Rheb, a highly conserved farnesylated G-protein

描述（由申请人提供）： 这笔赠款支持了我们的主要努力，以表征法尼基化蛋白质。我们在当前资助期间的研究发现了Rheb G蛋白的新特征。我们已经表明，Rheb代表一个独特的家庭的Ras超家族G蛋白，是高度保守的酵母，果蝇和人类。我们在粟酒裂殖酵母中的研究确定了Rheb是必不可少的，并且在细胞周期和细胞生长的调节中起作用。最近对果蝇Rheb的研究表明，Rheb可能在胰岛素/RS6 K信号转导中发挥作用。与这一想法相一致，我们已经证明Rheb能够激活哺乳动物细胞中的S6 K。在本申请中，我们提出首先评估Rheb是TOR信令的组件的模型。我们将使用S。粟酒裂殖酵母、果蝇和哺乳动物细胞中检测Rheb与TOR/S6 K功能的相互作用。此外，将评价Rheb与TSC的相互作用。TSC 2蛋白是特别感兴趣的，因为这种肿瘤抑制基因产物含有GAP（GTP酶激活蛋白）结构域，提高了TSC 2作为Rheb的GAP发挥功能的可能性。在第二个具体目标中，我们建议进行筛选和选择以鉴定影响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鉴定的任何基因的意义。Rheb的显性负突变可能导致我们鉴定Rheb的GEF（GDP/GTP交换因子）。将进行使用野生型和Rheb效应结构域突变体的酵母双杂交测定以鉴定Rheb的下游效应子。我们还将利用异源S。科学家们希望深入了解影响S.粟酒S.获得的粟酒裂殖酵母基因将用于鉴定和表征果蝇和哺乳动物对应物。最后，我们将尝试抑制由Rheb激活介导的S6 K激活。我们将重新检查之前的观察结果，即法尼基转移酶抑制剂（FTI）能够阻断哺乳动物细胞中S6 K的激活。本研究之后将对TSC突变细胞进行表征，以确定Rheb的可能活化及其对FTI的敏感性。这些实验对于理解Rheb如何影响Torsignalng具有重要意义。该研究对于癌症治疗也具有重要意义，因为我们的研究可能揭示FTI抑制肿瘤细胞中RhebFFOR/S6 K过度活化的能力。

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.