This proposal focuses on an emerging signal transduction paradigm which counters the prevailing view that the nucleus is a passive recipient of signals produced at the plasma membrane. Our laboratory and others have shown that receptor activation at the cell surface results in regulation of nuclear enzymes that produce and modify potent lipid signaling molecules. While this area has garnered increasing interest, we still understand neither the molecular aspects of the regulation nor the precise physiological roles of the induced lipid metabolism. The proposed studies aim at elucidating key elements of this signal transduction mechanism and its functional consequences. We find that the small molecular weight GTP-binding protein RhoA reciprocally regulates two signaling enzymes, diacylglycerol kinase-theta and phospholipase D, at the nucleus in response to alpha-thrombin stimulation of fibroblasts. We have termed this regulation the """"""""RhoA Switch"""""""". We predict that the resulting modulation of diacylglycerol and phosphatidic acid levels and molecular composition have important physiological consequences such as the regulation of nuclear localization and activity of protein kinase C-alpha. Two heterotrimeric GTP-binding proteins, Gq and G13, and the lipid kinase PI 3-kinase mediate thrombin activation of this pathway. We hypothesize that; alpha-thrombin, after initiating an early Gq-dependent increase in nuclear diacylglycerol kinase-theta activity, stimulates a G13/PI 3-kinase-dependent nuclear translocation of RhoA that acts as a temporal """"""""switch"""""""" to inhibit nuclear diacylglycerol kinase-theta while activating nuclear phospholipase D. We will test the hypothesis that Gq is essential for the induced increase in nuclear diacylglycerol kinase-theta activity, and identify the diacylglycerol-theta domains essential for its nuclear translocation in Aim I.
Aim II will determine the molecular mechanism of the RhoA Switch by testing whether PI 3-kinase activation is responsible for nuclear RhoA translocation. Further, we will determine the molecular interactions between RhoA, diacylglycerol kinase-theta and phospholipase D. The physiological consequences of the RhoA Switch will be examined in Aim III. We will focus on the effect of the Switch on nuclear protein kinase C-alpha activity, composition of nuclear phosphatidic acids, and growth. We believe that an understanding of nuclear lipid signaling is key to our ability to intervene in events such as wound repair and tissue remodeling.
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