mTOR·RICTOR Is the Ser473 Kinase for Akt/Protein Kinase B in 3T3-L1 Adipocytes*

By R. Hresko, M. Mueckler. Journal of Biological Chemistry, 2005

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The insulin-signaling pathway leading to the activation of Akt/protein kinase B has been well characterized except for a single step, the phosphorylation of Akt at Ser-473. Double-stranded DNA-dependent protein kinase (DNA-PK), ataxia telangiectasia mutated (ATM) gene product, integrin-linked kinase (ILK), protein kinase Cα (PKCα), and mammalian target of rapamycin (mTOR), when complexed to rapamycin-insensitive companion of mTOR (RICTOR), have all been identified as playing a critical role in Akt Ser-473 phosphorylation. However, the apparently disparate results reported in these studies are difficult to evaluate, given that different stimuli and cell types were examined and that all of the candidate proteins have never been systematically studied in a single system. Additionally, none of these studies were performed in a classical insulin-responsive cell type or tissue such as muscle or fat. We therefore examined each of these candidates in 3T3-L1 adipocytes. In vitro kinase assays, using different subcellular fractions of 3T3-L1 adipocytes, revealed that phosphatidylinositol 3,4,5-trisphosphate-stimulated Ser-473 phosphorylation correlated well with the amount of DNA-PK, mTOR, and RICTOR but did not correlate with levels of ATM, ILK, and PKCα. PKCα was completely absent from compartments with Ser-473 phosphorylation activity. Although purified DNA-PK could phosphorylate a peptide derived from Akt that contains amino acid Ser-473, it could not phosphorylate full-length Akt2. Vesicles immunoprecipitated from low density microsomes using antibodies directed against mTOR or RICTOR had phosphatidylinositol 3,4,5-trisphosphate-stimulated Ser-473 activity that was sensitive to wortmannin but not staurosporine. In contrast, immunopurified low density microsome vesicles containing ILK could not phosphorylate Akt on Ser-473 in vitro. Small interference RNA knockdown of RICTOR, but not DNA-PK, ATM, or ILK, suppressed insulin-activated Ser-473 phosphorylation and, to a lesser extent, Thr-308 phosphorylation in 3T3-L1 adipocytes. Based on our cell-free kinase and small interference RNA results, we conclude that mTOR complexed to RICTOR is the Ser-473 kinase in 3T3-L1 adipocytes.

Subfields: Immunology, Molecular Biology, Oncology

View Paper: https://www.semanticscholar.org/paper/d67a11b24fee103fa21c0fa9169ac5c1e969da04

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Citations in the corpus

Ablation in mice of the mTORC components raptor, rictor, or mLST8 reveals that mTORC2 is required for signaling to Akt-FOXO and PKCalpha, but not S6K1. (2006, background): “This is surprising because RNAi-mediated knockdown of rictor reduced T308 phosphorylation in tandem with that of S473 in multiple cell types (Hresko and Mueckler, 2005; Sarbassov et al., 2005).”
Active-Site Inhibitors of mTOR Target Rapamycin-Resistant Outputs of mTORC1 and mTORC2 (2009, background): “Targeting mTORC2 by RNA interference (RNAi) [6,14], homologous recombination [15–17], or longterm rapamycin treatment [18] results in loss of Akt hydrophobic motif phosphorylation (S473), strongly implicating mTORC2 as the kinase responsible for phosphorylation of this site.”
In brief (2011, neutral): “…that TORC2 is associated with membranes, including the endoplasmic reticulum (ER) and Golgi apparatus, and that mTORC2 isolated from ER microsomes phosphorylates Akt Ser473 in vitro (Drenan et al., 2004; Hresko and Mueckler, 2005; Liu and Zheng, 2007; Schroder et al., 2007; Sturgill et al., 2008).”
Roles of the Raf/MEK/ERK and PI3K/PTEN/Akt/mTOR pathways in controlling growth and sensitivity to therapy-implications for cancer and aging (2011, background): “Thus, mTORC2 can function as the elusive PDK-2 which phosphorylates Akt on S473 in response to growth factor stimulation [47].”
The Molecular Bases of Training Adaptation (2007, background): “2001b), evidence is emerging that mTOR also phosphorylates AKT when bound to its associated protein rictor suggesting mutual activation under certain conditions (Hresko and Mueckler 2005; Sarbassov et al. 2005b; Sarbassov et al. 2006).”
SIN1/MIP1 maintains rictor-mTOR complex integrity and regulates Akt phosphorylation and substrate specificity. (2006, background): “However, despite a lack of genetic evidence, recent biochemical studies have strongly suggested that TORC2 is the elusive PDK2 for Akt Ser473 (Hresko and Mueckler, 2005; Sarbassov et al., 2005b).”
A Novel Function of eIF2α Kinases as Inducers of the Phosphoinositide-3 Kinase Signaling Pathway (2007, background): “Upon recruitment to the membrane, Akt/PKB is phosphorylated at threonine (Thr) 308 by PDK1 and at serine (Ser) 473 by the FKBP and Rapamycin-associated protein (FRAP)/mTOR-Rictor complex (Scheid and Woodgett, 2003; Hresko and Mueckler, 2005; Sarbassov et al., 2005).”
Targets of curcumin (2011, background): “mTORC2 regulates the actin cytoskeleton by mediating phosphorylation state of PKCα [127], and modulates cell survival in response to growth factors by phosphorylating its downstream effector Akt at the hydrophobic motif site, S473 [131-133].”
The mTOR Signalling Pathway in Human Cancer (2012, background): “mTORC2 is activated by growth factors, phosphorylates PKC-α, AKT (on Ser473) and paxillin (focal adhesion-associated adaptor protein), and regulates the activity of the small GTPases Rac and Rho related to cell survival, migration and regulation of the actin cytoskeleton [12,38,39].”
mSin1 is necessary for Akt/PKB phosphorylation, and its isoforms define three distinct mTORC2s. (2006, background): “mTORC2 is a hydrophobic motif kinase for the cell-survival protein Akt/PKB [2, 3] and, here, we identify mSin1 as a component of mTORC2 but not mTORC1.”
Sphingolipids, insulin resistance, and metabolic disease: new insights from in vivo manipulation of sphingolipid metabolism. (2008, background): “The mammalian target of rapamycin (mTOR)-Rictor protein complex phosphorylates a regulatory serine (S473) on the C terminus of Akt/PKB (175, 176).”