Regulation of mTOR Complex 1 (mTORC1) by Raptor Ser863 and Multisite Phosphorylation

The rapamycin-sensitive mTOR complex 1 (mTORC1) promotes protein synthesis, cell growth, and cell proliferation in response to growth factors and nutritional cues. To elucidate the poorly defined mechanisms underlying mTORC1 regulation, we have studied the phosphorylation of raptor, an mTOR-interacting partner. We have identified six raptor phosphorylation sites that lie in two centrally localized clusters (cluster 1, Ser⁶⁹⁶/Thr⁷⁰⁶ and cluster 2, Ser⁸⁵⁵/Ser⁸⁵⁹/Ser⁸⁶³/Ser⁸⁷⁷) using tandem mass spectrometry and generated phosphospecific antibodies for each of these sites. Here we focus primarily although not exclusively on raptor Ser⁸⁶³ phosphorylation. We report that insulin promotes mTORC1-associated phosphorylation of raptor Ser⁸⁶³ via the canonical PI3K/TSC/Rheb pathway in a rapamycin-sensitive manner. mTORC1 activation by other stimuli (e.g. amino acids, epidermal growth factor/MAPK signaling, and cellular energy) also promote raptor Ser⁸⁶³ phosphorylation. Rheb overexpression increases phosphorylation on raptor Ser⁸⁶³ as well as on the five other identified sites (e.g. Ser⁸⁵⁹, Ser⁸⁵⁵, Ser⁸⁷⁷, Ser⁶⁹⁶, and Thr⁷⁰⁶). Strikingly, raptor Ser⁸⁶³ phosphorylation is absolutely required for raptor Ser⁸⁵⁹ and Ser⁸⁵⁵ phosphorylation. These data suggest that mTORC1 activation leads to raptor multisite phosphorylation and that raptor Ser⁸⁶³ phosphorylation functions as a master biochemical switch that modulates hierarchical raptor phosphorylation (e.g. on Ser⁸⁵⁹ and Ser⁸⁵⁵). Importantly, mTORC1 containing phosphorylation site-defective raptor exhibits reduced in vitro kinase activity toward the substrate 4EBP1, with a multisite raptor 6A mutant more strongly defective that single-site raptor S863A. Taken together, these data suggest that complex raptor phosphorylation functions as a biochemical rheostat that modulates mTORC1 signaling in accordance with environmental cues.