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

SRC 和 FAK 在商店经营的 CA 2 条目中的作用

基本信息

批准号：
6625106
负责人：
MITCHEL L VILLEREAL
金额：
$ 31.39万
依托单位：
UNIVERSITY OF CHICAGO
依托单位国家：
美国
项目类别：
财政年份：
1996
资助国家：
美国
起止时间：
1996-05-01 至 2005-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/6625106
关键词：
biological signal transduction calcium channel calcium flux enzyme activity focal adhesion kinase gene targeting genetically modified animals laboratory mouse protein tyrosine kinase protooncogene tissue /cell culture transfection western blottings

项目摘要

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.

在非兴奋性细胞中，内部Ca 2+储存的耗尽导致激活商店运营的渠道（SOC），在生理过程中的作用，如Ca 2+储存的再填充，分泌、基因转录调节、细胞周期控制和增殖和细胞凋亡的调节。直到最近，已知将Ca 2+储存耗尽与 SOC的激活基于大量药理学研究，暗示了多种潜在的调节途径。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，提出了酪氨酸激酶在SOC调节中的作用。期间在上一个赠款期间，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传导激活SOC的机制。在这个建议中，实验描述阐明c-src和FAK在SOC活化中的下游事件。这将扩展研究，证明钙池操作的Ca 2+进入（SOCE）在转基因src_小鼠的成纤维细胞中显著降低，但可以通过将野生型c-src转染到这些细胞中来恢复。实验具体建议1）表达一系列src突变体，以确定 c-src的哪些结构域是激活SOCE所必需的。此外， FAK、Grb 2和Shc，已知它们干扰来自整合素信号通路中的src/FAK复合物将表达，确定它们是否阻止SOCE的激活。主要假设是， c-src的酪氨酸激酶活性是必需的启动SOCE。2)确定哪些蛋白质参与HEK-293中的SOCE 细胞作为鉴定酪氨酸激酶活性的潜在靶点的手段 c-src和FAK。虽然有强有力的证据表明trp是一种SOC，在果蝇中，7种哺乳动物中哪一种trp仍不确定。迄今为止鉴定的同源物是SOC。在上一个资助期，被鉴定为在HEK-293细胞和反义构建体中表达对每一种都有具体规定。单个trp同源物的表达将是阻断，并通过fura-2和电生理学评估对SOCE的影响接近。3)确定HEK-293中负责SOCE的蛋白质是否当SOCE被激活时，细胞直接酪氨酸磷酸化。如果这如果证明是阴性的，他将研究辅助细胞的酪氨酸磷酸化，与Trp蛋白直接相互作用的蛋白质（例如钙调蛋白和 InaD的人类同源物，果蝇trp的调节蛋白）。的这些实验的成功结果将创造一个重大突破我们对SOC监管方式的理解。