The long-term goal of this proposal is to clarify the functional significance of the 2 closely related atypical protein kinase C (PKC) isoforms (iota and zeta) in apoptosis and in cell survival. Each of the atypical PKC isoforms appears to transduce distinctly different prosurvival signals, which oppose the induction of apoptosis by cytokines or DNA damaging anticancer agents. This proposal originated from the observation that PKC ( (zeta), but not PKC ( (iota), is proteolytically processed by caspases to the active catalytic domain, dubbed CAT (, and to RD (, the free regulatory domain, following the induction of apoptosis in mammalian cells. The 2 atypical PKC( have almost the same amino acid sequence except for a segment (-60 amino acids) of the regulatory domain.
The first aim i s to elucidate the roles of PKC (, CAT (, and RD (, in regulating apoptotic signals. PKC (, and ( mutants and a chimera, which has the unique segment of PKC (, replaced by the corresponding segment of PKC (, will be used to investigate the signals transduced by each atypical PKC isoform. The hypothesis that PKC (, CAT (, and RD (, have different subcellular locations will be tested. The efficacy of PKC t, and CAT ( to activate the transcription factor, nuclear factor KB, will be compared. Human breast carcinoma (MCF-7) cells, which express PKC (, will be used to examine the roles of endogenous PKC (, CAT (, and RD ( in the modulation of caspase activation and apoptosis. The possibility that RD (, induces apoptosis by antagonizing PKC (, will be tested.
The second aim i s to elucidate the biochemical basis of the regulation of the cellular levels of the PKC (, and CAT ( proteins by the ubiquitin proteasome system. The hypothesis that interaction with other proteins (e.g., heat shock proteins or phosphoinositide-dependent kinase-1) or the phosphorylation of specific residues of PKC (, influences its ubiquitination will be tested. Experiments will be done to determine whether CAT (, has transferable destabilizing elements. A cell-free system will be reconstituted that has key features of intracellular ubiquitination and degradation of CAT (. The reconstituted system will be used in conjunction with cultured cells to identify components of the ubiquitination complex and the lysine residues of CAT ( that are conjugated to ubiquitin.
