Rheb, a highly conserved farnesylated G-protein

描述(由申请人提供)： 这笔赠款支持了我们表征法尼化蛋白质的主要努力。我们在当前资助期间的研究发现了Rheb G蛋白的新特征。我们已经证明，Rheb代表了一个独特的Ras超家族G蛋白家族，在酵母、果蝇和人类中高度保守。我们在裂殖酵母中的研究证实，Rheb是必需的，并在细胞周期和细胞生长的调节中发挥作用。最近对果蝇Rheb的研究表明，Rheb可能在胰岛素/RS6K信号转导中发挥作用。与这一想法一致，我们已经证明Rheb能够在哺乳动物细胞中激活S6K。在这个应用中，我们建议首先评估RHEB是TOR信令的一个组件的模型。我们将利用S.pombe、果蝇和哺乳动物细胞来研究Rheb与TOR/S6K功能的相互作用。此外，还将评估RHEB与TSC的相互作用。TSC2蛋白特别令人感兴趣，因为这种肿瘤抑制基因产物含有GAP(GTPase激活蛋白)结构域，这增加了TSC2作为Rheb的GAP的可能性。在第二个具体目标中，我们建议进行筛选和选择，以确定影响Rheb功能的基因。筛选将在酵母中进行，因为酵母提供了一个简单而强大的系统来评估从筛选中识别出的任何基因的重要性。显性负性Rheb突变体可能导致对Rheb的全球环境基金(GDP/GTP交换因子)的鉴定。使用野生型和Rheb效应域突变体的酵母双杂交试验将用于鉴定Rheb的下游效应器。我们还将利用异源酿酒酵母系统来深入了解影响S.pombe Rheb的基因。获得的S.pombe基因将被用来鉴定和鉴定果蝇和哺乳动物的同类基因。最后，我们将尝试抑制Rheb激活介导的S6K激活。我们将重新验证先前的观察结果，即法尼基转移酶抑制剂(FTI)能够阻断哺乳动物细胞中S6K的激活。在这项研究之后，将对TSC突变细胞可能激活Rheb以及它们对FTI的敏感性进行表征。这些实验对于理解Rheb如何影响Torsignalng具有重要意义。这项研究也将对癌症治疗具有重要意义，因为我们的研究可能揭示FTI抑制肿瘤细胞中RhebFFOR/S6K过度激活的能力。

DESCRIPTION (provided by applicant): This grant has supported our major effort to characterize farnesylated proteins. Our study during the current funding period uncovered novel features of Rheb G-protein. We have shown that Rheb represents a unique family of the Ras-superfamily G-proteins that is highly conserved in yeast, Drosophila and human. Our studies in Schizosaccharomyces pombe established that Rheb is essential and plays a role in the regulation of cell cycle and cell growth. Recent studies on Drosophila Rheb suggested that Rheb may play a role in the insulin/RS6K signaling. Consistent with this idea, we have shown that Rheb is capable of activating S6K in mammalian cells. In this application, we propose first to evaluate the model that Rheb is a component of TOR signaling. We will utilize S. pombe, Drosophila and mammalian cells to examine interaction of Rheb with TOR/S6Kfunctions. In addition, interaction of Rheb with TSC will be evaluated. TSC2 protein is of particular interest, as this tumor suppressor gene product contains a GAP (GTPase activating protein) domain raising the possibility that TSC2 functions as a GAP for Rheb. In the second specific aim, we propose to carry outscreens and selections to identify genes affecting Rheb function. The screens will be carried out in yeast, since yeast provides a simple and powerful system to evaluate significance of any genes identified from the screen. Dominant negative Rheb mutants may lead us to the identification of GEF (GDP/GTP exchange factor) for Rheb. Yeast two-hybrid assays using the wild type and Rheb effector domain mutants will be carried out to identify downstream effectors of Rheb. We will also make use of a heterologous S. cerevisiaesystem to gain insight into genes affecting S. pombe Rheb. S. pombe genes obtained will be used to identify and characterize Drosophila and mammalian counterparts. Finally, we will attempt to inhibit S6K activation mediated by Rheb activation. We will reexamine a previous observation that farnesyl transferase inhibitor(FTI) is capable of blocking activation of S6K in mammalian cells. This study will be followed by the characterization of TSC mutant cells for possible activation of Rheb as well as their sensitivity to FTI. These experiments should have significant implication for understanding how Rheb affects Torsignalng. The study will also be of importance for cancer therapy, as our study may reveal the ability of FTIto inhibit over-activation of RhebFFOR/S6K in tumor cells.