Activation of rapamycin-sensitive TORC1 by endomembrane amino acid transporters

描述（由申请人提供）：雷帕霉素复合物1（TORC 1）的靶标在普遍保守的信号转导级联中起着重要作用，该信号转导级联响应营养素和生长因子信号以控制细胞生长和增殖。TORC 1级联的失调导致多种类型的人类恶性肿瘤。TORC 1的核心成员是Tor蛋白激酶，它是在酵母酿酒酵母中发现的，这是一种在阐明TORC 1信号级联中至关重要的模型系统。基于其有效的抗增殖活性，雷帕霉素被用于几个临床领域，包括免疫抑制，癌症化疗和介入心脏病学。尽管进行了大量研究，但TORC 1通路仍具有部分特征。我们发现了一个意想不到的作用，内膜囊泡运输系统在调节TORC 1信号在酵母中的蛋白质复合物的突变与囊泡运输和蛋白质分选的作用，结合突变的非必需的Tor 1激酶，使细胞无法生存或严重生长受损。我们证明，HOPS复合物是必需的，以提供有效的TORC 1信号和正常表达的TORC 1控制的基因的氨基酸稳态。这些研究还揭示了雷帕霉素作用机制的一个新方面：雷帕霉素绕过囊泡运输事件来激活TORC 1控制的反式激活因子。此外，我们发现，自我复合物（EGOC）中的突变会损害TORC 1信号传导，并且EGOC和TORC 1活性都是最佳细胞生长所必需的。EGOC具有进化上保守的氨基酸敏感性GTR亚基，并且这些（GTR 1、2和RagA-D蛋白）的直系同源物介导酵母、昆虫和哺乳动物细胞中的TORC 1活化。然而，氨基酸传感器的身份和氨基酸激活EGOC GT3的潜在机制尚不清楚。 有趣的是，TORC 1和EGOC共定位于内膜，包括内体和空泡的内膜，沿着空泡氨基酸转运蛋白。本研究的目的是：1）阐明内膜系统激活TORC 1信号的作用和潜在机制; 2）确定哺乳动物中保守的液泡氨基酸转运蛋白是否整合到传递氨基酸信号以激活TORC 1的分子级联中。我们的工作模型是内膜网络提供了一个平台，以促进分子相互作用，激活和启用TORC 1信号。与酵母TORC 1类似，哺乳动物TORC 1也定位于内膜，特别是溶酶体，其是酵母液泡的对应物。因此，我们提出，我们的研究将继续揭示基本的，保守的TORC 1信号传导方面，可以直接外推到哺乳动物模型，并指导药物干预选择TORC 1通路缺陷在人类癌症和其他疾病。

DESCRIPTION (provided by applicant): The target of rapamycin complex 1 (TORC1) plays a prominent role in a ubiquitously conserved signal transduction cascade that responds to nutrients and growth factor cues to control cell growth and proliferation. Dysregulation of the TORC1 cascade results in multiple types of human malignancies. The central TORC1 members are the Tor protein kinases, which were discovered in the yeast Saccharomyces cerevisiae, a model system that has been crucial in elucidating the TORC1 signaling cascade. Based on its potent antiproliferative activity, rapamycin is in use in several clinical areas including immunosuppression, cancer chemotherapy, and interventional cardiology. The TORC1 pathway remains partially characterized despite much study. We uncovered an unexpected role for the endomembrane vesicular trafficking system in regulating TORC1 signaling in yeast; mutations in protein complexes with roles in vesicular trafficking and protein sorting in combination with mutation of the nonessential Tor1 kinase, render cells inviable or severely growth impaired. We demonstrated that the HOPS complex is required to provide amino acid homeostasis for efficient TORC1 signaling and for normal expression of TORC1-governed genes. These studies also revealed a novel facet in the rapamycin mechanism of action: rapamycin bypasses vesicular trafficking events to activate TORC1- controlled transactivators. Moreover, we showed that mutations in the Ego complex (EGOC) compromise TORC1 signaling and both EGOC and TORC1 activity are required for optimal cell growth. The EGOC possesses evolutionary conserved amino acid-sensitive GTPase subunits and orthologs of these (Gtr1,2 and RagA-D proteins) mediate TORC1 activation in yeast, insect, and mammalian cells. However, the identity of the amino acid sensors and the underlying mechanisms by which amino acids activate EGOC GTPase are unknown. Intriguingly, TORC1 and the EGOC colocalize to endomembranes including those of endosomes and vacuoles, along with vacuolar amino acid transporters. The aims of this proposal are: 1) to elucidate the roles and underlying mechanism by which the endomembrane system enables TORC1 signaling and 2) to determine whether the vacuolar amino acid transporters, which are conserved in mammals, are integrated into the molecular cascade that conveys amino acid signals to evoke TORC1 activation. Our working model is that the endomembrane network provides a platform to facilitate molecular interactions that activate and enable TORC1 signaling. Similar to yeast TORC1, mammalian TORC1 is also localized to endomembranes, in particular lysosomes, which are the counterparts of yeast vacuoles. Thus, we submit that our research will continue to uncover fundamental, conserved TORC1 signaling aspects that could be directly extrapolated to mammalian models and guide pharmacological intervention in select TORC1 pathway defects in human cancer and other diseases.