Activation of atypical protein kinase C ζ by caspase processing and degradation by the ubiquitin-proteasome system

Atypical protein kinase C zeta (PKC zeta) is known to transduce signals that influence cell proliferation and survival. Here we show that recombinant human caspases can process PKC zeta at three sites in the hinge region between the regulatory and catalytic domains. Caspase-3, -6, -7, and -8 chiefly cleaved human PKC zeta at EETD down arrow G, and caspase-3 and -7 also cleaved PKC zeta at DGMD down arrow G and DSED down arrow L, respectively. Processing of PKC zeta expressed in transfected cells occurred chiefly at EETD down arrow G and DGMD down arrow G and produced carboxyl-terminal polypeptides that contained the catalytic domain. Epitope-tagged PKC zeta that lacked the regulatory domain was catalytically active following expression in HeLa cells. Induction of apoptosis in HeLa cells by tumor necrosis factor cu plus cycloheximide evoked the conversion of full-length epitope-tagged PKC zeta to two catalytic domain polypeptides and increased PKC zeta activity. A caspase inhibitor, zVAD-fmk, prevented epitope-tagged PKC zeta processing and activation following the induction of apoptosis. Induction of apoptosis in rat parotid C5 cells produced catalytic domain polypeptides of endogenous PKC zeta and increased PKC zeta activity. Caspase inhibitors prevented the increase in PKC zeta activity and production of the catalytic domain polypeptides. Treatment with lactacystin, a selective inhibitor of the proteasome, caused polyubiquitin-PKC zeta conjugates to accumulate in cells transfected with the catalytic domain or full-length PKC alpha, or with a PKC zeta mutant that was resistant to caspase processing. We conclude that caspases process PKC zeta to carboxyl-terminal fragments that are catalytically active and that are degraded by the ubiquitin-proteasome pathway.