Substrate specificity of nonreceptor tyrosine kinases

描述（由申请人提供）：非受体酪氨酸激酶如Src和Abl参与调节正常细胞的生长和分化。这些酶的活化形式与人类癌症的发生和发展有关。该项目的长期目标是了解非受体酪氨酸激酶在正常细胞中是如何调节的，以及酶的各种结构域如何促进底物磷酸化。有两个具体目标：1。许多重要的Src底物在多个酪氨酸残基上被持续磷酸化。第一种假设是，黏附蛋白p130Cas的多位点磷酸化对下游信号转导至关重要。这将通过构建缺乏磷酸化位点的Cas突变体来验证。这些突变体将作为Src底物在体外和来自cas缺陷小鼠的成纤维细胞中进行测试。2. Src家族激酶Hck在造血细胞生理中起着重要作用，但很少有细胞底物、激活剂或Hck效应物被发现。Pl的研究小组最近在U937单核细胞中筛选Hck SH3结构域结合蛋白时发现了WASP、WIP和ELMO1蛋白。第二种假设是，这些蛋白是Hck的底物，磷酸化在Hck信号传导中很重要。我们将在活化的U937和THP-1细胞中研究ELMO1的磷酸化。ELMO1在吞噬作用和Rac活化中的作用也将被研究。这一目标的最后一个组成部分将是研究与Hck的SH3结构域结合的蛋白质的一般调控特征。这些研究将为酪氨酸激酶的调控和底物特异性提供新的信息。这一信息可以为抗癌药物的开发奠定基础，这些抗癌药物可以破坏致癌酪氨酸激酶对底物的识别和细胞转化。

DESCRIPTION (provided by applicant): Nonreceptor tyrosine kinases such as Src and Abl are involved in regulating the growth and differentiation of normal cells. Activated forms of these enzymes have been implicated in the development and progression of human cancer. The long-term objectives of this project are to understand how nonreceptor tyrosine kinases are regulated in a normal cell, and how the various domains of the enzymes contribute to substrate phosphorylation. There are two specific aims: 1. Many important Src substrates are processively phosphorylated at multiple tyrosine residues. The first hypothesis is that multisite phosphorylation of the focal adhesion protein p130Cas is critical for downstream signal transduction. This will be tested by constructing Cas mutants lacking phosporylation sites. These mutants will be tested as Src substrates in vitro and in fibroblasts derived from Cas-deficient mice. 2. The Src family kinase Hck plays an important role in hematopoietic cell physiology, but very few cellular substrates, activators, or effectors for Hck have been identified. The Pl's group recently identified the proteins WASP, WIP, and ELMO1 in a screen for Hck SH3 domain binding proteins in U937 monocytic cells. The second hypothesis is that these proteins are substrates for Hck, and that phosphorylation is important in Hck signaling. Phosphorylation of ELMO1 will be studied in activated U937 and THP-1 cells. The involvement of ELMO1 in phagocytosis and in Rac activation will also be investigated. A final component of this aim will be to investigate the general regulatory features of proteins that bind to the SH3 domain of Hck. These studies will provide new information on the regulation and substrate specificity of tyrosine kinases. This information could form the basis for the development of anticancer agents that disrupt substrate recognition and cellular transformation by oncogenic tyrosine kinases.