The identification of the mTOR-regulated phosphoproteome and a mediator of feedback inhibition to P13K-Akt

Abstract

The mTOR protein kinase nucleates two complexes, mTORC1 and mTORC2. Collectively, the two complexes regulate processes important for cell growth and proliferation, including protein synthesis, autophagy, metabolism, and cytoskeletal maintenance. Despite this diverse array of cellular functions, few mTOR substrates are known. To address this deficit, we defined the mTOR-regulated phosphoproteome by quantitative mass spectrometry and characterized the primary sequence motif specificity of mTOR using positional scanning peptide libraries. We found that the phosphorylation response to insulin is largely mTOR-dependent and that mTOR regulates the phosphorylation of many proteins not presently appreciated to be linked to mTOR signaling. The mTOR kinase, moreover, exhibits a preference for proline, hydrophobic, and aromatic residues at the +1 position which is unique among all kinases previously profiled. Grbl 0 is an adaptor protein and negative regulator of growth factor signaling identified as an mTORC1 substrate that mediates the inhibition of P13K typical of cells lacking TSC2, a tumor suppressor and negative regulator of mTORC1. Phosphorylation of Grbl 0 is important for its inhibitory function as well as for its stability. While acute mTORC1 inhibition results in changes in Grb1O and IRS1 phosphorylation which partially reactivates Akt in TSC2-null cells, chronic mTORC1 inhibition causes Grbl 0 destabilization, IRS protein stabilization, and a complete resensitization of Akt to insulin and IGF-1. These changes in Grbl 0 and IRS protein abundance are likely to be the most important effects of mTOR inhibitors to consider in their clinical use. Finally, the discovery of Grbl 0 as an mTORC1 substrate validates our phosphoproteomic approach and suggests that the other potential downstream effectors we identified may also serve as starting points for new areas of investigation in mTOR biology.