Purification and characterization of a rabbit liver calmodulin-dependent protein kinase able to phosphorylate glycogen synthase.

A calmodulin-dependent protein kinase has been extensively purified from rabbit liver by the criterion of its ability to phosphorylate muscle glycogen synthase.The enzyme bound to phosphocellulose, DEAE-cellulose, and blue dextran-agarose.The enzyme also bound, in a Caz+-dependent manner, to a calmodulin-agarose affinity column.Expression of activity required the presence of both calmodulin and Ca", with half-maxim a l activation occurring at approximately 80 n M calmodulin.Trifluoperazine, 50 p ~, completely inhibited the enzyme.Enzyme activity was associated with two polypeptide species of apparent molecular weights 53,000 and 51,000.The molecular weight of the native enzyme was approximately 5O(r,OOO, as judged from gel filtration, and 275,000 as determined from sucrose density gradient sedimentation, suggesting an oligomeric structure.Incubation of the enzyme with ATP and Mg2+ led to phosphorylation of the constituent polypeptides and an accompanying decrease in electrophoretic mobilities in the presence o€ sodium dodecyl sulfate.The protein kinase had an apparent K,,, for ATP of 27 PM and had half-maximal activity at 0.75 mM Mg2+.The protein kinase phosphorylated muscle glycogen synthase to a stoichiometry of greater than 1 phosphate/ subunit with significant inactivation.Phosphate was introduced into at least two sites on the glycogen synthase.Another effective substrate f o r the protein kinase was the smooth muscle 20,000-dalton myosin light chain.The enzyme had modest activity toward phosvitin and casein, but importantly, had little activity toward cardiac myosin light chains, histone, and either skeletal muscle or liver glycogen phosphorylase.The enzyme is, therefore, distinguishable from the two best characterized calmodulin-stimulated protein kinases, phosphorylase kinase and myosin light chain kinase, and represents a distinct category of such enzymes.Multiple phosphorylation of the subunit of glycogen synthase (EC 2.4.1.11)occurs in vitro through the action of some five or more distinct protein kinases (see Refs. 1-4 for a review) and may afford a complex regulation of this ratelimiting enzyme of glycogen synthesis.Muscle enzymes have been most extensively studied, and three major classes of