Phosphorylation of smooth muscle myosin light chain kinase by protein kinase C. Comparative study of the phosphorylated sites.
Smooth muscle myosin light chain kinase is phosphorylated in vitro by protein kinase C purified from human platelets. When myosin light chain kinase which has calmodulin bound is phosphorylated by protein kinase C, 0.8-1.1 mol of phosphate is incorporated per mol of myosin light chain kinase with no effect on its enzyme activity. Phosphorylation of myosin light chain kinase with no calmodulin bound results in the incorporation of 2-2.4 mol of phosphate and significantly decreases the rate of myosin light chain kinase activity. The decrease in myosin light chain kinase activity is due to a 3.3-fold increase in the concentration of calmodulin necessary for the half-maximal activation of myosin light chain kinase. The sites phosphorylated by protein kinase C and the catalytic subunit of cAMP-dependent protein kinase were compared by two-dimensional peptide mapping following extensive tryptic digestion of phosphorylated myosin light chain kinase. The single site phosphorylated by protein kinase C when calmodulin is bound to myosin light chain kinase (site 3) is different from that phosphorylated by the catalytic subunit of cAMP-dependent protein kinase (site 1). The additional site that is phosphorylated by protein kinase C when calmodulin is not bound appears to be the same site phosphorylated by the catalytic subunit of cAMP-dependent protein kinase (site 2). These studies confirm the important role of site 2 in binding calmodulin to myosin light chain kinase. Sequential studies using both protein kinase C and the catalytic subunit of cAMP-dependent protein kinase suggest that the phosphorylation of site 1 also plays a part in decreasing the affinity of myosin light chain kinase for calmodulin.
Nishikawa
M
M
Shirakawa
S
S
Adelstein
R S
RS
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001665:Binding Sites; D002147:Calmodulin / Q000378:metabolism; D006801:Humans; D009130:Muscle, Smooth / Q000201:enzymology*; D009219:Myosin-Light-Chain Kinase; D010766:Phosphorylation; D011493:Protein Kinase C; D011494:Protein Kinases / Q000032:analysis / Q000378:metabolism* / Q000494:pharmacology*; D014422:Turkeys
D000596:Amino Acids; D002147:Calmodulin; D011494:Protein Kinases; D011493:Protein Kinase C; D009219:Myosin-Light-Chain Kinase