Rapamycin is a potent immunosuppressive drug that halts cell cycle progression in the G1 phase in IL-2 activated T-cells, certain tumor cell lines and in yeast. The major cellular target of rapamycin, acting in situ as a complex with its binding protein (FKBP-12), is a novel 280 KDa polypeptide, first identified in yeast as TOR, and subsequently in mammalian cells. TOR is a kinase that phosphorylates phosphatidyl inositides and perhaps proteins, and is homologous in structure to the Ataxia-Teleangiectasia gene product. Yeast TOR is situated on the cytoplasmic surface of the vacuole. Rapamycin treatment of yeast delocalizes TOR from the vacuole, and elicits a phenotype equivalent to that seen with deletion of both TOR genes, and which resembles closely the response to nutrient deprivation; specifically, protein synthesis initiation is quickly and strongly inhibited, followed by G1 arrest. In mammalian cells, rapamycin, acting through mTOR, rapid inhibits both the phosphorylation of the 40 S ribosomal protein S6 (by inhibiting the p70 S6 kinase) and the phosphorylation and inactivation of eIF-4E-BP1, a negative regulator of the mRNA Cap binding protein, eIF-4E. Both these actions are accompanied by a selective inhibition in mRNA translation, which appears to underlie the inhibition of cell cycle progression. The studies proposed will uncover the physiologic and biochemical mechanisms by which mTOR participates in the regulation of mammalian cell growth by signalling to the translational apparatus. The cellular localization of mTOR will be defined, and the domains responsible for its localization mapped. The cellular binding site will be characterized. The regulation and properties of the mTOR-associated kinase catalytic activities will be defined. The cellular proteins that bind to mTOR will be isolated and examined for their role as effectors of mTORs signalling function. mTOR-regulated protein kinases or phosphatases active on eIF- 4E-BP1 will be sought, and the contribution of the p70 S6 kinase to the rapamycin-sensitive components of mTOR signalling will be established. These studies will define a new signal transduction pathway controlling cell growth in lymphoid and other cells.
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