Coordinate Regulation Of Nuclear DGK-theta and PLD

描述（由申请人提供）：该提案侧重于一种新兴的信号转导范式，该范式与细胞核是质膜产生的信号的被动接受者的流行观点相反。我们的实验室和其他实验室已经表明，细胞表面的受体活化导致对产生和修饰有效脂质信号分子的核酶的调节。虽然这一领域已经获得了越来越多的兴趣，我们仍然不知道的分子方面的调节，也不精确的诱导脂质代谢的生理作用。拟议的研究旨在阐明这种信号转导机制及其功能后果的关键要素。我们发现，小分子量的GTP结合蛋白RhoA β调节两个信号酶，二酰甘油激酶-θ和磷脂酶D，在细胞核中的α-凝血酶刺激成纤维细胞。我们将这种调节称为“RhoA开关”。我们预测，由此产生的调制二酰甘油和磷脂酸的水平和分子组成具有重要的生理后果，如调节核定位和蛋白激酶C-α的活性。两种异源三聚体GTP结合蛋白Gq和G13以及脂质激酶PI 3-激酶介导该途径的凝血酶活化。我们假设，α-凝血酶在启动核二酰甘油激酶-θ活性的早期Gq依赖性增加后，刺激RhoA的G13/PI 3-激酶依赖性核转位，其充当时间“开关”以抑制核二酰甘油激酶-θ，同时激活核磷脂酶D。我们将检验Gq对于核二酰基甘油激酶-θ活性的诱导增加是必需的这一假设，并鉴定其在Aim I中核转位所必需的二酰基甘油-θ结构域。目的II通过检测PI 3-激酶的激活是否与核内RhoA易位有关来确定RhoA开关的分子机制。此外，我们将确定RhoA，二酰甘油激酶-θ和磷脂酶D之间的分子相互作用。RhoA开关的生理后果将在目标III中进行检查。我们将重点关注Switch对核蛋白激酶C-α活性、核磷脂酸组成和生长的影响。我们相信，了解核脂质信号传导是我们干预伤口修复和组织重塑等事件的关键。

DESCRIPTION (provided by applicant): 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.