Identity, regulation, and function of mTOR phosphorylation sites

描述(由申请人提供)：这项题为“mTOR磷酸化位点的识别、调节和功能”的提案的目的是了解雷帕霉素的哺乳动物靶标mTOR在培养细胞中受生理信号调控的机制。MTOR是一种进化保守的蛋白激酶，它与大量细胞蛋白结合形成不同的信号复合体。TOR复合体1(TORC1)是最具特征性的mTOR复合体，可被免疫抑制剂雷帕霉素抑制。TORC1整合了来自营养物质、生长因子和细胞压力的信号，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的TOR复合体2(TORC2)知之甚少，但该复合体调节肌动蛋白细胞骨架的组织。虽然mTOR信号受到严格调控，但生理信号直接调节TORC1或TORC2中mTOR活性的机制尚不清楚。由于磷酸化控制着许多TORC1调节蛋白的活性，这一提议的中心假设是mTOR本身的磷酸化响应细胞信号调节mTOR信号和生物学功能。事实上，我们已经通过串联质谱仪(MS2)确定了mTOR上七个新的丝氨酸/苏氨酸(Ser/Thr)磷酸化位点(P位点)。在这个提案中，我们将确定使用MS2从完整细胞中分离的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。我们提出了以下具体目标：1.确定mTOR上更多的磷酸化位点。2.确定mTOR磷酸化的调节。3.确定mTOR的磷酸化功能。失调的mTOR信号与多种人类疾病的病理生理学有关，包括II型糖尿病、肥胖症、心血管疾病和癌症。事实上，mTOR抑制药物目前被用于预防移植排斥反应和冠状动脉再狭窄，并正在作为抗肿瘤和抗糖尿病药物进行测试。鉴于mTOR抑制剂的治疗潜力，了解mTOR的调节是一个具有重大治疗价值和生物学意义的问题。

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.