SUBSTRATE SPECIFICITY OF NONRECEPTOR TYROSINE KINASES

劳斯肉瘤病毒引起的细胞转化和肿瘤形成是 依赖于病毒V-src基因的表达。 这个产品 基因是60千道尔顿的酪氨酸蛋白激酶，命名为v-Src。 同样，Abelson鼠白血病病毒的转化基因（ 导致体内B细胞淋巴瘤）编码酪氨酸蛋白激酶 命名为v-Abl。 该建议的重点是蛋白质-蛋白质相互作用的作用， v-Src和v-Abl酪氨酸激酶的功能。 有人建议 非受体酪氨酸的Src同源（SH）区域2和3 激酶在激酶活性的调节和 对细胞基质的识别。 v-Src和v-Abl的区域， 参与分子内或分子间识别 通过基于肽的光亲和标记实验。 肽底物 含有光活性氨基酸的这些酶的类似物 对苯甲酰基-Phe将用作亲和标记。 改质区域 内部和外部的SH 2和SH 3域将进一步研究， 定点诱变，突变酶将通过在 用含酪氨酸的合成肽进行体外磷酸化。 这些 结果将与三维模型相关联 结构的v-Src的基础上最近的晶体结构的催化 cAMP依赖性蛋白激酶的结构域。 确定决定因素， 赋予野生型识别的v-Src的蛋白质底物 和突变酶，合成肽“文库”将用于选择 这些肽通过v-Src产生最大磷酸化。 突变型v-Src激酶将用于研究体内底物识别。 用于这些研究的模型系统将是转染有以下的Rat-1细胞： 编码具有改变的特异性的激酶的v-Src构建体。 基板 突变酶的识别将通过三个标准进行评估： 对总酪氨酸磷酸化的影响， 磷脂酶C-γ和其他特异性底物， 细胞转化 这些研究的目的是描述在一个 分子水平的步骤导致形成的“信号 复合物”在信号转导和致癌作用中起核心作用， 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.