FUNCTION OF C-SRC AND RELATED KINASES IN CHROMAFFIN CELLS

C-SRC 及相关激酶在嗜铬细胞中的功能

• 批准号: 6311495
• 负责人: SARAH J PARSONS
• 金额: $ 1.8万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-05-01 至 2001-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6311495
• 关键词: affinity chromatography; biological signal transduction; chemical structure function; chromaffin cells; complementary DNA; gene expression; immunoprecipitation; laboratory mouse; laboratory rabbit; neoplastic transformation; nicotine; nucleic acid sequence; oncogenes; phosphorylation; polymerase chain reaction; protein purification; protein tyrosine kinase; secretion; tissue /cell culture; vaccinia virus

Pp60c-src is a 60 kDa nonreceptor tyrosine protein kinase that is found in highest concentrations in non-dividing cells specialized for exocytosis, such as adrenal chromaffin cells. Accumulation in post- mitotic cells is an unexpected finding, considering the popularly held notion that pp60c-src, by analogy with its viral transforming counterpart, pp60v-src, is considered to play a role in control of cellular proliferation. The overall goal of our studies is to elucidate the function(s) of pp60c-src and related kinases in normal cells in which they are found to be naturally abundant. Several lines of evidence indicate that pp60c-src plays a role in the secretory process of chromaffin cells. They include (a) the subcellular localization of pp60c-src to the secretory vesicle (chromaffin granule) membrane, (b) modulation of pp60c-src specific tyrosine kinase activity following secretagogue stimulation, and (c) enhanced secretory activity of cultured chromaffin cells that transiently overexpress c-src using vaccinia virus vectors. Surprisingly, overexpression of a kinase-defective c-src also enhances secretory activity, suggesting that at least part of the effect of ectopically-expressed c-src on secretion is mediated through the N- terminal regulatory domain of the molecule. However, the changes in phosphotyrosine content of cellular proteins, as well as the modulations of c-src kinase activity which occur following secretagogue treatment, indicate that the C-terminal catalytic domain may also be important for the effect of pp60c-src on secretion. The goals of this proposal, therefore, are to identify and characterize cellular proteins that interact with pp60c-src, either as regulators or substrates, and to determine which subdomains of pp60c-src bind these proteins and are responsible for src's effect in viral-mediated overexpression studies. This will be accomplished by (a) screening a panel of available antibodies for their ability to react with proteins that co-precipitate with src protein or domains of pp60c-src (b) purifying novel proteins that co-precipitate with either intact pp60c-src or domains of pp60c-src, and (c) generating monoclonal antibodies to unidentified substrates of tyrosine kinases in chromaffin cells, using phosphotyrosine immunoaffinity reagents to purify them. Identified proteins will be characterized with respect to their relationship with pp60c-src and their function in chromaffin cells. Immunologic and genetic reagents for selected proteins will be generated to facilitate these studies. Which N-terminal domains of pp60c-src are responsible for the enhanced secretory activity will be further investigated using viral expression vectors and correlated with the binding of specific proteins. Similar studies will be initiated with pp62c-yes and pp59fyn to test the degree of functional overlap between the different src family members. Through these studies we hope to clarify the mechanism by which pp60c-src and related kinases participate in chromaffin cell biology.

PP60C-SRC是60 kDa非受体酪氨酸蛋白激酶，发现 在非分散细胞中，专门的非分散细胞中的最高浓度 胞吐作用，例如肾上腺炎蛋白细胞。 积累 考虑到普遍存在的有丝分裂细胞是一个出乎意料的发现 PP60C-SRC的概念，类似于其病毒转化 同行PP60V-SRC被认为在控制中发挥作用 细胞增殖。 我们研究的总体目标是阐明 PP60C-SRC和相关激酶的正常细胞的功能，其中 发现它们自然丰富。 几条证据 表明PP60C-SRC在分泌过程中起作用 铬蛋白细胞。 它们包括（a） PP60C-SRC到分泌囊泡（Chromaffin颗粒）膜，（B） PP60C-SRC特异性酪氨酸激酶活性的调节 分泌刺激和（c）培养的分泌活动增强 使用牛ac病毒瞬时过表达C-SRC的铬蛋白细胞 向量。 令人惊讶的是，激酶缺陷的C-SRC的过表达 增强分泌活动，表明至少一部分效果 分泌的异位表达的C-SRC通过n-介导 分子的末端调节结构域。 但是，变化 细胞蛋白的磷酸酪氨酸含量以及调节 在促分析治疗后发生的C-SRC激酶活性 表明C末端催化结构域对 PP60C-SRC对分泌的影响。 该提议的目标， 因此，要识别并表征细胞蛋白 与PP60C-SRC相互作用，作为调节器或底物，并与 确定PP60C-SRC的哪些子域结合了这些蛋白质，并且是 负责SRC在病毒介导的过表达研究中的影响。 这将通过（a）筛选可用的面板来完成 抗体的能力与蛋白质反应的蛋白质反应能力 与PP60C-SRC（B）纯化新蛋白的SRC蛋白或域 与完整的PP60C-SRC或PP60C-SRC的域共沉淀， （c）生成针对身份不明的底物的单克隆抗体 磷酸蛋白细胞中的酪氨酸激酶，使用磷酸酪氨酸 免疫亲和力试剂净化它们。 确定的蛋白质将是 其与PP60C-SRC及其关系的特征 在铬蛋白细胞中的功能。 免疫学和遗传试剂 将生成选定的蛋白质以促进这些研究。 哪个 PP60C-SRC的N末端域负责增强 分泌活动将使用病毒表达进一步研究 向量，与特定蛋白质的结合相关。 相似的 研究将使用PP62C-YE和PP59FYN进行测试 不同SRC家族成员之间功能重叠。 通过 我们希望这些研究能够阐明PP60C-SRC和 相关激酶参与铬蛋白细胞生物学。