Ps. Leventhal et Pj. Bertics, ACTIVATION OF PROTEIN-KINASE-C BY SELECTIVE BINDING OF ARGININE-RICH POLYPEPTIDES, The Journal of biological chemistry, 268(19), 1993, pp. 13906-13913
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.