Rictor Acetylation Promotes mTORC2-Medicated Akt Phosophorylation

The Ser/Thr protein kinase mTOR is a critical regulator of cell growth, survival and metabolism in response to growth factors. mTOR functions in at least two distinct multiprotein complexes known as mTOR complex (mTORC) -1 and -2. mTORC2 phosphorylates Akt at S473, an event required for increased Akt kinase activity. Although it is known that the mTORC2 components Rictor and mSin1 are required for the stability and activity of mTORC2, little is known about post-translational modifications that regulate mTORC2. Work presented in this thesis identifies Rictor acetylation as a positive regulator of mTORC2-mediated phosphorylation of Akt at S473. Inhibition of deacetylases, including the NAD + - dependent Sirtuins, promotes Rictor acetylation and IGF1-mediated Akt phosphorylation at S473. While mapping the major acetylated regions contained within Rictor we identified two important regions: 1) a region critical for interaction with mSin1.1 and LST8 that subsequently promotes mTORC2 stabilization, and 2) an adjacent acetylated region localized between amino acids 1041 and 1137. Analysis of this acetylated region in Rictor identified 9 lysines that could potentially be post-translationally modified, each of which we divided into 4 distinct groups or modules (M1-M4). Site-directed mutagenesis (lysine to arginine) of each of the lysines located within two of the modules (M2 and M4) reduced Rictor acetylation and IGF1-dependent mTORC2 kinase activity. These results indicate that multiple-site acetylation of Rictor signals for increased activation of mTORC2 in response to IGF1 stimulation. To further examine iii whether other post-translational modifications contribute to Rictor acetylation, evidence provided here indicates that the O-linked N-acetylglucosamine (O- GlcNAc) moiety potentiates Rictor acetylation. This is biologically significant because heightened O-GlcNAcylation of Rictor not only increased acetylation, but promoted mTORC2-mediated phosphorylation of Akt at S473. Since Akt is known to promote metabolic pathways that generate metabolite signaling molecules required for acetylation and O-GlcNAc modification, post-translational modifications that target Rictor may comprise a positive feedback loop during conditions of nutrient abundance.

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