VAV FAMILY PROTEINS IN CELL SIGNALING AND CANCER

Vav家族是在脊椎动物(Vav、Vav-2和Vav-3)和线虫(C.elegans Vav)中具有代表性的一组新的癌蛋白。这些蛋白质作为酶催化Rho/RAC家族的GTP结合蛋白上的核苷酸交换，从而促进这些GTP酶从无活性(GDP结合)到活性(GTP结合)再到活性(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控。在没有刺激物的情况下，这些蛋白质由于低水平的磷酸化而保持不活跃。在细胞刺激后，Vav蛋白被酪氨酸残基直接磷酸化激活，从而刺激依赖于Rho/Rac的细胞内通路。一些证据表明，Vav蛋白的功能对于建立有效的发育和有丝分裂反应至关重要。因此，VaV基因通过同源重组的缺失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诱导了啮齿动物成纤维细胞的高水平细胞转化。最后，最近的结果表明，Vav途径在人类嗜淋巴病毒(如HIV和HTLV)的致病循环中具有一定的作用。我们实验室的长期目标是在生化、细胞和生物体水平上实现对该蛋白质家族的全面表征。为了实现这一目标，我们建议进行以下研究。在目标1中，我们将使用生化和结晶学技术来可视化在生理和肿瘤条件下触发Vav蛋白激活的构象变化。在目标2中，我们将进行定点突变研究，以确定介导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的方法来分离体内调节Vav蛋白活性的抑制和协同因子。在目标4中，我们将使用同源重组技术来研究Vav家族的每个成员以及整个Vav家族在发育和生理反应中的作用。这些研究将使我们第一次对Vav蛋白在信号转导过程中的工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。

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.