summary, these results offer strong support for a physiological role of Rho-kinase in regulation of MP activity.
• Feng, J., Ito, M., Kureishi, Y., Ichikawa, K., Amano, M., Isaka, N., Okawa, K., Iwamatsu, A., Kaibuchi, K., Hartshorne, D. J., & Nakano, T. (1999). Rho-associated kinase of chicken gizzard smooth muscle. Journal of Biological Chemistry, 274(6), 3744-3752.
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  PMID: 9920927;Abstract: Rho-associated kinase (Rho-kinase) from chicken gizzard smooth muscle was purified to apparent homogeneity (160 kDa on SDS-polyacrylamide gel electrophoresis) and identified as the ROKα isoform. Several substrates were phosphorylated. Rates with myosin phosphatase target subunit 1 (MYPT1), myosin, and the 20-kDa myosin light chain were higher than other substrates. Thiophosphorylation of MYPT1 inhibited myosin phosphatase activity. Phosphorylation of myosin at serine 19 increased actin-activated Mg±-ATPase activity, i.e. similar to myosin light chain kinase. Myosin phosphorylation was increased at higher ionic strengths, possibly by formation of 6 S myosin. Phosphorylation of the isolated light chain and myosin phosphatase was decreased by increasing ionic strength. Rhokinase was stimulated 1.5-2-fold by guanosine 5'-O-3(thio)triphosphate-RhoA, whereas limited tryptic hydrolysis caused a 5-6-fold activation, independent of RhoA. Several kinase inhibitors were screened and most effective were Y-27632, staurosporine, and H-89. Several lipids caused slight activation of Rho-kinase, but arachidonic acid (30-50 μM) induced a 5-6-fold activation, independent of RhoA. These results suggest that Rho-kinase of smooth muscle may be involved in the contractile process via phosphorylation of MYPT1 and myosin. Activation by arachidonic acid presents a possible regulatory mechanism for Rho-kinase.
• Feng, J., Ito, M., Nishikawa, M., Okinaka, T., Isaka, N., Hartshorne, D. J., & Nakano, T. (1999). Dephosphorylation of distinct sites on the 20 kDa myosin light chain by smooth muscle myosin phosphatase. FEBS Letters, 448(1), 101-104.
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  PMID: 10217418;Abstract: The dephosphorylation of the myosin light chain kinase and protein kinase C sites on the 20 kDa myosin light chain by myosin phosphatase was investigated. The myosin phosphatase holoenzyme and catalytic subunit, dephosphorylated Ser-19, Thr-18 and Thr-9, but not Ser-1/Ser-2. The role of non-catalytic subunits in myosin phosphatase was to activate the phosphatase activity. For Ser-19 and Thr-18, this was due to a decrease in K(m) and an increase in k(cat) and for Thr-9 to a decrease in K(m). Thus, the distinction between the various sites is a property of the catalytic subunit. Copyright (C) 1999 Federation of European Biochemical Societies.
• Hartshorne, D. J., & Hirano, K. (1999). Interactions of protein phosphatase type 1, with a focus on myosin phosphatase. Molecular and Cellular Biochemistry, 190(1-2), 79-84.
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  PMID: 10098973;Abstract: It has been established for many years that MLCK is regulated by the intracellular Ca2+ concentration via the formation of the Ca2+-calmodulin-MLCK complex. A more recent discovery has been that the myosin phosphatase may also be regulated. This is manifest at suboptimal Ca2+ levels where under certain conditions (e.g. stimulation with several agonists) the MP is inhibited. The net result being that the extent of myosin phosphorylation for a fixed Ca2+ level is increased, i.e. an enhanced Ca2+-sensitivity. Spurred by this intriguing discovery several laboratories began studies on MP with an emphasis to determine the regulatory, or inhibitory, mechanism. A similar preparation was obtained by 3 laboratories and consisted of a catalytic subunit, PP1δ, plus a large subunit (M130/133 for gizzard, M130 for bladder and M110 for rat aorta) and a smaller subunit of 20-21 kD. The isolated catalytic subunit has a much lower activity towards phosphorylated myosin than the holoenzyme, thus the non-catalytic subunits may serve as targeting proteins and in addition may play a regulatory role. Because of the difference in activities between the catalytic subunit and holoenzyme, one mechanism of regulation may involve dissociation of the trimeric complex, and such was proposed for the effect of arachidonic acid. Another suggested regulatory mechanism was that phosphorylation of the large subunit in its C-terminal half caused inhibition of phosphatase activity. The two mechanisms need not be mutually exclusive and in addition several kinases could influence the activity of the myosin phosphatase. In order to understand the molecular basis of phosphatase regulation it is necessary to determine the topography of the holoenzyme and identify sites of interaction between subunits and substrate. This work is in progress. Using various truncation mutants of M130/133 it has been determined that the binding sites for both PPlc and substrate are located within the N-terminal part of the molecule. The M20 subunit binds to the C-terminal end, although the functional significance of this is not established. Many questions remain to be answered concerning the biochemistry of the myosin phosphatase. An exciting and challenging focus will be to determine the mechanism(s) of regulation and to unravel the signaling cascade(s) that are initiated by agonist-receptor complex formation. In addition, the location of the MP is not known and it is important to establish which (if any) of the cytoskeletal elements are involved in binding to MP. Finally, it is assumed that the trimeric phosphatase, as discussed above, is specific for myosin dephosphorylation and does not act on other substrates. Because of the breadth of its distribution in different tissues and the wide range of proteins interacting with the ankyrin repeats it is possible that this phosphatase, or variants thereof, has roles in other cellular processes.
• Hirano, M., Niiro, N., Hirano, K., Nishimura, J., Hartshorne, D. J., & Kanaide, H. (1999). Expression, subcellular localization, and cloning of the 130-kDa regulatory subunit of myosin phosphatase in porcine aortic endothelial cells. Biochemical and Biophysical Research Communications, 254(2), 490-496.
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  PMID: 9918866;Abstract: In endothelial cells in situ and in primary culture, immunoblot analysis revealed an expression of the 180-kDa subunit of myosin phosphatase, similar to the myosin phosphatase targeting subunit (MYPT) of smooth muscle. Screening of an endothelial cell cDNA library yielded a clone encoding an NH2-terminal fragment of 89.6 kDa, closely related to smooth muscle MYPT1. Two isoforms differing by a central insert of 56 residues were detected. In growing cells, MYPT1 was localized on stress fiber, but at confluence the localization pattern changed and MYPT1 was distributed close to the cell membrane and at cell-cell contacts. The membrane localization of MYPT1 suggested a target other than myosin and raised the possibility that MYPT1 may be involved in dephosphorylation of alternative substrate(s). These distinct mechanisms would also be dependent on the growth state of the endothelial cells, i.e., regulation of actin-myosin interactions in growing cells and an unknown function in cells at confluence.
• Totsukawa, G., Yamakita, Y., Yamashiro, S., Hosoya, H., Hartshorne, D. J., & Matsumura, F. (1999). Activation of myosin phosphatase targeting subunit by mitosis-specific phosphorylation. Journal of Cell Biology, 144(4), 735-744.
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  PMID: 10037794;PMCID: PMC2132942;Abstract: It has been demonstrated previously that during mitosis the sites of myosin phosphorylation are switched between the inhibitory sites, Ser 1/2, and the activation sites, Ser 19/Thr 18 (Yamakita, Y., S. Yamashiro, and F. Matsumura. 1994. J. Cell Biol. 124:129-137; Satterwhite, L.L., M.J. Lohka, K.L. Wilson, T.Y. Scherson, L.J. Cisek, J.L. Corden, and T.D. Pollard. 1992. J. Cell Biol. 118:595-605), suggesting a regulatory role of myosin phosphorylation in cell division. To explore the function of myosin phosphatase in cell division, the possibility that myosin phosphatase activity may be altered during cell division was examined. We have found that the myosin phosphatase targeting subunit (MYPT) undergoes mitosis-specific phosphorylation and that the phosphorylation is reversed during cytokinesis. MYPT phosphorylated either in vivo or in vitro in the mitosis-specific way showed higher binding to myosin II (two- to threefold) compared to MYPT from cells in interphase. Furthermore, the activity of myosin phosphatase was increased more than twice and it is suggested this reflected the increased affinity of myosin binding. These results indicate the presence of a unique positive regulatory mechanism for myosin phosphatase in cell division. The activation of myosin phosphatase during mitosis would enhance dephosphorylation of the myosin regulatory light chain, thereby leading to the disassembly of stress fibers during prophase. The mitosis-specific effect of phosphorylation is lost on exit from mitosis, and the resultant increase in myosin phosphorylation may act as a Signal to activate cytokinesis.
• Fujioka, M., Takahashi, N., Odai, H., Araki, S., Ichikawa, K., Feng, J., Nakamura, M., Kaibuchi, K., Hartshorne, D. J., Nakano, T., & Ito, M. (1998). A new isoform of human myosin phosphatase targeting/regulatory subunit (MYPT2): cDNA cloning, tissue expression, and chromosomal mapping. Genomics, 49(1), 59-68.
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  PMID: 9570949;Abstract: Myosin phosphatase target subunit 1 (MYPT1), a subunit of myosin phosphatase, plays a pivotal role in the regulation of myosin phosphatase activity. Here we have cloned a novel isoform of MYPT1, termed MYPT2, from a human brain cDNA library screened with a cDNA fragment of rat MYPT1. Overlapping clones indicated an open reading frame of 3763 nucleotides and a predicted polypeptide of mass 110,398. Ankyrin repeats and leucine zipper motifs were identified for the sequences 57-316 and 956-982, respectively. Overall, the deduced amino acid sequence of MYPT2 was 61% identical to MYPT1. MYPT2 gene is transcribed abundantly in heart and skeletal muscle, while Western blots using an antibody specific for MYPT2 showed exclusive expression of MYPT2 in heart and brain. A recombinant of the N-terminal two- thirds of MYPT2 bound to the catalytic subunit of type 1 phosphatase (δ isoform) and increased activity toward phosphorylated myosin light chain. In situ hybridization localized the human MYPT2 gene on chromosome 1q32.1, compared to the chromosomal location 12q15-q21.2 for MYPT1. It is suggested that the products of the two gene families of myosin phosphatase target subunit may be localized differently among various tissues.
• Hartshorne, D. J. (1998). Myosin phosphatase: Subunits and interactions. Acta Physiologica Scandinavica, 164(4), 483-493.
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  PMID: 9887971;Abstract: Myosin phosphorylation is an important mechanism in regulating contractile activity of smooth muscle. The level of myosin phosphorylation depends on the balance of two enzymes, myosin light chain kinase and myosin phosphatase. Recently it has been discovered that myosin phosphatase can be regulated and this renewed interest in characterization of the phosphatase. It is suggested that the myosin phosphatase is composed of three subunits: a catalytic subunit of type 1 phosphatase (δ isoform; PP1cδ): and two non-catalytic subunits, large and small (M20). The large subunit is thought to be a targeting subunit and is termed myosin phosphatase target subunit (MYPT), There are several isoforms of MYPT and two genes have been identified on human chromosomes 1 and 12. A dominant feature of MYPT is a series of ankyrin repeats at the N-terminal end of the molecule and these may be involved in binding to the catalytic subunit and to substrate, phosphorylated myosin, In addition, at the N-terminal fringe of the ankyrin motifs is a consensus PP1c binding motif. The function of the M20 subunit is not established but is known to bind to the C-terminal end of MYPT. Various interactions between subunits that might be relevant for the regulation of phosphatase activity are discussed.
• Hartshorne, D. J., Ito, M., & Erdödi, F. (1998). Myosin light chain phosphatase: Subunit composition, interactions and regulation. Journal of Muscle Research and Cell Motility, 19(4), 325-341.
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  PMID: 9635276;Abstract: This review has presented some of the recent data on myosin phosphatase from smooth muscle. Although it is not conclusive, it is likely that most of the myosin phosphatase activity is represented by a holoenzyme composed of three subunits. These are: a catalytic subunit of 38 kDa of the type 1 phosphatase, probably the δ isoform (i.e. PP1cδ); a subunit of about 20 kDa whose function is not established; and a larger subunit that is thought to act as a target subunit. This is termed the myosin phosphatase target subunit, MYPT. Various isoforms of MYPT exist and the relatively minor distinctions are in the C-terminal leucine zipper motifs and/or with inserts in the central region. Many regions of the molecule are highly conserved, including the ankyrin repeats in the N-terminal part of the molecule and the sequence around the phosphorylation site. In addition, these isoforms all contain the four residue PP1c-binding motif (Arg/Lys-Val/Ile-Xaa-Phe). MYPT has been detected in a variety of cells and thus is riot unique to smooth muscle. With phosphorylated myosin as substrate, the phosphatase activity of PP1c is low and is enhanced on addition of MYPT. It is assumed that MYPT functions as a target subunit and binds to both PP1c and substrate. The N- terminal fragment of MYPT is responsible for the activation of PP1c activity, but how much of the N-terminal sequence is required is not established. An important point is that activation is not a general effect and is specific for myosin. It is not known if other substrates may be targeted to MYPT. There are two binding sites for PP1c on MYPT: a strong site in the N-terminal segment (containing the 4-residue motif) and a weaker site in the ankyrin repeats, possibly in repeats 5, 6 and 7. The location(s) of the myosin- binding sites on MYPT is controversial, and binding of myosin, or light chain, to both N- and C-terminal fragments has been reported. Regulation of myosin phosphatase activity involves changes in subunit interactions, although molecular mechanisms are not defined. There are basically two theories proposed for phosphatase inhibition (i.e. as seen in the agonist- induced increase in Ca2+ sensitivity). One hypothesis is that phosphorylation of Myosin light chain phosphatase MYPT (at residue 654 or 695 of the gizzard MYPT isoforms or an equivalent residue) inhibits the activity of the MP holoenzyme. The kinase involved is not established, but may be an unidentified endogenous kinase or a RhoA-activated kinase. The latter is an attractive possibility because there is convincing evidence that RhoA plays a crucial role in the Ca2+sensitizing process in smooth muscle. A second idea involves arachidonic acid. This is released via phospholipase A2 and could either interact directly with MYPT and cause dissociation of the holoenzyme (thus effectively reducing the phosphatase activity to that of the isolated catalytic subunit), or it could activate a kinase that would phosphorylate MYPT and inhibit the phosphatase. It is possible that MP activity may also be activated, for example, following increases in cAMP and/or cGMP. Evidence in support of this is very limited and under in vivo conditions the phosphorylation of MYPT by the respective kinases has not been demonstrated. There is, however, a tentative hypothesis based on in vitro data that phosphorylation of MYPT by PKA alters its cellular localization. This involves a shuttle between the dephosphorylated membrane-bound and inhibited state (at least towards P-myosin) to a phosphorylated cytosolic or cytoskeletal, and active state. The pathway(s) discussed above originates at the cell membrane and is carried via one or more messengers to the level of the contractile apparatus where it is manifested by regulation of phosphatase activity. Various components of the route have been identified, including RhoA and the atypical PKC isoforms, but more remain to be discovered. It is possible that more than one pathway, or cascade, is involved and these may reflect the variety of receptors involved. There is also the possibility of cross-talk between pathways. It is important to identify components of each pathway and their meeting points because not only will this be important for smooth muscle research, but it will also provide basic information on myosin II-based functions in other cell types.
• Murányi, A., Erdodi, F., Ito, M., Gergely, P., & Hartshorne, D. J. (1998). Identification and localization of myosin phosphatase in human platelets. Biochemical Journal, 330(1), 225-231.
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  PMID: 9461514;PMCID: PMC1219131;Abstract: Type 1 (PP1) and type 2A (PP2A) phosphatase activity was measured in three subcellular fractions of human platelets. About 80%, of the activity was in the high-speed supernatant. Western blots showed that the catalytic subunit of PP1 (PP1c), including α- and δ-isoforms, was present in each fraction, but the level of the catalytic subunit of PP2A was very low in the low-speed pellet (cytoskeletal fraction). Various antibodies detected a subunit similar to the 130 kDa subunit (M130) of myosin phosphatase (MP) of smooth muscle in the low- and the high-speed pellets of human platelets. PP1c and associated proteins were isolated by microcystin-Sepharose. Many proteins were separated from each fraction, including myosin, actin and PP1c. M130 was separated only from the low-speed and the high-speed pellets. Kinase activities were detected in the unbound fractions and fractions from the low- and high-speed pellets phosphorylated M130 and myosin respectively. Treatment of platelets with calyculin A increased the phosphorylation level of many proteins, including myosin heavy- and light-chains, and caused association of cytoskeletal proteins with the low-speed pellet. No marked change in the distribution of PP1c and M130 was detected. These results suggest that the MP in human platelets is composed of PP1c plus a subunit similar to M130 of the smooth muscle phosphatase.
• Siegman, M. J., Funabara, D., Kinoshita, S., Watabe, S., Hartshorne, D. J., & Butler, T. M. (1998). Phosphorylation of a twitchin-related protein controls catch and calcium sensitivity of force production in invertebrate smooth muscle. Proceedings of the National Academy of Sciences of the United States of America, 95(9), 5383-5388.
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  PMID: 9560285;PMCID: PMC20270;Abstract: 'Catch' is a condition of prolonged, highforce maintenance at resting intracellular Ca2+ concentration ([Ca2+]) and very low energy usage, occurring in invertebrate smooth muscles, including the anterior byssus retractor muscle (ABRM) of Mytilus edulis. Relaxation from catch is rapid on serotonergic nerve stimulation in intact muscles and application of cAMP in permeabilized muscles. This release of catch occurs by protein kinase A- mediated phosphorylation of a high (~600 kDa) molecular mass protein, the regulator of catch. Here, we identify the catch-regulating protein as a homologue of the mini-titin, twitchin, based on (i) a partial cDNA of the purified isolated protein showing 77% amino acid sequence identity to the kinase domain of Aplysia californica twitchin; (ii) a polyclonal antibody to a synthetic peptide in this sequence reacting with the phosphorylated catch- regulating protein band from permeabilized ABRM; and (iii) the similarity of the amino acid composition and molecular weight of the protein to twitchin. In permeabilized ABRM, at all but maximum [Ca2+], phosphorylation of twitchin results in a decreased calcium sensitivity of force production (half-maximum at 2.5 vs. 1.3 μM calcium). At a given submaximal force, with equal numbers of force generators, twitchin phosphorylation increased unloaded shortening velocity ≃2-fold. These data suggest that aspects of the catch state exist not only at resting [Ca2+], but also at higher submaximal [Ca2+]. The mechanism that gives rise to force maintenance in catch probably operates together, to some extent, with that of cycling myosin crossbridges.
• Tanaka, J., Ito, M., Feng, J., Ichikawa, K., Hamaguchi, T., Nakamura, M., Hartshorne, D. J., & Nakano, T. (1998). Interaction of myosin phosphatase target subunit 1 with the catalytic subunit of type 1 protein phosphatase. Biochemistry, 37(47), 16697-16703.
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  PMID: 9843438;Abstract: In the investigation of the sequences of myosin phosphatase target subunit 1 (MYPT1) involved in binding the substrate and catalytic subunit of protein phosphatase type 1 (PP1c), fragments of MYPT1 were prepared and characterized. The shortest fragment capable of full activation of PP1c contained the sequence of residues 1-295. Within this fragment, the N- terminal sequence of residues 1-38 is involved in activation of PP1c (k(cat)) and the ankyrin repeats (residues 39-295) were involved in substrate binding (K(m)). The ankyrin repeats alone (residues 39-295) and the C-terminal fragment of residues 667-1004 did not activate PP1c. Using gel filtration, an interaction with PP1c was detected for the sequences of residues 1-295, 17- 295, and 1-170. Affinity columns were prepared with various fragments to assess binding of PP1c. Binding to the column with residues 1-295 was strongest, followed by the binding to the column with residues 1-170. A weak interaction was observed with the column with residues 1-38. The column with residues 1-295 was used to isolate PP1c from gizzard. The purified PP1c was activated by MYPT1 and fragments to a greater extent than previous preparations. These results suggest that the N-terminal sequence (residues 1- 38) and the ankyrin repeats are involved in binding PP1c. The C-terminal ankyrin repeats appear to be dominant, but there is an interaction of PP1c with the N-terminal ankyrin repeats. The N-terminal peptide has two apparent functions, the binding of PP1c via the consensus binding sequence and activation of PP1c by the sequence of residues 1-16.
• Hirano, K., Hirano, M., & Hartshorne, D. J. (1997). Cloning and characterization of a protein phosphatase type 1-binding subunit from smooth muscle similar to the glycogen-binding subunit of liver. Biochimica et Biophysica Acta - Protein Structure and Molecular Enzymology, 1339(2), 177-180.
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  PMID: 9187237;Abstract: A yeast two-hybrid screen of a chicken gizzard cDNA library detected the interaction of the catalytic subunit of protein phosphatase type 1 with a novel subunit. Subsequent characterization established similarity (58%) to the rat liver glycogen-binding subunit. Northern analyses showed expression in a wide range of tissues.
• Hirano, K., Phan, B. C., & Hartshorne, D. J. (1997). Interactions of the subunits of smooth muscle myosin phosphatase. Journal of Biological Chemistry, 272(6), 3683-3688.
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  PMID: 9013623;Abstract: Myosin phosphatase from smooth muscle consists of a catalytic subunit (PP1c) and two non-catalytic subunits, M130 and M20. Interactions among PP1c, M20, and various mutants of M130 were investigated. Using the yeast two- hybrid system, PP1c was shown to bind to the NH 2-terminal sequence of M130, 1-511. Other interactions were detected, i.e. PP1c to PP1c, M20 to the COOH- terminal fragment of M130, and dimerization of the COOH-terminal fragment of M130. Mutants of M130 were constructed to localize the PP1c and light chain binding regions. Results from the two-hybrid system indicated two binding sites for PP1c on M130: one site in the NH 2-terminal 38 residues and a weaker site(s) in the ankyrin repeats region. Inhibition of PP1c activity with phosphorylase a by the M130 mutants also was consistent with the assignment of these two sites. Overlay assays showed binding of phosphorylated light chain to the ankyrin repeats, probably in the COOH- terminal repeats. Activation of PP1c with phosphorylated light chain required binding sites for PP1c and substrate, plus an additional sequence COOH- terminal to the ankyrin repeats. Thus, activation of phosphatase and binding of PP1c and substrate are properties of the NH 2-terminal one-third of M130.
• Ito, M., Feng, J., Tsujino, S., Inagaki, N., Inagaki, M., Tanaka, J., Ichikawa, K., Hartshorne, D. J., & Nakano, T. (1997). Interaction of smooth muscle myosin phosphatase with phospholipids. Biochemistry, 36(24), 7607-7614.
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  PMID: 9200713;Abstract: The 130 kDa myosin-binding subunit (MBS) of smooth muscle myosin phosphatase was detected in cytoskeletal, cytosolic, and membrane fractions of T24 cells. Also, MBS was distributed between cytoplasm and plasmalemma in mitotic REF52 cells. These observations prompted this study of the interaction(s) of phospholipids with myosin phosphatase. Using a sedimentation assay, gizzard myosin phosphatase bound to vesicles of acidic phospholipids, i.e. phosphatidylserine (PS), phosphatidylinositol, and phosphatidic acid (PA). Neutral phospholipids did not bind. Binding of PS to myosin phosphatase also was demonstrated by electrophoresis under nondenaturing conditions. Preferential binding of PA, compared to that of the other acidic phospholipids, was indicated. Interaction of acidic phospholipids with myosin phosphatase inhibited phosphatase activity toward phosphorylated myosin. The extent of PS binding with myosin phosphatase decreased on increasing ionic strength and Mg2+ concentration. MBS (M130/M133) and M20 were phosphorylated by protein kinase A to 3 and 1 mol of P/(mol of subunit), respectively. Phosphorylation of the holoenzyme decreased phospholipid binding with recovery of phosphatase activity. Using limited proteolysis of the holoenzyme and various mutants, it was shown that phospholipid binding was associated with the C-terminal part of MBS, Ser 667-Ile 1004, and M20. The phosphorylation site involved in regulation of phospholipid binding is within the C-terminal MBS sequence. These results suggest that myosin phosphatase may interact with membranes and that phosphorylation by protein kinase A could modify this interaction. This mechanism could be important in localization of myosin phosphatase and in targeting substrates at different loci.
• Murata, K., Hirano, K., Villa-Moruzzi, E., Hartshorne, D. J., & Brautigan, D. L. (1997). Differential localization of myosin and myosin phosphatase subunits in smooth muscle cells and migrating fibroblasts. Molecular Biology of the Cell, 8(4), 663-673.
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  PMID: 9247646;PMCID: PMC276117;Abstract: Myosin II light chains (MLC20) are phosphorylated by a Ca2+/calmodulin-activated kinase and dephosphorylated by a phosphatase that has been purified as a trimer containing the δ isoform of type 1 catalytic subunit (PP1Cδ), a myosin-binding 130-kDa subunit (M130) and a 20-kDa subunit. The distribution of M130 and PP1C as well as myosin II was examined in smooth muscle cells and fibroblasts by immunofluorescence microscopy and immunoblotting after differential extraction. Myosin and M130 colocalized with actin stress fibers in permeabilized cells. However, in nonpermeabilized cells the staining for myosin and M130 was different, with myosin mostly at the periphery of the cell and the M130 appearing diffusely throughout the cytoplasm. Accordingly, most M130 was recovered in a soluble fraction during permeabilization of cells, but the conditions used affected the solubility of both M130 and myosin. The PP1Cα isoform colocalized with M130 and also was in the nucleus, whereas the PP1Cδ isoform was localized prominently in the nucleus and in focal adhesions. In migrating cells, M130 concentrated in the tailing edge and was depleted from the leading half of the cell, where double staining showed myosin II was present. Because the tailing edge of migrating cells is known to contain phosphorylated myosin, inhibition of myosin LC20 phosphatase, probably by phosphorylation of the M130 subunit, may be required for cell migration.
• Nishio, H., Ichikawa, K., & Hartshorne, D. J. (1997). Evidence for myosin-binding phosphatase in heart myofibrils. Biochemical and Biophysical Research Communications, 236(3), 570-575.
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  PMID: 9245690;Abstract: Protein phosphatase was partially purified from myofibrils of bovine heart by sequential column chromatographies. The purified protein phosphatase was immunologically identified as a δ isoform of PP1 (PP1δ). The myosin-binding subunit (MBS) of myosin-binding phosphatase (MBP) in smooth muscle was co-purified with PP1δ at each step of the sequential column chromatographies. The immunoprecipitation experiment using the polyclonal antibody to MBS showed that PP1δ associates with MBS in the purified phosphatase. In addition, the myosin-binding assay showed that the purified phosphatase has the characteristics of binding to cardiac myosin. These data strongly suggest that MBP, the holoenzyme composed of PP1δ and MBS, is expressed in heart myofibrils.
• Siegman, M. J., Mooers, S. U., Chenqing, L. i., Narayan, S., Trinkle-Mulcahy, L., Watabe, S., Hartshorne, D. J., & Butler, T. M. (1997). Phosphorylation of a high molecular weight (-600 kDa) protein regulates catch in invertebrate smooth muscle. Journal of Muscle Research and Cell Motility, 18(6), 655-670.
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  PMID: 9429159;Abstract: A unique property of smooth muscle is its ability to maintain force with a very low expenditure of energy. This characteristic is highly expressed in molluscan smooth muscles, such as the anterior byssus retractor muscle (ABRM) of Mytilus edulis, during a contractile state called 'catch'. Catch occurs following the initial activation of the muscle, and is characterized by prolonged farce maintenance in the face of a low [Ca2+](i), high instantaneous stiffness, a very slow cross-brides cycling rate, and low ATP usage. In the intact muscle, rapid relaxation (release of catch) is initiated by serotonin, and mediated by an increase in cAMP and activation of protein kinase A. We sought to determine which proteins undergo a change in phosphorylation on a time-course that corresponds to the release of catch in permeabilized ABRM. Only one protein consistently satisfied this criterion. This protein, having a molecular weight of ~600 kDa and a molar concentration about 30 times lower than the myosin heavy chain, showed an increase in phosphorylation during the release of catch. Under the mechanical conditions studied (rest, activation, catch, and release of catch), changes in phosphorylation of all ether proteins, including myosin light chains, myosin heavy chain and paramyosin, are minimal compared with the cAMP-induced phosphorylation of the ~600 kDa protein. Under these conditions, somewhat less than one mole of phosphate is incorporated per mole of ~600 kDa protein. Inhibition of A kinase blocked both the cAMP induced increase in phosphorylation of the protein and the release of catch. In addition, irreversible thiophosphorylation of the protein prevented the development of catch. In intact muscle, the degree of phosphorylation of the protein increases significantly when catch is released with serotonin. In muscles pre-treated with serotonin, a net dephosphorylation of the protein occurs when the muscle is subsequently put into catch. We conclude that the phosphorylation state of the ~600 kDa protein regulates catch.
• Tsuchiya, T., Ikeda, N., Obara, K., & Hartshorne, D. J. (1997). A type 2A protein phosphatase from clam smooth muscle. Use of 4- methylumbelliferyl phosphate as substrate. Comparative Biochemistry and Physiology - B Biochemistry and Molecular Biology, 118(1), 17-21.
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  Abstract: 1) The major cytosolic protein phosphatase from clam (Meretrix lamarckii) smooth muscle was isolated by ion exchange and gel filtration chromatography. 2) The isolation procedures were facilitated by use of the sensitive fluorescent substrate, 4-methylumbelliferyl phosphate. 3) The isolated phosphatase was a type 2A enzyme, as indicated by subunit composition and antigenic properties of the catalytic subunit. 4) In spite of some differences between the organic and protein substrates, 4- methylumbelliferyl phosphate offers an convenient alternative to the use of radioisotopes in phosphatase assays.
• Hirano, K., Erdödi, F., Patton, J. G., & Hartshorne, D. J. (1996). Interaction of protein phosphatase type 1 with a splicing factor. FEBS Letters, 389(2), 191-194.
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  PMID: 8766827;Abstract: A gizzard cDNA library was screened by the two-hybrid system using as bait the δ isoform of the catalytic subunit of protein phosphatase 1 (PP1δ). Among the proteins identified was a fragment of the polypyrimidine tract-binding protein-associated splicing factor (PSF) and for 242 residues was 97.1% identical to the human isoforms. Binding of PSP and PP1δ was confirmed by inhibition of phosphatase activity and by an overlay technique. The PP1δ binding site was contained in the N-terminal 82 residues of the PSF fragment, PSP may therefore act as a PP1 target molecule in the spliceosome.
• Holden, H. M., Wesenberg, G., Raynes, D. A., Hartshorne, D. J., Guerriero Jr., V., & Rayment, I. (1996). Molecular structure of a proteolytic fragment of TLP20. Acta Crystallographica Section D: Biological Crystallography, 52(6), 1153-1160.
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  PMID: 15299576;Abstract: Myosin light-chain kinase is responsible for the phosphorylation of myosin in smooth muscle cells. In some tissue types, the C-terminal portion of this large enzyme is expressed as an independent protein and has been given the name telokin. Recently, an antibody directed against telokin was found to interact with a protein derived from the baculovirus Autographa californica nuclear polyhedrosis virus. This protein was biochemically characterized and given the name TLP20 for telokin-like protein of 20 000 molecular weight. The amino-acid sequence of TLP20 was determined on the basis of a cDNA clone and subsequent alignment searches failed to reveal any homology to telokin or to other known proteins. The three-dimensional structure of a proteolytic portion of TLP20 is reported here. Crystals employed in the investigation were grown from ammonium sulfate solutions at pH 6.0 and belonged to the space group P213 with unit-cell dimensions of a = b = c = 76.3 Å and one molecule per asymmetric unit. The structure was determined by multiple isomorphous replacement with three heavy-atom derivatives. Least-squares refinement of the model reduced the crystallographic R factor to 18.1% for all measured X-ray data from 30.0 to 2.2 Å. The overall fold of the molecule may be described as a seven-stranded antiparallel β-barrel flanked on the bottom by two additional β-strands and on top by an α-helix. Quite surprisingly, the three-dimensional structure of this β-barrel is not similar to telokin or to any other known protein.
• Ichikawa, K., Ito, M., & Hartshorne, D. J. (1996). Phosphorylation of the large subunit of myosin phosphatase and inhibition of phosphatase activity. Journal of Biological Chemistry, 271(9), 4733-4740.
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  PMID: 8617739;Abstract: The partially purified myosin-bound phosphatase had an associated protein kinase that phosphorylated the holoenzyme, primarily on the large (130-kDa) subunit. Phosphorylation of the 130-kDa subunit resulted in inhibition of phosphatase activity. The major site of phosphorylation was threonine 654 of the 130-kDa subunit or threonine 695 of the 133-kDa isoform. Phosphorylation of the large subunit did not dissociate the holoenzyme. Dephosphorylation of the large subunit was achieved by the holoenzyme, and addition of the catalytic subunit of the type 2A enzyme did not increase the rate of dephosphorylation. The associated kinase was inhibited by chelerythrine, with half-maximal inhibition at approximately 5 μM (in 150 μM ATP). The associated kinase phosphorylated two synthetic peptides, one corresponding to the sequence flanking the phosphorylated threonine, i.e. 648-661 of the 130- kDa subunit, and the other to a known protein kinase C substrate, i.e. a modified sequence from the autoinhibitory region of ε protein kinase C. The associated kinase was activated by arachidonic and oleic acid and to a lesser extent by myristic acid. The protein kinase that phosphorylated the 130-kDa subunit and resulted in inhibition of myosin phosphatase activity was not identified.
• Nishiyama, U., Ubukata, M., Magae, J., Kataoka, T., Erdödi, F., Hartshorne, D. J., Isono, K., Nagai, K., & Osada, H. (1996). Structure - Activity relationship within a series of degradation products of tautomycin. Bioscience, Biotechnology and Biochemistry, 60(1), 103-107.
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  PMID: 8824829;Abstract: Tautomycin, a protein serine/threonine phosphatase inhibitor, was chemically degraded, and five derivatives were investigated for their biological activities. None of them exerted any inhibitory effects on the activity of protein phosphatase types 1 and 2A. However, one derivative, named TM2a, induced a significant morphological change (bleb-formation) of human myeloid leukemia K562 cells. TM2b, the trimethyl ester of TM2, did not induce bleb-formation. Thus, the maleic anhydride structure played an important role in the biological activity. The biological properties of TM2a toward K562 cells resembled those of a phorbol ester, rather than of tautomycin. The phorbol ester-induced bleb formation was abrogated by a non-specific inhibitor of protein kinases, staurosporine, and by an inhibitor of protein kinase C (PKC), H-7, but TM2a-induced bleb formation was abrogated only by staurosporine. Enhanced phosphorylation of the two proteins was observed after their exposure to TM2a. This suggest that the effect was not due to any inhibition of protein phosphatase 1 or 2A, but rather to the activation of an unidentified kinase, possibly of the PKC family, or to inhibition of a protein phosphatase other than type 1 or 2A.
• Erdodi, F., Toth, B., Hirano, K., Hirano, M., Hartshorne, D. J., & Gergely, P. (1995). Endothall thioanhydride inhibits protein phosphatases-1 and -2A in vivo. American Journal of Physiology - Cell Physiology, 269(5 38-5), C1176-C1184.
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  PMID: 7491907;Abstract: The objective of this study was to relate the toxicity of several cantharidin-derivative pesticides with their abilities to inhibit protein phosphatases-1 (PP1) and -2A (PP2A). Cantharidin (CA), endothall, and endothall thioanhydride (ETA) inhibited the activity of PP1 and PP2A, and the potency sequence was CA > endothall > ETA in vitro. We determined the inhibitory potency of these pesticides on hepatic protein phosphatases by administration of the toxins into the portal vein of rats. The potency sequence of ETA > CA > endothall was established for the inhibition of PP1 and PP2A in vivo and shows close correlation with the sequence of relative toxicity. ETA predominantly targets PP1 for inhibition in liver, as revealed by assays specific for PP1 or PP2A. Studies using 3T3 fibroblasts showed that only ETA, but not CA or endothall, induced marked morphological changes. These effects included cell rounding and detachment as well as extensive reorganization of actin filaments and are characteristic for the cell- permeable phosphatase-inhibitory toxins. It is suggested that the in vivo effectiveness is related to enhanced uptake of ETA, because this is permeable across the plasmalemma.
• Hirano, K., Ito, M., & Hartshorne, D. J. (1995). Interaction of the ribosomal protein, L5, with protein phosphatase type 1. Journal of Biological Chemistry, 270(34), 19786-19790.
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  PMID: 7649987;Abstract: The two-hybrid system was used to screen for binding proteins of type 1 protein phosphatase. Two plasmids were constructed, one containing the cDNA of the δ isoform of the type 1 catalytic subunit and the other containing a chicken gizzard cDNA library. Yeast (Y190) were transformed with the plasmids and screened for interacting species. 35 positive clones were categorized into 19 gene groups. Most of these were not identified. One clone, however, contained a sequence identical to the C-terminal portion of the chicken ribosomal protein L5 and corresponded to nucleotide residues 606-975. L5 was isolated from rat liver ribosomes as the L5.5 S RNA complex. This activated phosphatase activity of a myosin-bound phosphatase and the isolated type 1 catalytic subunit using phosphorylated myosin light chains and phosphorylase α as substrates. In addition, it was found that phosphatase sedimented with ribosomal subunits containing L5 but did not sediment with those deficient in L5. These data indicate that L5 binds to the catalytic subunit of the type 1 protein phosphatase and may act as a target molecule for phosphatase in ribosomal function or other cell mechanisms.
• Kurisaki, T., Taylor, R. G., & Hartshorne, D. J. (1995). Effects of the protein phosphatase inhibitors, tautomycin and calyculin-A, on protein phosphorylation and cytoskeleton of human platelets. Cell Structure and Function, 20(5), 331-343.
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  PMID: 8581989;Abstract: Effects of the protein phosphatase inhibitors, tautomycin and calyculin A on protein phosphorylation and cytoskeleton of human platelets. It has been discovered recently that many cytotoxic compounds isolated from a variety of sources are potent phosphatase inhibitors. Two of these, tautomycin (TM) and calyculin-A (CL-A) were applied to human platelets to investigate the role of protein phosphorylation on cytoskeletal structure and function. Exposure to 10 μM TM or 0.1 μM CL-A induced marked morphological changes. The granules were centralized and surrounded by actin filaments, but there was no evidence of granule release. Myosin became more centralized, was occluded from the granulomere, but was not confined to the microfilament ring. These changes occurred without an increase in cytosolic Ca2+ concentrations, as determined by measurements with fura-2. TM and CL-A induced an overall increase in protein phosphorylation. Phosphorylation of the 20,000 dalton light chain of myosin increased markedly and multiple phosphorylation sites were indicated. cytoskeletons were prepared from control, thrombin- and TM-treated platelets, the latter prepared in the absence of external calcium. The major difference in protein composition was the increased content of myosin associated with the cytoskeleton from TM-treated platelets where the dominant phosphoprotein was the 20,000 dalton light chain. These results suggest that myosin phosphorylation drives the initial shape changes, and via a contractile process results in the formation of the microfilament ring and centralization of granules.
• Trinkle-Mulcahy, L., Ichikawa, K., Hartshorne, D. J., Siegman, M. J., & Butler, T. M. (1995). Thiophosphorylation of the 130-kDa subunit is associated with a decreased activity of myosin light chain phosphatase in α-toxin-permeabilized smooth muscle. Journal of Biological Chemistry, 270(31), 18191-18194.
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  PMID: 7629133;Abstract: Pretreatment of α-toxin-permeabilized smooth muscle with ATPγS (adenosine 5'-O-(thiotriphosphate)) under conditions resulting in minimal (< 1%) thiophosphorylation of the myosin light chain increases the subsequent calcium sensitivity of force output and myosin light chain phosphorylation. The change in calcium sensitivity results at least in part from a 5-fold decrease in myosin light chain phosphatase activity. One of the few proteins thiophosphorylated under these conditions is the 130-kDa subunit of myosin light chain phosphatase. These results suggest that thiophosphorylation of this subunit leads to a decrease in the activity of the phosphatase, and that phosphorylation and dephosphorylation of the subunit may play a role in regulating myosin light chain phosphatase activity.
• Matsushima, S., Huang, Y. -., Dudas, C. V., Guerriero Jr., V., & Hartshorne, D. J. (1994). Mutants of smooth muscle myosin light chain kinase at tryptophan 800. Biochemical and Biophysical Research Communications, 202(3), 1329-1336.
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  PMID: 8060310;Abstract: The importance of Trp 800 in the calmodulin-binding site of myosin light chain kinase was investigated. Truncation mutants from Leu 447 to the C-terminus were expressed in E. coli and these were modified by point mutations of Trp 800 to Gly, Cys, Leu and Tyr. Trp at this position was more effective than any of the other residues. The Leu mutant was partially active and its Km for calmodulin decreased from about 10 nM to 175 nM. The Tyr mutant had detectable activity but the other two mutants were inactive and did not bind calmodulin. Thus Trp at position 800 is critical. The activity of the Leu mutant at high calmodulin concentrations was less than the wild-type mutant, about 20%. This suggests that the binding of calmodulin does not release inhibition in an all-or-none mechanism and that other intramolecular interactions are important.
• Okubo, S., Ito, M., Takashiba, Y., Ichikawa, K., Miyahara, M., Shimizu, H., Konishi, T., Shima, H., Nagao, M., Hartshorne, D. J., & Nakano, T. (1994). A regulatory subunit of smooth muscle myosin bound phosphatase. Biochemical and Biophysical Research Communications, 200(1), 429-434.
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  PMID: 8166716;Abstract: The relationship between two putative myosin-binding subunits of smooth muscle myosin phosphatase was investigated. A monoclonal antibody (MoAb) to the 58 kD component of smooth muscle myosin-bound phosphatase (MBP) cross-reacted with a 130 kD protein in extracts of fresh chicken gizzards. The MoAb in combination with protein A immunoprecipitated from gizzard extracts a complex of the 130 kD protein plus the 38 kD catalytic subunit of the type 1δ protein phosphatase. It is proposed that the 130 kD component is a native subunit of MBP and that the 58 kD protein is its proteolytic degradation product. The distribution of the 130 kD component in chicken tissues was screened using the MoAb. An immunoreactive band of appropriate mass was detected in all tissues except liver and skeletal muscle. Higher concentrations of the 130 kD component were evident in the smooth muscle samples.
• Raynes, D. A., Hartshorne, D. J., & Guerriero Jr., V. (1994). Sequence and expression of a baculovirus protein with antigenic similarity to telokin. Journal of General Virology, 75(7), 1807-1809.
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  PMID: 7517434;Abstract: A protein from baculovirus-infected cells reacted with an antibody against the smooth muscle protein telokin. Because of this unusual similarity, the protein, termed telokin-like protein-20 (TLP20), was isolated and characterized. Its M(r) on denaturing polyacrylamide gels was 28K and the protein contained a high proportion of β structure. A cDNA for TLP20 was isolated and sequenced. The 3' non-coding sequence contained a region of high identity with the 5' end of two other baculovirus genes. The 5' non-coding region contains several baculovirus regulatory elements. Surprisingly, the derived amino acid sequence showed no homologies to telokin. The cDNA was cloned into a bacterial expression vector and the subsequently expressed protein had a slightly lower M(r) than the native protein, but cross-reacted with telokin antibody. This paper reports the characterization of a new baculovirus protein that shares some antigenic similarities to the smooth muscle protein telokin.
• Shimizu, H., Ito, M., Miyahara, M., Ichikawa, K., Okubo, S., Konishi, T., Naka, M., Tanaka, T., Hirano, K., Hartshorne, D. J., & Nakano, T. (1994). Characterization of the myosin-binding subunit of smooth muscle myosin phosphatase. Journal of Biological Chemistry, 269(48), 30407-30411.
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  PMID: 7982954;Abstract: A myosin phosphatase was purified from chicken gizzard smooth muscle. The holoenzyme is a trimer and consists of 130,000-, 38,000-, and 20,000-Da subunits (in agreement with the results of Alessi et al.: Alessi, D., MacDougall, L. K., Sola, M. M., Ikebe, M., and Cohen, P. (1992) Eur. J. Biochem. 210, 1023-1035). The catalytic subunit, 38,000 Da, is the type 1 δ isoform, and its derived amino acid sequence is identical to the rat isoform. The larger subunit bound to myosin and also interacted with the catalytic subunit. cDNA clones encoding the large subunit were isolated from chicken gizzard cDNA libraries. Overlapping clones indicated the presence of two isoforms, and open reading frames of 2889 and 3012 bases were obtained. These encoded proteins of 963 and 1004 amino acids, with masses of 106,700 and 111,600 Da, respectively. The insert in the larger isoform is in the center of the molecule, at residues 512-552. The N-terminal third of the molecule is composed of eight repeat sequences, similar to the cdc10/SW16 or ankyrin repeat. Myosin binding and binding to the catalytic subunit are properties of a 58,000-Da fragment that represents the N-terminal part of the molecule.
• Hirano, K., & Hartshorne, D. J. (1993). Phosphorylation of vimentin in the C-terminal domain after exposure to calyculin-A. European Journal of Cell Biology, 62(1), 59-65.
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  PMID: 8269979;Abstract: Exposure of 3T3 fibroblasts to the phosphatase inhibitor, calyculin-A, induces marked morphological changes and the formation of an aggregate of actin and myosin connected to the nucleus by intermediate filaments. Vimentin was isolated from this complex and shown to be phosphorylated. At least 4 phosphorylation sites were indicated. These sites were distinct from those phosphorylated by the cAMP-dependent protein kinase. Limited proteolysis was used to define the domains in which phosphorylation occurred. Vimentin was isolated from 32P-labeled calyculin-A-treated cells and digested with thrombin and α-chymotrypsin. Proteolysis with thrombin limited the phosphorylation to either the central core or C-terminal domain. Proteolysis with α-chymotrypsin indicated that the multiple phosphorylation sites were restricted to the C-terminal domain of vimentin
• Hosoya, N., Mitsui, M., Yazama, F., Ishihara, H., Ozaki, H., Karaki, H., Hartshorne, D. J., & Mohri, H. (1993). Changes in the cytoskeletal structure of cultured smooth muscle cells induced by calyculin-A. Journal of Cell Science, 105(4), 883-890.
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  PMID: 8227210;Abstract: Changes in the cytoskeletal structure of cultured AlO smooth muscle cells induced by calyculin-A (CL-A), a potent inhibitor of types 1 and 2A protein phosphatases, were analyzed using indirect fluorescence techniques. In the presence of 1×10-7 M CL-A the cells became round and subsequently detached from the substratum. The effect of CL-A was inhibited by a non-selective kinase inhibitor, K-252a, but not by EGTA. In rounded cells stress fibers were absent and staining for F-actin appeared in patches. Vinculin, one of the components of focal contacts, was localized at the periphery of control cells. CL-A treatment moved the focal contacts towards the inside of the cell along the stress fibers, and this was followed by the rounding up of the cell. In addition, rapid and marked changes in microtubule structure were observed in CL-A-treated cells. Many 'nicks' or 'gaps' were observed along the microtubules in the attached, spread cells. A filamentous network of microtubules was not observed in the detached cells, i.e. after longer exposure to CL-A. These results suggest that CL-A may change the structure of focal contacts, resulting in the rounding up of the cell, and inducing a microtubule-severing activity. These effects were independent of the external Ca2+ concentration. The changes in cytoskeletal structure may be caused by disturbing the balance of phosphorylation and dephosphorylation in the cell.
• Ikebe, M., Mitra, S., & Hartshorne, D. J. (1993). Cleavage at site A, Glu-642 to Ser-643, of the gizzard myosin heavy chain decreases affinity for actin. Journal of Biological Chemistry, 268(34), 25948-25951.
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  PMID: 7902357;Abstract: The actin-activated ATPase activities of subfragment 1 (S1) produced from gizzard myosin by papain or Staphylococcus aureus protease are different. The activity of the latter is lower, in spite of the presence of intact 20,000-dalton light chains. To study this difference, the S. aureus protease S1 was subjected to further proteolysis by papain. This second stage of proteolysis markedly increased actin-activated ATPase, due to a decrease in Kactin with no change in Vm and increased the affinity of S1 for actin in the presence of ATP. Treatment with papain caused degradation of the 20-kDa light chain, a decrease in the 26-kDa C-terminal domain of S1 and the 68-kDa fragment containing the N-terminal and central domains, and in the appearance and progressive increase of a 94-kDa fragment. The increase in actin-activated ATPase activity was due to the production of the 94-kDa fragment but not due to light chain degradation. Analyses of N-terminal sequences following papain digestion showed that the 94-kDa fragment was formed from a combination of the 68- and 26-kDa fragments. The bond formed probably involved the N-terminal residue of the 26-kDa fragment (Ser-643) and a side chain carboxyl (Glu-642) or amine (Glu-636). From the sequence data site A was identified as Glu-642-Ser-643. These results confirm the importance of site Ain actin-binding of gizzard myosin. It is suggested that the sequence Ser-643 and Val-659, as well as the 3 lysine residues, are important for actin binding.
• Kanoh, S., Ito, M., Niwa, E., Kawano, Y., & Hartshorne, D. J. (1993). Actin-binding peptide from smooth muscle myosin light chain kinase. Biochemistry, 32(34), 8902-8907.
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  PMID: 8364036;Abstract: The objective of this study was to localize the actin-binding site in the smooth muscle myosin light chain kinase. Limited proteolysis by thermolysin indicated that hydrolysis of the kinase at the N-terminal end of the molecule resulted in loss of actin-binding ability. Various methods of cleavage were investigated for the generation of a discrete actin-binding peptide. The method chosen was cleavage at the cysteine residues by the 5,5′-dithiobis(2-nitrobenzoic acid)-cyanide complex. This procedure yielded an actin-binding peptide of approximate Mr 17 000. The peptide was purified and shown to possess the actin-binding properties of the native myosin light chain kinase. The binding constant of the isolated peptide and parent enzyme to actin was estimated as 7.5 × 104 M-1. From the amino acid composition of the peptide and comparison with the sequence of gizzard myosin light chain kinase, it was suggested that the actin-binding site is located within the N-terminal sequence 1-114. Comparison with other actin-binding proteins shows some similarities to gizzard α-actinin and caldesmon. © 1993 American Chemical Society.
• Hartshorne, D. J., Matsushima, S., Ito, M., Guerriero, V., & Kawamura Jr., T. (1992). Myosin light chain kinase: Structure-function relationships. Japanese Journal of Pharmacology, 58(SUPPL. 2), 17P-22P.
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  PMID: 1507532;
• Hirano, K., Chartier, L., Taylor, R. G., Allen, R. E., Fusetani, N., Karaki, H., & Hartshorne, D. J. (1992). Changes in the cytoskeleton of 3T3 fibroblasts induced by the phosphatase inhibitor, calyculin-A. Journal of Muscle Research and Cell Motility, 13(3), 341-353.
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  PMID: 1326568;Abstract: Addition of the protein phosphatase inhibitor, calyculin-A, to 3T3 fibroblasts causes a marked change in cell morphology. Initially the cells become rounded, develop surface blebs and then detach from the substratum. In the detached cells an unusual ball-like structure is observed. This study focuses on the cytoskeleton during these calyculin-A-induced morphological changes. Stress fibres disappear as the cells begin to round and aggregates of actin are formed towards the apical surface of the cell. These aggregates condense, in the detached cells, to form the ball structure of approximately 3 μm diameter. Between the ball and the nucleus are cables of intermediate filaments that appear to be attached to the surface of the ball and to the nuclear lamina. Using a procedure designed for the isolation of nuclei the nucleus-ball complex can be obtained. Analysis of the nucleus-ball preparation by immunofluorescence and electron microscopy demonstrate that the ball contains actin and that intermediate filaments are located between the ball and the nucleus. In this preparation, the intermediate filaments also appear to attach to the surfaces of the ball and the nucleus. Electrophoretic analysis of the nucleus-ball preparation indicates that, in addition to actin, a major component of the ball is myosin. It is suggested that the formation of the ball is caused by an actin-myosin-based contractile process, initiated by the phosphorylation of myosin. The aggregation of the actomyosin draws together the intermediate filaments into the area between the ball and nucleus. This hypothesis requires that vimentin binds both to the nucleus and to some component of the ball. © 1992 Chapman & Hall.
• Holden, H. M., Ito, M., Hartshorne, D. J., & Rayment, I. (1992). X-ray structure determination of telokin, the C-terminal domain of myosin light chain kinase, at 2.8 Å resolution. Journal of Molecular Biology, 227(3), 840-851.
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  PMID: 1404391;Abstract: The three-dimensional structure of telokin, an acidic protein identical to the C-terminal portion of smooth muscle myosin light chain kinase from turkey gizzard, has been determined at 2.8 Å resolution and refined to a crystallographic R-factor of 19.5% for all measured X-ray data from 30 Å to 2.8 Å. Crystals used in the investigation belonged to the space group P3221, with one molecule per asymmetric unit and unit cell dimensions of a = b = 64.4 Å and c = 50.6 Å. Telokin contains 154 amino acid residues, 103 of which were visible in the electron density map. The overall molecular fold of telokin consists of seven strands of antiparallel β-pleated sheet that wrap around to form a barrel. There is also an extended tail of eight amino acid residues at the N terminus that does not participate in β-sheet formation. The β-barrel can be simply envisioned as two layers of β-sheet, nearly parallel to one another, with one layer containing four and the other three β-strands. This type of β-barrel, as seen in telokin, was first observed for the CH2 domain of an immunoglobulin fragment Fc. Telokin is an intracellular protein and, as such, does not contain the disulphide linkage between β-strands B and F normally observed in the immunoglobulin constant domains. It does, however, contain two cysteine amino acid residues (Cys63 and Cys115) that are situated at structurally identical positions to those forming the disulphide linkage in the immunoglobulin constant domain.
• Miki, M., Walsh, M. P., & Hartshorne, D. J. (1992). The mechanism of inhibition of the actin-activated myosin MgATPase by calponin. Biochemical and Biophysical Research Communications, 187(2), 867-871.
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  PMID: 1388358;Abstract: Calponin inhibits the actin-activated ATPase of smooth muscle myosin and thus has been proposed as a thin filament-based regulatory component in smooth muscle. To obtain information on the mechanism of inhibition by calponin we have used chemical modification of actin and cross-linking of actin and subfragment 1. Modification of Lys 61 of actin had no effect on the inhibition by calponin of acto-heavy meromyosin ATPase, i.e. different from tropomyosin-troponin. In addition, modification of the acidic N-terminal region of actin did not impair the ability of calponin to bind to F-actin. Finally, calponin was effective in inhibiting ATPase activity of cross-linked actosubfragment 1. Therefore the mechanism of inhibition by calponin is distinct from troponin-tropomyosin and caldesmon in that it does not involve either the N-terminal acidic region of actin nor the area around Lys 61 and does not fit a simple steric blocking model. © 1992.
• Okubo, S., Ito, M., Ichikawa, K., Konishi, T., Nakano, T., Kawamura, T., & Hartshorne, D. J. (1992). Smooth muscle myosin light chain phosphatase. Japanese Journal of Pharmacology, 58(SUPPL. 2), 267P.
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  PMID: 1324340;
• Anderson, T. A., Ito, M., Hartshorne, D. J., & Rayment, I. (1991). Crystallization and preliminary analysis of telokin, the C-terminal domain of myosin light chain kinase. Journal of Molecular Biology, 217(4), 621-623.
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  PMID: 2005615;Abstract: Telokin, an acidic protein related to the C-terminal portion of smooth muscle myosin light chain kinase from turkey gizzard has been crystallized in a form suitable for a high-resolution diffraction analysis. The crystals were grown from solutions of polyethylene glycol 8000 using the hanging-drop vapor diffusion method. They belong to the trigonal space group P3121 or P3221 with cell parameters a = 64.0 Å, c = 59.4 Å and diffract to at least 2.7 Å resolution.
• Chartier, L., Rankin, L. L., Allen, R. E., Kato, Y., Fusetani, N., Karaki, H., Watabe, S., & Hartshorne, D. J. (1991). Calyculin-A increases the level of protein phosphorylation and changes the shape of 3T3 fibroblasts. Cell Motility and the Cytoskeleton, 18(1), 26-40.
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  PMID: 1848484;
• Hartshorne, D. J. (1991). Definition of the inhibitory domain of smooth muscle myosin light chain kinase by site-directed mutagenesis. Biochemistry, 30(14), 3498-3503.
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  PMID: 2012809;Abstract: Site-directed mutagenesis of smooth muscle myosin light chain kinase was applied to define its autoinhibitory domain. Mutants were all initiated at Leu-447 but contained varying lengths of C-terminal sequence. Those containing the complete C-terminal sequence to Glu-972 possessed kinase activities that were calmodulin-dependent. Removal of the putative inhibitory domain by truncation to Thr-778 resulted in generation of a constitutively active (calmodulin-independent) species. Thus, the inhibitory domain lies to the C-terminal side of Thr-778. Truncation to Lys-793 and to Trp-800 also resulted in constitutively active mutants, although the specific activity of the latter was less than the other mutants. None of the truncated mutants bound calmodulin. For each mutant, the Km values with respect to ATP and to the 20000-dalton light chain were similar to values obtained with the native enzyme. The presence of the inhibitory domain was detected by activation of kinase activity following limited proteolysis with trypsin. Using this procedure, it was determined that the inhibitory domain was manifest only in the mutant truncated to Trp-800 and was absent from that ending at Lys-793. These results indicate that a critical region of the inhibitory domain is contained within the sequence Tyr-794 to Trp-800. This region overlaps with the calmodulin-binding site for five residues. Our assignment of the inhibitory sequence is consistent with autoinhibition via a pseudosubstrate domain. © 1991 American Chemical Society.
• Hori, M., Magae, J., Han, Y. -., Hartshorne, D. J., & Karaki, H. (1991). A novel protein phosphatase inhibitor, tautomycin Effect on smooth muscle. FEBS Letters, 285(1), 145-148.
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  PMID: 1648511;Abstract: The antibiotic, tautomycin, was found to be a potent inhibitor of protein phosphatases and equally effective for the type-1 and type-2A enzymes. For the catalytic subunits of the type-1 and type-2A phosphatases the IC50 value was 22 to 32 nM. For the phosphatase activity present in chicken gizzard actomyosin the IC50 value was 6 nM. Tautomycin had no effect on myosin light chain kinase activity. Tautomycin induced a Ca2+-independent contraction of intact and permeabilized smooth muscle fibers and this was accompanied by an increase in the level of myosin phosphorylation. Thus, tautomycin by virtue of its ability to inhibit phosphatase activity is a valuable addition for studying the role of protein phosphorylation. © 1991.
• Ikebe, M., Hewett, T. E., Martin, A. F., Chen, M., & Hartshorne, D. J. (1991). Cleavage of a smooth muscle myosin heavy chain near its c terminus by α-chymotrypsin: Effect on the properties of myosin. Journal of Biological Chemistry, 266(11), 7030-7036.
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  PMID: 1826682;Abstract: Limited proteolysis of gizzard myosin by α-chymotrypsin converted the heavy chain doublet pattern, seen by gel electrophoresis, to a single band. Light chain degradation was not observed and only minor cleavage occurred at other heavy chain sites. Using a polyclonal antibody raised against a unique sequence from the slower-migrating heavy chain (SM1) it was shown that this conversion was due to the loss of a peptide approximately 4000 daltons from the C terminus of SM1. The peptide was isolated and sequenced, and the cleavage site was identified between phenylalanine 1943 and alanine 1944. Addition of antibody before protease protected SM1 from cleavage. The following changes were observed (a) the Mg2+-dependence of actin-activated ATPase of digested phosphorylated myosin was altered and activity was relatively high at low Mg2+ levels, i.e. similar to phosphorylated heavy meromyosin; (b) the KCI dependence of Mg2+-ATPase of the digested myosin, particularly the phosphorylated form, showed an altered pattern consistent with the stabilization of the 6 S conformation; (c) the tendency for aggregation was increased by proteolysis of phosphorylated myosin. These results show that the C-terminal region of a gizzard myosin heavy chain can modify some of the properties of myosin. It is suggested that the observed modifications reflect an enhanced tendency of the digested myosin to aggregate.
• Ito, M., Guerriero Jr., V., & Hartshorne, D. J. (1991). Structure-function relationships in smooth muscle myosin light chain kinase. Advances in Experimental Medicine and Biology, 304, 3-10.
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  PMID: 1803904;
• Pearson, R. B., Ito, M., Morrice, N. A., Smith, A. J., Condron, R., Wettenhall, R. E., Kemp, B. E., & Hartshorne, D. J. (1991). Proteolytic cleavage sites in smooth muscle myosin-light-chain kinase and their relation to structural and regulatory domains. European Journal of Biochemistry, 200(3), 723-730.
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  PMID: 1915344;Abstract: Proteolysis of the smooth muscle myosin-light-chain kinase with either thermolysin or endoproteinase Lys-C cleaves the enzyme towards the amino-terminus between the first and second unc domains, unc-II-1 and unc-II-2, and in the calmodulin-binding domain. The thermolytic fragment extends 532 residues from Ser275 to Ala806 and is resistant to further digestion. It is catalytically inactive and does not bind calmodulin. Further proteolysis of the thermolytic fragment with trypsin generates a constitutively active fragment. Digestion with endoproteinase Lys-C initially results in an inactive fragment of 516 residues, Ala287 to Lys802. Further digestion with Lys-C endoproteinase results in a constitutively active 474-residue fragment with the same amino-terminus, but a carboxyl-terminus at Lys760, near Arg762, the last conserved residue of protein kinase catalytic domains. There is no cleavage in the acidic-residue-rich connecting peptide between the amino-terminus of the catalytic domain and the unc-I domain, nor within the unc-II or unc-I domains or between the adjacent unc-II-2 and unc-I domains. The pattern of cleavages by these proteases reflects well the predicted domain structure of the myosin-light-chain kinase and further delineates the regulatory pseudosubstrate region. A synthetic peptide corresponding to the pseudosubstrate sequence, MLCK (787-807) was a more potent inhibitor by three orders of magnitude than the overlapping peptide MLCK(777-793) proposed by Ikebe et al. (1989) [Ikebe, M., Maruta, S. and Reardon, S. (1989) J. Biol. Chem. 264, 6967-6971] to be important in autoregulation of the myosin-light-chain kinase.
• Eriksson, J. E., Toivola, D., Meriluoto, J. A., Karaki, H., Han, Y. G., & Hartshorne, D. (1990). Hepatocyte deformation induced by cyanobacterial toxins reflects inhibition of protein phosphatases. Biochemical and Biophysical Research Communications, 173(3), 1347-1353.
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  PMID: 2176489;Abstract: The cyclic peptide hepatotoxins microcystin-LR, 7-desmethyl-microcystin-RR and nodularin are potent inhibitors of the protein phosphatases type 1 and type 2A. Their potency of inhibition resembles calyculin-A and to a lesser extent okadaic acid. These hepatotoxins increase the overall level of protein phosphorylation in hepatocytes. Evidence is presented to indicate that in hepatocytes the morphological changes and effects on the cytoskeleton are due to phosphatase inhibition. The potency of these compounds in inducing hepatocyte deformation is similar to their potency in inhibiting phosphatase activity. These results suggest that the hepatotoxicity of these peptides is related to inhibition of phosphatases, and further indicate the importance of the protein phosphorylation in maintenance of structural and homeostatic integrity in these cells. © 1990 Academic Press, Inc.
• Ito, M., & Hartshorne, D. J. (1990). Phosphorylation of myosin as a regulatory mechanism in smooth muscle.. Progress in clinical and biological research, 327, 57-72.
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  PMID: 2181476;Abstract: Contractile activity of the smooth muscle cell is regulated by the concentration of intracellular Ca2+. The Ca2+ transients are sensed by the target protein, calmodulin, and via activation of myosin light chain kinase (by Ca2(+)-calmodulin) transmitted to the contractile apparatus. Phosphorylation of myosin increases its actin-activated ATPase activity and in smooth muscle fibers is thought to initiate contraction. The effects of phosphorylation on the conformation of myosin are not established, but at least two areas of the molecule are influenced by phosphorylation of the two light chains. These are at the actin-binding site and at the head-neck junction. The latter site is important in regulating ATPase activity and a working hypothesis is that phosphorylation increases flexibility at this site and facilitates cross-bridge cycling. The phosphorylation theory has extensive experimental support, and is accepted as a major regulatory component in smooth muscle. However, the simplest interpretation of this scheme cannot adequately account for the varied physiological responses. Either there are aspects of the phosphorylation theory that are not considered, or an additional regulatory mechanism is involved. Both possibilities are discussed.
• Itoh, T., Ikebe, M., Kargacin, G., Hartshorne, D., Kemp, B., & Fay, F. S. (1990). Modulators of myosin light chain kinase activity affect both [Ca+2] and contraction in single smooth muscle cells.. Progress in clinical and biological research, 327, 73-87.
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  PMID: 2108445;
• Sommerville, L. E., Henry, G. D., Sykes, B. D., & Hartshorne, D. J. (1990). Spin-echo ¹H NMR studies of differential mobility in gizzard myosin and its subfragments. Biochemistry, 29(48), 10855-10864.
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  PMID: 2271685;Abstract: The unexpectedly narrow resonances in the 1H NMR spectra of gizzard myosin, heavy mer-omyosin, and subfragment 1 were examined by spin-echo NMR spectroscopy. These resonances originated predominantly in the myosin heads, or subfragment 1 units. Smooth muscle myosin undergoes a dramatic change in hydrodynamic properties and can exist either as a folded (10S) or as an extended (6S) species. Factors that influence this transition, namely, ionic strength and phosphorylation (or thiophosphorylation), were varied in the NMR experiments. T2 relaxation experiments on dephosphorylated myosin indicated several components of different relaxation times that were not influenced by changes in ionic strength. Our experiments focused on the components with longer relaxation times, i.e., corresponding to nuclei with more mobility, and these were observed selectively in a spin-echo experiment. With dephosphorylated myosin and HMM, increases in ionic strength caused an increased intensity in several of the narrower resonances. The ionic strength dependence of these changes paralleled that for the 10S to 6S transition. With thiophosphorylated myosin and HMM, changes in ionic strength also influenced the intensities of the narrower resonances, and in addition changes in the 1H NMR spectrum due to thiophosphorylation were observed. The narrow resonances seen with myosin and HMM were observed with S1, but the spin-echo spectra of S1 were not influenced either by changes in ionic strength or by phosphorylation. These results suggest that a fraction of the 1H resonances in smooth muscle myosin and its fragments originates from both aliphatic and aromatic residues of increased mobility compared to the mobility expected from hydrodynamic properties of these proteins. In general, the intensities of these residues increase with increasing ionic strength, and this is consistent with an increase in the percentage of mobile residues during the 10S to 6S transition. Segmental flexibility appeared also to be influenced by phosphorylation within the 6S conformation. These changes were not detected in the isolated myosin heads and thus required a higher order of structure, either the subfragment 2 region or the interaction of myosin heads. © 1990 American Chemical Society.
• Watabe, S., & Hartshorne, D. J. (1990). Paramyosin and the catch mechanism. Comparative Biochemistry and Physiology -- Part B: Biochemistry and, 96(4), 639-646.
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  PMID: 2225769;Abstract: 1. 1. Catch is a mechanism found in many molluscan smooth muscles in which tension is maintained at relatively low energy cost. 2. 2. Paramyosin forms the core of thick filaments. In catch muscle paramyosin concentrations are high and the thick filaments are relatively long. 3. 3. The mechanism of catch is not understood, but the consensus is that tension during catch is borne by slowly-cycling cross-bridge attachments to actin. 4. 4. Stimulation by acetylcholine increases intracellular Ca2+ and initiates a contraction characterized by a relatively rapid cross-bridge cycling. Reduction of Ca2+ can lead to relaxation or catch. Relaxation occurs only when a second neurotransmitter, serotonin, is present. 5. 5. The catch state is released by serotonin, via activation of adenylate cyclase, increased levels of cAMP and phosphorylation of one or more contractile proteins, possibly paramyosin. Other targets for phosphorylation are discussed. 6. 6. The contractile cycle of catch muscles, therefore, is controlled by both Ca2+ and cAMP. © 1990.
• Abougou, J. -., Hagiwara, M., Hachiya, T., Terasawa, M., Hidaka, H., & Hartshorne, D. J. (1989). Phosphorylation of caldesmon. FEBS Letters, 257(2), 408-410.
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  PMID: 2555223;Abstract: The phosphorylation of caldesmon was studied to determine if kinase activity reflected either an endogenous kinase or caldesmon itself. Titration of kinase activity with calmodulin yielded maximum activity at substoichiometric ratios of calmodulin/caldesmon. The sites of phosphorylation on caldesmon for calcium/calmodulin-dependent protein kinase II and endogenous kinase were the same, but distinct from protein kinase C sites. Phosphorylation in the presence of Ca2+ and calmodulin resulted in a subsequent increase of endogenous kinase activity in the absence of Ca2+. These results suggest that caldesmon is not a protein kinase and that kinase activity in caldesmon preparations is due to calcium/calmodulindependent protein kinase II. © 1989.
• Hartshorne, D. J. (1989). Tribute to Emil Bozler: Frontiers in smooth muscle research, Emil Bozler international symposium. Journal of Muscle Research and Cell Motility, 10(6), 399-402.
• Hartshorne, D. J., Ito, M., & Ikebe, M. (1989). Myosin and contractile activity in smooth muscle.. Advances in experimental medicine and biology, 255, 269-277.
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  PMID: 2694808;
• Higashihara, M., Hartshorne, D. J., Craig, R., & Ikebe, M. (1989). Correlation of enzymatic properties and conformation of bovine erythrocyte myosin. Biochemistry, 28(4), 1642-1649.
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  PMID: 2541759;Abstract: Myosin was purified from bovine erythrocytes by chromatography on DEAE-cellulose, Sepharose CL-4B, hydroxylapatite, and DEAE-5PW. The yield was about 200 μg/L of packed cells. From SDS-polyacrylamide gels, the purity was estimated to be greater than 95%. The bovine erythrocyte myosin is composed of heavy chains of 200 kDa and light chains of 20 and 17 kDa, in a molar stoichiometry of 1. Myosin was also purified from human erythrocytes by the same method. The molecular weights of two light chains were 26K and 19.5K which confirmed the earlier reports [Fowler, V. M., Davis, J. Q., & Bennet, V. (1985) J. Cell Biol. 100, 47-55; Wong, A. J., Kiehart, D. P., & Pollard, T. D. (1985) J. Biol. Chem. 260, 46-49]. Phosphorylation by gizzard myosin light chain kinase, to a level of 1 mol of phosphate/mol of 20-kDa light chain, increased actin-activated ATPase, and the extent of activation was dependent on the MgCl2 concentration. Both Ca2+-ATPase and Mg2+-ATPase activities were dependent on KCl concentration and markedly decreased below 0.3 M KCl. Mg2+-ATPase of phosphorylated myosin, while more resistant to decreasing ionic strength, was also decreased below 0.2 M KCl. These results are similar to those obtained with smooth muscle myosin and suggest that the 10S-6S transition occurs. In confirmation of this, gel filtration, viscosity, and electron microscopy (rotary shadowing) show that erythrocyte myosin forms extended and folded conformations in high and low salt, respectively. It is proposed that each conformation is characterized by distinct enzymatic properties. These results suggest that the conformational transition (10 S-6 S) or some part of the transition is important in determining the biological properties of erythrocyte myosin. © 1989 American Chemical Society.
• Ishihara, H., Martin, B. L., Brautigan, D. L., Karaki, H., Ozaki, H., Kato, Y., Fusetani, N., Watabe, S., Hashimoto, K., Uemura, D., & Hartshorne, D. J. (1989). Calyculin A and okadaic acid: Inhibitors of protein phosphatase activity. Biochemical and Biophysical Research Communications, 159(3), 871-877.
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  PMID: 2539153;Abstract: Calyculin A and okadaic acid induce contraction in smooth muscle fibers. Okadaic acid is an inhibitor of phosphatase activity and the aims of this study were to determine if calyculin A also inhibits phosphatase and to screen effects of both compounds on various phosphatases. Neither compound inhibited acid or alkaline phosphatases, nor the phosphotyrosine protein phosphatase. Both compounds were potent inhibitors of the catalytic subunit of type-2A phosphatase, with IC50 values of 0.5 to 1 nM. With the catalytic subunit of protein phosphatase type-1, calyculin A was a more effective inhibitor than okadaic acid, IC50 values for calyculin A were about 2 nM and for okadaic acid between 60 and 500 nM. The endogenous phosphatase of smooth muscle myosin B was inhibited by both compounds with IC50 values of 0.3 to 0.7 nM and 15 to 70 nM, for calyculin A and okadaic acid, respectively. The partially purified catalytic subunit from myosin B had IC50 values of 0.7 and 200 nM for calyculin A and okadaic acid, respectively. The pattern of inhibition for the phosphatase in myosin B therefore is similar to that of the type-1 enzyme. © 1989.
• Ishihara, H., Ozaki, H., Sato, K., Hori, M., Karaki, H., Watabe, S., Kato, Y., Fusetani, N., Hashimoto, K., Uemura, D., & Hartshorne, D. J. (1989). Calcium-independent activation of contractile apparatus in smooth muscle by calyculin-A. Journal of Pharmacology and Experimental Therapeutics, 250(1), 388-396.
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  PMID: 2545866;Abstract: Calyculin-A (CL-A), a novel marine toxin isolated from Discodermia calyx, caused contraction in the smooth muscle of guinea pig taenia ceci and rat aorta in the presence or absence (with 1 mM ethylene glycol bis(β-aminoethyl ether)-N,N'-tetraacetic acid) of external Ca++ at concentrations ranging from 1 x 10-8 to 1 x 10-6 M. In the presence of external Ca++, the contraction induced by CL-A was accompanied by an increase in the cytosolic free Ca++ concentration ([Ca++](cyt)) as measured by the fluorescence indicator fura-2. Verapamil (3 x 10-6 M) inhibited the increase in [Ca++](cyt), but not tension development caused by CL-A. In the absence of external Ca++, CL-A still caused contraction without changing [Ca++](cyt). Thus, from studies with intact smooth muscle it was demonstrated that, in the absence of external Ca++, CL-A can induce a contraction that was not accompanied by an increase in [Ca++](cyt). In permeabilized taenia, CL-A caused contraction in the absence of Ca++ (with 2 mM ethylene glycol bis(β-aminoethyl ether)N,N'-tetraacetic acid) at concentrations similar to those required to contract intact tissue. This contraction was inhibited by the nonselective kinase inhibitors such as amiloride (1 x 10-3 M) and K-252a (2 x 10-5 M). Low concentrations of Ca++ (approximately 1 x 10-6 M) augmented the CL-A-induced contraction in the permeabilized taenia. In native actomyosin prepared from chicken gizzard CL-A induced phosphorylation of the 20 kDa myosin light chain (MLC) in the absence of Ca++. This phosphorylation was inhibited by amiloride and K-252a, but not by calmodulin (CaM) inhibitor, trifluoperazine (1 x 10-4 M), or protein kinase C inhibitor, polymyxin B (1 x 10-4 g/ml). CL-A was not effective as a CaM antagonist, or did it influence the activity of the MLC kinase measured under a variety of conditions. CL-A inhibited the dephosphorylation of MLC upon removal of Ca++ and ATP from the native actomyosin, indicating that CL-A inhibits myosin phosphatase. These data suggest that CL-A induces contraction through the direct activation of contractile elements which is attributable to the phosphorylation of MLC. This may be due either to the direct activation of Ca++/CaM independent MLC kinase or the unmasking of Ca++/CaM-independent MLC kinase activity via the inhibition of phosphatase activity. In addition, CL-A activates voltage-dependent Ca++ channels, but this effect appears to be independent of the CL-A induced contraction.
• Ito, M., Dabrowska, R., Guerriero Jr., V., & Hartshorne, D. J. (1989). Identification in turkey gizzard of an acidic protein related to the C-terminal portion of smooth muscle myosin light chain kinase. Journal of Biological Chemistry, 264(24), 13971-13974.
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  PMID: 2760053;Abstract: The isolation of an acidic protein, pI 4.5, that is abundant in turkey gizzard is described. Its apparent molecular weight measured by electrophoretic procedures is 24,000. This protein is phosphorylated by the catalytic subunit of the cAMP-dependent protein kinase and one phosphorylation site is indicated. From sequence determinations of tryptic peptides it is concluded that this protein is closely related to the C-terminal part of smooth muscle myosin light chain kinase. The initiation site for the protein is to the C-terminal side of the calmodulin-binding site. From the sequence data an estimated molecular weight is 18,000. This protein is expressed independently, as indicated by a blocked N terminus, and is probably the translation product of the 2.7-kilobase RNA detected previously in chicken gizzard (Guerriero, V., Jr., Russo, M.A., Olson, N.J., Purtkey, J.A., and Means, A.R. (1986) Biochemistry 25, 8372-8381). Because of its putative origin as the C-terminal end of smooth muscle myosin light chain kinase, it is termed 'telokin' (from a combination of kinase and the Greek telos, 'end').
• Ito, M., Pierce, P. R., Allen, R. E., & Hartshorne, D. J. (1989). Effect of monoclonal antibodies on the properties of smooth muscle myosin. Biochemistry, 28(13), 5567-5572.
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  PMID: 2528373;Abstract: Monoclonal antibodies were generated against turkey gizzard myosin, and their effects on some of the properties of myosin were assayed. Ca2+- and Mg2+-ATPase activities of myosin were enhanced by the anti-subfragment 2 antibodies at low ionic strength (i.e., with 10S myosin). Tryptic fragments of an anti-S2 IgM also activated these activities. Antibodies directed against subfragment 1 and light meromyosm had no effect. The Mg2+-ATPase activity of heavy meromyosin also was activated by an anti-S2 antibody. Actin-activated ATPase activity of phosphorylated myosin was enhanced by the anti-S2 IgM fragments at low MgCl2 concentrations. This increase was reflected by a 5-fold increase in Vmax and a slight decrease in the apparent dissociation constant for actin. The actin-activated ATPase of dephosphorylated myosin was not affected by intact anti-S2 antibody or its fragments. The rates of phosphorylation and dephosphorylation of the 20 000-dalton light chains were increased by interaction of myosin with anti-S2 antibody. Limited proteolysis of myosin was used as a conformational probe. Interaction of anti-S2 antibody with 10S myosin increased the extent of cleavage at the S1-S2 junction. Proteolysis of 6S myosin was rapid and was not influenced by anti-S2 antibody. Our interpretation of these results is that interaction of the anti-S2 antibodies with myosin alters the conformation in the S2 region and this in turn modifies some of the properties of myosin. This is consistent with the hypothesis that the S2 region of smooth muscle myosin is a determinant oif its biological properties. © 1989 American Chemical Society.
• Itoh, T., Ikebe, M., Kargacin, G. J., Hartshorne, D. J., Kemp, B. E., & Fay, F. S. (1989). Effects of modulators of myosin light-chain kinase activity in single smooth muscle cells. Nature, 338(6211), 164-167.
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  PMID: 2493140;Abstract: Phosphorylation of myosin light chains by a calmodulin-myosin light-chain kinase (MLCK) pathway is considered to be responsible for coupling increased calcium concentration with contraction in smooth muscle. This simple view has, however, recently been questioned. To test this hypothesis directly, we microinjected individual smooth muscle cells with modulators of the MLCK pathway while measuring contraction and calcium-ion concentration. Injection of a constitutively active proteolysed form of MLCK causes contraction but no change in calcium concentration. By contrast, injection of peptide inhibitors of MLCK blocks contraction in response to K+ depolarization, despite the fact that the change in calcium concentration in response to stimulation was enhanced over controls. These results provide a direct demonstration at the level of a single cell that activation of the calmodulin-MLCK pathway is both necessary and sufficient to trigger contraction of smooth muscle.
• Merkel, L., Ikebe, M., & Hartshorne, D. J. (1989). Interaction of smooth muscle tropomyosin and smooth muscle myosin. Effect on the properties of myosin. Biochemistry, 28(5), 2215-2220.
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  PMID: 2541778;Abstract: Several techniques were used to investigate the possibility that smooth muscle tropomyosin interacts with smooth muscle myosin. These experiments were carried out in the absence of actin. The Mg2+-ATPase activity of myosin was activated by tropomyosin. This was most marked at low ionic strength but also occurred at higher ionic strength with monomeric myosin. For myosin and HMM, the activation of Mg2+-ATPase by tropomyosin was greater at low levels of phosphorylation. There was no detectable effect of tropomyosin on the Mg2+-ATPase activity of S1. The KCl dependence of myosin viscosity was influenced by tropomyosin, and in the presence of tropomyosin, the 6S to 10S transition occurred at lower KCl concentrations. From the viscosity change, an approximate stoichiometry of 1:1 tropomyosin to myosin was estimated. The phosphorylation dependence of viscosity, which reflects the 10S-6S transition, also was altered in the presence of tropomyosin. An interaction between myosin and tropomyosin was detected by fluorescence measurements using tropomyosin labeled with dansyl chloride. These results indicate that an interaction occurs between myosin and tropomyosin. In general, the interaction is favored at low ionic strength and at low levels of phosphorylation. This interaction is not expected to be competitive with the formation of the actin-tropomyosin complex, but the possibility is raised that a direct interaction between myosin and tropomyosin bound to the thin filament could modify contractile properties in smooth muscle. © 1989 American Chemical Society.
• Rüegg, J., Zeugner, C., Strauss, J. D., Paul, R. J., Kemp, B., Chem, M., Li, A. -., & Hartshorne, D. J. (1989). A calmodulin-binding peptide relaxes skinned muscle from guinea-pig taenia coli. Pflügers Archiv European Journal of Physiology, 414(3), 282-285.
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  PMID: 2780212;Abstract: During smooth muscle activation the calcium calmodulin complex interacts with myosin light chain kinase (MLCK) whereby activating it. A synthetic peptide analogue (RS20) corresponding to the calmodulin recognition sequence of MLCK has been synthesized and previously found to inhibit the calmodulin stimulated light chain kinase activity. Here we studied the effect of this peptide on skinned fibers from guinea pig taenia coli. Maximal contractions induced by 30 μM Ca2+ at 0.1 μM calmodulin could be completely relaxed by the peptide at 1 μM. The inhibitory effect was accompanied by partial dephosphorylation only of the regulatory myosin light chain. Relaxation could be reversed by addition of calmodulin which also increased the extent of light chain phosphorylation.The calmodulin concentration required for reversing the inhibition depended on the concentration of the inhibitory peptide suggesting that the peptide competed with MLCK for the calmodulin binding site. As the calcium-calmodulin-peptide mixture constitutes a calmodulin buffer, our results suggest, that the peptide is a calmodulin antagonist unique in terms of its potency and that less than nanomolar concentrations of free calmodulin may be required for inducing smooth muscle contractions. © 1989 Springer-Verlag.
• Watabe, S., Tsuchiya, T., & Hartshorne, D. J. (1989). Phosphorylation of paramyosin. Comparative Biochemistry and Physiology -- Part B: Biochemistry and, 94(4), 813-821.
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  PMID: 2532591;Abstract: 1. 1. Myofibrils isolated from Mercenaria mercenaria were phophorylated by endogenous kinase. Over a range of ionic strengths only paramyosin was phosphorylated. 2. 2. Thiophosphorylation of paramyosin caused an inhibition of steady-state actin-activated ATPase activity of the myofibrils. 3. 3. It is proposed that the endogenous kinase is the catalytic subunit of the cAMP-dependent protein kinase. 4. 4. The sequence around the phosphorylation site was determined. 5. 5. The phosphorylation site probably is close to the C-terminus of the paramyosin molecule. © 1989.
• Ikebe, M., Koretz, J., & Hartshorne, D. J. (1988). Effects of phosphorylation of light chain residues threonine 18 and serine 19 on the properties and conformation of smooth muscle myosin. Journal of Biological Chemistry, 263(13), 6432-6437.
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  PMID: 2966156;Abstract: Smooth muscle myosin can be phosphorylated by myosin light chain kinase at the serine 19 and threonine 18 residues of the two 20,000-dalton light chains (Ikebe, M., Hartshorne, D.J., and Elizinga, M. (1986) J. Biol. Chem. 261, 36-39). These studies with myosin and heavy meromyosin (HMM) compare the effects induced by phosphorylation of serine 19 (M(2P) and HMM(2P)) and serine 19 plus threonine 18 (M(4P) and HMM(4P)). Formation of M(4P) altered the KCl dependence of viscosity and Mg2+-ATPase and higher values were maintained at lower ionic strengths, compared to M(2P) or dephosphorylated myosin (M0). This is consistent with the stabilization of the 6 S conformation. The tendency for aggregation, as judged by light scattering, followed the sequence M(4P) > M(2P) > M0. Filaments formed with M(4P) were more resistant to dissociation by ATP compared to filaments of M(2P). Phosphorylation of HMM(2P) doubled V(max) of actin-activated ATPase with little effect on the apparent affinity for actin. The Mg2+-ATPase of HMM(4P) exhibited a higher activity at low ionic strength compared to HMM(2P) and HMM0. Hydrodynamic differences were detected at low ionic strength in the presence of ATP by sedimentation velocity measurements with HMM(4P), HMM(2P), and HMM0. Proteolysis by papain indicated an increased susceptibility of the head-neck junction of HMM(4P) compared to HMM(2P). These data suggest that the phosphorylation of threonine 18 in addition to serine 19 change the conformation of myosin and HMM and this is associated with altered biological properties.
• Pearson, R. B., Wettenhall, R. E., Means, A. R., Hartshorne, D. J., & Kemp, B. E. (1988). Autoregulation of enzymes by pseudosubstrate prototypes: myosin light chain kinase. Science, 241(4868), 970-973.
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  PMID: 3406746;Abstract: The myosin light chain kinase requires calmodulin for activation. Tryptic cleavage of the enzyme generates an inactive 64-kilodalton (kD) fragment that can be further cleaved to form a constitutively active, calmodulin-independent, 61-kD fragment. Microsequencing and amino acid analysis of purified peptides after proteolysis of the 61- and 64-kD fragments were used to determine the amino-terminal and carboxyl-terminal sequences of the 64-kD fragment. Cleavage within the calmodulin-binding region at Arg505 generates the catalytically inactive 64-kD fragment, which is incapable of binding calmodulin. Further digestion removes a carboxyl-terminal fragment, including the pseudosubstrate sequence t-Lys-Lys-Tyr-Met-Ala-Arg-Arg-Lys-Trp-Gln-Lys-Thr-Gly-His-Ala-Val-Arg505 and results in a calmodulin-independent 61-kD fragment. Both the 61- and 64-kD fragments have the same primary amino-terminal sequences. These results provide direct support for the concept that the pseudosubstrate structure binds the active site and that the role of calmodulin is to modulate this interaction. Pseudosubstrates may be utilized in analogous ways by other allosterically regulated enzymes.
• Barsotti, R. J., Ikebe, M., & Hartshorne, D. J. (1987). Effects of Ca2+, Mg2+, and myosin phosphorylation on skinned smooth muscle fibers.. The American journal of physiology, 252(5 Pt 1), C543-554.
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  PMID: 3578506;Abstract: Isometric tension, unloaded shortening velocity (Vus), and myosin light chain phosphorylation were measured with skinned chicken gizzard fibers at various Ca2+ concentrations and at two concentrations of free Mg2+, 0.7 and 2.2 mM. At low free Mg2+, an increase in Ca2+ from pCa 8.0 to 6.4 resulted in an increase of all three parameters. Between pCa 6.4 and 5.0, isometric tension and phosphorylation remained constant but Vus continued to increase. At low free Mg2+, therefore, Vus showed a dependence both on phosphorylation and on Ca2+. At high free Mg2+, tension and Vus increased as phosphorylation increased and both were maximum at pCa 6.4, where phosphorylation became constant. Therefore, at high free Mg2+, Vus was dependent only on phosphorylation and did not show an additional Ca2+ dependence. Incubation of the Ca2+-independent kinase (approximately 3 microM) with skinned fibers under various conditions resulted in a constant level of phosphorylation (49-58%). At high free Mg2+ plus the Ca2+-independent kinase Vus was independent of Ca2+, whereas at low free Mg2+ Vus increased from pCa 6.4 to 5.0. These data are consistent with the hypothesis that Ca2+ binding to the Ca2+-Mg2+ sites of myosin increase Vus and that this occurs at Ca2+ concentrations higher than those necessary to saturate calmodulin.
• Barsotti, R. J., Ikebe, M., & Hartshorne, D. J. (1987). Effects of Ca²⁺, Mg²⁺, and myosin phosphorylation on skinned smooth muscle fibers. American Journal of Physiology - Cell Physiology, 252(5), 21/5.
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  Abstract: Isometric tension, unloaded shortening velocity (V(us)), and myosin light chain phosphorylation were measured with skinned chicken gizzard fibers at various Ca2+ concentrations and at two concentrations of free Mg2+, 0.7 and 2.2 mM. At low free Mg2+, an increase in Ca2+ from pCa 8.0 to 6.4 resulted in an increase of all three parameters. Between pCa 6.4 and 5.0, isometric tension and phosphorylation remained constant but V(us) continued to increase. At low free Mg2+, therefore, V(us) showed a dependence both on phosphorylation and on Ca2+. At high free Mg2+, tension and V(us) increased as phosphorylation increased and both were maximum at pCa 6.4, where phosphorylation became constant. Therefore, at high free Mg2+, V(us) was dependent only on phosphorylation and did not show an additional Ca2+ dependence. Incubation of the Ca2+-independent kinase (~3 μM) with skinned fibers under various conditions resulted in a constant level of phosphorylation (49-58%). At high free Mg2+ plus the Ca2+-independent kinase V(us) was independent of Ca2+, whereas at low free Mg2+ V(us) increased from pCa 6.4 to 5.0. These data are consistent with the hypothesis that Ca2+ binding to the Ca2+-Mg2+ sites of myosin increase V(us) and that this occurs at Ca2+ concentrations higher than those necessary to saturate calmodulin.
• Ikebe, M., Hartshorne, D. J., & Elzinga, M. (1987). Phosphorylation of the 20,000-dalton light chain of smooth muscle myosin by the calcium-activated, phospholipid-dependent protein kinase. Phosphorylation sites and effects of phosphorylation.. Journal of Biological Chemistry, 262(20), 9569-9573.
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  PMID: 3036866;Abstract: Smooth muscle heavy meromyosin (HMM) is phosphorylated by the Ca2+-activated phospholipid-dependent protein kinase, i.e. protein kinase C, at three sites on each 20,000-dalton light chain. Phosphorylation of three sites also is observed with isolated 20,000-dalton light chain and HMM subfragment 1. The phosphorylation sites are serine 1, serine 2, and threonine 9. Threonine is phosphorylated most rapidly followed by either serine 1 or 2. Phosphorylation of the third site occurs only on prolonged incubation. Phosphorylation is a random process. HMM phosphorylated at two sites per light chain by protein kinase C can be dephosphorylated, as shown using two phosphatase preparations. Increasing levels of phosphorylation of HMM by protein kinase C causes a progressive inhibition of the subsequent rate of phosphorylation of serine 19 by myosin light chain kinase and causes a progressive inhibition of actin-activated ATPase activity of HMM, prephosphorylated by myosin light chain kinase. Inhibition of ATPase activity is due to a decreased affinity of HMM for actin rather than a change in Vmax. Previous results with HMM and protein kinase C (Nishikawa, M., Sellers, J. R., Adelstein, R. S., and Hidaka, H. (1984) J. Biol. Chem. 259, 8808-8814) examined effects induced by phosphorylation of the threonine residues. Our results confirm these and consider also the influence of higher levels of phosphorylation by protein kinase C.
• Ikebe, M., Stepinska, M., Kemp, B. E., Means, A. R., & Hartshorne, D. J. (1987). Proteolysis of smooth muscle myosin light chain kinase. Formation of inactive and calmodulin-independent fragments.. Journal of Biological Chemistry, 262(28), 13828-13834.
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  PMID: 3654638;Abstract: Proteolysis by trypsin of gizzard myosin light chain kinase in the absence of Ca2+-calmodulin causes a biphasic effect on kinase activity. During the initial phase of proteolysis, Ca2+-calmodulin-dependent kinase activity is reduced over a thousand-fold. Further proteolysis, in the second phase, causes an increase in activity that is independent of Ca2+-calmodulin. Loss of activity is associated with the formation of a 64,000-dalton fragment. Calmodulin-independent activity is associated with the formation of a 61,000-dalton fragment. Procedures for the isolation of each fragment are outlined. Tryptic hydrolysis of the isolated 64,000-dalton peptide generates the 61,000-dalton peptide and increases calmodulin-independent activity. Km values for ATP and light chains for the native kinase and two fragments are the same, i.e. approximately 100 and 5 microM, respectively. Neither fragment binds to F-actin. Amino acid analyses of both fragments are given. Synthetic peptides corresponding to the calmodulin-binding regions of the smooth and skeletal muscle kinases are potent inhibitors of the 61,000-dalton fragment. These data demonstrate the existence of an inhibitory region that is suggested to be located between the active site and the calmodulin-binding site. Whether it is distinct from or at the N-terminal end of the calmodulin-binding site cannot be determined from these data.
• Ikebe, M., & Hartshorne, D. J. (1986). Proteolysis and actin-binding properties of 10S and 6S smooth muscle myosin: Identification of a site protected from proteolysis in the 10S conformation and by the binding of actin. Biochemistry, 25(20), 6177-6185.
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  PMID: 3790513;Abstract: It was shown previously [Ikebe, M., & Hartshorne, D. J. (1985) Biochemistry 24, 2380-2387] that the conformation of gizzard myosin, either 10S or 6S, influences proteolysis of myosin at two regions designated sites A and B. The studies reported here are focused on site A, which is located approximately 68 000 daltons from the N-terminus of the myosin heavy chain. With papain, Staphylococcus aureus protease, and actinidin, it is shown that the formation of 10S myosin reduces proteolysis at site A. Binding of actin to 6S myosin also hinders cleavage at site A for each of these proteases. To investigate binding of actin to 6S and 10S myosins, adenosine 5′-(β,γ-imidotriphosphate) (AMPPNP) is used as a substitute for ATP. In the presence of AMPPNP, it is shown that the 6S to 10S transition occurs and that 10S myosin binds actin with lower affinity than 6S myosin. For 6S myosin at high salt (0.35 M KCl) the dissocation constant of actin from the actin-myosin-nucleotide complex (K3) is approximately the same for phosphorylated (1.9 mol of P/mol of myosin) and dephosphorylated myosin, i.e., 1.3-2.4 μM, respectively. At lower ionic strength (0.17 M KCl) K3 for dephosphorylated myosin (10S myosin) is 42 μM and K3 for phosphorylated myosin (6S myosin) is 0.3 μM. These data show that the conformation of myosin influences the actin-myosin interaction. The constant (K4) for the dissociation of nucleotide from the actin-myosin-nucleotide complex varies slightly (in the range of 0.2-1.3 mM), but there is no marked change as a result of either a conformational change or phosphorylation. Since site A is masked, at least partly, by the formation of 10S myosin and by the binding of actin, this may indicate that site A is located at, or close to, the actin-binding site on the myosin heavy chain. © 1986 American Chemical Society.
• Ikebe, M., & Hartshorne, D. J. (1986). Reverse reaction of smooth muscle myosin light chain kinase. Formation of ATP from phosphorylated light chain plus ADP. Journal of Biological Chemistry, 261(18), 8249-8253.
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  PMID: 3522566;Abstract: Incubation of smooth muscle phosphorylated heavy meromyosin in the presence of myosin light chain kinase, calmodulin, ADP, and Ca2+ results in a decrease of the protein-bound phosphate. The dephosphorylation is not due to phosphatase activity and is dependent on the presence of ADP and the active ternary myosin light chain kinase complex. Using 32P-labeled phosphorylated 20,000-dalton light chains as the phosphate donor, the formation of ATP from ADP can be demonstrated. This reaction requires the presence of Ca2+, calmoculin, and myosin light chain kinase. These results indicate that myosin light chain kinase can catalyze a reverse reaction and form ATP from ADP and phosphorylated substrate. The rate of the reverse reaction, k(cat)/K(LC) ~ 0.21 min-1 μM-1, is considerably slower than the forward reaction under similar conditions and is therefore detectable only at relatively high concentrations of myosin light chain kinase. For the reverse reaction, K(m)(ADP) is approximately 30 μM and ATP is a competitive inhibitor, K(i)(ATP) ~ 88 μM. For the forward reaction, measured with both isolated light chains and intact myosin, K(m)(ATP) is approximately 100 μM and ADP is a competitive inhibitor, K(i)(ADP) ~ 140 μM (myosin) and 120 μM (light chains). Thus, the affinity of ATP for the forward and reverse reactions is similar, but the affinity of ADP is higher for the reverse reaction. From the light chain dependence of the two reactions, the following was calculated: forward, K(m) = 5 μM, k(cat) = 1720 min-1, and reverse, K(m) = 130 μM, k(cat) = 27 min-1. In contrast to the data obtained with isolated light chains, it is suggested that, with intact myosin as substrate, the K(m) term is primarily responsible for determining the rate of the reverse reaction. With light chains phosphorylated at serine 19 and threonine 18, it was shown that both sites act as a phosphate donor, although the reverse reaction for threonine 18 is slower than that for serine 19.
• Ikebe, M., Hartshorne, D. J., & Elzinga, M. (1986). Identification, phosphorylation, and dephosphorylation of a second site for myosin light chain kinase on the 20,000-dalton light chain of smooth muscle myosin. Journal of Biological Chemistry, 261(1), 36-39.
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  PMID: 3079756;Abstract: At relatively high concentrations of myosin light chain kinase, a second site on the 20,000-dalton light chain of smooth muscle myosin is phosphorylated. In this communication the site is identified and kinetics associated with its phosphorylation and dephosphorylation are described. The doubly phosphorylated 20,000-dalton light chain from turkey gizzard myosin was hydrolyzed with α-chymotrypsin and the phosphorylated peptide was isolated by reverse phase chromatography. Following amino acid analyses and partial sequence determinations the second site of phosphorylation is shown to be threonine 18. This site is distinct from the threonine residue phosphorylated by protein kinase C. The time courses of phosphorylation of serine 19 and threonine 18 in isolated light chains follow a single exponential indicating a random process, although the phosphorylation rates differ considerably. The values of k(cat/K(m) for serine 19 and threonine 18 for isolated light chains are 550 and 0.2 min-1 μM-1, respectively. With intact myosin, phosphorylation of serine 19 is biphasic; k(cat)/K(m) values are 22.5 and 7.5 min-1 μM-1 for the fast and slow phases, respectively. In contrast, phosphorylation of threonine 18 in intact myosin is a random, but markedly slower process, k(cat)/K(m) = 0.44 min-1 μM-1. Dephosphorylation of doubly phosphorylated myosin (~4 mol of phosphate/mol of myosin) and isolated light chains (~2 mol of phosphate/mol of light chain) follows a random process and dephosphorylation of the serine 19 and threonine 18 sites occurs at similar rates.
• Sommerville, L., & Hartshorne, D. (1986). Intracellular calcium and smooth muscle contraction. Cell Calcium, 7(5-6), 353-364.
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  PMID: 3545487;Abstract: Excitation-contraction coupling in smooth muscle involves many processes, some of which are outlined in this article. The total amount of Ca2+ released on excitation is considerably in excess of the free Ca2+ concentration and this implies a high capacity, high affinity Ca2+ buffer system. The two major Ca2+-binding proteins are calmodulin and myosin. Only calmodulin has the appropriate binding affinity to act as a component of the Ca2+-buffer system. The Ca2+-calmodulin complex activates myosin light chain kinase and thus is involved in the regulation of contractile activity. Phosphorylation of myosin stabilizes an active conformation and promotes cross bridge cycling and is essential for the initiation of contraction. During the initial contractile response phosphorylation correlates to tension development and velocity of shortening. However, as contraction continues the extent of myosin phosphorylation and velocity often decreases but tension is maintained. In general, the Ca2+ transient is reflected by the extent of phosphorylation that in turn correlates with shortening velocity. Maintenance of tension at low phosphorylation levels is not accounted for within our understanding of the phosphorylation theory and thus alternative regulatory mechanisms have been implicated. Some of the possibilities are discussed. © 1986.
• Ikebe, M., & Hartshorne, D. J. (1985). Effect of brief proteolysis of gizzard myosin on actomyosin ATPase activity. Federation Proceedings, 44(5), No. 6088.
• Ikebe, M., & Hartshorne, D. J. (1985). Effects of Ca²⁺ on the conformation and enzymatic activity of smooth muscle myosin. Journal of Biological Chemistry, 260(24), 13146-13153.
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  PMID: 2932435;
• Ikebe, M., & Hartshorne, D. J. (1985). Phosphorylation of smooth muscle myosin at two distinct sites by myosin light chain kinase. Journal of Biological Chemistry, 260(18), 10027-10031.
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  PMID: 3839510;Abstract: The 20,000-dalton light chain of turkey gizzard myosin is phosphorylated at two sites. Dual phosphorylation is observed when both intact myosin and isolated light chains are used as substrates. Phosphorylation of the second site is not observed at higher ionic strength (e.g. 0.35 M KCl). The first phosphorylation site (serine 19) is phosphorylated preferentially to the second site. The latter is phorphorylated more slowly than the first site, and its phosphorylation requires relatively high concentrations of myosin light chain kinase. It is suggested that myosin light chain kinase catalyzes the phosphorylation of both sites on the light chain, and several reasons are cited that make it unlikely that a contaminant kinase is involved. The second phosphorylation site is a threonine residue. Based on the results of limited proteolysis of the light chain, it is concluded that the threonine residue is close to serine 19, and possible locations are threonines 9, 10, and 18. At all concentrations of MgCl2, phosphorylation of the second site markedly increases the actin-activated ATPase activity of myosin and accelerates the superprecipitation response of myosin plus actin.
• Ikebe, M., & Hartshorne, D. J. (1985). Proteolysis of smooth muscle myosin by Staphylococcus aureus protease: Preparation of heavy meromyosin and subfragment 1 with intact 20 000-dalton light chains. Biochemistry, 24(9), 2380-2387.
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  PMID: 3158349;Abstract: The proteolysis of gizzard myosin by Staphylococcus aureus protease produces both heavy meromyosin and subfragment 1 in which the 20 000-dalton light chains are intact, and conditions are suggested for the preparation of each. Cleavage of the myosin heavy chain to produce subfragment 1 is dependent on the myosin conformation. Proteolysis of myosin in the 10S conformation yields predominantly heavy meromyosin, and myosin in the 6S conformation yields mostly subfragment 1 and some heavy meromyosin. Two sites are influenced by myosin conformation, and these are located at approximately 68 000 and 94 000 daltons from the N-terminus of the myosin heavy chain. The latter site is thought to be located at the subfragment 1-subfragment 2 junction, and cleavage at this site results in the production of subfragment 1. The time courses of phosphorylation of both heavy meromyosin and subfragment 1 can be fit by a single exponential. The actin-activated Mg2+-ATPase activity of heavy meromyosin is markedly activated by phosphorylation of the 20 000-dalton light chains. From the actin dependence of Mg2+-ATPase activity the following values are obtained: for phosphorylated heavy meromyosin, Vmax ∼ 5.6 s-1 and Ka (the apparent dissociation constant for actin) ∼ 2 mg/mL; for dephosphorylated heavy meromyosin, Vmax ∼ 0.2 s-1 and Ka ∼ 7 mg/mL. The actin-activated ATPase activity of subfragment 1 is not influenced by phosphorylation, and Vmax and Ka for both the phosphorylated and dephosphorylated forms are 0.4 s-1 and 5 mg/mL, respectively. The Mg2+- and Ca2+-ATPase activities of subfragment 1 are distinct from those of heavy meromyosin, and the Mg2+-ATPase activity of subfragment 1 is not affected by phosphorylation. It is clear from these results that the phosphorylation of the 20 000-dalton light chain of subfragment 1 is not required for actin-activated ATPase activity. It is suggested that regulation via phosphorylation involves the interaction of the myosin heads with other parts of the molecule, possibly in the subfragment 2 region. © 1985 American Chemical Society.
• Ikebe, M., & Hartshorne, D. J. (1985). The role of myosin phosphorylation in the contraction-relaxation cycle of smooth muscle. Experientia, 41(8), 1006-1010.
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  PMID: 3160603;Abstract: Considerable evidence from a variety of experimental procedures indicates that the phosphorylation of myosin is involved in the regulation of contractile activity in smooth muscle. Phosphorylation of the 20,000-dalton myosin light chains is required to initiate crossbridge cycling and this is consistent with the observation that the actin-activated Mg2+-ATPase activity of myosin is phosphorylation-dependent. In the simplest interpretation of this process it may be proposed that phosphorylation acts as an 'on-off' switch. Clearly this cannot explain the observed complexity of smooth muscle contractile behavior and such may imply either that additional mechanisms are involved or that the role of myosin phosphorylation is not fully appreciated. Recently it has been shown that monomeric smooth muscle myosin can exist in a 'folded' and an 'extended' conformation and that each form is characterized by distinct enzymatic properties. Under appropriate solvent conditions phosphorylation of myosin favors the extended conformation. It is tentatively suggest that this, or an analogous, transition might be involved in the regulation of the smooth muscle contractile apparatus, and this possibility is discussed. © 1985 Birkhäuser Verlag.
• Mrwa, U., Güth, K., Rüegg, J., Paul, R. J., Boström, S., Barsotti, R., & Hartshorne, D. (1985). Mechanical and biochemical characterization of the contraction elicited by a calcium-independent myosin light chain kinase in chemically skinned smooth muscle. Experientia, 41(8), 1002-1006.
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  PMID: 3160602;Abstract: The contraction induced by a Ca2+-independent myosin light chain kinase (MLCK-) was characterized in terms of isometric force (Fo), immediate elastic recoil (SE), unloaded shortening velocity (Vus), shortening under a constant load and ATPase activity of chemically skinned smooth muscle preparations. These parameters were compared to those measured in a Ca2+-induced contraction to assess the nature of cross bridge interaction in the MLCK-induced contraction. Fo developed in chicken gizzard fibers as well as SE were similar in contractions elicited by either agent. Vus in the contraction induced by MLCK-(0.36 mg/ml) was similar though averaged 39.3±8.9% less than Vus induced by Ca2+ (1.6x10-6M) in the control fibers. Addition of Ca2+ (1.6x10-6M) to a contraction induced by MLCK-resulted in small increases in both Fo and Vus. Shortening under a constant load was similar for both types of contractions. The contraction induced by MLCK-was accompanied by an increased rate of ATP hydrolysis. The MLCK-induced contraction is thus kinetically similar though not identical to a contraction induced by Ca2+. We conclude that with respect to actin-myosin interaction, MLCK- and Ca2+-induced contractions are similar. © 1985 Birkhäuser Verlag.
• Srivastava, S., Ikebe, M., & Hartshorne, D. J. (1985). Trinitrophenylation of smooth muscle myosin. Biochemical and Biophysical Research Communications, 126(2), 748-755.
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  PMID: 3156591;Abstract: The reaction of trinitrobenzenesulfonate with gizzard myosin was studied. The initial phase of the reaction involved two residues and at this level of modification the following was observed: the Mg2+-ATPase of myosin, the actin-activated ATPase of phosphorylated myosin and the phosphorylation kinetics of myosin were not affected. However, trinitrophenylation did induce an activation of the actin-activated ATPase of dephosphorylated myosin and in this respect mimicked the effect of light chain phosphorylation. The Mg2+-dependence of actin-activated ATPase also is altered on trinitrophenylation. These alterations of enzymatic properties could be at least partly explained by the finding that trinitrophenylation favored the 6S conformation of myosin. © 1985.
• Gagelmann, M., Mrwa, U., Bostrom, S., Rüegg, J., & Hartshorne, D. (1984). Effect of Ca²⁺-independent myosin light chain kinase on different skinned smooth muscle fibers. Pflügers Archiv European Journal of Physiology, 401(1), 107-109.
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  PMID: 6548012;Abstract: In various skinned smooth muscle fiber preparations, (porcine carotid artery, rat tail artery, chicken gizzard and Taenia coli from guinea pig) a Ca2+-independent myosin light chain kinase (MLCK) initiated a contraction in absence of Ca2+. While the Ca2+ insensitive MLCK was effective on the vertebrate smooth muscles it did not act on the invertebrate skinned skeletal muscle preparation from Limulus and anterior byssus retractor muscle from Mytilus edulis. The results indicate that in vertebrate smooth muscles phosphorylation is sufficient for activation and that there is no obligatory role for an additional mechanism in initiation of contraction. © 1984 Springer-Verlag.
• Ikebe, M., & Hartshorne, D. J. (1984). Conformation-dependent proteolysis of smooth-muscle myosin. Journal of Biological Chemistry, 259(19), 11639-11642.
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  PMID: 6384209;Abstract: The folded 10 S conformation of turkey gizzard myosin is more resistant to proteolysis by papain than the extended 6 S conformation. These findings confirm those of Onishi and Watanabe (Onishi, H., and Watanabe, S. (1984) J. Biochem. (Tokyo) 95, 899-902). In addition, we suggest that the effect of phosphorylation on heavy-chain digestion by papain is related to the dependence of conformation on phosphorylation and not to a direct effect of phosphorylation itself. Proteolysis by Staphylococcus aureus protease and trypsin also is slower for 10 S compared to the 6 S conformation. Heavy chain hydrolysis by α-chymotrypsin is not dependent on myosin conformation. Filamentous myosin and heavy meromyosin are more resistant to papain proteolysis in the dephosphorylated compared to the phosphorylated states. The different sensitivities to proteolysis probably are caused by changes in the subfragment 1-subfragment 2 region of the molecule rather than at the heavy meromyosin-light meromyosin junction. These changes are induced as part of the 6 S-10 S transition and occur in monomeric and filamentous myosin and in heavy meromyosin. These more subtle alterations in the head-neck junctions of the molecule may be more important in modifying myosin enzymatic activity than the actual interaction of the tail and neck regions of the molecule.
• Ikebe, M., Barsotti, R. J., Hinkins, S., & Hartshorne, D. J. (1984). Effects of magnesium chloride on smooth muscle actomyosin adenosine-5′-triphosphatase activity, myosin conformation, and tension development in glycerinated smooth muscle fibers. Biochemistry, 23(21), 5062-5068.
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  PMID: 6238628;Abstract: The contractile system of smooth muscle exhibits distinctive responses to varying Mg2+ concentrations in that maximum adenosine-5′-triphosphatase (ATPase) activity of actomyosin requires relatively high concentrations of Mg2+ and also that tension in skinned smooth muscle fibers can be induced in the absence of Ca2+ by high Mg2+ concentrations. We have examined the effects of MgCl2 on actomyosin ATPase activity and on tension development in skinned gizzard fibers and suggest that the MgCl2-induced changes may be correlated to shifts in myosin conformation. At low concentrations of free Mg2+ (≤1 mM) the actin-activated ATPase activity of phosphorylated turkey gizzard myosin is reduced and is increased as the Mg2+ concentration is raised. The increase in Mg2+ (over a range of 1-10 mM added MgCl2) induces the conversion of 10S phosphorylated myosin to the 6S form, and it was found that the proportion of myosin as 10S is inversely related to the level of actin-activated ATPase activity. Activation of the actin-activated ATPase activity also occurs with dephosphorylated myosin but at higher MgCl2 concentrations, between 10 and 40 mM added MgCl2. Viscosity and fluorescence measurements indicate that increasing Mg2+ levels over this concentration range favor the formation of the 6S conformation of dephosphorylated myosin, and it is proposed that the 10S to 6S transition is a prerequisite for the observed activation of ATPase activity. With glycerinated chicken gizzard fibers high MgCl2 concentrations (6-20 mM) promote tension in the absence of Ca2+. MgCl2-induced tension is not associated with an increased extent of myosin phosphorylation, and this is supported by the observation that in the presence of ITP tension can also be developed by high concentrations of MgCl2. © 1984 American Chemical Society.
• Ikebe, M., Hinkins, S., & Hartshorne, D. J. (1983). Correlation of enzymatic properties and conformation of smooth muscle myosin. Biochemistry, 22(19), 4580-4587.
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  PMID: 6138093;Abstract: In the presence of adenosine 5′-triphosphate (ATP) and 1-10 mM MgCl2, the relative viscosity (ηrel) of dephosphorylated gizzard myosin is reduced markedly over a range of KCl from 0.35 to 0.15 M. Sedimentation patterns show that the decrease in ηrel is due to the conversion of the 6S to 10S forms of myosin. Under similar conditions, ηrel of phosphorylated myosin is not altered, and at 0.2 M KCl, the 10S form is not observed. In 1 and 2 mM MgCl2 and
• Ikebe, M., Hinkins, S., & Hartshorne, D. J. (1983). Correlation of intrinsic fluorescence and conformation of smooth muscle myosin. Journal of Biological Chemistry, 258(24), 14770-14773.
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  PMID: 6654892;Abstract: The addition of ATP to turkey gizzard myosin causes an enhancement of the intrinsic tryptophan fluorescence. The level of fluorescence enhancement is determined by the myosin conformation. The transition of myosin from the folded (10 S) state to the extended (6 S) state is accompanied by a decrease in the fluorescence level. Phosphorylation-dephosphorylation of myosin does not directly influence fluorescence and will induce changes only if the myosin conformation is altered. Under the appropriate conditions, phosphorylation of myosin favors the transition of 10 S to 6 S. The phosphorylation dependence of the associated fluorescence decrease is not linear, and it is proposed that the phosphorylation of both light chains is required for the full transition. The tryptophan residues involved respond to the binding of ATP at the hydrolytic sites. Since the fluorescence properties of gizzard myosin are influenced by the myosin conformation, it is reasonable to assume that the active sites are also modified by the shape of the myosin molecule.
• Persechini, A., & Hartshorne, D. J. (1983). Ordered phosphorylation of the two 20 000 molecular weight light chains of smooth muscle myosin. Biochemistry, 22(2), 470-476.
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  PMID: 6687432;Abstract: The time courses of phosphorylation of the Mr 20 000 light chains by purified myosin light chain kinase plus calmodulin were determined. In confirmation of an earlier report [Persechini, A., & Hartshorne, D. J. (1981) Science (Washington, D.C.) 213, 1383-1385], a steady-state kinetic analysis indicates that the phosphorylation occurs in an ordered manner; i.e., at a phosphorylation level of 0.5 mol of 32P incorporated per mol of bound Mr 20 000 light chain, each myosin molecule would have one phosphorylated head. The kinetic parameters obtained for the phosphorylation of the more reactive myosin head are similar to those determined by using isolated light chains. It is suggested that the ordered, or sequential, phosphorylation, and the different reactivities of the two Mr 20 000 light chains, is the result of preexisting asymmetry of the myosin molecule. Similar patterns of myosin phosphorylation are obtained in both the absence and presence of skeletal muscle actin. © 1983 American Chemical Society.
• Srivastava, S., & Hartshorne, D. J. (1983). Conversion of a Ca²⁺-dependent myosin light chain kinase from skeletal muscle to a Ca²⁺-independent form. Biochemical and Biophysical Research Communications, 110(2), 701-708.
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  PMID: 6687681;Abstract: The Ca2+- and calmodulin-dependent myosin light chain kinase of rabbit skeletal muscle was converted to a Ca2+-independent form by limited proteolysis with α-chymotrypsin. The conditions prevailing during proteolysis are important and the loss of Ca2+-dependence was achieved best by hydrolysis of the Ca2+-calmodulin-kinase complex. The lack of Ca2+- and calmodulin-dependence was found using both myosin and isolated light chains as substrates. The specific activity of the Ca2+-independent form (Mr approximately 65,000) was similar to that of the native enzyme, i.e., 2 to 5 μmol phosphate transferred min-1 mg-1 kinase. The 65,000-dalton fragment was phosphorylated by the catalytic subunit of the cAMP-dependent protein kinase and approximately 0.8 moles phosphate were incorporated per fragment. © 1983.
• Walsh, M. P., Bridenbaugh, R., Kerrick, W. G., & Hartshorne, D. J. (1983). Gizzard Ca²⁺-independent myosin light chain kinase: Evidence in favor of the phosphorylation theory. Federation Proceedings, 42(1), 45-50.
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  PMID: 6848377;Abstract: Limited digestion of calmodulin (CaM)-dependent myosin light chain kinase from turkey gizzard with α-chymotrypsin in the presence of bound CaM generated an 80,000-dalton kinase fragment that was fully active in the absence of Ca2+. This kinase catalyzed specific Ca2+-independent phosphorylation of the 20,000-dalton light chain of myosin using isolated light chains, intact myosin, and actomyosin. Phosphorylation of myosin in the absence of Ca2+ allowed us to dissociate myosin phosphorylation from other potential Ca2+-dependent regulatory mechanisms, thus permitting an evaluation of the postulated central role of myosin phosphorylation in the regulation of smooth muscle contraction. Ca2+-independent myosin phosphorylation was found to cause loss of Ca2+ sensitivity of actin-activated myosin ATPase activity in a crude actomyosin preparation, and tension development in skinned smooth muscle fibers in the absence of Ca2+. Myosin phosphorylation is, therefore, the key event in actin activation of ATPase activity and initiation of contraction in skinned chicken gizzard fibers.
• Walsh, M. P., Hinkins, S., Dabrowska, R., & Hartshorne, D. J. (1983). Smooth muscle myosin light chain kinase. Methods in Enzymology, Vol. 99, 279-288.
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  PMID: 6358787;
• Walsh, M. P., Hinkins, S., Muguruma, M., & Hartshorne, D. J. (1983). Identification of two forms of myosin light chain kinase in turkey gizzard. FEBS Letters, 153(1), 156-160.
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  PMID: 6687459;Abstract: Two forms of myosin light chain kinase from turkey gizzard are separable by ion-exchange chromatography. One is the well-characterized 13 000 Mr enzyme. Purification of the second form by affinity chromatography on calmodulin-Sepharose showed it to consist of two polypeptide chains of Mr 136 000 and 141 000. This form of the enzyme required Ca2+ and calmodulin for activity, was specific for the Mr 20 000 light chain of myosin, and appeared to phosphorylate the same site on the light chain as the Mr 130 000 enzyme. The low-Mr gizzard kinase may be a proteolytic fragment of a higher-Mr species or these may represent different isoenzymes. © 1983.
• Dabrowska, R., Hinkins, S., Walsh, M. P., & Hartshorne, D. J. (1982). The binding of smooth muscle myosin light chain kinase to actin. Biochemical and Biophysical Research Communications, 107(4), 1524-1531.
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  PMID: 6982712;Abstract: Myosin light chain kinase isolated from turkey gizzards binds to skeletal muscle actin. The binding is not influenced significantly by the presence, or absence, of Ca2+, calmodulin, gizzard tropomyosin and Mg2+-ATP. The myosin light chain kinase is removed from the actin filaments as the ionic strength is increased. The possibility of non-specific binding to actin is considered unlikely since the interaction is not affected by the presence of excess bovine serum albumin and also by the finding that the Ca2+-independent form of the myosin light chain kinase does not bind to actin. The binding of myosin light chain kinase to gizzard myosin is not as marked, and under conditions similar to those used to demonstrate binding to actin only about 10% of the kinase is associated with the myosin aggregates. © 1982 Academic Press, Inc.
• Hartshorne, D. J., & Mrwa, U. (1982). Regulation of smooth muscle actomyosin. Blood Vessels, 19(1), 1-18.
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  PMID: 7037073;Abstract: In smooth muscle it is generally accepted that at the level of the contractile apparatus regulation is achieved by activating a dormant state in the presence of Ca2+. This event initiates the contractile process which is manifest by an increased cross-bridge cycling rate and the development of tension, or in biochemical terms, by an increase in the activation by actin of the Mg2+-ATPase activity of myosin. A controversy exists, however, on the identity of the activator, and this review considers the two proposed possibilities. One theory is based on the phosphorylation and dephosphorylation of the 20,000 dalton molecular weight myosin light chains. It is assumed that in the phosphorylated state the Mg2+-ATPase activity of myosin can be activated by actin whereas dephosphorylated myosin cannot be activated by actin. Phosphorylation of myosin, and hence the activation of the contractile apparatus, is achieved by a myosin light chain kinase, and it has been shown that the calcium dependence of the phosphorylation reaction resides with one of the two components of this enzyme, namely calmodulin. Inactivation of the contractile apparatus is brought about by a second enzyme, the myosin light chain phosphatase. There is considerable experimental evidence in support of the phosphorylation theory and it appears that it must be at least a part of the regulatory system. However, it cannot be concluded that phosphorylation-dephosphorylation of myosin is the sole regulatory mechanism, and recent results have indicated that additional factors may be involved. The second theory to account for the activation of the contractile apparatus is not based on the phosphorylation of the myosin molecule and is thought to be due to a system called leiotonin. This system, composed of two subunits leiotonin A and C, is thought to be associated with the thin filaments. Its mode of action has not been established.
• Persechini, A., & Hartshorne, D. J. (1982). Cooperative behavior of smooth muscle myosin. Federation Proceedings, 41(12), 2868-2872.
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  PMID: 6215263;
• Walsh, M. P., Bridenbaugh, R., Hartshorne, D. J., & Kerrick, W. G. (1982). Phosphorylation-dependent activated tension in skinned gizzard muscle fibers in the absence of Ca2+.. Journal of Biological Chemistry, 257(11), 5987-5990.
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  PMID: 6896202;Abstract: A Ca2+-insensitive myosin light chain kinase was prepared and used in experiments with skinned gizzard fibers. In the absence of Ca2+, this kinase activated isometric force development. The force development was associated with phosphorylation of the 20,000-dalton myosin light chains and addition of Ca2+ did not activate the fibers further. Pretreatment of the fibers with Ca2+-insensitive myosin light chain kinase and adenosine 5'-O-(3-thiotriphosphate) in the absence of Ca2+ caused thiophosphorylation of the light chains and, on the addition of ATP, an activation of isometric tension was observed. The subsequent addition of Ca2+ did not increase force development. A comparison of Ca2+-activated tension in the skinned gizzard muscle fibers with Ca2+-insensitive myosin light chain kinase-activated tension suggests that the phosphorylation of the myosin light chains is the dominant factor in the development of tension in smooth muscle.
• Walsh, M. P., Dabrowska, R., Hinkins, S., & Hartshorne, D. J. (1982). Calcium-independent myosin light chain kinase of smooth muscle. Preparation by limited chymotryptic digestion of the calcium ion dependent enzyme, purification, and characterization. Biochemistry, 21(8), 1919-1925.
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  PMID: 6896283;Abstract: Limited α-chymotryptic digestion of Ca2+-, calmodulin-dependent myosin light chain kinase partially purified from smooth muscle (turkey gizzard) yielded a Ca2+-independent form of the enzyme. Digestion to yield the Ca2+independent kinase required the enzyme complexed with Ca2+-calmodulin; when digestion was performed on the apoenzyme, i.e., in the absence of Ca2+, the dependence of kinase activity on Ca2+ was retained. The Ca2+-independent kinase was purified by ion-exchange chromatography and shown to have an apparent molecular weight of ∼80 000. The specific activity of the freshly prepared enzyme was 6.5 ± 0.2 μmol of Pi incorporated min-1 mg-1 in the presence of Ca2+ and 8.3 ± 0.3 μmol min-1 mg-1 in the absence of Ca2+, using the isolated light chains of gizzard myosin as the substrate. The Ca2+-independent enzyme also phosphorylated the 20 000-dalton light chains of purified myosin and crude actomyosin from turkey gizzard. The Km of the Ca2+-independent kinase for Mg2+-ATP (54 μM) was not significantly different from that of the native, Ca2+-dependent enzyme (68 μM). These observations indicate maintenance of the integrity of the active site after digestion with α-chymotrypsin. It is suggested that the loss of Ca2+ sensitivity of the kinase after limited proteolysis is due to loss of the calmodulin-binding site from the 80 000-dalton fragment. The two sites of phosphorylation by the cyclic AMP dependent protein kinase were also removed by the chymotryptic hydrolysis. © 1982 American Chemical Society.
• Walsh, M. P., Hinkins, S., Flink, I. L., & Hartshorne, D. J. (1982). Bovine stomach myosin light chain kinase: Purification, characterization, and comparison with the turkey gizzard enzyme. Biochemistry, 21(26), 6890-6896.
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  PMID: 6897613;Abstract: Myosin light chain kinase has been purified from bovine rumen by a simple and rapid procedure involving extraction from washed "myofibrils" with a high Mg2+ concentration, ammonium sulfate fractionation, Affi-Gel Blue chromatography, and ion-exchange chromatography. A similar method was successful in purification of the kinase from turkey gizzard. The bovine stomach enzyme exhibits many properties in common with the turkey gizzard enzyme: kinase activity is dependent on Ca2+ and calmodulin, with an apparent Kd for calmodulin of 1.3 × 10-9 M; bovine stomach myosin light chain kinase exhibits Ca2+-dependent interaction with immobilized calmodulin; bovine stomach and turkey gizzard kinases appear to catalyze phosphorylation of the same site on the 20000-dalton light chain of smooth muscle myosin; and bovine stomach myosin light chain kinase is a substrate of the cAMP-dependent protein kinase with phosphorylation of two sites in the apoenzyme and one site in the holoenzyme (calmodulin-kinase complex). The rate of phosphorylation of the bovine stomach enzyme is significantly faster than that of the turkey gizzard enzyme in both the presence and absence of bound calmodulin. As in the case of turkey gizzard myosin light chain kinase, phosphorylation of both sites on the bovine stomach enzyme by cAMP-dependent protein kinase reduces the affinity of the kinase for calmodulin. The amino acid composition of bovine stomach myosin light chain kinase is very similar to that of the turkey gizzard and chicken gizzard enzyme. The major difference between turkey gizzard and bovine stomach myosin light chain kinases is the molecular weight of the apoenzyme: the turkey gizzard enzyme has Mr 130000 and the bovine stomach enzyme has Mr 155000 as determined by 0.1% dodecyl sulfate-7.5-20% polyacrylamide gradient gel electrophoresis. © 1982 American Chemical Society.
• Hartshorne, D. J., & Siemankowski, R. F. (1981). Regulation of smooth muscle actomyosin.. Annual Review of Physiology, 43, 519-530.
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  PMID: 6260022;
• Persechini, A., & Hartshorne, D. J. (1981). Erratum: (Science (1383)). Science, 214(4519), 390-.
• Persechini, A., & Hartshorne, D. J. (1981). Phosphorylation of smooth muscle myosin: Evidence for cooperativity between the myosin heads. Science, 213(4514), 1383-1385.
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  PMID: 6455737;
• Persechini, A., Mrwa, U., & Hartshorne, D. J. (1981). Effect of phosphorylation on the actin-activated atpase activity of myosin. Biochemical and Biophysical Research Communications, 98(3), 800-805.
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  PMID: 6452877;Abstract: The purpose of this study was to test the hypothesis that the phosphorylation of myosin is solely responsible for the activation of the Mg2+-ATPase activity of gizzard actomyosin. Using a washed natural actomyosin and a reconstituted actomyosin it was shown that phosphorylation alone caused only a slight activation of ATPase activity. Full activity was obtained only when proteins in addition to the myosin light chain kinase were added. It is evident from these results that: 1) there is no simple relationship between the extent of myosin phosphorylation and the specific Mg2+-ATPase activity of actomyosin and 2) in order for full activation by actin of the Mg2+-ATPase activity of phosphorylated myosin additional factors are required. © 1981.
• Walsh, M. P., Hinkins, S., & Hartshorne, D. J. (1981). Phosphorylation of smooth muscle actin by the catalytic subunit of the cAMP-dependent protein kinase. Biochemical and Biophysical Research Communications, 102(1), 149-157.
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  PMID: 6272789;Abstract: Partially purified smooth muscle (chicken gizzard) actomyosin contains two major substrates of cAMP-dependent protein kinase: a protein of Mr = 130,000, identified as the calmodulin-dependent myosin light chain kinase, and a protein of Mr = 42,000. This latter protein was shown by a variety of electrophoretic procedures to be actin. Purified smooth muscle actin also was phosphorylated by the catalytic subunit of cAMP-dependent protein kinase. The rate of phosphorylation of smooth muscle actin was significantly enhanced by depolyjerization of actin. A maximum of 2.0 mol phosphate could be incorporated per mol G-actin. Skeletal muscle F-actin was not significantly phosphorylated by protein kinase; however, skeletal G-actin is a substrate for the protein kinase although its rate of phosphorylation was significantly slower than that of smooth muscle G-actin. © 1981.
• Walsh, M. P., Persechini, A., Hinkins, S., & Hartshorne, D. J. (1981). Is smooth muscle myosin a substrate for the cAMP-dependent protein kinase?. FEBS Letters, 126(1), 107-110.
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  PMID: 6894576;
• Hartshorne, D. J. (1980). Biochemical basis for contraction of vascular smooth muscle. Chest, 78(1 SUPPL.), 140-149.
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  PMID: 6447058;Abstract: Some of the current facts and theories concerning the contractile mechanism in smooth muscle are summarized. The review is divided into two major sections. One deals with the components of the contractile apparatus (the thick and thin filaments), and the protein components of each filament type are described briefly. The other is devoted to the Ca2+-dependent mechanism of regulation in smooth muscle, and this is restricted to those components that control the activity of the contractile apparatus. There are basically two theories that have been proposed as the regulatory mechanism in smooth muscle. One theory is that the regulatory mechanism is located on the thin filaments and is functional via some modification of the thin filament proteins. This system is termed leiotonin. The second theory is that regulation is achieved by the phosphorylation and dephosphorylation of the light chains of myosin. Since the latter theory represents the author's bias and in general is more widely accepted, it is considered in more detail. A cyclic scheme is presented to illustrate the role of the myosin light chain kinase in the activation and contraction of smooth muscle and the role of the myosin light chain phosphatase in the relaxation process.
• Hartshorne, D. J., & Persechini, A. J. (1980). Phosphorylation of myosin as a regulatory component in smooth muscle.. Annals of the New York Academy of Sciences, 356, 130-141.
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  PMID: 6112943;
• Mrwa, U., & Hartshorne, D. J. (1980). Phosphorylation of smooth muscle myosin and myosin light chains. Federation Proceedings, 39(5), 1564-1568.
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  PMID: 7364053;Abstract: The most popular theory to account for the regulation of the contractile activity of smooth muscle, at the contractile protein level, is based on the phosphorylation and dephosphorylation of the myosin molecule. The enzymes involved are a myosin light chain kinase and a phosphatase, respectively. In this communication a method is given for the purification of the kinase. Using the purified kinase in combination with calmodulin, the pH dependence and rates of P transfer were examined. An Arrhenius plot of phosphorylation rates indicated that Q10 is approximately 2. The rates of P transfer to myosin light chains at 25 C and 37 C were about 15 and 34 μmol.min-1.mg-1 kinase, respectively. It is shown also that the rate of phosphorylation of isolated myosin light chains is significantly faster than the rate obtained when whole myosin is used as the phosphate acceptor, the latter being at least an order of magnitude slower. This difference in rates was not due entirely to the difference in physical states of the two substrates since at an increased ionic strength, were myosin was soluble, the rate of phosphorylation of the light chain fraction was still considerably faster than the rate of phosphorylation of whole myosin.
• Yerna, M. -., Dabrowska, R., Hartshorne, D. J., & Goldman, R. D. (1979). Calcium-sensitive regulation of actin-myosin interactions in baby hamster kidney (BHK-21) cells. Proceedings of the National Academy of Sciences of the United States of America, 76(1), 184-188.
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  PMID: 154671;PMCID: PMC382901;
• Yerna, M. J., Hartshorne, D. J., & Goldman, R. D. (1979). Isolation and characterization of baby hamster kidney (BHK-21) cell modulator protein.. Biochemistry, 18(4), 673-678.
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  PMID: 217421;Abstract: A Ca2+-dependent modulator protein has been isolated from BHK-21 cells. The purification requires heat treatment, ion-exchange chromatography, and gel filtration. The protein appears homogenous on sodium dodecyl sulfate--polyacrylamide and isoelectric focusing gels. The protein comigrates with purified smooth muscle and brain modulators. BHK-21 modulator is characterized by a high content of aspartic and glutamic acids and by a high phenylalanine/tyrosine ratio. It lacks both cysteine and tryptophan. The protein is effective in activating brain-modulator-deficient phosphodiesterase. It can also be used in assay systems to generate Ca2+-sensitive actin activation of both BHK-21 and smooth muscle myosins. Therefore, it is proposed that the BHK-21 modulator protein is a component of the Ca2+-dependent mechanism involved in the regulation of actin--myosin interactions in BHK-21 cells.
• Yerna, M., Dabrowska, R., Hartshorne, D. J., & Goldman, R. D. (1979). Erratum: Calcium-sensitive regulation of actin-myosin interactions in baby hamster kidney (BHK-21) cells (Proceedings of the National Academy of Sciences of the United States of America (1979) 76 (184-188)). Proceedings of the National Academy of Sciences of the United States of America, 76(2), 2485-.
• Dabrowska, R., & Hartshorne, D. J. (1978). A Ca²⁺- and modulator-dependent myosin light chain kinase from non-muscle cells. Biochemical and Biophysical Research Communications, 85(4), 1352-1359.
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  PMID: 743302;Abstract: A myosin light chain kinase has been obtained in a partially purified form from human blood platelets and bovine brain. The kinase from both sources required Ca2+ and the modulator protein for its activity. The subunit molecular weight is approximately 105,000 daltons. These kinases are therefore similar to the smooth muscle kinase (Dabrowska, R., Aromatorio, D., Sherry, J. M. F., and Hartshorne, D. J. (1977) Biochem. Biophys. Res. Commun. 78, 1263-1272). It is suggested that the role of the myosin light chain kinase is similar in both muscle and non-muscle cells and serves to activate the contractile apparatus, via the phosphorylation of myosin, in response to an increase in the intracellular free Ca2+ concentration. © 1978.
• Dabrowska, R., M., J., Aromatorio, D. K., & Hartshorne, D. J. (1978). Modulator protein as a component of the myosin light chain kinase from chicken gizzard. Biochemistry, 17(2), 253-258.
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  PMID: 202300;Abstract: The Ca2+-dependent regulation of smooth muscle actomyosin involves a myosin light chain kinase (ATP:myosin light chain phosphotransferase). It has been shown (Dabrowska, R., Aromatorio, D., Sherry, J. M. F., and Hartshorne, D. J. 1977, Biochem. Biophys. Res. Commun. 78, 1263) that the kinase is composed of two proteins of approximate molecular weights 105 000 and 17 000. In this communication it is demonstrated that the 17 000 component is the modulator protein. This conclusion is based on: (1) the identical behavior of the 17 000 kinase component and modulator protein in assays of actomyosin Mg2+-ATPase activity, phosphorylation of myosin, and phosphodiesterase activity, and, (2) the similarity of the 17 000 kinase component and the modulator protein with respect to amino acid composition, absorption spectrum, and electrophoresis in urea-polyacrylamide gels. It is shown also that the modulator protein from smooth muscle and troponin C are distinct proteins. © 1978 American Chemical Society.
• Hartshorne, D. J. (1978). Roles of calcium and phosphorylation in the regulation of the activity of gizzard myosin. Biochemistry, 17(21), 4411-4418.
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  PMID: 152641;Abstract: In the theory of regulation of smooth-muscle actomyosin involving phosphorylation, it is generally accepted that the Mg2+-ATPase activity of phosphorylated myosin is activated by actin, whereas the activity of the unphosphorylated myosin is not. The role of Ca2+, however, is not clear and here there are basically two possibilities; i.e., either Ca2+ acts only to activate the myosin light-chain kinase or, that in addition to the sites on the myosin light-chain kinase, other regulatory sites are involved, presumably on the myosin molecule. To distinguish between these possibilities, three types of experiments were performed: (1) Gizzard myosin was phosphorylated and then separated from the myosin light-chain kinase and phosphatase by chromatography on Sepharose 4-B. The actin-activated Mg2+-ATPase activity of this stable phosphorylated myosin was not inhibited by the removal of Ca2+. (2) Adenosine 5′-O-(3-thiotriphosphate) was used as a substrate for the myosin light-chain kinase. The resultant thiophosphorylated myosin is resistant to hydrolysis by the phosphatase and, therefore, the myosin was trapped in the thiophosphorylated state. It was found that as the proportion of thiophosphorylated myosin increased the inhibition in the absence of Ca2+ of the actin-activated ATPase activity decreased; i.e., Ca2+ sensitivity was progressively lost. (3) The rate of inhibition of the actin-activated ATPase activity of phosphorylated myosin (in a system containing kinase and phosphatase) following the removal of Ca2+ was measured and found to correlate well with the rate of removal of phosphate groups from the myosin light chains. It is concluded that the dominant role of Ca2+ is to activate the myosin light-chain kinase. No evidence for the implication of additional regulatory sites was obtained, and thus the actin-activated ATPase activity of gizzard myosin is dependent only on its state of phosphorylation. It follows that the actin-activated ATPase activity of phosphorylated myosin is not inhibited directly as a result of the removal of Ca2+ but is inhibited by the removal of the phosphate groups from the myosin light chains. © 1978 American Chemical Society.
• Yerna, M. J., Aksoy, M. O., Hartshorne, D. J., & Goldman, R. D. (1978). BHK21 myosin: isolation, biochemical characterization and intracellular localization. Journal of Cell Science, Vol. 31, 411-429.
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  PMID: 149798;
• Dabrowska, R., Aromatorio, D., Sherry, J. M., & Hartshorne, D. J. (1977). Composition of the myosin light chain kinase from chicken gizzard. Biochemical and Biophysical Research Communications, 78(4), 1263-1272.
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  PMID: 144501;Abstract: The Ca2+-dependent protein kinase (ATP:myosin light chain phosphotransferase) from chicken gizzard smooth muscle requires two proteins for enzymatic activity. These have approximate molecular weights of 105,000 and 17,000 daltons. The isolation procedure for each component is described. Neither component alone markedly alters either the actin-moderated ATPase activity or the phosphorylation of myosin. Activation of ATPase activity by a combination of the two components occurred only in the presence of Ca2+ and was always accompanied by the phosphorylation of myosin. The simultaneous activation of ATPase activity and myosin phosphorylation establishes a direct correlation between the two events. © 1977.
• Kobayashi, R., Goldman, R. D., Hartshorne, D. J., & Field, J. B. (1977). Purification and characterization of myosin from bovine thyroid. Journal of Biological Chemistry, 252(22), 8285-8291.
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  PMID: 914871;Abstract: Myosin and actin were isolated from bovine thyroid and characterized. New methodology was used to purify myosin since classical methods did not remove thyroglobulin contamination. Thyroid extracts were prepared using 0.3 M ammonium acetate with 2 mM CaCl2. After blue dextran/Sepharose affinity chromatography, the myosin was purified using gel filtration with a discontinuous buffer system as described by Pollard et al. (Pollard, T.D., Thomas, S. M., and Niederman, R. (1974) Anal. Biochem. 60, 258-266). The final product was more than 95% pure as indicated by electron microscopy and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Thyroid myosin resembles myosins from other tissues in regard to subunit size, morphology, ATPase activity, and the activation of Mg2+ATPase by actin. It contains a heavy chain of 200,000 daltons, two light chains of 20,500 and 16,500 daltons, and forms bipolar thick filaments in 0.1 M KCl and Mg2+. Skeletal muscle and thyroid F-actin stimulate thyroid myosin ATPase 2- to 3-fold in the presence of KCl and Mg2+. Ouabain and sodium azide have little or no effect on the ATPase activity indicating the absence of plasma membrane (Na+ + K+) ATPase and minimal contamination by mitochondrial ATPase. Thyroid myosin ATPase activity differed qualitatively from myosins obtained from other tissues. In contrast to the Ca2+ and EDTA activation and Mg2+ inhibition of other vertebrate myosins, the ATPase activity of thyroid myosin is not affected by EDTA and inhibited by Ca2+ and Mg2+. The EDTA/K+ATPase pH curves of thyroid myosin are very different from skeletal muscle myosin. The optimal salt concentration is somewhat lower than that of other myosins. Thyroid actin was purified by repolymerization after extraction from an acetone powder of actomyosin. The electron microscopic appearance of the actin is similar to that of skeletal F-actin.
• Seamon, K. B., Hartshorne, D. J., & Bothner-By, A. A. (1977). Ca²⁺ and Mg²⁺ dependent conformations of troponin C as determined by ¹H and ¹⁹F nuclear magnetic resonance. Biochemistry, 16(18), 4039-4046.
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  PMID: 20928;Abstract: The effects of pH and pCa variation on the 250-MHz proton magnetic resonance spectrum of troponin C have been examined. Assignments of resonances in the aromatic region to histidine-125, and to tyrosines-10 and -109, have been made. The tyrosine peak assignments are confirmed by the spectrum of a derivative of troponin C in which the tyrosine rings have been iodinated. Two upfield shifted phenylalanine resonances in the spectrum of the Ca2+-free protein have been identified. Three distinct conformational states of the protein are revealed by Ca2+ titration. These correspond to states of the protein with: (1) no Ca2+ bound; (2) Ca2+ bound only at one or both of the high affinity binding sites; and (3) Ca2+ bound at both high and one or both of the low affinity binding sites. The upfield phenylalanine resonances and an upfield aliphatic resonance are shifted and broadened as the third binding site (first low affinity site) is occupied by Ca2+. This change is not effected by Mg2+, which is shown to produce spectral changes characteristic of the binding of metal only at the high affinity sites. A fluorinated derivative was prepared by reacting the protein with bromotrifluoroacetone, a sulfhydryl alkylating reagent. The fluorine resonance of this derivative shifts downfield upon adding Ca2+. The same shift is effected by the addition of Mg2+ indicating that the fluorine probe is sensitive to conformational changes induced by binding of metal at the high affinity sites only.
• Aksoy, M. O., Williams, D., Sharkey, E. M., & Hartshorne, D. J. (1976). A relationship between Ca²⁺ sensitivity and phosphorylation of gizzard actomyosin. Biochemical and Biophysical Research Communications, 69(1), 35-41.
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  PMID: 130907;Abstract: Ca2+ regulated actomyosin from chicken gizzard is phosphorylated to a significant extent (∼2.5 moles P per 106g. actomyosin) only in the presence of Ca2+. The Ca2+ dependence of phosphorylation is similar to that shown by the ATPase activity. When the actomyosin is desensitized to the effects of Ca2+ the phosphorylating system is removed. Reconstitution of Ca2+ sensitivity is accompanied by an increase in the extent of phosphorylation. These results suggest a relationship between phosphorylation and the Ca2+ sensitive ATPase activity of gizzard actomyosin. © 1976 Academic Press, Inc.
• Górecka, A., Aksoy, M. O., & Hartshorne, D. J. (1976). The effect of phosphorylation of gizzard myosin on actin activation. Biochemical and Biophysical Research Communications, 71(1), 325-331.
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  PMID: 962924;Abstract: Gizzard myosin is phosphorylated by a kinase found in chicken gizzards. The 20,000 dalton light chains are the only subunits to show an appreciable extent of 32P incorporation. Phosphorylation requires trace amounts of Ca2+. The Mg2+-ATPase activity of gizzard myosin in the phosphorylated form is activated to an appreciable extent by skeletal actin, whereas the activation of the non-phosphorylated myosin is verylow. These results suggest that the Ca2+-sensitive regulatory mechanism of gizzard actomyosin is mediated via a kinase. In the presence of Ca2+ the onset of contraction and the resultant increase of the Mg2+-ATPase activity we suggest is due, at least partly, to the phosphorylation of the 20,000 dalton light chains. Whether or not Ca2+ binding by myosin is also essential remains to be established. © 1976.
• Poo, W. J., & Hartshorne, D. J. (1976). Actin crosslinked with glutaraldehyde: Evidence to suggest an active role for actin in the regulatory mechanism. Biochemical and Biophysical Research Communications, 70(2), 406-412.
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  PMID: 132931;Abstract: Actin crosslinked with glutaraldehyde retains the ability to activate the Mg2+-ATPase activity of heavy meromyosin subfragment 1, but the resultant ATPase activity is not controlled by the regulatory proteins, troponin and tropomyosin. Fluorescent energy transfer measurements imply that the crosslinked actin is frozen in the active state. These results indicate that the conformation of actin is important in the regulatory mechanism, and suggest that actin plays a more active role in this mechanism than thought previously. © 1976.
• Driska, S., & Hartshorne, D. J. (1975). The contractile proteins of smooth muscle. Properties and components of a Ca²⁺-sensitive actomyosin from chicken gizzard. Archives of Biochemistry and Biophysics, 167(1), 203-212.
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  PMID: 236728;Abstract: The preparation and characterization of a Ca2+-sensitive actomyosin from chicken gizzard is described. The pH curve of the Mg2+ ATPase activity of the actomyosin was dominated by the activity of the myosin component, and this gave rise to the acid and alkaline optima. Skeletal muscle myosin showed a similar curve. Both the activation of myosin ATPase by actin, and the Ca2+ sensitivity were confined to the neutral pH region. The subunit composition of the Ca2+-sensitive actomyosin was interesting in that no components corresponding to skeletal muscle troponin were obvious. It is suggested that the activity of gizzard actomyosin is regulated by a protein on the thin filaments with a subunit weight of ~130,000. © 1975.
• Fuchs, F., Hartshorne, D. J., & Barns, E. M. (1975). ATPase activity and superprecipitation of skeletal muscle actomyosin of frog and rabbit: Effect of temperature on calcium sensitivity. Comparative Biochemistry and Physiology -- Part B: Biochemistry and, 51(2), 165-170.
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  PMID: 124644;Abstract: 1. 1. In the presence of Mg2+, but not Ca2+, the activation energy of rabbit skeletal actomyosin ATPase was constant from 0 to 40°C. With the addition of a trace amount of Ca2+, the activation energy was reduced by almost a factor of three at temperatures greater than 20°C. 2. 2. For frog skeletal actomyosin ATPase the activation energy decreased by a factor of two at temperatures greater than 15°C. This reduction in activation energy occurred both in the presence and absence of Ca2+ and, therefore, did not require the binding of Ca2+ to troponin, as was the case for rabbit actomyosin. 3. 3. At temperatures greater than 15°C there was a progressive reduction in the Ca2+ requirement for actomyosin superprecipitation. In the presence of 1 mM MgATP as substrate the Ca2+ sensitivity of frog actomyosin was eliminated at 30-35°C while that of rabbit actomyosin was eliminated at 40-45°C. 4. 4. A simple model to explain the effect of temperature on Ca2+ sensitivity is proposed and the implication of these findings for the mechanical behavior of muscle is considered. © 1975.
• Daniel, J. L., & Hartshorne, D. J. (1974). The reaction of myosin with N-ethylmaleimide in the presence of ADP. BBA - Bioenergetics, 347(2), 151-159.
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  PMID: 4276217;Abstract: Myosin reacted with N-ethylmaleimide in the presence of ADP lost its ability to be activated by actin. Subfragment 1 behaved similarly. About 2 moles of N-ethylmaleimide per mole of Subfragment 1 were required to eliminate actin activation of the Mg2+-ATPase activity. At the point at which actin activation was lost the K+-EDTA-ATPase activity was also lost, but the Ca2+-activated ATPase activity was increased. Kinetic measurements indicated that the labelling with N-ethylmaleimide in the presence of ADP reduced V (the ATPase activity at infinite actin concentration) but did not effect Kapp (which is related to the dissociation constant of the actin-Subfragment 1 complex). The Mg2+-activated activity of the reacted myosin alone remained unaltered and the ability to bind actin was retained. We propose that the N-ethylmaleimide labelling blocked the actin activation by preventing the accelerated release of hydrolysis products from the myosin. © 1974.
• Daniel, J. L., & Hartshorne, D. J. (1973). Comparison of the reaction of N-ethylmaleimide with myosin and heavy meromyosin subfragment 1. Biochemical and Biophysical Research Communications, 51(1), 125-131.
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  PMID: 4266972;Abstract: Myosin reacted at low ionic strength with NEM forms an actomyosin which is Ca++ insensitive. With HMM S-1 the reaction with NEM causes a marked loss of the actin activated ATPase activity and the Ca++ sensitivity is reduced but not eliminated. The presence of actin during the sulfhydryl reaction does not significantly alter this result. HMM S-1 prepared from myosin previously desensitized by NEM regains Ca++ sensitivity. These results indicate that the conformations of myosin and HMM S-1 are different and could reflect a difference between insoluble (filamentous) myosin and myosin, or its fragments, in solution. © 1973.
• Daniel, J. L., & Hartshorne, D. J. (1972). Sulfhydryl groups of natural actomyosin essential for the Ca^2--sensitive response: location and properties. BBA - Protein Structure, 278(3), 567-576.
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  PMID: 4263859;Abstract: 1. 1. Certain sulfhydryl groups in natural actomyosin are essential for Ca2+ sensitivity. These are located on the heavy chains of the myosin molecule. 2. 2. Reaction of these sulfhydryl groups is influenced by ATP and ATP analogs. ATP exhibits a pronounced protective effect, with ADP and pyrophosphate being less effective. 3. 3. The critical sulfhydryl groups are most susceptible to reaction at low ionic strength and in the presence of actin. 4. 4. The reaction of this site does not produce the same effects as the reaction of the S1 sulfhydryl groups. 5. 5. The inhibition of ATPase activity by troponin B or troponin B plus tropomyosin is also removed under conditions similar to those which eliminate Ca2+ sensitivity. © 1972.
• Hartshorne, D. J., Barns, E. M., Parker, L., & Fuchs, F. (1972). The effect of temperature on actomyosin. BBA - Bioenergetics, 267(1), 190-202.
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  PMID: 4259758;Abstract: 1. The effect of temperature on the ATPase activity of actomyosin systems was studied. The Arrhenius plot for natural actomyosin was curved between o °C and 40 °C for the Mg2+-activated ATPase activity in the presence of calcium. A similar plot in the absence of calcium was linear over the same temperature range. The Arrhenius plot of the Mg2+-activated activity of desensitized actomyosin in both the presence and absence of calcium was linear up to 40 °C. The addition of troponin B and tropomyosin to desensitized actomyosin did not alter the linearity of the plots. However, when troponin A was added (to complete the regulatory system) a non-linear plot resulted. It is proposed that the characteristic curvature of the Arrhenius plots for the Mg2+-activated ATPase activity of natural actomyosin and myofibrils is a consequence of the binding of calcium to troponin A. 2. At temperatures above 40 °C all of the systems studied demonstrated a nonlinearity, which was not influenced to any great extent by the regulatory proteins. 3. As the temperature was increased above 20 °C calcium sensitivity became less and at 50 °C was lost. It is unlikely that this effect was due to either the oxidation of critical sulfhydryl groups or to an alteration of the calcium binding characteristics of troponin. A possible explanation is suggested. © 1972.
• Hartshorne, D. J., & Pyun, H. Y. (1971). Calcium binding by the troponin complex, and the purification and properties of troponin A. BBA - Protein Structure, 229(3), 698-711.
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  PMID: 5555217;Abstract: The percentage of tropomyosin and troponin in rabbit psoas muscle was estimated to be 4.2% and 5.6% of the myofibrillar protein, respectively. This suggests that on the thin filament there are two molecules of tropomyosin per 390 Å, and probably two molecules of the troponin complex. Approximately 60% of the calcium bound by troponin was accounted for by the component which conferred the calcium sensitivity, troponin A. The binding data for troponin A indicated two classes of binding sites. The Scatchard plots were approximated by assuming that each class contained 4.8 sites/105 g protein and that the high affinity binding constant was equal to 5.4 · 106 M-1 and the low affinity constant was equal to 2 · 105 M-1. Troponin A was purified by column chromatography and it was found that both the calcium binding ability and activity in the assays with troponin B were confined to one component. This was characterized by its high mobility on disc electrophoresis. The purified troponin A was roughly spherical, and had a molecular weight of approximately 18 500. It is unusual in that it contained a high proportion of phenylalanine relative to tyrosine. The excess of negatively charged amino acids would also suggest that at neutral pH it was a polyanion. On a weight basis the purified troponin A bound approximately 50% more calcium than the unpurified troponin A. © 1971.
• Hartshorne, D. J. (1970). Interactions of desensitized actomyosin with tropomyosin, troponin A, troponin B, and polyanions.. Journal of General Physiology, 55(5), 585-601.
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  PMID: 4245689;PMCID: PMC2203015;
• Hartshorne, D. J., & Daniel, J. L. (1970). The importance of sulfhydryl groups for the calcium-sensitive response of natural actomyosin. BBA - Bioenergetics, 223(1), 214-217.
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  PMID: 4249477;
• Parker, L., Pyun, H. Y., & Hartshorne, D. J. (1970). The inhibition of the adenosine triphosphatase activity of the subfragment 1-actin complex by troponin plus tropomyosin, troponin B plus tropomyosin and troponin B. BBA - Bioenergetics, 223(2), 453-456.
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  PMID: 4251402;
• Hartshorne, D. J., & Mueller, H. (1969). The preparation of tropomyosin and troponin from natural actomyosin. BBA - Protein Structure, 175(2), 301-319.
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  PMID: 4238075;Abstract: The procedure described by Bailey for the preparation of tropomyosin B has been modified to yield either a complex of tropomyosin and troponin or the individual proteins. This complex, the EGTA-sensitizing factor has been implicated in the relaxation mechanism of muscle. The starting material, in the modified procedure, was purified natural actomyosin which had been treated with ethanol and ether. Subsequent high ionic strength extraction of this material yielded the EGTA-sensitizing factor complex. Impurities were removed on ammonium sulfate fractionation, and the complex was isolated in the fraction precipitated between 40 to 60% ammonium sulfate saturation. Troponin was obtained as the isoelectric supernatant of the complex over the pH range of 3.5 to 4.6. Tropomyosin was isolated from the isoelectric precipitate by ammonium sulfate fractionation between the limits of 53 to 60% saturation. This separation depended critically on the protein concentration. In the sedimentation velocity analyses the separation was manifested by a transformation of the hypersharp boundary of the EGTA-sensitizing factor complex into two broader boundaries representative of tropomyosin and troponin. The separation was also monitored by the effect of relevant fractions on the ATPase activity of synthetic actomyosin. © 1969.
• Hartshorne, D. J., Theiner, M., & Mueller, H. (1969). Studies on troponin. BBA - Protein Structure, 175(2), 320-330.
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  PMID: 4238076;Abstract: Troponin was isolated from preparations of the ethylene glycol bis-(β-aminoethyl ether)-N,N′-tetraacetic acid (EGTA)-sensitizing factor over the pH range of 3.5-4.6. The variations among troponins were due essentially to differences in the proportion and properties of the troponin A component. This was dictated by the choice of the source fraction (EGTA-sensitizing factor) and by the efficiency of recovery of troponin A in troponin by the isoelectric fractionation. A higher yield of troponin A was obtained at the alkaline edge of the given pH range. The relative proportion of troponin A in troponin was reflected by the A278 mμ:A260 mμ ratio and by the effect troponin had on the Mg2+-activated ATPase activity of synthetic actomyosin. The inhibitory effect of the EGTA-sensitizing factor on the Mg2+-activated ATPase activity of synthetic actomyosin in the presence of EGTA, was due to an interaction of troponin B and tropomyosin. Ultracentrifugal analysis revealed that this interaction was reflected by the formation of a hypersharp boundary different from the boundaries of troponin B or tropomyosin alone. Inhibition of the ATPase activity and complex formation was maximal at a ratio of tropomyosin to troponin B of approx. 1:1 (w/w). It is proposed that troponin A is responsible for the calcium sensitivity of the EGTA-sensitizing factor. © 1969.
• Stromer, M. H., Hartshorne, D. J., Mueller, H., & Rice, R. V. (1969). The effect of various protein fractions on Z- and M-line reconstitution.. Journal of Cell Biology, 40(1), 167-178.
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  PMID: 5782443;PMCID: PMC2107591;
• Hartshorne, D. J., & Mueller, H. (1968). Fractionation of troponin into two distinct proteins. Biochemical and Biophysical Research Communications, 31(5), 647-653.
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  PMID: 4233121;Abstract: The protein complex which governs the calcium sensitivity of natural actomyosin (Ebashi, 1963) has been shown to consist of two components, tropomyosin and troponin (Ebashi and Kodama, 1966; Hartshorne and Mueller, 1967). In the present study troponin has been separated into two distinct proteins, as judged by amino acid analyses. The fractionation was achieved at low pH and high ionic strength. One of the fractions, termed troponin B, effected a calcium insensitive inhibition of synthetic actomyosin ATPase activity, which was enhanced by the addition of tropomyosin. The other fraction, troponin A, conferred calcium sensitivity to the troponin B - tropomyosin system. The separation was reversible and all the properties of the source troponin were regained upon mixing troponin A and B in the correct proportions. © 1968.
• Hartshorne, D. J., & Mueller, H. (1967). Separation and recombination of the ethylene glycol bis (beta-aminoethyl ether)-N,N'-tetraacetic acid-sensitizing factor obtained from a low ionic strength extract of natural actomyosin.. Journal of Biological Chemistry, 242(13), 3089-3092.
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  PMID: 4226222;
• Hartshorne, D. J., Perry, S. V., & Schaub, M. C. (1967). A protein factor inhibiting the magnesium-activated adenosine triphosphatase of desensitized actomyosin.. Biochemical Journal, 104(3), 907-913.
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  PMID: 4227823;PMCID: PMC1271232;
• Schaub, M. C., Hartshorne, D. J., & Perry, S. V. (1967). Effect of tropomyosin on the calcium-activated adenosine triphosphatase of actomyosin [28]. Nature, 215(5101), 635-636.
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  PMID: 4227837;
• Schaub, M. C., Hartshorne, D. J., & Perry, S. V. (1967). The adeonosine-triphosphatase activity of desensitized actomyosin.. Biochemical Journal, 104(1), 263-269.
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  PMID: 4226811;PMCID: PMC1270571;
• Stromer, M. H., Hartshorne, D. J., & Rice, R. V. (1967). Removal and reconstitution of Z-line material in a striated muscle.. Journal of Cell Biology, 35(3), C23-28.
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  PMID: 6064377;PMCID: PMC2107159;
• Dreizen, P., Hartshorne, D. J., & Stracher, A. (1966). The subunit structure of myosin. I. Polydispersity in 5 M guanidine.. Journal of Biological Chemistry, 241(2), 443-448.
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  PMID: 5903737;
• Hartshorne, D. J., Perry, S. V., & Davies, V. (1966). A factor inhibiting the adenosine triphosphatase activity and the superprecipitation of actomyosin [21]. Nature, 209(5030), 1352-1353.
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  PMID: 4224839;
• Hartshorne, D. J., & Stracher, A. (1965). Deuterium-hydrogen exchange of muscle proteins. Biochemistry, 4(10), 1917-1923.
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  Abstract: At apparent pH values of 6.3 the percentage of hard-to-exchange peptide hydrogens for the proteins light meromyosin fraction I, tropomyosin, actin, myosin, and heavy meromyosin bore little apparent relationship to their reported helical contents. Heavy meromyosin contained the highest percentage of hard-to-exchange peptide hydrogens. Increasing pH increased the exchange rate for all of these proteins. The effect of pH on the exchange rates of the highly helical proteins, light meromyosin fraction I, and tropomyosin was more marked than for the proteins heavy meromyosin and myosin. From the dissimilarity of the pH dependence of exchange of heavy meromyosin it is proposed that some conformation other than the α helix is partly responsible for the higher percentage of unexchanged peptide hydrogens in this protein. Sodium dodecyl sulfate increased the exchange rates for all the proteins but also altered considerably the physical properties of the studied proteins, heavy meromyosin and light meromyosin fraction I.
• Hartshorne, D., & Morales, M. (1965). Enzymatic modification of myosin by disulfide exchange. Biochemistry, 4(1), 18-22.
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  PMID: 14285236;Abstract: With various compounds, and under various conditions, there is studied the exchange between simple disulfides and the SH groups of myosin, and between disulfide myosin and various thiols. The first of these exchanges "modifies" myosin, as regards its nucleoside triphosphatase activity, and the second exchange partially "regenerates" the original myosin. Using 35S-labeled dithiopropionate to modify myosin, and "cold" thiopropionate to regenerate it, one finds that when maximal ATPase activity is reached during modification there is a much greater number of reacted SH groups than when a similar maximal ATPase activity is reached on regeneration. This result is interpretable on the assumption that SH reaction and modification are distinct processes, and therefore not always "tightly coupled.".
• Hartshorne, D., & Greenberg, D. M. (1964). Studies on liver threonine dehydrogenase. Archives of Biochemistry and Biophysics, 105(1), 173-178.
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  PMID: 14165492;Abstract: A soluble preparation of l-threonine dehydrogenase was obtained from bull frog liver mitochondria, and the enzyme was partially purified. The Km of the substrate was found by a colorimetric method to be 7.5 × 10-3 M, and by a spectrophotometric method to be 6.1 × 10-3 M. The optimum activity of the enzyme was at pH 7.8. No metal ion requirements for the l-threonine dehydrogenase were detected. © 1964.
• HARTSHORNE, D. J., & PERRY, S. V. (1962). A chromatographic and electrophoretic study of sarcoplasm from adult--and foetal-rabbit muscles.. The Biochemical journal, 85, 171-177.
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  PMID: 13953171;PMCID: PMC1243927;

Others

• Hartshorne, D. J., Goll, ., & Winzerling, . (2010, Fall). Protein NSC 665.