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: 6790506
• 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/6790506
• 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（线虫 Vav）中具有代表性。这些蛋白质作为酶催化 Rho/Rac 家族 GTP 结合蛋白上的核苷酸交换，从而促进这些 GTP 酶从非活性（GDP 结合）状态转变为活性（GTP 结合）状态，再到活性（GTP 结合）状态。 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，这些蛋白质由于磷酸化水平低而保持失活。 细胞刺激后，Vav 蛋白通过酪氨酸残基的直接磷酸化而被激活，从而刺激 Rho/Rac 依赖性细胞内途径。多项证据表明，Vav 蛋白的功能对于有效的发育和有丝分裂反应至关重要。因此，通过同源重组删除 vav 基因会导致淋巴发育受损、淋巴细胞减少以及成熟 B 和 T 淋巴细胞的免疫反应缺陷。 此外，Vav 和 Vav-2 功能获得突变体的异位表达可诱导啮齿动物成纤维细胞发生高水平的细胞转化。最后，最近的结果表明 Vav 途径与人类嗜淋巴细胞病毒（例如 HIV 和 HTLV）的致病周期有关。 我们实验室的长期目标是在生化、细胞和有机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，我们提出以下研究。 在目标 1 中，我们将使用生化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 GTPPase 相互作用的结构决定因素。 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。 这些研究应该让我们第一次对 Vav 蛋白在信号转导过程中的运作方式有一个统一的认识。 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。