Direct regulation of mTORC1 and mTORC2 by the IKK-related kinases TBK1 and IKKe

描述（由申请人提供）：雷帕霉素（mTOR）的机制靶点，一种进化上保守的蛋白激酶，协调信号传导网络以调节基本细胞过程，包括细胞生长和增殖、细胞代谢、细胞存活、免疫和衰老。mTOR复合物（mTORC）信号传导的失调导致无数疾病，包括糖尿病、肥胖症、心血管疾病和肿瘤发生。实际上，临床医生使用mTOR抑制剂用于器官移植后的免疫抑制、用于血管成形术后冠状动脉支架再狭窄的抑制和用于肾癌的治疗。尽管mTOR具有明确的生理和治疗重要性，但我们对细胞mTOR调节的科学知识存在根本性差距，特别是关于调节mTOR活性以响应不同信号的全套分子途径和机制。来自我们实验室的令人兴奋的工作揭示了mTOR磷酸化在mTORC 1功能中起着重要且以前未被认识的作用。使用磷酸化特异性抗体和体外激酶组筛选作为创新工具，我们发现非经典IKK（IkB激酶）相关激酶TBK 1和IKKe直接在S2159上磷酸化mTOR，这在体外，培养细胞和体内都发生。我们的初步数据显示， 几种细胞类型（即MEF; HEK 293; RAW264.7巨噬细胞）响应病原体相关的炎症信号（即细菌LPS; dsRNA）和生长因子亚类（即EGF但不是胰岛素），TBK 1/IKKe促进mTORC 1和mTORC 2信号传导。重要的是，TBK 1/IKKE驱动的mTORC 1信号传导需要mTOR S2159磷酸化并诱导IFNb产生，这是宿主对微生物感染应答的重要早期事件。这些初步数据提出了令人兴奋的假设，即TBK 1和IKKe作为新的mTORC 1/2激活剂发挥作用，mTORC 1/2代表新的TBK 1和IKKe底物。在目标1中，我们将阐明TBK 1/IKKe促进mTORC 1和mTORC 2信号传导的生化机制;在目标2中，我们将鉴定介导TBK 1/IKKe对mTORC 1和mTORC 2作用的上游信号传导中间体;在目标3中，我们将了解TBK 1/IKKe对mTORC 1和mTORC 2作用控制的细胞功能。这项在培养细胞中的研究代表了识别新型药物靶点和开发合理设计的治疗剂以治疗与异常mTOR和TBK 1/IKKe网络作用相关的临床疾病的重要第一步，如炎症和自身免疫性疾病，糖尿病，肥胖和癌症。

DESCRIPTION (provided by applicant): The mechanistic target of rapamycin (mTOR), an evolutionarily-conserved protein kinase, coordinates signaling networks to regulate fundamental cellular processes including cell growth and proliferation, cell metabolism, cell survival, immunity, and ageing. Dysregulation of mTOR complex (mTORC) signaling contributes to myriad diseases including diabetes, obesity, cardiovascular disorders, and tumorigenesis. Indeed, clinicians employ mTOR inhibitors for immunosuppression after organ transplantation, for suppression of coronary artery stent restenosis after angioplasty, and for treatment of kidney cancer. Despite the clear physiologic and therapeutic importance of mTOR, fundamental gaps exist in our scientific knowledge of cellular mTOR regulation, especially with regard to the full set of molecular pathways and mechanisms that regulate mTOR activity in response to diverse signals. Exciting work from our laboratory revealed that mTOR phosphorylation plays an important and previously unrecognized role in mTORC1 function. Using phospho specific antibodies and an in vitro kinome screen as innovative tools, we discovered that the non-canonical IKK (IkB kinase)-related kinases TBK1 and IKKe phosphorylate mTOR directly on S2159, which occurs in vitro, in cultured cells, and in vivo. Our preliminary data indicate that in several cell types (i.e. MEFs; HEK293; RAW264.7 macrophages) in response to pathogen-associated inflammatory signals (i.e. bacterial LPS; dsRNA) and a subset of growth factors (i.e. EGF but not insulin), TBK1/IKKe-promotes mTORC1 and mTORC2 signaling. Importantly, TBK1/IKKe-driven mTORC1 signaling requires mTOR S2159 phosphorylation and induces IFNb production, an important early event in the host response to microbial infection. These preliminary data present the exciting hypotheses that TBK1 and IKKe function as novel mTORC1/2 activators and that mTORC1/2 represent novel TBK1 and IKKe substrates. In Aim 1 we will elucidate biochemical mechanisms by which TBK1/IKKe promotes mTORC1 and mTORC2 signaling; in Aim 2 we will identify upstream signaling intermediates that mediate the action of TBK1/IKKe on mTORC1 and mTORC2; and in Aim 3 we will understand cellular functions controlled by TBK1/IKKe action on mTORC1 and mTORC2. This research in cultured cells represents an essential first step towards the identification of novel drug targets and the development of rationally-designed therapeutic agents to treat clinical disorders linked to aberrant mTOR and TBK1/IKKe network action, such as inflammatory and autoimmune diseases, diabetes, obesity, and cancer.