ABSTRACT
Two fusion proteins in which the regulatory domains of human protein kinase Calpha (Ralpha; amino acids 1-270) or mouse protein kinase Cepsilon (Repsilon; 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 Calpha (PKCalpha) and other protein kinases in vitro. Both GST-Ralpha and GST-Repsilon but not GST itself potently inhibited the activities of lipid-activated rat brain PKCalpha. 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-Ralpha and GST-Repsilon, on a molar basis, were 100-200-fold more potent inhibitors of PKCalpha activity than was the pseudosubstrate peptide PKC19-36. In addition, a GST-Ralpha fusion protein in which the first 32 amino acids of Ralpha were deleted (including the pseudosubstrate sequence from amino acids 19-31) was an effective competitive inhibitor of PKCalpha activity. The three GST-R fusion proteins also inhibited protamine-activated PKCalpha and proteolytically activated PKCalpha (PKM), two lipid-independent forms of PKCalpha; 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 PKCalpha 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 PKCalpha catalytic activity through direct interactions. These data indicate that the R domains of PKCalpha and PKCepsilon are specific inhibitors of protein kinase Calpha activity and suggest that regions of the R domain outside the pseudosubstrate sequence contribute to autoinhibition of the enzyme.
Subject(s)
Isoenzymes/chemistry , Isoenzymes/metabolism , Protein Kinase C/chemistry , Protein Kinase C/metabolism , Protein Kinases/metabolism , Amino Acid Sequence , Animals , Binding Sites , Binding, Competitive , Calcium-Calmodulin-Dependent Protein Kinases/metabolism , Casein Kinase II , Cyclic AMP-Dependent Protein Kinases/metabolism , Cyclic GMP-Dependent Protein Kinases/metabolism , Glutathione Transferase/biosynthesis , Humans , Isoenzymes/biosynthesis , Kinetics , Mice , Molecular Sequence Data , Myosin-Light-Chain Kinase/metabolism , Peptide Fragments/chemistry , Protein Kinase C/biosynthesis , Protein Kinase C-alpha , Protein Kinase C-epsilon , Protein Serine-Threonine Kinases/metabolism , Rats , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/metabolismABSTRACT
The regulatory (R) domain of PKC alpha fused to glutathione-S-transferase (GST-R alpha) competitively inhibited PKC activity associated with extracts of Y1 mouse adrenocortical tumor cells and the activities of several specific PKC isozymes. GST-R alpha did not inhibit the activities of cAMP-dependent protein kinase, cGMP-dependent protein kinase or calmodulin-dependent myosin light chain kinase. GST-R alpha inhibited PKC activities 20 times more potently than did a synthetic peptide corresponding to the pseudosubstrate sequence of PKC alpha. In intact yeast cells, the R domain prevented PKC beta-1-induced inhibition of growth and cytokinesis. These results indicate that the R domain of PKC alpha acts as a specific, dominant inhibitor of PKC activity, and suggest that the PKC alpha R domain may provide a useful genetic tool to assess the roles of PKC in various signal transduction processes.
