Molecular Mechanism of Insulin Resistance: Role of mTOR Signaling Pathways

150 p.Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 2008.In Chapter IV, I describe another investigation of mTOR signaling mechanisms. In this study I initially set out to identify nuclear export signals in mTOR. A systematic scan of the mTOR sequence revealed 16 peptides conforming to the canonical leucine-rich nuclear export signal, of which 3 were found by reporter assays to contain LMB-sensitive and leucine-dependent nuclear export activity. Unexpectedly, mTOR proteins with those conserved leucines mutated to alanines were unable to enter the nucleus. Further investigation revealed that the L982A/L984A and L1287A/L1289A mutations likely induced a global structural change in mTOR, whereas the L545A/L547A mutation directly impaired the nuclear import of the protein---potentially regulated by a nucleocytoplasmic shuttling signal. The loss of nuclear import was accompanied by the significantly reduced ability of the L545A/L547A mutant to activate S6K1 in cells. Most importantly, when nuclear import was restored in the L545A/L547A mutant by the addition of an exogenous NLS, signaling to S6K1 was rescued. Taken together, these observations suggest the existence of a nuclear shuttling signal in mTOR, and provide definitive evidence for the requirement of mTOR nuclear import in its cytoplasmic signaling to S6K1. (Abstract shortened by UMI.).U of I OnlyRestricted to the U of I community idenfinitely during batch ingest of legacy ETD