VAV FAMILY PROTEINS IN CELL SIGNALING AND CANCER

细胞信号传导和癌症中的 VAV 家族蛋白

• 批准号: 6647693
• 负责人: XOSE R BUSTELO
• 金额: $ 18万
• 依托单位: UNIVERSITY OF SALAMANCA
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-06-01 至 2005-09-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6647693
• 关键词: X ray crystallography; biological signal transduction; cell proliferation; conformation; expression cloning; gene targeting; guanine nucleotide binding protein; guanosinetriphosphatases; intermolecular interaction; laboratory mouse; neoplastic transformation; oncogenes; oncoproteins; phosphoproteins; phosphorylation; protein structure function; protooncogene; site directed mutagenesis

The Vav family is a new group of oncoproteins with known representative in vertebrates (Vav, Vav-2, and Vav-3) and nematodes (C. Elegans Vav). These proteins work as enzymes that catalyze the exchange of nucleotides on GTP-binding proteins of the Rho/Rac family, thereby facilitating the transition of these GTPases from their inactive (GDP-bound) to their active (GTP-bound) to their active (GTP- bound) state. The enzyme activity of Vav proteins is tightly regulated during signal transduction. In the absence of stimuli, these proteins remain inactive due to low phosphorylation levels. After cell stimulation, Vav proteins become activated by direct phosphorylation on tyrosine residues, leading to the stimulation of Rho/Rac-dependent intracellular pathways. Several lines of evidence demonstrate that the function of Vav proteins is crucial for mounting effective developmental and mitogenic responses. Thus, the deletion of the vav gene by homologous recombination results in impaired lymphoid development, lymphopenia, and in defective immune responses of mature B-and T-lymphocytes. Furthermore, the ectopic express of gain-of-function mutants of Vav and Vav-2 induces high levels of cellular transformation in rodent fibroblasts. Finally, recent results have implicated the Vav pathway in the pathogenic cycle of human lymphotropic viruses such as HIV and HTLV. The long-term objective of our laboratory is to achieve a comprehensive characterization of this protein family at the biochemical, cellular, and organism level. To materialize this objective, we propose the following studies. In Aim 1, we will use biochemical and crystallographic techniques to visualize the conformational changes that trigger the activation of Vav proteins during physiological and tumorigenic conditions. In Aim 2, we will perform site-directed mutagenesis studies to characterize the structural determinants that mediate the interaction of Vav proteins with Rho/Rac GTPPases. In Aim 3, we will utilize expression cloning approaches to isolate inhibitory and synergistic factors that modulate the activity if Vav proteins in vivo. In Aim 4, we will use homologous recombination technology to investigate the role of each Vav family member, and of the Vav family as a whole, in developmental and physiological responses. These studies should give us for the first time should give us for the first time a unified vision of the modus operandi of Vav proteins during signal transduction. This will in turn contribute to a better understanding of the intracellular pathways that mediate the proliferation of normal and cancer cells.

Vav家族是一组新的癌蛋白，已知在脊椎动物（Vav、Vav-2和Vav-3）和线虫（C. Elegans Vav）。这些蛋白质作为催化Rho/Rac家族的GTP结合蛋白上的核苷酸交换的酶起作用，从而促进这些GTP酶从其失活（GDP结合）到其活性（GTP结合）再到其活性（GTP结合）状态的转变。 Vav蛋白的酶活性在信号转导过程中受到严格调节。 在没有刺激的情况下，这些蛋白质由于低磷酸化水平而保持无活性。 在细胞刺激后，Vav蛋白通过酪氨酸残基上的直接磷酸化而被激活，导致Rho/Rac依赖性细胞内途径的刺激。一些证据表明，Vav蛋白的功能是至关重要的安装有效的发育和促有丝分裂反应。因此，通过同源重组缺失vav基因导致淋巴发育受损、淋巴细胞减少症以及成熟B淋巴细胞和T淋巴细胞的免疫应答缺陷。 此外，Vav和Vav-2的功能获得性突变体的异位表达在啮齿动物成纤维细胞中诱导高水平的细胞转化。最后，最近的研究结果表明，Vav途径参与了人类嗜淋巴细胞病毒（如HIV和HTLV）的致病循环。 我们实验室的长期目标是在生物化学，细胞和生物体水平上实现该蛋白质家族的全面表征。 为了实现这一目标，我们提出了以下研究。 在目标1中，我们将使用生物化学和晶体学技术来可视化在生理和致瘤条件下触发Vav蛋白激活的构象变化。 在目标2中，我们将进行定点诱变研究，以表征介导Vav蛋白与Rho/Rac GTP酶相互作用的结构决定簇。 在目标3中，我们将利用表达克隆方法来分离调节体内Vav蛋白活性的抑制因子和协同因子。 在目标4中，我们将使用同源重组技术来研究每个Vav家族成员以及整个Vav家族在发育和生理反应中的作用。 这些研究应该给我们第一次应该给我们第一次一个统一的看法的方式，在信号转导过程中的Vav蛋白。 这反过来将有助于更好地了解介导正常细胞和癌细胞增殖的细胞内途径。