Two fusion proteins in which the regulatory domains of human protein kinase C␣ (R␣; amino acids 1-270) or mouse protein kinase C⑀ (R⑀; 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␣ (PKC␣) and other protein kinases in vitro. Both GST-R␣ and GST-R⑀ but not GST itself potently inhibited the activities of lipid-activated rat brain PKC␣. 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␣ and GST-R⑀, on a molar basis, were 100 -200-fold more potent inhibitors of PKC␣ activity than was the pseudosubstrate peptide PKC 19 -36 . In addition, a GST-R␣ fusion protein in which the first 32 amino acids of R␣ were deleted (including the pseudosubstrate sequence from amino acids 19 -31) was an effective competitive inhibitor of PKC␣ activity. The three GST-R fusion proteins also inhibited protamine-activated PKC␣ and proteolytically activated PKC␣ (PKM), two lipid-independent forms of PKC␣; however, the IC 50 values for inhibition were 1 order of magnitude greater than the IC 50 values obtained in the presence of lipid. These results suggest that part of the inhibitory effect of the GST-R fusion proteins on lipidactivated PKC␣ 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␣ catalytic activity through direct interactions. These data indicate that the R domains of PKC␣ and PKC⑀ are specific inhibitors of protein kinase C␣ activity and suggest that regions of the R domain outside the pseudosubstrate sequence contribute to autoinhibition of the enzyme.

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This study demonstrates that the isolated regulatory (R) domain (amino acids 1-270) of human protein kinase C alpha (PKC alpha) is a potent inhibitor of PKC beta-I activity in a yeast expression system. The PKC alpha R domain fused to glutathione-S-transferase competitively inhibited the activity of yeast-expressed rat PKC beta-I in vitro (Ki = 0.2 microns) and was 400-fold more potent than a synthetic pseudosubstrate peptide corresponding to amino acids 19-36 from PKC alpha. In contrast, the fusion protein did not affect the activity of the purified catalytic subunit of cAMP-dependent protein kinase. The PKC alpha R domain (without glutathione-S-transferase [GST]) also was tested for its ability to inhibit PKC beta-I activity in vivo, in a yeast strain expressing rat PKC beta-I. Upon treatment with a PKC-activating phorbol ester, yeast cells expressing rat PKC beta-I were growth-inhibited and a fraction of the cells appeared as long chains. Coexpression of the R domain with rat PKC beta-I blocked the phorbol ester-induced inhibition of yeast cell growth and the phorbol ester-dependent alterations in yeast cell morphology. These results indicate that the R domain of PKC alpha acts as a dominant inhibitor of PKC activity in vivo and thus provides a useful genetic tool to assess the roles of PKC in various signal transduction processes.

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.