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

描述(申请人提供)：雷帕霉素(MTOR)的机制靶点，一种进化保守的蛋白激酶，协调信号网络来调节基本的细胞过程，包括细胞生长和增殖、细胞新陈代谢、细胞存活、免疫和衰老。MTOR复合体调节失调(MTORC)信号转导通路参与多种疾病，包括糖尿病、肥胖、心血管疾病和肿瘤的发生。事实上，临床医生将mTOR抑制剂用于器官移植后的免疫抑制、血管成形术后冠状动脉支架再狭窄的抑制以及肾癌的治疗。尽管mTOR具有明确的生理和治疗重要性，但我们对细胞mTOR调控的科学知识仍存在根本差距，特别是在调节mTOR活性以响应不同信号的一整套分子通路和机制方面。我们实验室令人兴奋的工作揭示了mTOR磷酸化在mTORC1功能中扮演着一个以前未被认识到的重要角色。使用磷酸化特异性抗体和体外动态组筛选作为创新工具，我们发现非规范的IKK(IKK)相关的激酶TbK1和IKKϵ直接磷酸化S2159上的mTOR，这发生在体外、培养细胞和体内。我们的初步数据表明，在几种细胞类型(即MEF；HEK293；RAW264.7巨噬细胞)中，Tbk1/IKKϵ-在病原体相关的炎症信号(例如细菌内毒素；双链RNA)和一组生长因子(即表皮生长因子而不是胰岛素)的响应下，促进mTORC1mTORC2信号转导。重要的是，tbk1/ikkϵ驱动的mTORC 1信号需要mTOR S2159的磷酸化，并诱导干扰素的产生，这是宿主对微生物感染反应的一个重要的早期事件。这些初步数据提出了令人兴奋的假设，即tBK1和IKKϵ作为新的mTORC 1/2激活剂发挥作用，mTORC 1/2代表新的tBK1和IKKϵ底物。在目标1中，我们将阐明tBK1/IKKϵ促进mTORC 1和mTORC2信号转导的生化机制；在目标2中，我们将确定介导tBK1/IKKϵ对mTORC 1和mTORC2作用的上游信号中间体；在目标3中，我们将了解tbk1/IKKϵ作用于mTORC 1和mTORC2所调控的细胞功能。这项在培养细胞中的研究是确定新的药物靶点和开发合理设计的治疗剂的关键的第一步，用于治疗与mTOR和Tbk1/ikkϵ网络作用相关的临床疾病，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 IKKϵ 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/IKKϵ-promotes mTORC1 and mTORC2 signaling. Importantly, TBK1/IKKϵ-driven mTORC1 signaling requires mTOR S2159 phosphorylation and induces IFNß production, an important early event in the host response to microbial infection. These preliminary data present the exciting hypotheses that TBK1 and IKKϵ function as novel mTORC1/2 activators and that mTORC1/2 represent novel TBK1 and IKKϵ substrates. In Aim 1 we will elucidate biochemical mechanisms by which TBK1/IKKϵ promotes mTORC1 and mTORC2 signaling; in Aim 2 we will identify upstream signaling intermediates that mediate the action of TBK1/IKKϵ on mTORC1 and mTORC2; and in Aim 3 we will understand cellular functions controlled by TBK1/IKKϵ 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/IKKϵ network action, such as inflammatory and autoimmune diseases, diabetes, obesity, and cancer.