STUDIES OF A NOVEL SH2/SH3-CONTAINING CELLULAR ONCOGENE

含SH2/SH3的新型细胞癌基因的研究

• 批准号: 2700580
• 负责人: Wei Li
• 金额: $ 19.22万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-08-15 至 1999-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2700580
• 关键词: DNA binding protein; PC12 cells; SDS polyacrylamide gel electrophoresis; affinity chromatography; antisense nucleic acid; binding proteins; biological signal transduction; cell transformation; chemical binding; colony stimulating factor; epidermal growth factor; gene mutation; growth factor receptors; high performance liquid chromatography; immunoprecipitation; insulin receptor; neoplastic cell; northern blottings; oncogenes; platelet derived growth factor; protein signal sequence; protein structure function; protein tyrosine kinase; receptor binding; western blottings

The specific and high affinity interaction between phosphotyrosyl- containing peptides and Src-homology 2 (SH2) domains appears to be a mechanism by which the receptor tyrosine kinases are linked to downstream signaling pathways. The function of such interaction is thought for the receptors to recruit cellular signaling molecules to form functional networks. Nck, a class II SH2- and SH3-(Src-homology 3) containing signaling molecule, is a ubiquitously expressed cellular oncogene, and a common target for varieties of cell surface receptors. Elevated expression of Nck causes cell transformation in culture and tumor formation in nude mice. Alterations (deletion, insertion, inversion, or translocation) at its chromosomal location are associated with a number of human inherited disorders and neoplasias. While the mechanism of Nck action remains to be resolved, these findings clearly indicate that Nck is involved in control of cell growth. The goal of research herein is towards elucidating the biological function and the mechanism of action of Nck. Accomplishment of this study may not only shed light on the specific function of Nck, but also the mechanism of the action of the class II SH2 and SH3 signaling molecules in general. We will be collaborating with Larry Rohrschneider (Fred Hutchinson Cancer Research Center, Seattle), Ed Skolnik and Joseph Schlessinger (New York University Medical Center, New York) on Nck binding to CSF- l receptor, IRS-1 (insulin receptor substrate-1) and EGF receptors, respectively. The binding sites of Nck SH2 domain in these tyrosine kinase receptors/substrate will be identified in intact cells by using cell lines expressing either the wild-type or the mutant receptors/substrate. The effects of the mutations at the Nck binding sites in the receptors/substrate and interfering mutations in the SH2 domain of Nck on the signaling by the receptors will be evaluated. Anti-PY, GST-Nck and anti-Nck antibody affinity columns will be used to purify a novel p60-kDa phosphotyrosine protein which specifically mediates the interaction between Nck and EGF receptor, and function and specificity of the p60 protein will be assessed. Using purified and radiolabeled Nck SH3 domains as the probe to screen the pLox mouse embryo cDNA expression library, we have identified partial sequences of two novel genes, NS3P1 and NS3P2 (Nck SH3 binding protein 1 and 2). These proteins associate with Nck in intact cells. We will obtain the full-length cDNA of the NS3Ps and study their role in Nck-mediated cell transformation. Finally, the expression and function of Nck and NS3P genes in Nck chromosomal location (3q21-24) alteration-associated neoplastic cells will be investigated.

磷酸酪氨酰-2，4-三甲基-2，4-三甲基-4，4，4，4，5，6-三甲基-4，4，4，4-三甲基-4，4，4，4，6-三甲基-4，4，4，4，4，4，6-三甲基-4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4 包含多肽和Src同源2(SH2)结构域似乎是一种 受体酪氨酸激酶与下游连接的机制 信号通路。这种相互作用的作用被认为是 受体招募细胞信号分子形成功能 网络。NCK，一个第II类SH2-和SH3-(Src-同源3)，包含 信号分子，是一种普遍表达的细胞癌基因， 各种细胞表面受体的共同靶点。提升的表情 NCK在培养中引起细胞转化并在裸鼠体内形成肿瘤 老鼠。的更改(删除、插入、倒置或移位) 它的染色体位置与许多人类遗传性疾病有关 疾病和肿瘤。而NCK的作用机制仍有待于 结果，这些发现清楚地表明NCK参与了控制 对细胞生长的影响。这项研究的目的是为了阐明 NCK的生物学功能及其作用机制。成就 这项研究不仅可能揭示Nck的具体功能，而且 第二类SH2和SH3信号的作用机制 一般的分子。 我们将与拉里·罗施奈德(弗雷德·哈钦森癌症公司)合作 西雅图研究中心)、埃德·斯科尔尼克和约瑟夫·施莱辛格(纽约 纽约大学医学中心)关于NCK与脑脊液-L受体结合的研究， IRS-1(胰岛素受体底物-1)和EGF受体。这个 NCK SH2结构域在这些酪氨酸激酶中的结合部位 用细胞系鉴定完整细胞中的受体/底物 表达野生型或突变型受体/底物。这个 NCK结合位点突变对人胆囊虫感染的影响 NCK的受体/底物及其SH2结构域的干扰突变 将对受体发出的信号进行评估。抗PY、GST-NCK和 抗NCK抗体亲和柱将用于纯化一种新的p60-kDa 特异性介导相互作用的磷酸酪氨酸蛋白 NCK和EGF受体之间的关系以及p60的功能和特异性 将对蛋白质进行评估。使用纯化和放射性标记的NCK SH3结构域 作为筛选plox小鼠胚胎cdna表达文库的探针 鉴定了两个新基因NS3P1和NS3P2(Nck)的部分序列 SH3结合蛋白1和2)。这些蛋白质与NCK结合在一起。 细胞。我们将获得NS3Ps的全长cDNA，并对其进行研究 在NCK介导的细胞转化中的作用。最后，表达式和 Nck和NS3P基因在Nck染色体定位中的作用(3q21-24) 与改变相关的肿瘤细胞将被研究。