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Mechanism of Signal Transduction by Nonreceptor Protein-tyrosine Kinases

非受体蛋白酪氨酸激酶的信号转导机制

基本信息

批准号：
04670150
负责人：
SASAKI Terukatsu
金额：
$ 1.28万
依托单位：
Sapporo Medical University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1992
资助国家：
日本
起止时间：
1992 至 1993
项目状态：
已结题

项目摘要

1. The Src homology 3 domain (SH3) is a protein domain involved in the assembly of proteins participating in the signal transduction by protein-tyrosine kinases. A cDNA clone encoding a new protein ligand to the SH3 domain of Fyn PTK was isolated. The clone was identified by screening mouse embryo cDNA library with Fyn・SH3/GST fusion protein as a probe. The new SH3 ligand protein is also expressed in adult brain. The protein contains two proline-rich sequences with a specificity in binding to Fyn and Src SH3 domains, a new SH3 domain, and potential ligand sequences for SH2 domains. Antibody specific to the newly identified protein immunoprecipitated and immunoblotted a protein from the teratocarcinoma cell lysate and from the lysate of COS-7 cells transfected with the cloned cDNA.The protein was tyrosine-phosphorylated by Fyn expressed from cotransfected fyn cDNA in COS-7 cells. 2. A 70-kDa protein with high affinity to Fyn SH3 was found in the Jurkat cell lysate by affinity binding to Fyn SH3/GST fusion protein. The protein was eluted from SDS-PAGE gels, digested with trypsin, and the peptides were separated. The results of gas-phase microsequencing of the peptides indicated that the protein is closely related to GAP-binding phosphoprotein, p62. The SH3 domains are known to bind to proline-rich sequences. The proline-rich sequences in p62 were expressed as GST fusion proteins and the binding of the GST fusion proteins to various SH3/GST fusion proteins were examined. The results indicate that each proline-rich sequence has a specific affinity to a subset of SH3 domains.

1. Src 同源 3 结构域 (SH3) 是参与蛋白酪氨酸激酶信号转导的蛋白组装的蛋白结构域。分离出编码 Fyn PTK 的 SH3 结构域的新蛋白质配体的 cDNA 克隆。以Fyn・SH3/GST融合蛋白为探针，筛选小鼠胚胎cDNA文库，鉴定该克隆。新的SH3配体蛋白也在成人大脑中表达。该蛋白包含两个富含脯氨酸的序列，与 Fyn 和 Src SH3 结构域具有特异性结合，一个新的 SH3 结构域，以及 SH2 结构域的潜在配体序列。对新鉴定的蛋白质具有特异性的抗体对来自畸胎癌细胞裂解物和转染克隆 cDNA 的 COS-7 细胞裂解物的蛋白质进行免疫沉淀和免疫印迹。该蛋白质被 COS-7 细胞中共转染的 fyn cDNA 表达的 Fyn 酪氨酸磷酸化。 2. 通过与 Fyn SH3/GST 融合蛋白的亲和结合，在 Jurkat 细胞裂解物中发现了与 Fyn SH3 高亲和力的 70-kDa 蛋白。从 SDS-PAGE 凝胶上洗脱蛋白质，用胰蛋白酶消化，然后分离肽。肽的气相微测序结果表明该蛋白与GAP结合磷蛋白p62密切相关。已知 SH3 结构域与富含脯氨酸的序列结合。 p62 中富含脯氨酸的序列被表达为 GST 融合蛋白，并检查了 GST 融合蛋白与各种 SH3/GST 融合蛋白的结合。结果表明，每个富含脯氨酸的序列对 SH3 结构域的子集具有特定的亲和力。