The mammalian target of rapamycin (mTOR) is a highly conserved serine/threonine protein kinase which controls cell growth, and is an important drug target for cancer therapy. mTOR is part of the phosphatidylinositol 3-kinase (PI3K)/Akt/Rheb signaling pathway; genetic alterations in this pathway lead to benign tumors and contribute to malignancy. Rheb, a member of the Ras family of small GTPases, directly interacts with the mTOR catalytic domain, independently of Rheb's nucleotide-loaded state. When Rheb changes from an inactive GDP-bound, to an active GTP-bound state, a conformational change occurs in its switch I region, the region which binds mTOR. We hypothesize that Rheb GTP loading induces a conformational change in mTOR leading to kinase activation. mTOR's catalytic activity may also be regulated by interactions between multiple, poorly characterized domains flanking the catalytic domain in the C-terminus. Deuterium exchange mass spectrometry (DXMS) involves hydrogen/deuterium exchange of protein backbone amide hydrogens, followed by rapid and sensitive localization of deuterated residues. It is a powerful method to study protein domain structure and dynamics, because exchange rates of a protein's amide protons are proportional to their solvent accessibility, and changes in exchange rates reflect dynamic changes in protein conformation and/or changes resulting from protein binding interactions. We propose using DXMS to: (i) map the interaction interface between Rheb and mTOR, and probe for Rheb-GTP-induced conformational changes in mTOR; and (ii) to determine the boundaries between domains in mTOR's C-terminal region, and study their interaction and function in regulating kinase activity. Specific residues involved in the Rheb/mTOR interface and in inter-domain contacts identified by DXMS will be confirmed by site-directed mutagenesis; the mutants' effects on mTOR conformation, Rheb interaction, and kinase activity will be determined. These studies will provide a better understanding of the mechanisms regulating mTOR activity, and lay the foundation for future crystallographic and detailed structural analyses of mTOR. During this work, the principal investigator will be assisted by several senior investigators, and the award will allow him to transition to an independent investigative career. ? ? ?
