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STUDIES OF A NOVEL SH2/SH3-CONTAINING CELLULAR ONCOGENE

含SH2/SH3的新型细胞癌基因的研究

基本信息

批准号：
2895193
负责人：
Wei Li
金额：
$ 21.93万
依托单位：
UNIVERSITY OF SOUTHERN CALIFORNIA
依托单位国家：
美国
项目类别：
财政年份：
1995
资助国家：
美国
起止时间：
1995-08-15 至 2001-04-30
项目状态：
已结题

项目摘要

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- 含有肽和Src-同源2（SH 2）结构域的蛋白质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功能被认为是受体来募集细胞信号分子，网络. Nck，一种II类SH 2-和SH 3-（Src-同源3），含有信号分子，是一种普遍表达的细胞癌基因，多种细胞表面受体的共同靶点。升高的表达 Nck在培养中引起细胞转化并在裸细胞中形成肿瘤小鼠改变（缺失、插入、倒位或易位）其染色体位置与许多人类遗传性疾病和瘤形成。虽然Nck的作用机制仍有待进一步研究解决，这些结果清楚地表明，Nck参与控制，细胞的生长。本文研究的目的是为了阐明 Nck的生物学功能和作用机制。完成这项研究不仅可以阐明Nck的特异性功能， II类SH 2和SH 3信号传导的作用机制一般的分子。我们将与拉里Rohrschneider（弗雷德哈钦森癌症研究中心，西雅图），艾德斯科尔尼克和约瑟夫施莱辛格（纽约大学医学中心，纽约）对Nck与CSF-1受体结合的研究， IRS-1（胰岛素受体底物-1）和EGF受体。的这些酪氨酸激酶中Nck SH 2结构域的结合位点将通过使用细胞系在完整细胞中鉴定受体/底物表达野生型或突变型受体/底物。的 Nck结合位点突变的影响，受体/底物和Nck的SH 2结构域中的干扰突变，将评估受体的信号传导。抗PY、GST-Nck和抗Nck抗体亲和柱将用于纯化新的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 SH 3结构域作为筛选pLox小鼠胚胎cDNA表达文库的探针，已经鉴定了两个新基因NS 3 P1和NS 3 P2（Nck）的部分序列 SH 3结合蛋白1和2）。这些蛋白质与完整的Nck结合，细胞我们将获得NS 3 Ps的全长cDNA，并研究其在细胞中的表达。在NCK介导的细胞转化中的作用。最后，表达式和 Nck和NS 3 P基因在Nck染色体定位（3q 21 -24）中的功能将研究改变相关的肿瘤细胞。