Abstract
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.
Highlights
From the Laboratory of Molecular Cardiology,National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda
The single site phosphorylated by that protein kinase C, similar to CAMP-dependent protein protein kinase C when calmodulin is bound to myosin kinase, has broad substrate specificity and both kilight chain kinase is different from thatphos- nases can often phosphorylate the same protein [11].Therephorylated by the catalytic subunitof CAMP-depend- fore, we decided to study the phosphorylation of smooth ent protein kinase
Previous results established that phosphorylation of site 1 alone by the catalytic subunit of CAMP-dependentprotein kinase had no effect on calmodulin binding
Summary
From the Laboratory of Molecular Cardiology,National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda. We were interested in whether protein kinase is not bound appears tobe the same siptehosphorylated 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. Sequentialstudies using both protein kinase C and the catalytic subunoift CAMP-dependent. The phosphorylation of myosin in these cells is catalyzed by a substrate-specific enzyme, myosin light chain kinase, the activity of which is absolutely dependent on the binding of a calcium-calmodulin complex [2, 3]. (ParrInstrument Co., Moline, IL).Thedebris was removed by centrifugation a t 20,000 X g for 20 min, and the resulting supernatant
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