Phosphorylation of the Protein Phosphatase Type 1 Inhibitor Protein CPI-17 by Protein Kinase C

  • Michael P. Walsh
  • Marija Susnjar
  • Jingti Deng
  • Cindy Sutherland
  • Enikő Kiss
  • David P. Wilson
Part of the Methods in Molecular Biology book series (MIMB, volume 365)


CPI-17 is a cytosolic protein of 17 kDa that becomes a potent inhibitor of certain type 1 protein serine/threonine phosphatases, including smooth muscle myosin light-chain phosphatase (MLCP), when phosphorylated at Thr38. Several protein kinases are capable of phosphorylating CPI-17 at this site in vitro; however, in intact tissue, compelling evidence only exists for phosphorylation by protein kinase C (PKC). Agonist-induced activation of heterotrimeric G proteins of the Gq/11 family via seven-transmembrane domain-containing, G protein-coupled receptors results in phospholipase Cβ-mediated hydrolysis of membrane phosphatidylinositol 4,5-bisphosphate to generate inositol 1,4,5-trisphosphate (IP3) and 1,2-diacylglycerol (DAG). IP3 triggers Ca2+ release from the sarcoplasmic reticulum. DAG and Ca2+ together activate classical isoforms of PKC, and DAG activates novel PKC isoforms without a requirement for Ca2+. Activated PKC phosphorylates CPI-17 at Thr38, enhancing its potency of inhibition of MLCP approx 1000-fold. The myosin light-chain kinase (MLCK) : MLCP activity ratio is thereby increased at the prevailing cytosolic free-Ca2+ concentration ([Ca2+]i), resulting in an increase in phosphorylation of the 20-kDa light chains of myosin II (LC20) catalyzed by Ca2+- and calmodulin-dependent MLCK and contraction of the smooth muscle. Physiologically, this mechanism can account for some instances of Ca2+ sensitization of smooth muscle contraction (i.e., an increase in force in response to agonist stimulation without a change in [Ca2+]i).

Key Words

CPI-17 protein kinase C myosin light-chain phosphatase myosin light-chain kinase Ca2+ sensitization smooth muscle contraction 


  1. 1.
    Cohen, P. (2002) The origins of protein phosphorylation. Nature Cell Biol. 4, E127–E130.PubMedCrossRefGoogle Scholar
  2. 2.
    Manning, G., Whyte, D. B., Martinez, R., Hunter, T., and Sudarsanam, S. (2002) The protein kinase complement of the human genome. Science 298, 1912–1934.PubMedCrossRefGoogle Scholar
  3. 3.
    Alonso, A., Sasin, J., Bottini, N., et al. (2004) Protein tyrosine phosphatases in the human genome. Cell 117, 699–711.PubMedCrossRefGoogle Scholar
  4. 4.
    Honkanen, R. E. (2005) Serine/threonine protein phosphatase inhibitors with antitumor activity, in Handbook of Experimental Pharmacology: Inhibitors of Protein Kinases and Protein Phosphatases (Pinna, L. A., ed.), Springer-Verlag, Berlin, Vol. 167, pp. 295–317.CrossRefGoogle Scholar
  5. 5.
    Allen, B. G. and Walsh, M. P. (1994) The biochemical basis of the regulation of smooth-muscle contraction. Trends Biochem. Sci. 19, 362–368.PubMedCrossRefGoogle Scholar
  6. 6.
    Kamm, K. E. and Stull, J. T. (2001) Dedicated myosin light-chain kinases with diverse cellular functions. J. Biol. Chem. 276, 4527–4530.PubMedCrossRefGoogle Scholar
  7. 7.
    Hartshorne, D. J., Ito, M., and Erdődi, F. (2004) Role of protein phosphatase type 1 in contractile functions: myosin phosphatase. J. Biol. Chem. 279, 37,211–37,214.PubMedCrossRefGoogle Scholar
  8. 8.
    Oliver, C. J. and Shenolikar, S. (1998) Physiological importance of protein phosphatase inhibitors. Front. Biosci. 3, D961–D972.PubMedGoogle Scholar
  9. 9.
    Eto, M., Ohmori, T., Suzuki, M., Furuya, K., and Morita, F. (1995) A novel protein phosphatase-1 inhibitory protein potentiated by protein kinase C. Isolation from porcine aorta media and characterization. J. Biochem. (Tokyo) 118, 1104–1107.Google Scholar
  10. 10.
    Eto, M., Senba, S., Morita, F., and Yazawa, M. (1997) Molecular cloning of a novel phosphorylation-dependent inhibitory protein of protein phosphatase-1 (CPI-17) in smooth muscle: its specific localization in smooth muscle. FEBS Lett. 410, 356–360.PubMedCrossRefGoogle Scholar
  11. 11.
    Woodsome, T. P., Eto, M., Everett, A., Brautigan, D. L., and Kitazawa, T. (2001) Expression of CPI-17 and myosin phosphatase correlates with Ca2+ sensitivity of protein kinase C-induced contraction in rabbit smooth muscle. J. Physiol. 535, 553–564.PubMedCrossRefGoogle Scholar
  12. 12.
    Senba, S., Eto, M., and Yazawa, M. (1999) 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. (Tokyo) 125, 354–362.Google Scholar
  13. 13.
    Eto, M., Karginov, A., and Brautigan, D. L. (1999) A novel phosphoprotein inhibitor of protein type-1 phosphatase holoenzymes. Biochemistry 38, 16,952–16,957.PubMedCrossRefGoogle Scholar
  14. 14.
    Liu, Q. R., Zhang, P. W., Zhen, Q., Walther, D., Wang, X. B., and Uhl, G. R. (2002) KEPI, a PKC-dependent protein phosphatase 1 inhibitor regulated by morphine. J. Biol. Chem. 277, 13,312–13,320.PubMedCrossRefGoogle Scholar
  15. 15.
    Koyama, M., Ito, M., Feng, J., et al. (2000) Phosphorylation of CPI-17, an inhibitory phosphoprotein of smooth muscle myosin phosphatase, by Rho-kinase. FEBS Lett. 475, 197–200.PubMedCrossRefGoogle Scholar
  16. 16.
    Hamaguchi, T., Ito, M., Feng, J., et al. (2000) Phosphorylation of CPI-17, an inhibitor of myosin phosphatase, by protein kinase N. Biochem. Biophys. Res. Commun. 274, 825–830.PubMedCrossRefGoogle Scholar
  17. 17.
    Deng, J. T., Sutherland, C., Brautigan, D. L., Eto, M., and Walsh, M. P. (2002) Phosphorylation of the myosin phosphatase inhibitors, CPI-17 and PHI-1, by integrin-linked kinase. Biochem. J. 367, 517–524.PubMedCrossRefGoogle Scholar
  18. 18.
    MacDonald, J. A., Eto, M., Borman, M. A., Brautigan, D. L., and Haystead, T. A. J. (2001) Dual Ser and Thr phosphorylation of CPI-17, an inhibitor of myosin phosphatase, by MYPT-associated kinase. FEBS Lett. 493, 91–94.PubMedCrossRefGoogle Scholar
  19. 19.
    Erdődi, F., Kiss, E., Walsh, M. P., et al. (2003) Phosphorylation of protein phosphatase type-1 inhibitory proteins by integrin-linked kinase and cyclic nucleotide-dependent protein kinases. Biochem. Biophys. Res. Commun. 306, 382–387.PubMedCrossRefGoogle Scholar
  20. 20.
    Takizawa, N., Koga, Y., and Ikebe, M. (2002) Phosphorylation of CPI17 and myosin binding subunit of type 1 protein phosphatase by p21-activated kinase. Biochem. Biophys. Res. Commun. 297, 773–778.PubMedCrossRefGoogle Scholar
  21. 21.
    Kitazawa, T., Eto, M., Woodsome, T. P., and Brautigan, D. L. (2000) 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. 275, 9897–9900.PubMedCrossRefGoogle Scholar
  22. 22.
    Eto, M., Kitazawa, T., Yazawa, M., Mukai, H., Ono, Y., and Brautigan, D. L. (2001) Histamine-induced vasoconstriction involves phosphorylation of a specific inhibitor protein for myosin phosphatase by protein kinase C α and δ isoforms. J. Biol. Chem. 276, 29,072–29,078.PubMedCrossRefGoogle Scholar
  23. 23.
    Niiro, N., Koga, Y., and Ikebe, M. (2003) Agonist-induced changes in the phosphorylation of the myosin-binding subunit of myosin light-chain phosphatase and CPI17, two regulatory factors of myosin light-chain phosphatase, in smooth muscle. Biochem. J. 369, 117–128.PubMedCrossRefGoogle Scholar
  24. 24.
    Kitazawa, T., Eto, M., Woodsome, T. P., and Khalequzzamana, M. D. (2003) Phosphorylation of the myosin phosphatase targeting subunit and CPI-17 during Ca2+ sensitization in rabbit smooth muscle. J. Physiol. 546, 879–889.PubMedCrossRefGoogle Scholar
  25. 25.
    Wilson, D. P., Susnjar, M., Kiss, E., Sutherland, C., and Walsh, M. P. (2005) Thromboxane A2-induced contraction of rat caudal arterial smooth muscle involves activation of Ca2+ entry and Ca2+ sensitization: Rho-associated kinase-mediated phosphorylation of MYPT1 at Thr-855 but not Thr-697. Biochem. J. 389, 763–774.PubMedCrossRefGoogle Scholar
  26. 26.
    Eto, M., Kitazawa, T., and Brautigan, D. L. (2004) Phosphoprotein inhibitor CPI-17 specificity depends on allosteric regulation of protein phosphatase-1 by regulatory subunits. Proc. Natl. Acad. Sci. USA 101, 8888–8893.PubMedCrossRefGoogle Scholar
  27. 27.
    Li, L., Eto, M., Lee, M. R., Morita, F., Yazawa, M., and Kitazawa, T. (1998) Possible involvement of the novel CPI-17 protein in protein kinase C signal transduction of rabbit arterial smooth muscle. J. Physiol. 508, 871–881.PubMedCrossRefGoogle Scholar
  28. 28.
    Somlyo, A. P. and Somlyo, A. V. (2003) Ca2+ sensitivity of smooth muscle and nonmuscle myosin II: modulated by G proteins, kinases, and myosin phosphatase. Physiol. Rev. 83, 1325–1358.PubMedGoogle Scholar
  29. 29.
    Ohki, S., Eto, M., Kariya, E., et al. (2001) Solution NMR structure of the myosin phosphatase inhibitor protein CPI-17 shows phosphorylation-induced conformational changes responsible for activation. J. Mol. Biol. 314, 839–849.PubMedCrossRefGoogle Scholar
  30. 30.
    Allen, B. G., Andrea, J. E., and Walsh, M. P. (1994) Identification and characterization of protein kinase Cζ-immunoreactive proteins. J. Biol. Chem. 269, 29,288–29,298.PubMedGoogle Scholar
  31. 31.
    Walsh, M. P., Valentine, K. A., Ngai, P. K., Carruthers, C. A., and Hollenberg, M. D. (1984) Ca2+-dependent hydrophobic interaction chromatography. Isolation of a novel Ca2+-binding protein and protein kinase C from bovine brain. Biochem. J. 224, 117–127.PubMedGoogle Scholar
  32. 32.
    Ngai, P. K., Carruthers, C. A., and Walsh, M. P. (1984) Isolation of the native form of chicken gizzard myosin light-chain kinase. Biochem. J. 218, 863–870.PubMedGoogle Scholar
  33. 33.
    Weber, L. P., Van Lierop, J. E., and Walsh, M. P. (1999) Ca2+-independent phosphorylation of myosin in rat caudal artery and chicken gizzard myofilaments. J. Physiol. 516, 805–824.PubMedCrossRefGoogle Scholar
  34. 34.
    Andrea, J. E. and Walsh, M. P (1992) Protein kinase C of smooth muscle. Hypertension 20, 585–595.PubMedGoogle Scholar
  35. 35.
    Winder, S.J. and Walsh, M.P. (1990) Smooth muscle calponin. Inhibition of actomyosin MgATPase and regulation by phosphorylation. J. Biol. Chem. 265, 10,148–10,155.PubMedGoogle Scholar

Copyright information

© Humana Press Inc. 2007

Authors and Affiliations

  • Michael P. Walsh
    • 1
  • Marija Susnjar
    • 1
  • Jingti Deng
    • 1
  • Cindy Sutherland
    • 1
  • Enikő Kiss
    • 1
  • David P. Wilson
    • 1
  1. 1.Smooth Muscle Research Group, Department of Biochemistry & Molecular BiologyUniversity of CalgaryCalgaryCanada

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