Integrity of mTORC2 is dependent on the rictor Gly-934 site

Abstract

Growth factor signaling coupled to activation of the phosphatidylinositol-3-OH kinase (PI3K)/Akt pathway plays a crucial role in the regulation of cell proliferation and survival. The key regulatory kinase of Akt has been identified as mammalian target of rapamycin complex 2 (mTORC2), which functions as the PI3K-dependent Ser-473 kinase of Akt. This kinase complex is assembled by mTOR and its essential components rictor, Sin1 and mLST8. The recent genetic screening study in Caenorhabditis elegans has linked a specific point mutation of rictor to an elevated storage of fatty acids that resembles the rictor deficiency phenotype. In our study, we show that in mammalian cells the analogous single rictor point mutation (G934E) prevents the binding of rictor to Sin1 and the assembly of mTORC2, but this mutation does not interfere with the binding of the rictor-interacting protein Protor. A substitution of the rictor Gly-934 residue to a charged amino acid prevents formation of the rictor/Sin1 heterodimer. The cells expressing the rictor G934E mutant remain deficient in the mTORC2 signaling, as detected by the reduced phosphorylation of Akt on Ser-473 and a low cell proliferation rate. Thus, although a full length of rictor is required to interact with its binding partner Sin1, a single amino acid of rictor Gly-934 controls its interaction with Sin1 and assembly of mTORC2.

Regulatory effects of mTORC2 complexes in type I IFN signaling and in the generation of IFN responses

“…Rictor is an essential component of mTORC2 and is required for assembly and function of this complex (22)(23)(24)(25)(27)(28)(29), whereas mTORC2 phosphorylates serine 473 site in the hydrophobic motif of AKT (22)(23)(24)29). In initial studies, we examined the ability of IFN-α to induce AKT phosphorylation in MEFs with targeted disruption of the Rictor gene (30).…”

Rictor Negatively Regulates High-Affinity Receptors for IgE-Induced Mast Cell Degranulation

“…LAD2 cells were transfected with human myc-tagged rictor in a pRK5 plasmid kindly supplied by Dr. D. D. Sarbassov (39) as described (40). Briefly, 2 × 10 6 LAD2 cells were transfected using the Amaxa Nucleofector II system and then plated out in StemPro-34 medium supplemented with SCF and 100 ng/ml biotinylated human IgE for 6 h. This time point was chosen to achieve near maximal expression of rictor without discernible effects on cell morphology or viability.…”

Autoregulation of the Mechanistic Target of Rapamycin (mTOR) Complex 2 Integrity Is Controlled by an ATP-dependent Mechanism

“…It is conceivable that phosphorylation of the Ser-260 site on SIN1 by mTOR hinders a recognition site responsible for the disintegration of mTORC2 and lysosomal degradation of SIN1. It has been recently reported that a proper protein folding is critical in the assembly of mTORC2 and that a single amino acid substitution on rictor has precluded the assembly of mTORC2 (23). Because the endoplasmic reticulum has been identified as the main localization site of mTORC2 (27), this phosphorylation-dependent degradation pathway might be linked to proper protein folding monitoring by endoplasmic reticulum quality control that tags mTORC2 containing the non-phosphorylated form of SIN1 for disintegration and lysosomal degradation.…”

“…5A), the stable expression of SIN1 also indicates an important role of its Ser-260 site in controlling the integrity of mTORC2. Interestingly, regulation of the mTORC2 integrity by SIN1 phosphorylation on Ser-260 is independent of its binding partner rictor (23)(24)(25), because the SIN1 phosphomutants did not show any differences in binding to rictor. We also found that the abundance of mTORC2 induced by mimicking the mTOR-dependent phosphorylation of SIN1 carried out its functional activity not only by the elevated basal phosphorylation of Akt but also by a higher cell proliferation rate (Fig.…”