No abstract

Atypical protein kinase C (PKC) is known to transduce signals that influence cell proliferation and survival. Here we show that recombinant human caspases can process PKC at three sites in the hinge region between the regulatory and catalytic domains. Caspase-3, -6, -7, and -8 chiefly cleaved human PKC at EETD2G, and caspase-3 and -7 also cleaved PKC at DGMD2G and DSED2L, respectively. Processing of PKC expressed in transfected cells occurred chiefly at EETD2G and DGMD2G and produced carboxyl-terminal polypeptides that contained the catalytic domain. Epitopetagged PKC that lacked the regulatory domain was catalytically active following expression in HeLa cells. Induction of apoptosis in HeLa cells by tumor necrosis factor ␣ plus cycloheximide evoked the conversion of full-length epitope-tagged PKC to two catalytic domain polypeptides and increased PKC activity. A caspase inhibitor, zVAD-fmk, prevented epitope-tagged PKC processing and activation following the induction of apoptosis. Induction of apoptosis in rat parotid C5 cells produced catalytic domain polypeptides of endogenous PKC and increased PKC activity. Caspase inhibitors prevented the increase in PKC activity and production of the catalytic domain polypeptides. Treatment with lactacystin, a selective inhibitor of the proteasome, caused polyubiquitin-PKC conjugates to accumulate in cells transfected with the catalytic domain or full-length PKC, or with a PKC mutant that was resistant to caspase processing. We conclude that caspases process PKC to carboxyl-terminal fragments that are catalytically active and that are degraded by the ubiquitinproteasome pathway.

No abstract

We show that bryostatin 1 (Bryo) rapidly produces an inactive, incompetent 76-kDa form of protein kinase C-alpha (PKC-alpha) in the LLC-MK2 line of renal epithelial cells. Bryo, like phorbol 12-myristate 13-acetate (PMA), acutely activated PKC, as indicated by autophosphorylation and translocation of PKC-alpha, the predominant PMA-sensitive isoform expressed by the cells. Bryo concomitantly increased the 32P labeling of 80-kDa PKC-alpha by autophosphorylation and produced a 76-kDa form of PKC-alpha that lacked detectable 32P. The 76-kDa form was in the particulate rather than the cytosolic fraction, which suggests that it was produced from activated kinase. Alkaline phosphatase treatment of immunoprecipitated PKC-alpha converted the 80-kDa form to 76 kDa, but it had no effect on the mobility of the 76-kDa form, suggesting that it was not phosphorylated. Pulse-chase labeling of PKC-alpha with [35S]Met/Cys indicated that there is a precursor-product relationship between the 80- and 76-kDa forms, respectively. Inhibition of protein synthesis had no effect on the production of 76-kDa PKC-alpha by Bryo. PMA also produced 76-kDa PKC-alpha but was less potent and efficacious than Bryo. Bryo produced a more rapid loss of 80-kDa PKC-alpha protein and total Ca(2+)- and phospholipid-dependent PKC activity than PMA. The 76-kDa form is inactive and incompetent because it lacked detectable 32P under conditions that strongly autophosphorylated the 80-kDa form. We suggest that dephosphorylation predisposes PKC to proteolysis, and greater production of the 76-kDa form explains the more efficient downregulation of the kinase by Bryo vs. PMA.

Treatment of HeLa cells with tumour necrosis factor alpha (TNFalpha) induced caspase processing of ectopic PKC (protein kinase C) zeta, which converted most of the holoenzyme into the freed kinase domain and increased immune-complex kinase activity. The goal of the present study was to determine the basis for the increased kinase activity that is associated with caspase processing of PKC zeta. Atypical PKC iota is largely identical with PKC zeta, except for a 60-amino-acid segment that lacks the caspase-processing sites of the zeta isoform. Replacement of this segment of PKC zeta with the corresponding segment of PKC iota prevented caspase processing and activation of the kinase function. Processing of purified recombinant PKC zeta by caspase 3 in vitro markedly increased its kinase activity. Caspase processing activated PKC zeta in vitro or intracellularly without increasing the phosphorylation of Thr410 of PKC zeta, which is required for catalytic competency. The freed kinase domain of PKC zeta had a much shorter half-life than the holoenzyme in transfected HeLa cells and in non-transfected kidney epithelial cells. Treatment with TNF-alpha shortened the half-life of the kinase domain protein, and proteasome blockade stabilized the protein. Studies of kinase-domain mutants indicate that a lack of negative charge at Thr410 can shorten the half-life of the freed kinase domain. The present findings indicate that the freed kinase domain has substantially higher kinase activity and a much shorter half-life than the holoenzyme because of accelerated degradation by the ubiquitin-proteasome system.