PKCdelta is a key enzyme in the regulation of cell apoptosis in various cellular systems. PKCdelta acts as a pro or anti-apoptotic kinase depending on the specific cell type and apoptotic stimulus, however the mechanisms underlying its diverse effects are not understood. In this proposal we seek to understand the molecular mechanisms involved in the regulation of cell apoptosis by PKCdelta focusing on gliomas as a cellular system. Gliomas exhibit deregulated cell apoptosis due to altered apoptotic pathways that favor cell survival. In a recent study we demonstrated that PKCdelta expression is reciprocally correlated with the degree of malignancy in gliomas. Moreover, PKCdelta regulates the apoptosis of these cells in a stimulus-specific manner. The main hypothesis of this proposal is that PKCdelta is a major regulator of glioma cell apoptosis and that the pro and anti-apoptotic effects of PKCdelta in glioma cells are determined by its activation, phosphorylation on distinct tyrosine residues and by its subcellular localization. To test this hypothesis we will employ different apoptotic stimuli and will first examine the role of PKCdelta activity in its pro and anti-apoptotic effects by using a PKCdelta inhibitory peptide, a PKCdelta KD mutant and siRNAs directed against PKCdelta mRNA. Tyrosine phosphorylation of PKCdelta is often induced by apoptotic stimuli. Therefore, the tyrosine phosphorylation of PKCdelta will be studied in response to the various apoptotic stimuli employed in this study. The specific tyrosine residues and the tyrosine kinases involved in their phosphorylation will be identified and their role in the PKCdelta apoptotic effects will be determined. The translocation of PKCdelta in response to the different apoptotic stimuli will be examined using GFP-tagged PKCdelta and the role of PKCdelta tyrosine phosphorylation in the translocation of PKCdelta will be explored using PKCdelta tyrosine mutants. The role of PKCdelta localization in its apoptotic effects will be studied using a PKCdelta mutant in which the NLS was mutated and by employing vectors targeting PKCdelta to the nucleus, ER, cytosol and mitochondria. Finally, the cleavage of PKCdelta and the roles of the cleaved regulatory and catalytic fragments will be studied for their effects on the apoptotic function of PKCdelta. To identify proteins and signaling pathways that mediate the pro and anti-apoptotic effects of PKCdelta the effect of PKCdelta on the levels, activation and phosphorylation of different apoptosis-related proteins and on the activation of signaling pathways associated with cell apoptosis and survival (members of the MAP kinase family and Akt) will be determined. The roles of tyrosine phosphorylation and subcellular localization of PKCdelta in the activation of these proteins will be then evaluated. Another important factor in delineating the function of PKCdelta in glioma cell apoptosis is identifying its binding partners. This will be done using GST-PKCdelta and GST-PKCdelta mutants fusion proteins to identify PKCdelta binding proteins in glioma cells stimulated with different apoptotic stimuli and by screening glioma cDNA libraries using the yeast two hybrid system. The results of this study will enhance our understanding of the factors contributing to the divergent effects of PKCdelta on cell apoptosis and of the role of PKCdelta in the regulation of glioma cell apoptosis. A better understanding of the molecular mechanisms underlying the diverse effects of PKCdelta in gliomas may enable us to predict the tumor response to therapy based on its molecular profile and may lead to novel approaches for altering the sensitivity of gliomas to specific therapeutic agents.
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