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.

Pp 60 c-src是一种60 kDa的非受体酪氨酸蛋白激酶， 在非分裂细胞中浓度最高， 胞吐作用，如肾上腺嗜铬细胞。 后积累 有丝分裂细胞是一个意想不到的发现，考虑到普遍持有的 pp 60 c-src的概念，通过类比其病毒转化， 对应物pp 60 v-src被认为在控制 细胞增殖 我们研究的总体目标是阐明 pp 60 c-src和相关激酶在正常细胞中的功能， 它们被发现是天然丰富的。 若干条证据 表明pp 60 c-src在分泌过程中起作用 嗜铬细胞 它们包括（a）亚细胞定位的 pp 60 c-src至分泌囊泡（嗜铬颗粒）膜，（B） PP 60 c-src特异性酪氨酸激酶活性的调节 促分泌素刺激，和（c）培养的细胞的分泌活性增强 使用牛痘病毒瞬时过表达c-src的嗜铬细胞 向量。 令人惊讶的是，激酶缺陷型c-src的过表达也 增强分泌活性，这表明至少部分效果 外源性表达的c-src对分泌的影响是通过N- 分子的末端调节结构域。 然而， 磷酸酪氨酸含量的细胞蛋白质，以及调制 促分泌素处理后发生的c-src激酶活性， 表明C-末端催化结构域也可能对 pp 60 c-src对分泌的影响。 这项提案的目标， 因此，是为了识别和表征细胞蛋白质， 与pp 60 c-src相互作用，作为调节剂或底物， 确定pp 60 c-src的哪些亚结构域结合这些蛋白质， 负责src在病毒介导的过表达研究中的作用。 这将通过以下方式实现：（a）筛选一组可用的 抗体与共沉淀的蛋白质反应的能力 用src蛋白或pp 60 c-src（B）的结构域纯化新蛋白 其与完整PP 60 C-SRC或PP 60 C-SRC结构域共沉淀， 和（c）产生针对以下未鉴定底物的单克隆抗体： 嗜铬细胞中的酪氨酸激酶，使用磷酸酪氨酸 免疫亲和试剂来纯化它们。 已识别的蛋白质将被 其特征在于它们与PP 60 C-SRC的关系及其 在嗜铬细胞中起作用。 免疫和遗传试剂，用于 将产生选定的蛋白质以促进这些研究。 这 pp 60 c-src的N-末端结构域负责增强 将使用病毒表达进一步研究分泌活性 载体，并与特定蛋白质的结合相关。 类似 研究将开始与pp 62 c-yes和pp 59 fyn测试程度 不同src家族成员之间的功能重叠。 通过 这些研究我们希望阐明pp 60 c-src和 相关激酶参与嗜铬细胞生物学。