Site-specific dephosphorylation of smooth muscle myosin light chain kinase by protein phosphatases 1 and 2A.
Smooth muscle myosin light chain kinase is phosphorylated at two sites (A and B) by different protein kinases. Phosphorylation at site A increases the concentration of Ca2+/calmodulin required for kinase activation. Diphosphorylated myosin light chain kinase was used to determine the site-specificity of several forms of protein serine/threonine phosphatase. These phosphatases readily dephosphorylated myosin light chain kinase in vitro and displayed differing specificities for the two phosphorylation sites. Type 2A protein phosphatase specifically dephosphorylated site A, and binding of Ca2+/calmodulin to the kinase had no effect on dephosphorylation. The purified catalytic subunit of type 1 protein phosphatase dephosphorylated both sites in the absence of Ca2+/calmodulin but only dephosphorylated site A in the presence of Ca2+/calmodulin. A protein phosphatase fraction was prepared from smooth muscle actomyosin by extraction with 80 mM MgCl2. On the basis of sensitivity to okadaic acid and inhibitor 2, this activity was composed of multiple protein phosphatases including type 1 activity. This phosphatase fraction dephosphorylated both sites in the absence of Ca2+/calmodulin. However, dephosphorylation of both sites A and B was completely blocked in the presence of Ca2+/calmodulin. These results indicate that two phosphorylation sites of myosin light chain kinase are dephosphorylated by multiple protein serine/threonine phosphatases with unique catalytic specificities.
Nomura
M
M
Department of Physiology, University of Texas Southwestern Medical Center, Dallas 75235.
Stull
J T
JT
Kamm
K E
KE
Mumby
M C
MC
D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000494:pharmacology; D002147:Calmodulin / Q000494:pharmacology; D002417:Cattle; D002645:Chickens; D000242:Cyclic AMP / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D005895:Gizzard, Avian / Q000201:enzymology; D007700:Kinetics; D009130:Muscle, Smooth / Q000201:enzymology*; D009206:Myocardium / Q000201:enzymology; D009219:Myosin-Light-Chain Kinase / Q000378:metabolism*; D010749:Phosphoprotein Phosphatases / Q000378:metabolism*; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism
D002147:Calmodulin; D000242:Cyclic AMP; D011494:Protein Kinases; D009219:Myosin-Light-Chain Kinase; D010749:Phosphoprotein Phosphatases; D002118:Calcium