Identity, regulation, and function of mTOR phosphorylation sites

描述（由申请人提供）：本提案题为“mTOR磷酸化位点的身份、调节和功能”，其目的是了解mTOR（雷帕霉素的哺乳动物靶标）在培养细胞中受生理信号调节的机制。mTOR是一种进化上保守的蛋白激酶，与许多细胞蛋白结合形成不同的信号复合物。TOR复合物1（TORC 1）是最好表征的mTOR复合物，并且被免疫抑制药物雷帕霉素抑制。TORC 1整合来自营养素、生长因子和细胞应激的信号，以控制基本的细胞过程，如蛋白质生物合成和细胞生长/大小。虽然对最近描述的雷帕霉素不敏感TOR复合物2（TORC 2）知之甚少，但该复合物调节肌动蛋白细胞骨架的组织。虽然mTOR信号被严格调节，但生理信号直接调节TORC 1或TORC 2中mTOR活性的机制尚不清楚。由于磷酸化控制许多TORC 1调节蛋白的活性，因此该提议的中心假设是mTOR本身响应细胞信号的磷酸化调节mTOR信号传导和生物学功能。事实上，我们已经确定了七个新的丝氨酸/苏氨酸（Ser/Thr）磷酸化位点（P-网站）的mTOR串联质谱（MS 2）。在本提案中，我们将确定使用MS 2从完整细胞分离的mTOR上的主要体内磷酸化位点，并进行旨在了解这些P位点的调节和功能的实验。我们提出以下具体目标：1。鉴定mTOR上的其他磷酸化位点。2.确定mTOR磷酸化的调节。3.确定mTOR磷酸化的功能。失调的mTOR信号传导涉及几种人类疾病的病理生理学，包括II型糖尿病、肥胖症、心血管疾病和癌症。事实上，mTOR抑制药物目前用于预防移植排斥和冠状动脉再狭窄，并正在作为抗肿瘤和抗糖尿病药物进行测试。考虑到治疗潜力的mTOR抑制剂，理解mTOR的调节代表了主要治疗价值以及生物学重要性的问题。

DESCRIPTION (provided by applicant): The goal of this proposal, entitled "Identity, regulation, and function of mTOR phosphorylation sites", is to understand the mechanisms by which mTOR, the mammalian target of rapamycin, is regulated by physiological signals in cultured cells. mTOR is an evolutionary conserved protein kinase that associates with numerous cellular proteins to form distinct signaling complexes. TOR complex 1 (TORC1) is the best characterized mTOR complex and is inhibited by the immunosuppressive drug rapamycin. TORC1 integrates signals derived from nutrients, growth factors, and cellular stress to control fundamental cellular processes such as protein biosynthesis and cell growth/size. While less is known about the more recently described rapamycin-insensitive TOR complex 2 (TORC2), this complex regulates the organization of the actin cytoskeleton. While mTOR signaling is tightly regulated, the mechanisms by which physiological signals directly modulate mTOR activity in either TORC1 or TORC2 are not known. As phosphorylation controls the activity of many TORC1 regulatory proteins, the central hypothesis of this proposal is that phosphorylation of mTOR itself in response to cellular signals regulates mTOR signaling and biological function. Indeed, we have identified seven novel serine/threonine (Ser/Thr) phosphorylation sites (P-sites) on mTOR by tandem mass spectrometry (MS2). In this proposal we will identify the major in vivo phosphorylation sites on mTOR isolated from intact cells using MS2 and perform experiments directed towards understanding the regulation and function of these P-sites. We propose the following specific aims: 1. Identify additional phosphorylation sites on mTOR. 2. Determine the regulation of mTOR phosphorylation. 3. Determine the function of mTOR phosphorylation. Dysregulated mTOR signaling is implicated in the pathophysiology of several human diseases including type II diabetes, obesity, cardiovascular disease, and cancer. Indeed, mTOR-inhibitory drugs are currently employed to prevent transplant rejection and coronary restenosis and are being tested as anti-tumor and anti-diabetic agents. Given the therapeutic potential mTOR inhibitors, understanding the regulation of mTOR represents a problem of major therapeutic value as well as biological importance.