Phosphorylation of CPI-17, an inhibitor of myosin phosphatase, by protein kinase N

CPI-17 is a phosphorylation-dependent inhibitory protein for smooth muscle myosin phosphate, Phosphorylation at Thr(38), in vitro, by protein kinase C or Rho-kinase enhances the inhibitory potency toward myosin phosphatase, P hosphorylation of CPI-17 by protein kinase N (PKN), a fatty acid- and Rho-a ctivated serine/threonine kinase, and its effect on smooth muscle myosin ph osphatase activity were investigated. CPI-17 was phosphorylated by GST-PKN- CAT, a constitutively active GST-fusion fragment of PKN, to 1.46 mol of P/m ol of CPI-17, in vitro, The K-m value of CPI-17 for PKN was 0.96 mu M. Phos phorylation of PKN dramatically increased the inhibitory effect of CPI-17 o n myosin phosphatase activity. The major and inhibitory phosphorylation sit e was identified as Thr(38) using a point mutant of CPI-17 and a phosphoryl ation-state specific antibody. Thus, CPI-17 is a substrate of PKN and might be involved in the Ca2+ sensitization of smooth muscle contraction as a do wnstream effector of Rho and/or arachidonic acid. (C) 2000 Academic Press.