Skip to main content
NCBI home page
Biochemical Journal logoLink to Biochemical Journal
. 2002 Jul 1;365(Pt 1):79–87. doi: 10.1042/BJ20011295

Integrin-linked kinase phosphorylates the myosin phosphatase target subunit at the inhibitory site in platelet cytoskeleton.

Eniko Kiss 1, Andrea Murányi 1, Csilla Csortos 1, Pál Gergely 1, Masaaki Ito 1, David J Hartshorne 1, Ferenc Erdodi 1
PMCID: PMC1222641  PMID: 11931630

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'-[gamma-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 not 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-MYPT1(667-1004)). 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.

Full Text

The Full Text of this article is available as a PDF (280.1 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bauer M., Retzer M., Wilde J. I., Maschberger P., Essler M., Aepfelbacher M., Watson S. P., Siess W. Dichotomous regulation of myosin phosphorylation and shape change by Rho-kinase and calcium in intact human platelets. Blood. 1999 Sep 1;94(5):1665–1672. [PubMed] [Google Scholar]
  2. Blockmans D., Deckmyn H., Vermylen J. Platelet activation. Blood Rev. 1995 Sep;9(3):143–156. doi: 10.1016/0268-960x(95)90020-9. [DOI] [PubMed] [Google Scholar]
  3. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  4. Cohen P., Alemany S., Hemmings B. A., Resink T. J., Strålfors P., Tung H. Y. Protein phosphatase-1 and protein phosphatase-2A from rabbit skeletal muscle. Methods Enzymol. 1988;159:390–408. doi: 10.1016/0076-6879(88)59039-0. [DOI] [PubMed] [Google Scholar]
  5. Davies S. P., Reddy H., Caivano M., Cohen P. Specificity and mechanism of action of some commonly used protein kinase inhibitors. Biochem J. 2000 Oct 1;351(Pt 1):95–105. doi: 10.1042/0264-6021:3510095. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Deng J. T., Van Lierop J. E., Sutherland C., Walsh M. P. Ca2+-independent smooth muscle contraction. a novel function for integrin-linked kinase. J Biol Chem. 2001 Feb 8;276(19):16365–16373. doi: 10.1074/jbc.M011634200. [DOI] [PubMed] [Google Scholar]
  7. Erdödi F., Csortos C., Sparks L., Murányi A., Gergely P. Purification and characterization of three distinct types of protein phosphatase catalytic subunits in bovine platelets. Arch Biochem Biophys. 1992 Nov 1;298(2):682–687. doi: 10.1016/0003-9861(92)90466-a. [DOI] [PubMed] [Google Scholar]
  8. Erdödi F., Tóth B., Hirano K., Hirano M., Hartshorne D. J., Gergely P. Endothall thioanhydride inhibits protein phosphatases-1 and -2A in vivo. Am J Physiol. 1995 Nov;269(5 Pt 1):C1176–C1184. doi: 10.1152/ajpcell.1995.269.5.C1176. [DOI] [PubMed] [Google Scholar]
  9. Feng J., Ito M., Ichikawa K., Isaka N., Nishikawa M., Hartshorne D. J., Nakano T. Inhibitory phosphorylation site for Rho-associated kinase on smooth muscle myosin phosphatase. J Biol Chem. 1999 Dec 24;274(52):37385–37390. doi: 10.1074/jbc.274.52.37385. [DOI] [PubMed] [Google Scholar]
  10. Feng J., Ito M., Kureishi Y., Ichikawa K., Amano M., Isaka N., Okawa K., Iwamatsu A., Kaibuchi K., Hartshorne D. J. Rho-associated kinase of chicken gizzard smooth muscle. J Biol Chem. 1999 Feb 5;274(6):3744–3752. doi: 10.1074/jbc.274.6.3744. [DOI] [PubMed] [Google Scholar]
  11. Fox J. E. The platelet cytoskeleton. Thromb Haemost. 1993 Dec 20;70(6):884–893. [PubMed] [Google Scholar]
  12. Fujioka M., Takahashi N., Odai H., Araki S., Ichikawa K., Feng J., Nakamura M., Kaibuchi K., Hartshorne D. J., Nakano T. A new isoform of human myosin phosphatase targeting/regulatory subunit (MYPT2): cDNA cloning, tissue expression, and chromosomal mapping. Genomics. 1998 Apr 1;49(1):59–68. doi: 10.1006/geno.1998.5222. [DOI] [PubMed] [Google Scholar]
  13. Fujita A., Saito Y., Ishizaki T., Maekawa M., Fujisawa K., Ushikubi F., Narumiya S. Integrin-dependent translocation of p160ROCK to cytoskeletal complex in thrombin-stimulated human platelets. Biochem J. 1997 Dec 15;328(Pt 3):769–775. doi: 10.1042/bj3280769. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hamaguchi T., Ito M., Feng J., Seko T., Koyama M., Machida H., Takase K., Amano M., Kaibuchi K., Hartshorne D. J. Phosphorylation of CPI-17, an inhibitor of myosin phosphatase, by protein kinase N. Biochem Biophys Res Commun. 2000 Aug 11;274(3):825–830. doi: 10.1006/bbrc.2000.3225. [DOI] [PubMed] [Google Scholar]
  15. Hartshorne D. J., Ito M., Erdödi F. Myosin light chain phosphatase: subunit composition, interactions and regulation. J Muscle Res Cell Motil. 1998 May;19(4):325–341. doi: 10.1023/a:1005385302064. [DOI] [PubMed] [Google Scholar]
  16. Hirano K., Phan B. C., Hartshorne D. J. Interactions of the subunits of smooth muscle myosin phosphatase. J Biol Chem. 1997 Feb 7;272(6):3683–3688. doi: 10.1074/jbc.272.6.3683. [DOI] [PubMed] [Google Scholar]
  17. Ichikawa K., Ito M., Hartshorne D. J. Phosphorylation of the large subunit of myosin phosphatase and inhibition of phosphatase activity. J Biol Chem. 1996 Mar 1;271(9):4733–4740. doi: 10.1074/jbc.271.9.4733. [DOI] [PubMed] [Google Scholar]
  18. Kameshita I., Fujisawa H. A sensitive method for detection of calmodulin-dependent protein kinase II activity in sodium dodecyl sulfate-polyacrylamide gel. Anal Biochem. 1989 Nov 15;183(1):139–143. doi: 10.1016/0003-2697(89)90181-4. [DOI] [PubMed] [Google Scholar]
  19. Kawai T., Matsumoto M., Takeda K., Sanjo H., Akira S. ZIP kinase, a novel serine/threonine kinase which mediates apoptosis. Mol Cell Biol. 1998 Mar;18(3):1642–1651. doi: 10.1128/mcb.18.3.1642. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Kimura K., Ito M., Amano M., Chihara K., Fukata Y., Nakafuku M., Yamamori B., Feng J., Nakano T., Okawa K. Regulation of myosin phosphatase by Rho and Rho-associated kinase (Rho-kinase) Science. 1996 Jul 12;273(5272):245–248. doi: 10.1126/science.273.5272.245. [DOI] [PubMed] [Google Scholar]
  21. Kitazawa T., Eto M., Woodsome T. P., Brautigan D. L. Agonists trigger G protein-mediated activation of the CPI-17 inhibitor phosphoprotein of myosin light chain phosphatase to enhance vascular smooth muscle contractility. J Biol Chem. 2000 Apr 7;275(14):9897–9900. doi: 10.1074/jbc.275.14.9897. [DOI] [PubMed] [Google Scholar]
  22. Koyama M., Ito M., Feng J., Seko T., Shiraki K., Takase K., Hartshorne D. J., Nakano T. Phosphorylation of CPI-17, an inhibitory phosphoprotein of smooth muscle myosin phosphatase, by Rho-kinase. FEBS Lett. 2000 Jun 23;475(3):197–200. doi: 10.1016/s0014-5793(00)01654-9. [DOI] [PubMed] [Google Scholar]
  23. Kurisaki T., Taylor R. G., Hartshorne D. J. Effects of the protein phosphatase inhibitors, tautomycin and calyculin-A, on protein phosphorylation and cytoskeleton of human platelets. Cell Struct Funct. 1995 Oct;20(5):331–343. doi: 10.1247/csf.20.331. [DOI] [PubMed] [Google Scholar]
  24. MacDonald J. A., Borman M. A., Murányi A., Somlyo A. V., Hartshorne D. J., Haystead T. A. Identification of the endogenous smooth muscle myosin phosphatase-associated kinase. Proc Natl Acad Sci U S A. 2001 Feb 27;98(5):2419–2424. doi: 10.1073/pnas.041331498. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. 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. 1999 May 14;451(1):81–84. doi: 10.1016/s0014-5793(99)00550-5. [DOI] [PubMed] [Google Scholar]
  26. Murányi A., Erdodi F., Ito M., Gergely P., Hartshorne D. J. Identification and localization of myosin phosphatase in human platelets. Biochem J. 1998 Feb 15;330(Pt 1):225–231. doi: 10.1042/bj3300225. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Murányi A., Zhang R., Liu F., Hirano K., Ito M., Epstein H. F., Hartshorne D. J. Myotonic dystrophy protein kinase phosphorylates the myosin phosphatase targeting subunit and inhibits myosin phosphatase activity. FEBS Lett. 2001 Mar 30;493(2-3):80–84. doi: 10.1016/s0014-5793(01)02283-9. [DOI] [PubMed] [Google Scholar]
  28. Nakai K., Suzuki Y., Kihira H., Wada H., Fujioka M., Ito M., Nakano T., Kaibuchi K., Shiku H., Nishikawa M. Regulation of myosin phosphatase through phosphorylation of the myosin-binding subunit in platelet activation. Blood. 1997 Nov 15;90(10):3936–3942. [PubMed] [Google Scholar]
  29. Niiro N., Ikebe M. Zipper-interacting protein kinase induces Ca(2+)-free smooth muscle contraction via myosin light chain phosphorylation. J Biol Chem. 2001 May 30;276(31):29567–29574. doi: 10.1074/jbc.M102753200. [DOI] [PubMed] [Google Scholar]
  30. Paul B. Z., Daniel J. L., Kunapuli S. P. Platelet shape change is mediated by both calcium-dependent and -independent signaling pathways. Role of p160 Rho-associated coiled-coil-containing protein kinase in platelet shape change. J Biol Chem. 1999 Oct 1;274(40):28293–28300. doi: 10.1074/jbc.274.40.28293. [DOI] [PubMed] [Google Scholar]
  31. Senba S., Eto M., Yazawa M. Identification of trimeric myosin phosphatase (PP1M) as a target for a novel PKC-potentiated protein phosphatase-1 inhibitory protein (CPI17) in porcine aorta smooth muscle. J Biochem. 1999 Feb;125(2):354–362. doi: 10.1093/oxfordjournals.jbchem.a022294. [DOI] [PubMed] [Google Scholar]
  32. Shimizu H., Ito M., Miyahara M., Ichikawa K., Okubo S., Konishi T., Naka M., Tanaka T., Hirano K., Hartshorne D. J. Characterization of the myosin-binding subunit of smooth muscle myosin phosphatase. J Biol Chem. 1994 Dec 2;269(48):30407–30411. [PubMed] [Google Scholar]
  33. Somlyo A. P., Somlyo A. V. Signal transduction by G-proteins, rho-kinase and protein phosphatase to smooth muscle and non-muscle myosin II. J Physiol. 2000 Jan 15;522(Pt 2):177–185. doi: 10.1111/j.1469-7793.2000.t01-2-00177.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Suzuki Y., Yamamoto M., Wada H., Ito M., Nakano T., Sasaki Y., Narumiya S., Shiku H., Nishikawa M. Agonist-induced regulation of myosin phosphatase activity in human platelets through activation of Rho-kinase. Blood. 1999 May 15;93(10):3408–3417. [PubMed] [Google Scholar]
  35. Swärd K., Dreja K., Susnjar M., Hellstrand P., Hartshorne D. J., Walsh M. P. Inhibition of Rho-associated kinase blocks agonist-induced Ca2+ sensitization of myosin phosphorylation and force in guinea-pig ileum. J Physiol. 2000 Jan 1;522(Pt 1):33–49. doi: 10.1111/j.1469-7793.2000.0033m.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Tan I., Ng C. H., Lim L., Leung T. Phosphorylation of a novel myosin binding subunit of protein phosphatase 1 reveals a conserved mechanism in the regulation of actin cytoskeleton. J Biol Chem. 2001 Apr 3;276(24):21209–21216. doi: 10.1074/jbc.M102615200. [DOI] [PubMed] [Google Scholar]
  37. Totsukawa G., Yamakita Y., Yamashiro S., Hartshorne D. J., Sasaki Y., Matsumura F. 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. J Cell Biol. 2000 Aug 21;150(4):797–806. doi: 10.1083/jcb.150.4.797. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Totsukawa G., Yamakita Y., Yamashiro S., Hosoya H., Hartshorne D. J., Matsumura F. Activation of myosin phosphatase targeting subunit by mitosis-specific phosphorylation. J Cell Biol. 1999 Feb 22;144(4):735–744. doi: 10.1083/jcb.144.4.735. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Toyoda H., Nakai K., Omay S. B., Shima H., Nagao M., Shiku H., Nishikawa M. Differential association of protein Ser/Thr phosphatase types 1 and 2A with the cytoskeleton upon platelet activation. Thromb Haemost. 1996 Dec;76(6):1053–1062. [PubMed] [Google Scholar]
  40. Tóth A., Kiss E., Gergely P., Walsh M. P., Hartshorne D. J., Erdödi F. Phosphorylation of MYPT1 by protein kinase C attenuates interaction with PP1 catalytic subunit and the 20 kDa light chain of myosin. FEBS Lett. 2000 Nov 3;484(2):113–117. doi: 10.1016/s0014-5793(00)02138-4. [DOI] [PubMed] [Google Scholar]
  41. Tóth A., Kiss E., Herberg F. W., Gergely P., Hartshorne D. J., Erdödi F. Study of the subunit interactions in myosin phosphatase by surface plasmon resonance. Eur J Biochem. 2000 Mar;267(6):1687–1697. doi: 10.1046/j.1432-1327.2000.01158.x. [DOI] [PubMed] [Google Scholar]
  42. Watanabe Y., Ito M., Kataoka Y., Wada H., Koyama M., Feng J., Shiku H., Nishikawa M. Protein kinase C-catalyzed phosphorylation of an inhibitory phosphoprotein of myosin phosphatase is involved in human platelet secretion. Blood. 2001 Jun 15;97(12):3798–3805. doi: 10.1182/blood.v97.12.3798. [DOI] [PubMed] [Google Scholar]
  43. Yoganathan T. N., Costello P., Chen X., Jabali M., Yan J., Leung D., Zhang Z., Yee A., Dedhar S., Sanghera J. Integrin-linked kinase (ILK): a "hot" therapeutic target. Biochem Pharmacol. 2000 Oct 15;60(8):1115–1119. doi: 10.1016/s0006-2952(00)00444-5. [DOI] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES