| An important control of mammalian cell growth and proliferation is at the level of protein synthesis. Translation initiation is regulated in part by a signal transduction cascade involving mTOR (mammalian target of rapamycin) that results in the phosphorylation of p70S6K1 and 4E-BPI. The biochemical properties of mTOR and the regulatory mechanisms of its signaling, however, were largely unknown, and are the focus of my investigations reported here.; Systematic deletions of mTOR were carried out and their effects on mTOR subcellular localization, catalytic activity, and ability to signal to p70 S6K1 and 4E-B P1 were examined. Endogenous mTOR distributed between the cytosol and intracellular membrane, and the two pools of protein displayed similar intrinsic kinase activity. The mid-region of mTOR, spanning approximately amino acids 500–2000, contained several segments that were each sufficient for membrane targeting of this protein. The entire C-terminal ∼1000 amino acids were absolutely required for intrinsic kinase activity of mTOR, suggesting a biochemical mechanism unprecedented by any other known kinase. Meanwhile, the N-terminal ∼1400 amino acids were shown to be necessary for mTOR to signal to both p70S6K1 and 4E-BP1, presumably by recruiting multiple effectors.; Unexpectedly, mTOR has been found to shuttle between the nucleus and the cytoplasm, and this shuttling is required for its cytoplasmic signaling. I have further investigated the regulation and function of mTOR cytoplasmic-nuclear shuttling. A systematic screen of mTOR revealed a rev-like NES sequence within mTOR at amino acids 1281–1289, which displayed robust nuclear export activity in a reporter system. Mutation of the conserved leucines within this export site resulted in a loss of downstream signaling in vivo, despite retention of mTOR catalytic activity. Interestingly, the mTOR 1281–1289L/A mutant showed cytoplasmic staining similar to wild type mTOR, and did not accumulate in the nucleus upon LMB treatment. This suggests that the 1281–1289L/A mutations in mTOR disrupt nucleocytoplasmic shuttling activity. The nucleocytoplasmic shuttling of mTOR is likely a mechanism to lessen the probability of aberrant signaling, a necessary control for a protein that has an essential role in cell growth and proliferation. |
