Substrate specificity of nonreceptor tyrosine kinases

描述（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展和进展有关。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如何调节，以及酶的各个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处被原磷酸化。第一个假设是黏着斑蛋白p130 Cas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突变体来测试。这些突变体将作为Src底物在体外和来源于Cas缺陷小鼠的成纤维细胞中进行测试。2. Src家族激酶Hck在造血细胞生理学中起着重要作用，但很少有Hck的细胞底物、激活剂或效应物被鉴定。Pl的小组最近在U937单核细胞中筛选Hck SH 3结构域结合蛋白时鉴定了蛋白WASP、WFP和ELMO 1。第二个假设是这些蛋白质是Hck的底物，并且磷酸化在Hck信号传导中是重要的。将在活化的U937和THP-1细胞中研究ELMO 1的磷酸化。还将研究ELMO 1参与吞噬作用和Rac激活。该目标的最后一个组成部分是研究与Hck的SH 3结构域结合的蛋白质的一般调节特征。这些研究将提供有关酪氨酸激酶的调节和底物特异性的新信息。这一信息可能成为开发抗癌药物的基础，这些药物可以破坏致癌酪氨酸激酶的底物识别和细胞转化。

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.