The proposal aims at determining the mechanisms of regulation of protein kinase C (PKC) which has emerged as a critical enzyme in signal transduction, cell regulation, cell differentiation, and cell senescence. We have recently discovered a novel mechanism of long term activation of the beta isoform of PKC at the transcriptional level independent of diacylglycerol. Therefore, our long term goals are to evaluate the hypothesis that transcriptional regulation of PKC beta is an important mechanism for the long term regulation of the activity of this signal transduction system and its role in cell differentiation and senescence. This proposal aims at determining the mechanism of transcriptional upregulation of PKC beta. To this end, we have successfully cloned the human gene for PKC beta and its 5' flanking region.
The specific aims are addressed at: 1) identifying and characterizing the 5' regulatory region of the gene for PKC beta by determining the transcription start site (by S1 nuclease and primer extension analysis); identifying important cis- regulatory elements (using a Luciferase reporter gene and transient transfection studies), and characterizing cis-regulatory elements by gel shifting and DNAaseI footprinting studies; 2) determining effects of TNFalpha and retinoic acid on protein kinase Cbeta activity and transcription rates (using phorbol binding, phosphorylation studies, Northern blot analysis, nuclear run-on studies and evaluating message stability) and 3) determining mechanisms of induction by vitamin D3, retinoic acid, and TNFalpha by identifying cis-regulatory elements, sequencing, and studying DNA/protein interaction. These studies will begin to define a novel and important mechanism that may emerge as a critical pathway for long term regulation of cell differentiation and senescence.
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