MITOGENIC SIGNALING THROUGH RAL A AND PHOSPHOLIPASE D

这项提议试图理解 Rala/磷脂酶D(PLD)信号通路在促有丝分裂信号中发挥作用。这个 申请人的实验室已经证明PLD活性在细胞中升高 用各种有丝分裂刺激治疗，这一途径是一种 V-Src、v-Raf、v-Ras转化的关键调节因子及其过度表达 表皮生长因子受体。在正常静止细胞中，PLD与GDP结合是复杂的 Rala并定位于细胞膜。这种酶在以下情况下被激活 拉拉GTP交流和Arf GTP到综合体的招募，两项活动 被认为是由RAS的激活引起的。但是，Arf GTP不支持 直接与Rala/PLD复合体结合，并提高细胞膜PLD活性 RAS转化的细胞需要加入胞浆。这些发现表明 需要一个额外的因素来完全激活PLD。可编程逻辑器件 将磷脂酰胆碱水解为磷脂酸(PA)。PA参与了 许多生物过程，包括细胞内小泡的形成和 信号分子的激活，但它在促进有丝分裂中的确切作用 信令不被理解。来自申请人实验室的初步证据 提示PLD在EGF受体内吞作用中可能起一定作用。两个目标是 建议进一步研究PLD的激活机制和其 推测在内吞作用中的作用。第一个目标是提纯和表征一个 新近发现的低分子PLD刺激因子(PLD-SF) 在转化和分裂的细胞中升高。第二个目标将测试 PLD通过促进细胞内信号传导调节细胞内信号的假说 受体介导的内吞作用产生“信号囊泡”。

This proposal seeks to understand the role that the RalA/phospholipase D (PLD) signaling pathway plays in mitogenic signaling. The applicant's laboratory has demonstrated that PLD activity is elevated in cells treated with a variety of mitogenic stimuli, and that this pathway is a critical mediator of transformation by v-Src, v-Raf, v-Ras, and overexpression of the EGF receptor. In normal quiescent cells, PLD is complexed with GDP-bound RalA and localized to cellular membranes. The enzyme becomes activated upon RalA GTP exchange and recruitment of Arf GTP to the complex, both events thought to be brought about by activation of Ras. However, Arf GTP does not bind the RalA/PLD complex directly, and elevated PLD activity in membranes of Ras-transformed cells requires the addition of cytosol. These findings suggest that an additional factor is needed for complete activation of PLD. PLD hydrolyzes phosphatidylcholine to phosphatidic acid (PA). PA is involved in many biological processes, including intracellular vesicle formation and activation of signaling molecules, but its exact role in promoting mitogenic signaling is not understood. Preliminary evidence from the applicant's lab suggests that PLD may play a role in EGF receptor endocytosis. Two aims are proposed to further investigate both the mechanism of PLD activation and its putative role in endocytosis. The first aim is to purify and characterize a recently discovered low molecular weight PLD-stimulating factor (PLD-SF) that is elevated in transformed and dividing cells. The second aim will test the hypothesis that PLD regulates intracellular signaling by facilitating receptor-mediated endocytosis to generate "signaling vesicles."