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Protein kinase network in the regulation of phosphorylation and dephosphorylation of smooth muscle myosin light chain

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Abstract

The contraction of smooth muscle is regulated primarily by intracellular Ca2+ signal. It is well established that the elevation of the cytosolic Ca2+ level activates myosin light chain kinase, which phosphorylates 20 kDa regulatory myosin light chain and activates myosin ATPase. The simultaneous measurement of cytosolic Ca2+ concentration and force development revealed that the alteration of the Ca2+-sensitivity of the contractile apparatus as well as the Ca2+ signal plays a critical role in the regulation of smooth muscle contraction. The fluctuation of an extent of myosin phosphorylation for a given change in Ca2+ concentration is considered to contribute to the major mechanisms regulating the Ca2+-sensitivity. The level of myosin phosphorylation is determined by the balance between phosphorylation and dephosphorylation. The phosphorylation level for a given Ca2+ elevation is increased either by Ca2+-independent activation of phosphorylation process or inhibition of dephosphorylation. In the last decade, the isolation and cloning of myosin phosphatase facilitated the understanding of regulatory mechanism of dephosphorylation process at the molecular level. The inhibition of myosin phosphatase can be achieved by (1) alteration of hetrotrimeric structure, (2) phosphorylation of 110 kDa regulatory subunit MYPT1 at the specific site and (3) inhibitory protein CPI-17 upon its phosphorylation. Rho-kinase was first identified to phosphorylate MYPT1, and later many kinases were found to phosphorylate MYPT1 and inhibit dephosphorylation of myosin. Similarly, the phosphorylation of CPI-17 can be catalysed by multiple kinases. Moreover, the myosin light chain can be phosphorylated by not only authentic myosin light chain kinase in a Ca2+-dependent manner but also by multiple kinases in a Ca2+-independent manner, thus adding a novel mechanism to the regulation of the Ca2+-sensitivity by regulating the phosphorylation process. It is now clarified that the protein kinase network is involved in the regulation of myosin phosphorylation and dephosphorylation. However, the physiological role of each component remains to be determined. One approach to accomplish this purpose is to investigate the effects of the dominant negative mutants of the signalling molecule on the smooth muscle contraction. In this regards, a protein transduction technique utilizing the cell-penetrating peptides would provide a useful tool. In the preliminary study, we succeeded in introducing a fragment of MYPT1 into the arterial strips, and found enhancement of contraction.

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References

  1. Hartshorne DJ: Biochemistry of the contractile process in smooth muscle. In: L.R. Johnson (ed). Physiology of the Gastrointestinal Tract. Raven Press, New York, 1987, pp 423-482

    Google Scholar 

  2. Kanaide H: Measurement of [Ca2+]i in smooth muscle strips using front surface fluorometry. In: D.G. Lambert (ed). Methods in Molecular Biology. Humana Press Inc., Totowa, NJ, 1998, pp 269-277

    Google Scholar 

  3. Hirano K, Hirano M, Abe S, Kanaide H: Cytosolic calcium transients in vascular smooth muscle. In: N. Sperelakis, H. Kuriyama (eds). Ion Channels of Vascular Smooth Muscle Cells and Endothelial Cells. Elsevier, New York, 1991, pp 93-105

    Google Scholar 

  4. Somlyo AP, Somlyo AV: Signal transduction and regulation in smooth muscle. Nature 372: 231-236, 1994

    Google Scholar 

  5. Morgan JP, Morgan KG: Stimulus-specific patterns of intracellular calcium levels in smooth muscle of ferret portal vein. J Physiol 351: 155-167, 1984

    Google Scholar 

  6. Somlyo AP, Somlyo AV: From pharmacomechanical coupling to G-proteins and myosin phosphatase. Acta Physiol Scand 164: 437-448, 1998

    Google Scholar 

  7. Somlyo AP, Wu X, Walker LA, Somlyo AV: Pharmacomechanical coupling: The role of calcium, G-proteins, kinases and phosphatases. Rev Physiol Biochem Pharmacol 134: 201-234, 1999

    Google Scholar 

  8. Hartshorne DJ, Ito M, Erdodi F: Myosin light chain phosphatase: Subunit composition, interactions and regulation. J Muscle Res Cell Motil 19: 325-341, 1998

    Google Scholar 

  9. Takai A: Effects of protein phosphatase inhibitors on smooth muscle. In: T.B. Bolton, T. Tomita (eds). Smooth Muscle Excitation. Academic Press, 1996, pp 277-290

  10. Cohen P: The structure and regulation of protein phosphatases. Ann Rev Biochem 58: 453-508, 1989

    Google Scholar 

  11. Alessi D, MacDougall LK, Sola MM, Ikebe M, Cohen P: The control of protein phosphatase-1 by targetting subunits. The major myosin phosphatase in avian smooth muscle is a novel form of protein phosphatase-1. Eur J Biochem 210: 1023-1035, 1992

    Google Scholar 

  12. Shirazi A, Iizuka K, Fadden P, Mosse C, Somlyo AP, Somlyo AV, Haystead TA: Purification and characterization of the mammalian myosin light chain phosphatase holoenzyme. The differential effects of the holoenzyme and its subunits on smooth muscle. J Biol Chem 269: 31598-31606, 1994

    Google Scholar 

  13. Shimizu H, Ito M, Miyahara M, Ichikawa K, Okubo S, Konishi T, Naka M, Tanaka T, Hirano K, Hartshorne DJ, Nakano T: Characterization of the myosin-binding subunit of smooth muscle myosin phosphatase. J Biol Chem 269: 30407-30411, 1994

    Google Scholar 

  14. Chen YH, Chen MX, Alessi DR, Campbell DG, Shanahan C, Cohen P, Cohen PTW: Molecular cloning of cDNA encoding the 110 and 21 kDa regulatory subunits of smooth muscle protein phosphatase 1M. FEBS Lett 356: 51-55, 1994

    Google Scholar 

  15. Hartshorne DJ, Hirano K: Interactions of protein phosphatase type 1, with a focus on myosin phosphatase. Mol Cell Biochem 190: 79-84, 1999

    Google Scholar 

  16. Deng JT, Van Lierop JE, Sutherland C, Walsh MP: Ca2+-independent smooth muscle contraction. A novel function for integrin-linked kinase. J Biol Chem 276: 16365-16373, 2001

    Google Scholar 

  17. Niiro N, Ikebe M: Zipper-interacting protein kinase induces Ca2+-free smooth muscle contraction via myosin light chain phosphorylation. J Biol Chem 276: 29567-29574, 2001

    Google Scholar 

  18. Amano M, Ito M, Kimura K, Fukata Y, Chihara K, Nakano T, Matsuura Y, Kaibuchi K: Phosphorylation and activation of myosin by Rho-associated kinase (Rho-kinase). J Biol Chem 271: 20246-20249, 1996

    Google Scholar 

  19. Van Eyk JE, Arrell DK, Foster DB, Strauss JD, Heinonen TY, Furmaniak-Kazmierczak E, Cote GP, Mak AS: Different molecular mechanisms for Rho family GTPase-dependent, Ca2+-independent contraction of smooth muscle. J Biol Chem 273: 23433-23439, 1998

    Google Scholar 

  20. Ichikawa K, Ito M, Hartshorne DJ: Phosphorylation of the large subunit of myosin phosphatase and inhibition of phosphatase activity. J Biol Chem 271: 4733-4740, 1996

    Google Scholar 

  21. Kimura K, Ito M, Amano M, Chihara K, Fukata Y, Nakafuku M, Yamamori B, Feng J, Nakano T, Okawa K, Iwamatsu A, Kaibuchi K: Regulation of myosin phosphatase by Rho and Rho-associated kinase (Rho-kinase). Science 273: 245-248, 1996

    Google Scholar 

  22. Trinkle-Mulcahy L, Ichikawa K, Hartshorne DJ, Siegman MJ, Butler TM: Thiophosphorylation of the 130 kDa subunit is associated with a decreased activity of myosin light chain phosphatase in alpha-toxin-permeabilized smooth muscle. J Biol Chem 270: 18191-18194, 1995

    Google Scholar 

  23. Narumiya S: The small GTPase Rho: cellular functions and signal transduction. J Biochem 120: 215-228, 1996

    Google Scholar 

  24. Kaibuchi K, Kuroda S, Amano M: Regulation of the cytoskeleton and cell adhesion by the Rho family GTPases in mammalian cells. Ann Rev Biochem 68: 459-486, 1999

    Google Scholar 

  25. Broustas CG, Grammatikakis N, Eto M, Dent P, Brautigan DL, Kasid U: Phosphorylation of the myosin-binding subunit of myosin phosphatase by Raf-1 and inhibition of phosphatase activity. J Biol Chem 277: 3053-3059, 2002

    Google Scholar 

  26. Muranyi A, Zhang R, Liu F, Hirano K, Ito M, Epstein HF, Hartshorne DJ: Myotonic dystrophy protein kinase phosphorylates the myosin phosphatase targeting subunit and inhibits myosin phosphatase activity. FEBS Lett 493: 80-84, 2001

    Google Scholar 

  27. MacDonald JA, Borman MA, Muranyi A, Somlyo AV, Hartshorne DJ, Haystead TA: Identification of the endogenous smooth muscle myosin phosphatase-associated kinase. Proc Natl Acad Sci USA 98: 2419-2424, 2001

    Google Scholar 

  28. Kitazawa T, Masuo M, Somlyo AP: G protein-mediated inhibition of myosin light-chain phosphatase in vascular smooth muscle. Proc Natl Acad Sci USA 88: 9307-9310, 1991

    Google Scholar 

  29. Hirano K, Phan BC, Hartshorne DJ: Interactions of the subunits of smooth muscle myosin phosphatase. J Biol Chem 272: 3683-3688, 1997

    Google Scholar 

  30. Ichikawa K, Hirano K, Ito M, Tanaka J, Nakano T, Hartshorne DJ: Interactions and properties of smooth muscle myosin phosphatase. Biochemistry 35: 6313-6320, 1996

    Google Scholar 

  31. Johnson DF, Moorhead G, Caudwell FB, Cohen P, Chen YH, Chen MX, Cohen PTW: Identification of protein-phosphatase-1-binding domains on the glycogen and myofibrillar targetting subunits. Eur J Biochem 239: 317-325, 1996

    Google Scholar 

  32. Gong MC, Fuglsang A, Alessi D, Kobayashi S, Cohen P, Somlyo AV, Somlyo AP: Arachidonic acid inhibits myosin light chain phosphatase and sensitizes smooth muscle to calcium. J Biol Chem 267: 21492-21498, 1992

    Google Scholar 

  33. Toth A, Kiss E, Gergely P, Walsh MP, Hartshorne DJ, Erdodi F: Phosphorylation of MYPT1 by protein kinase C attenuates interaction with PP1 catalytic subunit and the 20 kDa light chain of myosin. FEBS Lett 484: 113-117, 2000

    Google Scholar 

  34. Zhou Y, Hirano K, Sakihara C, Nishimura J, Kanaide H: NH2-terminal fragments of the 130 kDa subunit of myosin phosphatase increases the Ca2+ sensitivity of porcine renal artery. J Physiol 516: 55-65, 1999

    Google Scholar 

  35. Zhou Y, Nishimura J, Hirano K, Kanaide H: The exogenously added small subunit of smooth muscle myosin phosphatase increases the Ca2+ sensitivity of the contractile apparatus in the permeabilized porcine renal artery. Biochem Biophys Res Commun 254: 158-163, 1999

    Google Scholar 

  36. Shin H-M, Je H-D, Gallant C, Tao TC, Hartshorne DJ, Ito M, Morgan KG: Differential association and localization of myosin phosphatase subunits during agonist-induced signal transduction in smooth muscle. Circ Res 90: 546-553, 2002

    Google Scholar 

  37. Ito M, Feng J, Tsujino S, Inagaki N, Inagaki M, Tanaka J, Ichikawa K, Hartshorne DJ, Nakano T: Interaction of smooth muscle myosin phosphatase with phospholipids. Biochemistry 36: 7607-7614, 1997

    Google Scholar 

  38. Takahashi N, Ito M, Tanaka J, Nakano T, Kaibuchi K, Odai H, Takemura K: Localization of the gene coding for myosin phosphatase, target subunit 1 (MYPT1) to human chromosome 12q15–q21. Genomics 44: 150-152, 1997

    Google Scholar 

  39. Hirano M, Niiro N, Hirano K, Nishimura J, Hartshorne DJ, Kanaide H: Expression, subcellular localization, and cloning of the 130 kDa regulatory subunit of myosin phosphatase in porcine aortic endothelial cells. Biochem Biophys Res Commun 254: 490-496, 1999

    Google Scholar 

  40. Johnson D, P. C, Chen MX, Chen YH, Cohen PTW: Identification of the region on the M110 subunit of protein phosphatase 1M that interact with M21 subunit and with myosin. Eur J Biochem 244: 931-939, 1997

    Google Scholar 

  41. Feng J, Ito M, Ichikawa K, Isaka N, Nishikawa M, Hartshorne DJ, Nakano T: Inhibitory phosphorylation site for Rho-associated kinase on smooth muscle myosin phosphatase. J Biol Chem 274: 37385-37390, 1999

    Google Scholar 

  42. Kawano Y, Fukata Y, Oshiro N, Amano M, Nakamura T, Ito M, Matsumura F, Inagaki M, Kaibuchi K: Phosphorylation of myosin-binding subunit (MBS) of myosin phosphatase by Rho-kinase in vivo. J Cell Biol 147: 1023-1037, 1999

    Google Scholar 

  43. Murata-Hori M, Suizu F, Iwasaki T, Kikuchi A, Hosoya H: ZIP kinase identified as a novel myosin regulatory light chain kinase in HeLa cells. FEBS Lett 451: 81-84, 1999

    Google Scholar 

  44. Ishizaki T, Mackawa M, Fujisawa K, Okawa K, Iwamatsu A, Fujita A, Watanabe N, Saito Y, Kakizuka A, Morii N, Narumiya S: The small GTP-binding protein Rho binds to and activates a 160 kDa Ser/Thr protein kinase homologous to myotonic dystrophy kinase. EMBO J 15: 1885-1893, 1996

    Google Scholar 

  45. Shimizu M, Wang W, Walch ET, Dunne PW, Epstein HF: Rac-1 and Raf-1 kinases, components of distinct signaling pathways, activate myotonic dystrophy protein kinase. FEBS Lett 475: 273-277, 2000

    Google Scholar 

  46. Kolch W: Meaningful relationships: the regulation of the Ras/Raf/MEK/ERK pathway by protein interactions. Biochem J 351: 289-305, 2000

    Google Scholar 

  47. Kolch W, Heidecker G, Lloyd P, Rapp UR: Raf-1 protein kinase is required for growth of induced NIH/3T3 cells. Nature 349: 426-428, 1991

    Google Scholar 

  48. Kyriakis JM, App H, Zhang XF, Banerjee P, Brautigan DL, Rapp UR, Avruch J: Raf-1 activates MAP kinase-kinase. Nature 358: 417-421, 1992

    Google Scholar 

  49. Leevers SJ, Paterson HF, Marshall CJ: Requirement for Ras in Raf activation is overcome by targeting Raf to the plasma membrane. Nature 369: 411-414, 1994

    Google Scholar 

  50. Uehata M, Ishizaki T, Satoh H, Ono T, Kawahara T, Morishita T, Tamakawa H, Yamagami K, Inui J, Maekawa M, Narumiya S: Calcium sensitization of smooth muscle mediated by a Rho-associated protein kinase in hypertension. Nature 389: 990-994, 1997

    Google Scholar 

  51. Amano M, K. C, Nakamura N, Kaneko T, Matsuura Y, Kaibuchi K: The COOH terminus of rho-kinase negatively regulates rho-kinase activity. J Biol Chem 274: 32418-32424, 1999

    Google Scholar 

  52. Pfitzer G, Sonntag-Bensch D, Brkic-Koric D: Thiophosphorylation-induced Ca2+ sensitization of guinea-pig ileum contractility is not mediated by Rho-associated kinase. J Physiol 533: 651-664, 2001

    Google Scholar 

  53. Cohen PTW: Protein phosphatase 1-targeted in many directions. J Cell Sci 115: 241-256, 2002

    Google Scholar 

  54. Eto M, Ohmori T, Suzuki M, Furuya K, Morita F: A novel protein phosphatase-1 inhibitory protein potentiated by protein kinase C. Isolation from porcine aorta media and characterization. J Biochem 118: 1104-1107, 1995

    Google Scholar 

  55. Eto M, Senba S, Morita F, Yazawa M: Molecular cloning of a novel phosphorylation-dependent inhibitory protein of protein phosphatase-1 (CPI17) in smooth muscle: Its specific localization in smooth muscle. FEBS Lett 410: 356-360, 1997

    Google Scholar 

  56. Hamaguchi T, Ito M, Feng J, Seko T, Koyama M, Machida H, Takase K, Amano M, Kaibuchi K, Hartshorne DJ, Nakano T: Phosphorylation of CPI-17, an inhibitor of myosin phosphatase, by protein kinase N. Biochem Biophys Res Commun 274: 825-830, 2000

    Google Scholar 

  57. Koyama M, Ito M, Feng J, Seko T, Shiraki K, Takase K, Hartshorne DJ, Nakano T: Phosphorylation of CPI-17, an inhibitory phosphoprotein of smooth muscle myosin phosphatase, by Rho-kinase. FEBS Lett 475: 197-200, 2000

    Google Scholar 

  58. MacDonald JA, Eto M, Borman MA, Brautigan DL, Haystead TA: Dual Ser and Thr phosphorylation of CPI-17, an inhibitor of myosin phosphatase, by MYPT-associated kinase. FEBS Lett 493: 91-94, 2001

    Google Scholar 

  59. Kureishi Y, Kobayashi S, Amano M, Kimura K, Kanaide H, Nakano T, Kaibuchi K, Ito M: Rho-associated kinase directly induces smooth muscle contraction through myosin light chain phosphorylation. J Biol Chem 272: 12257-12260, 1997

    Google Scholar 

  60. Itoh T, Ikebe M, Kargacin GJ, Hartshorne DJ, Kemp BE, Fay FS: Effects of modulators of myosin light-chain kinase activity in single smooth muscle cells. Nature 338: 164-167, 1989

    Google Scholar 

  61. Schwarze SR, Dowdy SF: In vivo protein transduction: Intracellular delivery of biologically active proteins, compounds and DNA. Trends Pharmacol Sci 21: 45-48, 2000

    Google Scholar 

  62. Schwarze SR, Hruska KA, Dowdy SF: Protein transduction: Unrestricted delivery into all cells? Trends Cell Biol 10: 290-295, 2000

    Google Scholar 

  63. Watson K, Edwards RJ: HIV-1 trans-activating (Tat) protein: Both a target and a tool in therapeutic approaches. Biochem Pharmacol 58: 1521-1528, 1999

    Google Scholar 

  64. Lindgren M, Hallbrink M, Prochiantz A, Langel U: Cell-penetrating peptides. Trends Pharmacol Sci 21: 99-103, 2000

    Google Scholar 

  65. Silhol M, Tyagi M, Giacca M, Lebleu B, Vives E: Different mechanisms for cellular internalization of the HIV-1 Tat-derived cell penetrating peptide and recombinant proteins fused to Tat. Eur J Biochem 269: 494-501, 2002

    Google Scholar 

  66. Nagahara H, Vocero-Akbani AM, Snyder EL, Ho A, Latham DG, Lissy NA, Becker-Hapak M, Ezhevsky SA, Dowdy SF: Transduction of full-length TAT fusion proteins into mammalian cells: TAT-p27Kip1 induces cell migration. Nature Med 4: 1449-1452, 1998

    Google Scholar 

  67. Sauzeau V, Le Mellionnec E, Bertoglio J, Scalbert E, Pacaud P, Loirand G: Human urotensin II-induced contraction and arterial smooth muscle cell proliferation are mediated by RhoA and Rho-kinase. Circ Res 88: 1102-1104, 2001

    Google Scholar 

  68. Chew T-L, Masaracchia RA, Goeckeler ZM, Wysolmerski RB: Phosphorylation of non-muscle myosin II regulatory light chain by p21-activated kinase (γ-PAK). J Muscle Res Cell Motil 19: 839-854, 1998

    Google Scholar 

  69. Komatsu S, Hosoya H: Phosphorylation by MAPKAP kinase 2 activates Mg2+-ATPase activity of myosin II. Biochem Biophys Res Commun 223: 741-745, 1996

    Google Scholar 

  70. Suizu F, Ueda K, Iwasaki T, Murata-Hori M, Hosoya H: Activation of actin-activated MgATPase activity of myosin II by phosphorylation with MAPK-activated protein kinase-1b (RSK-2). J Biochem 128: 435-440, 2000

    Google Scholar 

  71. Murata-Hori M, Fumoto K, Fukuta Y, Iwasaki T, Kikuchi A, M. T, Hosoya H: Myosin II regulatory light chain as a novel substrate for AIM-1, and aurora/IpI1p-related kinase from rat. J Biochem 128: 903-907, 2000

    Google Scholar 

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  1. Division of Molecular Cardiology, Research Institute of Angiocardiology, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka, 812-8582, Japan

    Katusya Hirano, Dmitry N. Derkach, Mayumi Hirano, Junji Nishimura & Hideo Kanaide

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  1. Katusya Hirano
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Hirano, K., Derkach, D.N., Hirano, M. et al. Protein kinase network in the regulation of phosphorylation and dephosphorylation of smooth muscle myosin light chain. Mol Cell Biochem 248, 105–114 (2003). https://doi.org/10.1023/A:1024180101032

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