The long range goals of the proposal are to improve understanding of the processing of the atypical zeta isoform of protein kinase C (PKCzeta ) by caspases and the ubiquitin (Ub)-proteasome systern and to clarify the roles of PKCzeta in apoptosis, cell survival, malignant transformation, and cell proliferation. The proposal stems from two novel observations: first, purified recombinant human caspase-3 efficiently processes PKCzeta in vitro to C-terminal fragments (CFzeta) which contain the catalytic domain; and second, induction of apoptosis produced CFzeta in cultured mammalian cells and markedly increased the kinase activity of PKCzeta immunocomplexes from the cells. The proposal has three specific aims.
The first aim i s to test the hypothesis that proteolytic processing by caspase is an alternative mechanism by which PKCzeta is activated intracellularly. To test this idea a PKCzeta mutant, which is neither phosphorylated nor activated by an upstream kinase, will be constructed and used along with kinase dead mutants of full-length and CFzeta in transfection experiments.
The second aim i s to elucidate the role of caspase processing of PKCzeta in apoptosis and/or cell survival. The hypothesis that CFzeta is sufficient to induce or modulate apoptosis or cell survival will be tested by transfection of cells with CFzeta and a catalytically inactive CFzeta mutant. Stimulus-evoked translocation of PKCzeta and CFzeta to plasma and nuclear membranes will be examined in cells. The last aim is to test the hypothesis that PKCzeta and/or CFzeta are degraded by the Ub-proteasome system and to increase understanding of the tumover and down-regulation of PKCzeta PKCzeta has putative destabilizing PEST elements in its regulatory and catalytic domains. The formation of Ub-PKCzeta and Ub-CFzeta conjugates will be explored in vitro and intracellularly. Experiments will be done to determine whether a PEST element or another destabilizing signal is necessary and/or sufficient for ubiquitinylation and degradation of PKCzeta.
