The N-terminal pseudosubstrate site within the protein kinase Cα (PKCα)-regulatory domain has long been regarded as the major determinant for autoinhibition of catalytic domain activity. Previously, we observed that the PKC-inhibitory capacity of the human PKCα-regulatory domain was only reduced partially on removal of the pseudosubstrate sequence [Parissenti, Kirwan, Kim, Colantonio and Schimmer (1998) J. Biol. Chem. 273, 8940–8945]. This finding suggested that one or more additional region(s) contributes to the inhibition of catalytic domain activity. To assess this hypothesis, we first examined the PKC-inhibitory capacity of a smaller fragment of the PKCα-regulatory domain consisting of the C1a, C1b and V2 regions [GST-Rα39–177: this protein contained the full regulatory domain of human PKCα fused to glutathione S-transferase (GST), but lacked amino acids 1–38 (including the pseudosubstrate sequence) and amino acids 178–270 (including the C2 region)]. GST-Rα39–177 significantly inhibited PKC in a phorbol-independent manner and could not bind the peptide substrate used in our assays. These results suggested that a region within C1/V2 directly inhibits catalytic domain activity. Providing further in vivo support for this hypothesis, we found that expression of N-terminally truncated pseudosubstrate-less bovine PKCα holoenzymes in yeast was capable of inhibiting cell growth in a phorbol-dependent manner. This suggested that additional autoinhibitory force(s) remained within the truncated holoenzymes that could be relieved by phorbol ester. Using tandem PCR-mediated mutagenesis, we observed that mutation of amino acids 33–86 within GST-Rα39–177 dramatically reduced its PKC-inhibitory capacity when protamine was used as substrate. Mutagenesis of a broad range of sequences within C2 (amino acids 159–242) also significantly reduced PKC-inhibitory capacity. Taken together, these observations support strongly the existence of multiple regions within the PKCα-regulatory domain that play a direct role in the inhibition of catalytic domain activity.

Abbreviations used: DAG, diacylglycerol; GST, glutathione S-transferase; PKC, protein kinase C; GST-Rα, regulatory domain of human PKCα fused to GST; mAb, monoclonal antibody.

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