Phosphorylation of synaptosomal cytoplasmic proteins: Inhibition of calcium-activated, phospholipid-dependent protein kinase (protein kinase c) by bay k 8644.
The phosphorylation of specific substrates of calcium-activated, phospholipid-dependent protein kinase (protein kinase C) was examined in striatal synaptosomal cytoplasm. The phosphoprotein substrata were termed group C phosphoprotems and were divided into two subgroups: group C(1) phosphoproteins (P83, P45A, P21 and P18) were found in both cytoplasm and synaptosomal membranes and, although stimulated by phosphatidylserine, only required exogamous calcium for their labeling; group C(2) phosphoproteins (P120, P96, P21.5, P18.5 and P16) were found predominantly in the cytoplasm and were absolutely dependent upon exogenous calcium and phosphatidylserme for their labeling. Several criteria were used to identify these proteins as specific protein kinase C substrates: (a) their phosphorylation was stimulated to a greater extent by Ca(2+) /phosphatidylserine/diolein than by Ca(2+) alone or Cal(2+) /calmodulin (group C(1)) or was completely dependent upon Ca(2+) /phosphatdylserine/diolein (group C(2)); (b) supermaximal concentrations of the cAMP-dependent protein kinase inhibitor were without effect; (c) their phosphorylation was stimulated by oleic acid, which selectively activates protein kinase C in the absence of Ca(2+); (d) NaCl, which inhibited cAMP- and Ca(2+)/calmodulindependent phosphorylation, slightly increased phosphorylation of group C(1) and slightly decreased phosphorylation of group C(2) phosphoproteins. Maximal phosphorylation of P96 and other group C phosphoproteins occurred within 60 s and was followed by a slow decay rate while substrata of calmodulin-dependent protein kinase were maximally labeled within 20-30 s and rapidly dephosphorylated. The phosphorylation of all group C phosphoproteins was inhibited by the calcium channel agomst BAY K 8644, however, group C(2) phosphoproteins were considerably more sensitive. The IC(50) for inhibition of P96 labeling was 19 ?M. but for P83 was 190 ?M. Group B phosphoproteins were also slightly inhibited, and the IC(50) for P63 was 290 ?M. No inhibitory effects of another dihydropyridine, nifedipine, or of verapamil were detected in this concentration range. BAY K 8644 did not displace [(3)H]phorbol-12,13-dibutyrate binding, nor was the inhibition decreased by increasing phosphatidylserine concentrations. BAY K 8644 had no effect on the rate of dephosphorylation of any phosphoprotein, indicating that it is unlikely to inhibit a protein phosphatase. BAY K 8644 may, therefore, prove to be a valuable tool for discriminating protein kinase C activity from the activity of other protein kinases. We conclude that BAY K 8644 interacts either with a specific subgroup of protein kinase C substrata or with one of two putative forms of protein kinase C.
Robinson
P J
PJ
Merrell Dow Research Institute, 2110 E. Galbraith Road, Cincinnati, OH 01145215, U.S.A.
Lovenberg
W
W