FUNCTION OF C-SRC AND RELATED KINASES IN CHROMAFFIN CELLS

Pp60c-src是一种60 kDa的非受体酪氨酸蛋白激酶 在未分裂的细胞中浓度最高，专门用于 胞吐功能，如肾上腺嗜铬细胞。在后积累- 有丝分裂细胞是一个意想不到的发现，考虑到普遍认为的 Pp60c-src的概念，类比于其病毒转化 对应的pp60v-src被认为在控制 细胞增殖。我们研究的总体目标是阐明 正常细胞中pp60c-src及其相关蛋白的功能(S) 它们被发现是天然丰富的。有几条证据 表明pp60c-src在细胞的分泌过程中起作用。 嗜铬细胞。它们包括：(A)亚细胞定位 Pp60c-src到分泌囊(嗜铬粒)膜，(B) 对pp60c-src特异性酪氨酸激酶活性的调节 促分泌剂刺激，以及(C)提高培养物的分泌活性 利用痘苗病毒瞬时过表达c-src的嗜铬细胞 向量。令人惊讶的是，激酶缺陷的c-src的过度表达也 增强分泌活动，表明至少部分效果 异位表达的c-src对分泌的影响是通过N- 该分子的末端调节域。然而，其中的变化 细胞蛋白质的磷酸酪氨酸含量及其调节 在促分泌剂治疗后发生的c-src激酶活性， 表明C-末端催化结构域也可能对 Pp60c-src对细胞分泌的影响。这项提案的目标是， 因此，是为了鉴定和表征细胞蛋白质 与pp60c-src相互作用，作为调节剂或底物，并 确定pp60c-src的哪些亚域与这些蛋白质结合 负责src在病毒介导的过度表达研究中的作用。 这将通过以下方式实现：(A)筛选可用的 抗体与共沉淀的蛋白质反应的能力 用src蛋白或pp60c-src(B)结构域纯化新蛋白 其与完整pp60c-src或pp60c-src结构域共沉淀， 以及(C)产生针对未知底物的单抗 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。已确定的蛋白质将是 关于它们与pp60c-src的关系以及它们的 在嗜铬细胞中发挥作用。免疫和遗传试剂 选定的蛋白质将被产生以促进这些研究。哪一个 Pp60c-src的N-末端结构域负责增强的 将通过病毒表达进一步研究分泌活性 载体，并与特定蛋白的结合相关。类似 将用pp62c-yes和pp59fyn启动研究，以测试学位 不同SRC家族成员之间的功能重叠。穿过 我们希望通过这些研究阐明pp60c-src和pp60c-src的作用机制。 相关的蛋白激酶参与嗜铬细胞生物学。

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.