ACTIVATION OF PROTEIN-KINASE-C BY SELECTIVE BINDING OF ARGININE-RICH POLYPEPTIDES

Protein substrates for protein kinase C (PKC) have phosphorylation dom ains that are typically rich in the basic amino acids arginine and lys ine. However, arginine-rich proteins may interact with PKC differently than lysine-rich proteins, i.e. lysine-rich histone requires phosphol ipid and Ca2+ to be phosphorylated whereas the arginine-rich protein, protamine, can by pass these effector requirements. We have studied th e interaction of PKC with protamine, histone, poly-L-arginine, and pol y-L-lysine to better understand the role of basic protein domains in P KC activation, effector dependence, and substrate specificity. Using a microtiter binding assay, PKC was found to bind tightly to protamine and poly-L-arginine, but not to histone or poly-L-lysine, in the absen ce of phospholipid, Ca2+, and MgATP. Furthermore, poly-L-arginine was much more potent than poly-L-lysine at inhibiting protamine phosphoryl ation; i. e. 1-2 nM poly-L-arginine was sufficient to cause 50% inhibi tion of protamine phosphorylation, whereas over 300 muM poly-L-lysine was needed to reach 50% inhibition. Autophosphorylation of PKC in the absence of activators was potently stimulated by protamine and poly-L- arginine, but not by histone or poly-L-lysine, suggesting selective st imulation of PKC by arginine-rich polypeptides. Double-reciprocal plot s of protamine phosphorylation using either a mixture of isozymes (alp ha/beta/gamma) or isolated PKC-beta were parabolic, and analysis of th e kinetic data on velocity/[protamine] versus velocity plots indicated positive cooperativity with respect to protamine. These findings are consistent with those from autophosphorylation experiments in that PKC appears to be selectively stimulated by arginine-rich polypeptides. T hese results suggest that PKC can preferentially bind arginine-rich pr oteins in the absence of phospholipid and Ca2+. This interaction appea rs to be distal to the catalytic site and thus binding of arginine-ric h proteins may allosterically activate PKC. Selective stimulation of P KC by arginine-rich proteins may be a mechanism by which protamine can bypass activator requirements. Furthermore, control of PKC activity b y activator-independent binding of arginine-rich polypeptides suggests that altering access to certain cellular proteins may be a mechanism for PKC regulation in vivo.