TYROSINE KINASES AND CAPACITATIVE CALCIUM ENTRY

在大多数不可兴奋的细胞中，内部钙存储的耗尽导致 质膜Ca~(2+)进入途径的激活 在补充体内钙离子储备和维持体内钙离子储备方面发挥着重要作用 细胞内钙离子浓度持续升高。目前， 内部存储器耗尽发出激活信号的机制 这种钙离子进入的途径还不完全清楚。然而，来自我们的工作 实验室表明，酪氨酸激酶在这一信号转导中起作用。 途径，并指出c-src是酪氨酸激酶的首选候选基因。 牵涉其中。在这个提案中，我们描述了验证该假设的实验 C-src是连接钙离子储存耗竭的酪氨酸激酶。 激活钙离子进入途径。我们将测试c-src的参与情况。 通过改变c-src的表达水平并研究 缓激肽和钙池刺激下钙离子内流的变化 耗尽。我们将使用表达反义技术和成纤维细胞 从c-src阴性的转基因小鼠检测降低c-src的效果 SRC级别。我们还将表示v-src并确定这是否可以 在没有缓激肽刺激的钙池中激活钙内流 耗尽 我们将继续研究酪氨酸激酶的刺激作用。 用thapsigargin耗尽钙离子库后的活性。因为我们的 初步实验表明，c-src可能被激活以响应 Ca2+池耗尽，我们首先将重点放在c-src上。我们会 免疫共沉淀法从对照细胞和其钙离子储存有 被各种方法耗尽，并通过in测量c-src活性 体外激活酶分析。我们的假设是钙离子储备库耗尽会导致 C-src酪氨酸激酶活性的激活。 我们描述了电生理实验来描述通道的特征 由缓激肽激活，钙池耗竭，c-src过度表达。 我们建议检验这样一种假设，即具有类似于 钙释放激活的钙通道(ICRAC)，之前在 在成纤维细胞中，肥大细胞和淋巴细胞被缓激肽激活 刺激和Ca~(2+)储存耗竭是通过依赖于 C-src的激活。

In most nonexcitable cells, the depletion of internal Ca2+ stores leads to the activation of a plasma membrane Ca2+ entry pathway which plays an important role both for refilling internal Ca2+ stores and for maintaining a prolonged elevation of cytosolic Ca2+ concentration. At present, the mechanism by which depletion of internal stores signals the activation of this Ca2+ entry pathway is not fully understood. However, work from our laboratory suggests that tyrosine kinases play a role in this signalling pathway and points to c-src as a prime candidate for the tyrosine kinase involved. In this proposal, we describe experiments to test the hypothesis that c-src is the tyrosine kinase coupling Ca2+ store depletion to activation of a Ca2+ entry pathway. We will test for involvement of c-src by varying the level of c-src expression and investigating the impact of these changes on Ca2+ entry stimulated by bradykinin and Ca2+ pool depletion. We will use expression antisense techniques and fibroblasts from c-src negative, transgenic mice to test for the effect of reducing c- src levels. We will also express v-src and determine whether this can activate Ca2+ entry in the absence of bradykinin stimulation ofr Ca2+ pool depletion We will continue our investigation of the stimulation of tyrosine kinase activity following Ca2+ pool depletion with thapsigargin. Since our preliminary experiments suggest that c-src may be activated in response to Ca2+ pool depletion, we will focus initially on c-src. We will immunoprecipitate c-src from control cells and cells whose Ca2+ stores have been depleted by a variety of methods and measure c-src activity by in vitro kinase assays. Our hypothesis is that Ca2+ store depletion leads to the activation of c-src tyrosine kinase activity. We describe electrophysiological experiments to characterize the channels activated by bradykinin, Ca2+ pool depletion and overexpression of c-src. We propose to test the hypothesis that a channel with properties similar to the Ca2+-release-activated Ca2+ channel (Icrac), previously described in mast cells and lymphocytes, is activated in fibroblasts by both bradykinin stimulation and Ca2+ store depletion via a mechanism that is dependent on activation of c-src.