Phosphorylation and activation of smooth muscle myosin light chain kinase by MAP kinase and cyclin-dependent kinase-1.
Smooth muscle myosin light chain kinase (MLCK) features several consensus sites of phosphorylation by proline-directed protein serine/threonine kinases. The phosphorylation of MLCK by two proline-directed kinases isolated from sea star oocytes, i.e., p44mpk (Mpk, a mitogen-activated protein kinase homologue) and cyclin-dependent kinase-1 (CDK1, also known as p34cdc2), was investigated. Chicken gizzard MLCK was phosphorylated on seryl and threonyl residues by both Mpk and CDK1. Phosphorylation of MLCK to 0.6 mol Pi/mol by Mpk increased the Vmax of phosphotransferase activity towards a synthetic peptide corresponding to residues 11-23 of the 20-kDa light chain of myosin by 1.6-fold. Phosphorylation of MLCK to 1.0 mol Pi/mol by CDK1 increased the Vmax by 2.3-fold. Phosphorylation by either kinase had no significant effect on the concentration of calmodulin required for half-maximal activation of MLCK. Analysis of the phosphorylation of synthetic peptides containing consensus phosphorylation sites for Mpk and CDK1 indicated that the major site of phosphorylation in MLCK by Mpk was Ser-834, and by CDK1 was Thr-283. Both of these sites are located outside the calmodulin-binding site (residues 796-815), consistent with the observation that phosphorylation by Mpk or CDK1 was unaffected by the presence of bound Ca2+/calmodulin. These results indicate that MLCK activity may be regulated by phosphorylation catalyzed by proline-directed kinases, possibly directed at Thr-40 and Thr-43 at the amino terminus of MLCK.
Morrison
D L
DL
Department of Medicine, University of British Columbia, Vancouver, Canada.
Sanghera
J S
JS
Stewart
J
J
Sutherland
C
C
Walsh
M P
MP
Pelech
S L
SL
D000818:Animals; D001665:Binding Sites; D016203:CDC2 Protein Kinase / Q000378:metabolism*; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism; D002645:Chickens; D004789:Enzyme Activation; D009130:Muscle, Smooth / Q000201:enzymology*; D009219:Myosin-Light-Chain Kinase / Q000737:chemistry / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism; D010766:Phosphorylation; D010768:Phosphoserine / Q000378:metabolism; D010769:Phosphothreonine / Q000378:metabolism; D010770:Phosphotransferases / Q000378:metabolism
D002147:Calmodulin; D010446:Peptide Fragments; D010769:Phosphothreonine; D010768:Phosphoserine; D010770:Phosphotransferases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D009219:Myosin-Light-Chain Kinase; D016203:CDC2 Protein Kinase