The insulin/IGF system evolved in metazoans to coordinate nutrient utilization with cell growth and proliferation, both in development and adult life. Control by the insulin/IGF system is superimposed upon and cross-regulated by phylogenetically older, nutrient-driven signaling pathways. A paradigm for such dual control is the giant protein kinase known as TOR. First identified in yeast, there TOR regulates transcription, ribosomal biogenesis, mRNA translation and protein turnover in response to nutrient availability. In mammalian cells TOR retains these functions, but regulation by the insulin/IGF system is superimposed on regulation by amino acids, especially leucine. In both yeast and man TOR functions in two physically distinct and independently regulated complexes;TOR complex 1, which contains the polypeptides raptor, the substrate binding subunit and lst8, is primarily concerned with insulin/IGF and nutrient regulation of growth, and is the focus of this proposal. The proximate regulator of mTORC1 is the small GTPase Rheb; insulin/IGF, through the kinase Akt, suppresses the GTPase activator function of the Tuberous Sclerosis heterodimer promoting the conversion of Rheb to the active state.
In AIM1, we propose to elucidate in detail the mechanism by which Rheb-GTP activates mTOR complex 1. We hypothesize a two step process;first, Rheb-GTP interacts with the mTOR catalytic domain and with FKBP38, an endogenous inhibitor of mTOR, to convert the mTOR catalytic domain into an active form. We will define the mechanism of this conversion and recreate this regulation in vitro using purified components. The second step involves the mTOR-catalyzed phosphorylation of itself and of raptor, which enhances the access of substrates to raptor. We will define the regulation of these phosphorylations and establish their contribution to the overall activation of mTORC1 signaling. Little is known about the mechanism by which intracellular leucine regulates mTORC1, apart from the ability of leucine withdrawal to interfere with Rheb-GTP activation of mTORC1.
In AIM2 we will use genome-wide RNA interference to generate a catalog of cellular components required for the ability of mTORC1 to promote phosphorylation of the 40S ribosomal protein S6. This will uncover many previously unappreciated inputs to mTORC1;we will then use more refined secondary assays to focus on elements most likely to mediate leucine regulation. Recently some of the genes that confer susceptibility to type 2 diabetes in the general population have been identified. Among them is an RNA binding protein IMP2, first discovered by its ability to bind to an IGF2 mRNA expressed primarily in fetal life, whose translation is regulated by mTORC1. We find that translation of most IMP2-associated mRNAs is regulated by mTORC1.
In AIM3 we propose to identify IMP2-associated RNAs, the mechanisms by which mTORC1 regulates the IGF2 mRNA transcript and the role of IMP2 in this regulation. A deeper understanding of IMP2 function and regulation will contribute to the understanding of how IMP2, perhaps through the regulation of IGF2 or other to-beidentified RNAs, confers susceptibility to type 2 diabetes.
The TOR complex 1 is a protein kinase that is major determinant of the development and growth of both skeletal muscle and beta cells. We will define how insulin and the amino acid leucine jointly control the activity of TOR complex 1. We will also determine how TOR complex 1 controls the translation of RNAs that bind to the protein IMP2, variants of whose gene appear to confer susceptibility to type 2 diabetes.
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