SUBSTRATE SPECIFICITY OF NONRECEPTOR TYROSINE KINASES

非受体酪氨酸激酶的底物特异性

• 批准号: 2099215
• 负责人: W Todd MILLER
• 金额: $ 9.77万
• 依托单位: STATE UNIVERSITY NEW YORK STONY BROOK
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-09-01 至 1998-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2099215
• 关键词: active sites; affinity labeling; cell transformation; chemical models; enzyme activity; enzyme inhibitors; enzyme structure; enzyme substrate analog; enzyme substrate complex; intermolecular interaction; phosphorylation; photoactivation; protein tyrosine kinase; site directed mutagenesis

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.

劳斯肉瘤病毒的细胞转化和肿瘤形成是 依赖于病毒 v-src 基因的表达。 这个产品的 基因是一种 60 千道尔顿的酪氨酸蛋白激酶，称为 v-Src。 同样，阿贝尔森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 指定为v-Abl。 该提案重点关注蛋白质-蛋白质相互作用在 v-Src 和 v-Abl 酪氨酸激酶的功能。 已建议 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 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将被识别 通过基于肽的光亲和标记实验。 肽底物 这些含有光活性氨基酸的酶的类似物 对苯甲酰基-Phe 将用作亲和标记。 修改区域 SH2和SH3域的内部和外部将进一步研究 定点诱变，突变酶将通过 in 进行测试 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 基于最新催化晶体结构的 v-Src 结构 cAMP 依赖性蛋白激酶的结构域。 确定决定因素 v-Src 的蛋白质底物，可被野生型识别 和突变酶，合成肽“文库”将用于选择 那些通过 v-Src 产生最大磷酸化的肽。 突变的 v-Src 激酶将用于研究体内底物识别。 这些研究的模型系统将是转染 v-Src 构建编码具有改变的特异性的激酶。 基材 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，对磷酸化的影响 磷脂酶 C-gamma 和其他特定底物，以及对 细胞转化。 这些研究的目的是描述 分子水平导致“信号传导”形成的步骤 复合物”在信号转导和致癌中发挥核心作用 转变。