Inhibitory properties of the regulatory domains of human protein kinase Cα and mouse protein kinase Cε

Two fusion proteins in which the regulatory domains of human protein kinase C alpha (R alpha; amino acids 1-270) or mouse protein kinase C epsilon (R epsilon; amino acids 1-385) were linked in frame with glutathione S-transferase (GST) were examined for their abilities to inhibit the catalytic activities of protein kinase C alpha (PKC alpha; and other protein kinases in vitro, Both GST-R alpha and GST-R epsilon but not GST itself potently inhibited the activities of lipid-activated rat brain PKC alpha, In contrast, the fusion proteins had little or no inhibitory effect on the activities of the Ser/Thr protein kinases cAMP-dependent protein kinase, cGMP-dependent protein kinase, casein kinase II, myosin light chain kinase, and mitogen activated protein kinase or on the src Tyr kinase, GST-R alpha and GST-R epsilon, on a molar basis, were 100-200-fold more potent inhibitors of PKC alpha activity than was the pseudosubstrate peptide PKC19-36, In addition, a GST-R alpha fusion protein in which the first 32 amino acids of R alpha were deleted (including the pseudosubstrate sequence from amino acids 19-31) was an effective competitive inhibitor of PKC alpha activity, The three GST-R fusion proteins also inhibited protamine-activated PKC alpha and proteolytically activated PKC alpha (PKM), two lipid-independent forms of PKC alpha; however, the IC50 values for inhibition were 1 order of magnitude greater than the IC50 values obtained in the presence of lipid, These results suggest that part of the inhibitory effect of the GST-R fusion proteins on lipid-activated PKC alpha may have resulted from sequestration of lipid activators, Nonetheless, as evidenced by their abilities to inhibit the lipid-independent forms of the enzyme, the GST-R fusion proteins also inhibited PKC alpha catalytic activity through direct interactions, These data indicate that the R domains of PKC alpha and PKC epsilon are specific inhibitors of protein kinase C alpha activity and suggest that regions of the R domain outside the pseudosubstrate sequence contribute to autoinhibition of the enzyme.