ROLE OF SRC AND FAK IN STORE-OPERATED CA 2+ ENTRY

在不可兴奋的细胞中，内部钙离子储存的耗尽导致 对于商店运营的通道(SOC)的激活起着重要的作用 在生理过程中的作用，如钙离子储存的再充盈，调节 分泌，基因转录的调节，细胞周期的控制和 增殖和细胞凋亡的调控。直到最近，仅有的一点 已知钙离子储存耗竭的信号机制 SOC的激活是基于一些药理学研究，这些研究 牵涉到多种潜在的调控途径。例如，基于 一系列酪氨酸激酶和酪氨酸磷酸酶抑制剂的研究 酪氨酸激酶在SOC调节中的作用被提出。在.期间 在之前的资助期，一种分子方法为 C-src和粘着斑激酶(FAK)参与SOC的调节。 据我们所知，这使得酪氨酸激酶途径成为第一个同时具有这两种功能的途径 药理学和分子数据支持其参与信号转导 激活SOC的机制。在这份提案中，描述了实验 目的：阐明c-src和FAK下游在SOC激活中的作用。 这将扩展证明商店操作的钙离子进入的研究 (SOCE)在转基因src小鼠的成纤维细胞中显著减少，但 可以通过将野生型c-src导入这些细胞来恢复。实验 1)表达一系列src突变体以确定 哪些c-src结构域是激活SOCE所必需的。此外，突变型的 FAK、Grb2和Shc，它们已知会干扰来自 整合素信号通路中的src/fak复合体将被表达为 确定它们是否阻止激活SOCE。主要的假设是 C-src的酪氨酸激酶活性对于 激活SOCE。2)确定HEK-293中哪些蛋白质参与SOCE 细胞作为识别酪氨酸激酶活性潜在靶点的手段 C-src和FAK。虽然有强有力的证据表明TRP是一个SOC 果蝇，7种哺乳动物色氨酸中的哪一种仍不确定 到目前为止已鉴定的同系物是SOC。在上一次授权期内，有4个色氨酸同源物 经鉴定在HEK-293细胞和反义载体中均有表达 每一种都有自己的特色。单独的色氨酸同源物的表达将是 用Fura-2和电生理方法评价阻滞剂对SOCE的影响 接近了。3)确定HEK-293中与SOCE有关的蛋白质 当SOCE被激活时，细胞直接被酪氨酸磷酸化。如果这个 结果是阴性的，他将研究附件的酪氨酸磷酸化 直接与色氨酸蛋白相互作用的蛋白质(例如，钙调蛋白和 Inad的人类同源物，一种果蝇Trp的调节蛋白)。这个 这些实验的成功结果将创造一个重大突破 在我们对如何监管SOC的理解中。

In nonexcitable cells, the depletion of internal Ca2+ stores leads to the activation of store-operated channels (SOCs) which play an important role in physiological processes such as refilling of Ca2+ stores, regulation of secretion, regulation of gene transcription, control of cell cycle and proliferation, and regulation of apoptosis. Until recently, what little was known about the signaling mechanism for coupling depletion of Ca2+ stores to activation of SOCs was based on a number of pharmacological studies which implicated a variety of potential regulatory pathways. For example, based on studies with a series of tyrosine kinase and tyrosine phosphatase inhibitors, a role for tyrosine kinases in the regulation of SOCs was proposed. During the previous grant period, a molecular approach provided evidence for the involvement of c-src and focal adhesion kinase (FAK) in the regulation of SOCs. To our knowledge, this makes the tyrosine kinase pathway the first to have both pharmacological and molecular data supporting its involvement in the signaling mechanism for activation of SOCs. In this proposal, experiments are described to elucidate the events downstream of c-src and FAK in the activation of SOCs. This will extend the studies which demonstrate that store-operated Ca2+ entry (SOCE) is dramatically reduced in fibroblasts from transgenic src_ mice, but can be restored by transfecting wild type c-src into these cells. Experiments are proposed specifically to 1) express a series of src mutants to determine which domains of c-src are necessary to activate SOCE. In addition, mutants of FAK, Grb2 and Shc, which are known to interfere with signaling downstream from the src/FAK complex in the integrin signaling pathway, will be expressed to determine whether they block activation of SOCE. The primary hypothesis is that the tyrosine kinase activity of c-src is both necessary and sufficient to activate SOCE. 2) Determine which proteins are involved in SOCE in HEK-293 cells as a means of identifying potential targets of tyrosine kinase activity of c-src and FAK. While there is strong evidence that trp is a SOC in Drosophila, there is still uncertainty concerning which of the 7 mammalian trp homologs identified to date are SOCs. In the last grant period 4 trp homologs were identified as being expressed in HEK-293 cells and antisense constructs specific for each were made. The expression of individual trp homologs will be blocked and the effect on SOCE assessed by fura-2 and electrophysiological approaches. 3) Determine whether the protein responsible for SOCE in HEK-293 cells is directly tyrosine phosphorylated when SOCE is activated. If this proves negative, he will investigate the tyrosine phosphorylation of accessory proteins which directly interact with trp proteins (e.g. calmodulin and the human homolog of InaD, a regulatory protein for Drosophilia trp). The successful outcome of these experiments would create a significant breakthrough in our understanding of how SOCs are regulated.