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。这些酶的激活形式与人类癌症的发生和发展有关。这个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，以及这些酶的不同结构域是如何促进底物磷酸化的。有两个特定的目的：1.许多重要的Src底物在多个酪氨酸残基上被连续磷酸化。第一个假设是粘着斑蛋白p130Cas的多位点磷酸化对下游信号转导至关重要。这将通过构建缺少磷酸化位点的CaS突变体来进行测试。这些突变体将在体外作为Src底物进行测试，并在来自CaS缺陷小鼠的成纤维细胞中进行测试。2.Src家族激酶HCK在造血细胞生理学中起着重要作用，但对HCK的细胞底物、激活物或效应物的研究很少。最近，Pl‘s团队在对U937单核细胞中的HCK SH3结构域结合蛋白的筛选中发现了WASP、WIP和ELMO1蛋白。第二个假设是这些蛋白是HCK的底物，并且磷酸化在HCK信号转导中很重要。ELMO1的磷酸化将在激活的U937和THP-1细胞中进行研究。ELMO1在吞噬和RAC激活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。这些信息可以为开发通过致癌酪氨酸激酶干扰底物识别和细胞转化的抗癌药物奠定基础。

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.