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

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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.