SUBSTRATE SPECIFICITY OF NONRECEPTOR TYROSINE KINASES

Rous肉瘤病毒的细胞转化和肿瘤形成 依赖于病毒v-src基因的表达。它的产物是 该基因是一个60kodalton的酪氨酸蛋白激酶，命名为v-Src。 同样，Abelson小鼠白血病病毒的转化基因(即 在体内引起B细胞淋巴瘤)编码一种酪氨酸蛋白激酶 指定为v-Abl。 这项建议侧重于蛋白质-蛋白质相互作用在 V-Src和v-Abl酪氨酸激酶的功能。有人建议说， 非受体酪氨酸的Src同源(SH)区2和3 蛋白水解酶在调节蛋白激酶活性和蛋白合成中都发挥着重要作用。 细胞底物的识别。V-Src和v-Abl的区域 参与分子内或分子间识别的基因将被确定 通过基于多肽的光亲和标记实验。多肽底物 这些含有光活性氨基酸的酶的类似物 以对苯甲酰苯丙氨酸为亲和标记物。改良区 SH2和SH3结构域的内部和外部将进一步研究 定点突变，突变的酶将通过In 含酪氨酸合成肽的体外磷酸化。这些 结果将与三维模型相关联 从催化剂的最新晶体结构看V-SrC的结构 CAMP依赖蛋白激酶的结构域。确定中的决定因素 野生型识别v-Src的蛋白质底物 以及突变酶、合成肽的“文库”将被用来选择 那些能使v-Src最大磷酸化的多肽。 突变的v-Src激酶将用于体内底物识别的研究。 这些研究的模型系统将是转基因的大鼠-1细胞 V-Src结构编码具有改变的特异性的激酶。底物 突变酶的识别能力将通过三个标准进行评估： 对总酪氨酸磷酸化的影响，对磷酸化的影响 磷脂酶C-伽马和其他特定底物，以及对 细胞转化。这些研究的目标是描述一个 分子水平上通向形成“信号传递”的步骤 在信号转导和致癌中起中心作用的复合体 转型。

Cellular transformation and tumor formation by Rous sarcoma virus is dependent on the expression of the viral v-src gene. The product of this gene is a 60-kilodalton tyrosine protein kinase designated as v-Src. Likewise, the transforming gene of Abelson murine leukemia virus (which causes B-cell lymphomas in vivo) encodes a tyrosine protein kinase designated as v-Abl. This proposal focuses on the role of protein-protein interactions in the function of the v-Src and v-Abl tyrosine kinases. It has been suggested that the Src homology (SH) regions 2 and 3 of nonreceptor tyrosine kinases are important both in the regulation of kinase activity and in the recognition of cellular substrates. Regions of v-Src and v-Abl which are involved in intra- or intermolecular recognition will be identified by peptide-based photoaffinity labelling experiments. Peptide substrate analogs for these enzymes containing the photoactive amino acid p-benzoyl-Phe will be used as the affinity labels. Modified regions inside and outside the SH2 and SH3 domains will be studied further by site-directed mutagenesis, and the mutant enzymes will be tested by in vitro phosphorylation with tyrosine-containing synthetic peptides. These results will be correlated with a model of the three-dimensional structure of v-Src based on the recent crystal structure of the catalytic domain of the cAMP-dependent protein kinase. To identify determinants in protein substrates for v-Src which confer recognition by the wild-type and mutant enzymes, synthetic peptide "libraries" will be used to select those peptides which give maximal phosphorylation by v-Src. Mutant v-Src kinases will be used to study in vivo substrate recognition. The model system for these studies will be Rat-1 cells transfected with v-Src constructs encoding kinases with altered specificity. Substrate recognition by the mutant enzymes will be assessed by three criteria: effects on total tyrosine phosphorylation, effects on phosphorylation of phospholipase C-gamma and other specific substrates, and effects on cellular transformation. The goal of these studies is to describe at a molecular level the steps leading to the formation of the "signalling complexes" which play a central role in signal transduction and oncogenic transformation.