Revised Manuscript Received July 23, 1993® abstract: Protein kinase C (PKC) is a family of closely related phospholipid-dependent protein kinases, A fully active, phospholipid-independent catalytic fragment of PKC is produced by limited proteolysis of the enzyme. The catalytic fragment allows a simplified assay system for the analysis of PKC inhibitors that interact with the catalytic domain. Recently, we reported that VV-myristoylation of the synthetic peptide substrate Arg-Lys-Arg-Thr-Leu-Arg-Arg-Leu (RKRTLRRL) transformed a peptide that completely lacked inhibitory activity against the histone kinase reactions of PKC and itscatalytic fragment into a peptide that potently inhibited both of these reactions. VV-Myristoylation did not alter the potency of the peptide as a PKC substrate, and the basis for the acquisition of inhibitory activity against the catalytic fragment by VV-myristoylation of the peptide remained unclear. In this report, we propose a mechanism for catalytic fragment inhibition by the VV-myristoylated peptide that is based on a comparison of the inhibitory potencies of several nonphosphorylatable analogs of VV-myristoyl-RKRTLRRL, a kinetic analysis of the inhibition of the histone kinase activity of the catalytic fragment by nonphosphorylatable VV-myristoyl-RKRTLRRL analogs, and an analysis of the inhibitory effects of the VV-myristoylated peptide series on the intrinsic ATPase activity of PKC. Our results support a mechanism in which the VV-myristoylated peptides inhibit the catalytic fragment by binding to PKCfree, but not to the complex PKC-ATP, at the protein-substrate binding site. The ability to bind PKCfree distinguishes the VV-myristoylated peptides from histone substrate and dead-end synthetic peptide inhibitors, because the latter agents appear to bind only to PKC-ATP. A serious limitation observed with inhibitory oligopeptide substrate analogs of protein kinases is that they often compete with protein substrates only weakly, if at all. VV-Myristoylation of oligopeptide substrate analogs of PKC may overcome this limitation by allowing the inhibitory peptides to bind to a form of PKC that does not bind protein substrates such as histone, so that inhibition can be achieved without direct competition with the protein substrate. Thus, VV-myristoylation of oligopeptide substrate analogs is a promising approach for the development of potent PKC inhibitors that exploit the substrate selectivity of the enzyme.

Protein kinase C (PKC) is a family of at least 10 closely related phospholipid-dependent isozymes that can be divided into Ca2+-dependent (conventional) and Ca2+-independent (nonconventional) subfamilies (Nishizuka, 1992; Parker, 1992). Most PKC isozymes are activated by the secondmessenger srt-1, 2-diacylglycerol. PKC activation has been implicated in various biological and pathogenic processes, eg, muscle contraction, neurotransmission, and carcinogenesis (Kuo et al., 1989; Nishizuka, 1992; Rotenberg & Weinstein, 1991). Specific, cell-permeable inhibitors of the PKC isozyme family and of its individual members are needed to define the role of this isozyme family in these various processes (Huang,