Protor-1 is required for efficient mTORC2-mediated activation of SGK1 in the kidney

Abstract

The mTOR (mammalian target of rapamycin) protein kinase is an important regulator of cell growth and is a key target for therapeutic intervention in cancer. Two complexes of mTOR have been identified: complex 1 (mTORC1), consisting of mTOR, Raptor (regulatory associated protein of mTOR) and mLST8 (mammalian lethal with SEC13 protein 8) and complex 2 (mTORC2) consisting of mTOR, Rictor (rapamycin-insensitive companion of mTOR), Sin1 (stress-activated protein kinase-interacting protein 1), mLST8 and Protor-1 or Protor-2. Both complexes phosphorylate the hydrophobic motifs of AGC kinase family members: mTORC1 phosphorylates S6K (S6 kinase), whereas mTORC2 regulates phosphorylation of Akt, PKCα (protein kinase Cα) and SGK1 (serum- and glucocorticoid-induced protein kinase 1). To investigate the roles of the Protor isoforms, we generated single as well as double Protor-1- and Protor-2-knockout mice and studied how activation of known mTORC2 substrates was affected. We observed that loss of Protor-1 and/or Protor-2 did not affect the expression of the other mTORC2 components, nor their ability to assemble into an active complex. Moreover, Protor knockout mice display no defects in the phosphorylation of Akt and PKCα at their hydrophobic or turn motifs. Strikingly, we observed that Protor-1 knockout mice displayed markedly reduced hydrophobic motif phosphorylation of SGK1 and its physiological substrate NDRG1 (N-Myc downregulated gene 1) in the kidney. Taken together, these results suggest that Protor-1 may play a role in enabling mTORC2 to efficiently activate SGK1, at least in the kidney.

Ubl4A is required for insulin-induced Akt plasma membrane translocation through promotion of Arp2/3-dependent actin branching

“…The beads were then washed and the immunocomplexes subjected to immunoblotting analysis. The immunocomplex kinase assay was performed as described previously (36), in which mTOR was immunoprecipitated by anti-mTOR antibody and validated by immunoblotting of Rictor, a component of mTORC2. The mRORC2 immunocomplex was incubated for 10 min at 30°C with purified unactive (unphorsphorylated) Akt protein (1 μg; Millipore) in a kinase reaction buffer containing nonradioactive ATP (100 μM).…”

Mechanistically distinct cancer-associated mTOR activation clusters predict sensitivity to rapamycin

“…PRAS40 and regulatory associated protein of mTOR (RAPTOR) are specific to mTORC1 (5-10), whereas RIC-TOR, mSIN1, and PROTOR1/2 are specific to mTORC2 (2,(11)(12)(13)(14)(15). mTORC1 integrates nutrient and growth factor signaling to promote anabolic metabolism, such as protein synthesis and lipid synthesis (16)(17)(18), and to inhibit catabolic pathways, such as lysosome biogenesis and autophagy (2,(19)(20)(21)(22)(23)(24)(25).…”

The Target of Rapamycin: Structure and Functions

“…Knockdown of these two proteins did not cause significant disruption of both complex integrity and kinase activity of mTORC2. Protor1 is required for the phosphorylation of SGK1, but not of Akt and PKC, specifically in mouse kidney (Pearce et al 2011). These findings suggest that this non-conserved interactor might regulate mTORC2 function in a tissue-and target-specific manner (Pearce et al 2011).…”

“…Protor1 is required for the phosphorylation of SGK1, but not of Akt and PKC, specifically in mouse kidney (Pearce et al 2011). These findings suggest that this non-conserved interactor might regulate mTORC2 function in a tissue-and target-specific manner (Pearce et al 2011). DEPTOR (DEPDC6, DEP domain-containing protein 6), is a negative regulator of mTOR that associates with both mTORC1 and mTORC2 (Peterson et al 2009;Proud 2009).…”