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
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 365)

Abstract

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 

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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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