The objective of this proposal is to define the contribution of the protein kinase C (PKC) signaling pathway in cellular survival. PKC is a Ser/Thr kinase, which has been implicated in numerous cellular responses, including survival. The PKC substrate protein kinase D (PKD) is also a Ser/Thr kinase, which is distantly related to PKCs, and in the previous funding period we showed that PKD is critical for increased survival in cells exposed to ROS (reactive oxygen species, e.g., H202). We also showed that activation of PKD in response to stress requires two events: 1) phosphorylation of PKD in the PH domain by the tyrosine kinase Abl, and 2) phosphorylation in the PKD activation loop, mediated by PKC. PKD increases cellular survival by activating the transcription factor NF-KappaB. We therefore propose a model in which the PKC-PKD pathway is important for cellular survival in response to oxidative stress. In the proposal, experiments are designed to test the hypothesis that signaling through the PKC-PKD signaling pathway is necessary for the survival of cells exposed to ROS. The selective phosphorylation of PKD by PKCdelta in response to stress will be determined, and the contribution of the PKD PH domain will be investigated. We will examine the mechanism by which PKD activates the canonical NF-KappaB pathway. The role of NF-KappaB-induced genes, including A20, in mediating increased survival in cells exposed to ROS will also be evaluated. Finally, we will also determine the contribution of the PKC-PKD pathway in the phosphorylation of the Forkhead transcription factor FOXO3a, which also promotes cell survival. The results of these studies will provide important new information about a previously uncharacterized signaling pathway which is activated in cells exposed to ROS, and which mediates cellular survival through the transcription factors NF-KappaB and FOXO3a. We anticipate that the combination of biochemical and molecular genetic approaches will contribute to a greater understanding of the mechanisms which govern cellular survival in response to oxidative stress. ROS have been implicated in a variety of human pathologies such as atherosclerosis, diabetes, cancer, and arthritis. In addition, ROS are believed to be major causative factors in aging. A better understanding of the signaling pathways activated in response to ROS holds the potential for the development of novel therapeutic interventions for these pathologies.
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