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. 2002 Oct 15;367(Pt 2):517–524. doi: 10.1042/BJ20020522

Phosphorylation of the myosin phosphatase inhibitors, CPI-17 and PHI-1, by integrin-linked kinase.

Jing Ti Deng 1, Cindy Sutherland 1, David L Brautigan 1, Masumi Eto 1, Michael P Walsh 1
PMCID: PMC1222907  PMID: 12144526

Abstract

Integrin-linked kinase (ILK) has been implicated in Ca(2+)- independent contraction of smooth muscle via its ability to phosphorylate myosin. We investigated the possibility that this kinase might also phosphorylate and regulate the myosin light-chain phosphatase inhibitor proteins CPI-17 [protein kinase C (PKC)-dependent phosphatase inhibitor of 17 kDa] and PHI-1 (phosphatase holoenzyme inhibitor-1), known substrates of PKC. Both phosphatase inhibitors were phosphorylated by ILK in an in-gel kinase assay and in solution. A Thr-->Ala mutation at Thr(38) of CPI-17 and Thr(57) of PHI-1 eliminated phosphorylation by ILK. Phosphopeptide mapping, phospho amino acid analysis and immunoblotting using phospho-specific antibodies indicated that ILK predominantly phosphorylated the site critical for potent inhibition, i.e. Thr(38) of CPI-17 or Thr(57) of PHI-1. CPI-17 and PHI-1 thiophosphorylated by ILK at Thr(38) or Thr(57) respectively inhibited myosin light-chain phosphatase (MLCP) activity bound to myosin, whereas the site-specific mutants CPI-17-Thr(38)Ala and PHI-1-Thr(57)Ala, treated with ILK under identical conditions, like the untreated wild-type proteins had no effect on the phosphatase. Consistent with these effects, both thiophospho-CPI-17 and -PHI-1 induced Ca(2+) sensitization of contraction of Triton X-100-demembranated rat-tail arterial smooth muscle, whereas CPI-17-Thr(38)Ala and PHI-1-Thr(57)Ala treated with ILK in the presence of adenosine 5'-[gamma-thio]triphosphate failed to evoke a contractile response. We conclude that ILK may activate smooth-muscle contraction both directly, via phosphorylation of myosin, and indirectly, via phosphorylation and activation of CPI-17 and PHI-1, leading to inhibition of MLCP.

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Selected References

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  1. Dadd C. A., Cook R. G., Allis C. D. Fractionation of small tryptic phosphopeptides by alkaline PAGE followed by amino acid sequencing. Biotechniques. 1993 Feb;14(2):266–273. [PubMed] [Google Scholar]
  2. Delcommenne M., Tan C., Gray V., Rue L., Woodgett J., Dedhar S. Phosphoinositide-3-OH kinase-dependent regulation of glycogen synthase kinase 3 and protein kinase B/AKT by the integrin-linked kinase. Proc Natl Acad Sci U S A. 1998 Sep 15;95(19):11211–11216. doi: 10.1073/pnas.95.19.11211. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Deng J. T., Parsons P. G. Solid phase immunoassay for high molecular weight alkaline phosphatase in human sera using a specific monoclonal antibody. Clin Chim Acta. 1988 Sep 15;176(3):291–301. doi: 10.1016/0009-8981(88)90188-x. [DOI] [PubMed] [Google Scholar]
  4. 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]
  5. Eto M., Karginov A., Brautigan D. L. A novel phosphoprotein inhibitor of protein type-1 phosphatase holoenzymes. Biochemistry. 1999 Dec 21;38(51):16952–16957. doi: 10.1021/bi992030o. [DOI] [PubMed] [Google Scholar]
  6. Eto M., Kitazawa T., Yazawa M., Mukai H., Ono Y., Brautigan D. L. Histamine-induced vasoconstriction involves phosphorylation of a specific inhibitor protein for myosin phosphatase by protein kinase C alpha and delta isoforms. J Biol Chem. 2001 Jun 7;276(31):29072–29078. doi: 10.1074/jbc.M103206200. [DOI] [PubMed] [Google Scholar]
  7. 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. 1995 Dec;118(6):1104–1107. doi: 10.1093/oxfordjournals.jbchem.a124993. [DOI] [PubMed] [Google Scholar]
  8. 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. 1997 Jun 30;410(2-3):356–360. doi: 10.1016/s0014-5793(97)00657-1. [DOI] [PubMed] [Google Scholar]
  9. Etter E. F., Eto M., Wardle R. L., Brautigan D. L., Murphy R. A. Activation of myosin light chain phosphatase in intact arterial smooth muscle during nitric oxide-induced relaxation. J Biol Chem. 2001 Jul 18;276(37):34681–34685. doi: 10.1074/jbc.M104737200. [DOI] [PubMed] [Google Scholar]
  10. Fresu M., Bianchi M., Parsons J. T., Villa-Moruzzi E. Cell-cycle-dependent association of protein phosphatase 1 and focal adhesion kinase. Biochem J. 2001 Sep 1;358(Pt 2):407–414. doi: 10.1042/0264-6021:3580407. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Gratecos D., Fischer E. H. Adenosine 5'-O(3-thiotriphosphate) in the control of phosphorylase activity. Biochem Biophys Res Commun. 1974 Jun 18;58(4):960–967. doi: 10.1016/s0006-291x(74)80237-8. [DOI] [PubMed] [Google Scholar]
  12. 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]
  13. 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]
  14. Hayashi Y., Senba S., Yazawa M., Brautigan D. L., Eto M. Defining the structural determinants and a potential mechanism for inhibition of myosin phosphatase by the protein kinase C-potentiated inhibitor protein of 17 kDa. J Biol Chem. 2001 Aug 21;276(43):39858–39863. doi: 10.1074/jbc.M107302200. [DOI] [PubMed] [Google Scholar]
  15. Kamm K. E., Stull J. T. Dedicated myosin light chain kinases with diverse cellular functions. J Biol Chem. 2000 Nov 28;276(7):4527–4530. doi: 10.1074/jbc.R000028200. [DOI] [PubMed] [Google Scholar]
  16. 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]
  17. Kitazawa T., Takizawa N., Ikebe M., Eto M. Reconstitution of protein kinase C-induced contractile Ca2+ sensitization in triton X-100-demembranated rabbit arterial smooth muscle. J Physiol. 1999 Oct 1;520(Pt 1):139–152. doi: 10.1111/j.1469-7793.1999.00139.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. 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]
  19. Li L., Eto M., Lee M. R., Morita F., Yazawa M., Kitazawa T. Possible involvement of the novel CPI-17 protein in protein kinase C signal transduction of rabbit arterial smooth muscle. J Physiol. 1998 May 1;508(Pt 3):871–881. doi: 10.1111/j.1469-7793.1998.871bp.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. MacDonald J. A., Eto M., Borman M. A., Brautigan D. L., Haystead T. A. Dual Ser and Thr phosphorylation of CPI-17, an inhibitor of myosin phosphatase, by MYPT-associated kinase. FEBS Lett. 2001 Mar 30;493(2-3):91–94. doi: 10.1016/s0014-5793(01)02277-3. [DOI] [PubMed] [Google Scholar]
  21. Murata K., Hirano K., Villa-Moruzzi E., Hartshorne D. J., Brautigan D. L. Differential localization of myosin and myosin phosphatase subunits in smooth muscle cells and migrating fibroblasts. Mol Biol Cell. 1997 Apr;8(4):663–673. doi: 10.1091/mbc.8.4.663. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Murányi Andrea, MacDonald Justin A., Deng Jing Ti, Wilson David P., Haystead Timothy A. J., Walsh Michael P., Erdodi Ferenc, Kiss Eniko, Wu Yue, Hartshorne David J. Phosphorylation of the myosin phosphatase target subunit by integrin-linked kinase. Biochem J. 2002 Aug 15;366(Pt 1):211–216. doi: 10.1042/BJ20020401. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Oliver C. J., Shenolikar S. Physiologic importance of protein phosphatase inhibitors. Front Biosci. 1998 Sep 1;3:D961–D972. doi: 10.2741/a336. [DOI] [PubMed] [Google Scholar]
  24. Perrie W. T., Perry S. V. An electrophoretic study of the low-molecular-weight components of myosin. Biochem J. 1970 Aug;119(1):31–38. doi: 10.1042/bj1190031. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Persechini A., Hartshorne D. J. Phosphorylation of smooth muscle myosin: evidence for cooperativity between the myosin heads. Science. 1981 Sep 18;213(4514):1383–1385. doi: 10.1126/science.6455737. [DOI] [PubMed] [Google Scholar]
  26. Saward L., Zahradka P. Angiotensin II activates phosphatidylinositol 3-kinase in vascular smooth muscle cells. Circ Res. 1997 Aug;81(2):249–257. doi: 10.1161/01.res.81.2.249. [DOI] [PubMed] [Google Scholar]
  27. 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]
  28. Takahashi T., Taniguchi T., Konishi H., Kikkawa U., Ishikawa Y., Yokoyama M. Activation of Akt/protein kinase B after stimulation with angiotensin II in vascular smooth muscle cells. Am J Physiol. 1999 Jun;276(6 Pt 2):H1927–H1934. doi: 10.1152/ajpheart.1999.276.6.H1927. [DOI] [PubMed] [Google Scholar]
  29. Walsh M. P., Valentine K. A., Ngai P. K., Carruthers C. A., Hollenberg M. D. Ca2+-dependent hydrophobic-interaction chromatography. Isolation of a novel Ca2+-binding protein and protein kinase C from bovine brain. Biochem J. 1984 Nov 15;224(1):117–127. doi: 10.1042/bj2240117. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Weber L. P., Van Lierop J. E., Walsh M. P. Ca2+-independent phosphorylation of myosin in rat caudal artery and chicken gizzard myofilaments. J Physiol. 1999 May 1;516(Pt 3):805–824. doi: 10.1111/j.1469-7793.1999.0805u.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Wilson David P., Sutherland Cindy, Walsh Michael P. Ca2+ activation of smooth muscle contraction: evidence for the involvement of calmodulin that is bound to the triton insoluble fraction even in the absence of Ca2+. J Biol Chem. 2001 Nov 13;277(3):2186–2192. doi: 10.1074/jbc.M110056200. [DOI] [PubMed] [Google Scholar]
  32. Winder S. J., Walsh M. P. Smooth muscle calponin. Inhibition of actomyosin MgATPase and regulation by phosphorylation. J Biol Chem. 1990 Jun 15;265(17):10148–10155. [PubMed] [Google Scholar]
  33. Woodsome T. P., Eto M., Everett A., Brautigan D. L., Kitazawa T. Expression of CPI-17 and myosin phosphatase correlates with Ca(2+) sensitivity of protein kinase C-induced contraction in rabbit smooth muscle. J Physiol. 2001 Sep 1;535(Pt 2):553–564. doi: 10.1111/j.1469-7793.2001.t01-1-00553.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Wu C., Dedhar S. Integrin-linked kinase (ILK) and its interactors: a new paradigm for the coupling of extracellular matrix to actin cytoskeleton and signaling complexes. J Cell Biol. 2001 Nov 5;155(4):505–510. doi: 10.1083/jcb.200108077. [DOI] [PMC free article] [PubMed] [Google Scholar]

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