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. Phosphorylation of CPI-17 by protein kinase N (PKN), a fatty acid- and Rho-activated serine/threonine kinase, and its effect on smooth muscle myosin phosphatase 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/mol of CPI-17, in vitro. The K(m) value of CPI-17 for PKN was 0.96 microM. Phosphorylation of PKN dramatically increased the inhibitory effect of CPI-17 on myosin phosphatase activity. The major and inhibitory phosphorylation site was identified as Thr(38) using a point mutant of CPI-17 and a phosphorylation-state specific antibody. Thus, CPI-17 is a substrate of PKN and might be involved in the Ca(2+) sensitization of smooth muscle contraction as a downstream effector of Rho and/or arachidonic acid.
First Department of Internal Medicine, Mie University School of Medicine, Tsu, Mie, 514-8507, Japan.
T
T
Hamaguchi
M
M
Ito
J
J
Feng
T
T
Seko
M
M
Koyama
H
H
Machida
K
K
Takase
M
M
Amano
K
K
Kaibuchi
D J
DJ
Hartshorne
T
T
Nakano
D000818:Animals; D009124:Muscle Proteins / Q000378:metabolism*; D009131:Muscle, Smooth, Vascular / Q000378:metabolism*; D043263:Myosin-Light-Chain Phosphatase; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D013552:Swine
D009124:Muscle Proteins; D010750:Phosphoproteins; C061570:protein kinase N; D011493:Protein Kinase C; D010749:Phosphoprotein Phosphatases; D043263:Myosin-Light-Chain Phosphatase