Evidence for Direct Activation of mTORC2 Kinase Activity by Phosphatidylinositol 3,4,5-Trisphosphate*
Xiaoqing Gan
Jiyong Wang
Bing Su
Dianqing Wu
To whom correspondence should be addressed. E-mail:dan.wu@yale.edu.
Received 2010 Oct 15; Revised 2011 Feb 7; Issue date 2011 Apr 1.
Abstract
mTORC2 (mammaliantargetofrapamycincomplex2) plays important roles in signal transduction by regulating an array of downstream effectors, including protein kinase AKT. However, its regulation by upstream regulators remains poorly characterized. Although phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3) is known to regulate the phosphorylation of AKT Ser473, the hydrophobic motif (HM) site, by mTORC2, it is not clear whether PtdIns(3,4,5)P3 can directly regulate mTORC2 kinase activity. Here, we used two membrane-docked AKT mutant proteins, one with and the other without the pleckstrin homology (PH) domain, as substrates for mTORC2 to dissect the roles of PtdIns(3,4,5)P3 in AKT HM phosphorylation in cultured cells andin vitro kinase assays. In HEK293T cells, insulin and constitutively active mutants of small GTPase H-Ras and PI3K could induce HM phosphorylation of both AKT mutants, which was blocked by the PI3K inhibitor LY294002. Importantly, PtdIns(3,4,5)P3 was able to stimulate the phosphorylation of both AKT mutants by immunoprecipitated mTOR2 complexes in anin vitro kinase assay. In bothin vivo andin vitro assays, the AKT mutant containing the PH domain appeared to be a better substrate than the one without the PH domain. Therefore, these results suggest that PtdIns(3,4,5)P3 can regulate HM phosphorylation by mTORC2 via multiple mechanisms. One of the mechanisms is to directly stimulate the kinase activity of mTORC2.
Keywords: AKT PKB, mTOR Complex (mTORC), Phosphatidylinositol, Phosphatidylinositol Signaling, Phosphatidylinositol 3-Kinase
Introduction
mTOR (mammaliantargetofrapamycin) is an evolutionarily conserved Ser/Thr protein kinase, which plays an integral role in coordinating cell growth and division in response to growth factors, nutrients, and other microenvironmental changes of the cell (1,2). This kinase is found in mTORC1 and mTORC2, two structurally and functionally distinct complexes, which are also conserved from yeasts to mammals. In addition to shared mTOR and mLST8, distinct components of these two complexes have been reported: Raptor and PRAS40 for mTORC1 (3–7) and Rictor, SIN1, and PRR5/PRR5L for mTORC2 (8–12). Two mTOR complexes have distinct cellular functions and are regulated differently. The mTORC1 activity is sensitive to inhibition by rapamycin, which regulates diverse cellular processes, including protein synthesis, ribosome biogenesis, transcription, and autophagy, some of which are regulated through its direct substrates S6 kinases and the eukaryotic initiation factor 4E-binding protein 1 (13–16).
The mTORC2 activity is resistant to rapamycin, at least in short term treatment (17,18). It phosphorylates the hydrophobic motif (HM)2 sites of several AGC kinases, including AKT, PKC, and SGK1, to activate their kinase activities (19–23). Studies using cells derived from Rictor, mSin1, or mLST8 knock-out mice show that intact mTORC2 is necessary for AKT HM phosphorylation (12,24–26). In addition, mTORC2 phosphorylates the turn motif site of AKT and regulates AKT stability, but this phosphorylation is independent of growth factor regulation (19,21). Moreover, mTORC2 can also regulate actin cytoskeleton reorganization through yet to be characterized mechanisms (10,22,27,28). Many extracellular stimuli, including growth factors, G protein-coupled receptor ligands, and cytokines, stimulate AKT HM phosphorylation in a PI3K-dependent manner. The PI3K-mediated regulation of AKT HM phosphorylation was initially attributed to PtdIns(3,4,5)P3-mediated membrane translocation and conformational changes of AKT. However, recent evidence suggests that the mTORC2 kinase can be directly regulated. The mTORC2 protein immunoprecipitated from growth factor-treated cells shows higher kinase activity in anin vitro kinase assay, and growth factors was initially reported to stimulate the phosphorylation of Ser2481 of mTOR presented in mTORC2 but not mTORC1 (29). However, more recently insulin was shown to stimulate the phosphorylation of Ser2481 of mTOR associated with both mTORC1 and -2 in a wortmannin-dependent manner (30). Furthermore, mTORC2 phosphorylates SGK1 in response to growth factors even though SGK1 lacks a PH domain and is activated independently of membrane recruitment. Nevertheless, it is not known how mTORC2 kinase activity is regulated.
In this report, we characterized the role of PI3K and its product PtdIns(3,4,5)P3 in regulation of AKT HM phosphorylation using two membrane-docked AKT mutant proteins. We found that PtdIns(3,4,5)P3 regulates AKT HM phosphorylation via at least three mechanisms. We confirmed that membrane translocation and conformation changes may contribute to PtdIns(3,4,5)P3-stimulated AKT HM phosphorylation, but importantly, we demonstrated for the first time that PtdIns(3,4,5)P3 could directly stimulate the intrinsic mTORC2 kinase activity.
EXPERIMENTAL PROCEDURES
Materials
Reagents were obtained from the following sources: protein A/G-PLUS-Sepharose and the antibody to Ras from Santa Cruz Biotechnology; the antibodies to mTOR, phospho-mTOR (Ser2481), AKT, and phospho-AKT (Ser473) from Cell Signaling Technology; Lipofectamine Plus transfection reagent and insulin from Invitrogen; LY294002 from ALEXIS Biochemicals; 1,2-dioleoyl-sn-glycero-3 (phospho-l-serine), 1,2-dioleoyl-sn-glycero-3-phospho- (1′-myo-inositol 3′,4′,5′-trisphosphate) (18:1 PI(3,4,5)P3), and 1,2-dioleoyl-sn-glycero-3-phospho-(1′-myo-inositol-4′,5′-bisphosphate) (18:1 PI(4,5)P2) from Avanti Polar Lipid.
Cell Culture and Stimulation
HEK293T cells were maintained in DMEM with 4.5 g/liter glucose supplemented with 10% FBS. Transient transfection was performed using the Lipofectamine Plus reagent. Three hours post transfection, cells were changed to serum-free DMEM medium for overnight. Before insulin (2 μg/ml) or LY294002 (10 μm) treatment, the cells were incubated with phosphate-buffered saline for 60 min.
Preparation of GST Fusion Protein
Human GST-AKT(KT) and GST-ΔPH-AKT(KT) were expressed inEscherichia coli BL21(DE3). After isopropyl-1-thio-β-d-galactopyranoside (100 μm) induction at 22 °C for 24 h, proteins were extracted with a buffer containing 20 mm Tris-HCl, pH 7.5, 50 mm NaCl, 0.1% Triton X-100, 1 mm phenylmethylsulfonyl fluoride, and 0.5 mg/ml lysozyme. After high speed centrifugation, the supernatant was loaded on glutathione-agarose beads and eluted with the buffer containing 20 mm Tris-HCl, pH 7.5, 50 mm NaCl, 0.1% Triton X-100, 5% glycerol, and 5 mm reduced glutathione. Human GST-PKCα was acquired from SignalChem.
Immunoprecipitation and Kinase Assays
Immunoprecipitation of mTOR complexes and kinase assays were performed as described previously (23). HEK293T cell extracts harvested from a 10-cm plate were used for each immunoprecipitation condition. The cells were lysed on ice for 20 min in the lysis buffer (40 mm HEPES, pH 7.5, 120 mm NaCl, 0.3% CHAPS, 1 mm EDTA, 10 mm pyrophosphate, 10 mm glycerophosphate, 50 mm NaF, and EDTA-free protease inhibitors). After centrifugation, the supernatant was incubated with the Rictor antibody at 4 °C for 90 min, followed by incubation with protein A/G-PLUS-agarose for another hour. Immunocomplexes were washed four times in the lysis buffer and twice with mTORC2 kinase buffer (25 mm HEPES, pH 7.5, 100 mm potassium acetate, 2 mm MgCl2). For the kinase assay reaction, immunocomplexes were incubated in a final volume of 30 μl for 30 min at 37 °C in the mTORC2 kinase buffer containing 500 ng of GST fusion substrates and 500 μm ATP. The reaction was terminated by adding 10 μl of 4× SDS sample buffer, followed by Western analysis.
RESULTS AND DISCUSSION
The ultimate goal of this study was to examine the effect of PtdIns(3,4,5)P3 on mTORC2 kinase activity. Although the HM site of AKT (Ser473) is an excellent substrate for mTORC2 kinase, simple examination of HM phosphorylation of the full-length AKT may not allow us to assess the effect of PtdIns(3,4,5)P3 on mTORC2 kinase activity. This is because PtdIns(3,4,5)P3 has a role in AKT recruitment to plasma membranes, where mTORC2 phosphorylates AKT HM. To eliminate this membrane translocation effect on AKT, we attached the myristylation signal to AKT. To avoid possible autophosphorylation of AKT HM, we also mutated Lys179, which is involved in ATP binding, and Thr308, which contributes to kinase activation after its phosphorylation by phosphoinositide-dependent kinase-1. The resultant AKT mutant, designated Myr-AKT(KT) (Fig. 1A), was expressed in HEK293T cells that were stimulated with insulin and/or treated with the PI3K inhibitor LY294002. The phosphorylation of Myr-AKT(KT) HM detected by an phospho-Thr473 AKT antibody was enhanced by insulin and inhibited by LY294002 (Fig. 1B). This result suggests that PtdIns(3,4,5)P3 has a role more than substrate translocation in regulating HM phosphorylation.
FIGURE 1.
Insulin and Ras regulate membrane-docked HM phosphorylation via PI3K.A, schematic representation of AKT mutants.B, effects of LY294002 (LY) on insulin-induced HM phosphorylation of Myr-AKT(KT) and Myr-ΔPH-AKT(KT) expressed in HEK293T cells. The transfected cells were incubated with serum-free medium for overnight and HBSS for 1 h before they were stimulated with insulin (2 μg/ml) for 30 min with or without pretreatment of LY294002 (15 min).NS, nonspecific band;Endo p473-AKT, endogenous p-473AKT.C andD, the roles of Ras and PI3K in the regulation of HM phosphorylation of Myr-ΔPH-AKT(KT). HEK293 cells were cotransfected with Myr-ΔPH-AKT(KT) and H-Ras-G12V, H-Ras-S17N, or PI3K-CAAX (PI3K*). All of the experiments were repeated at least three times. Quantification of the phosphorylated AKT contents normalized against total AKT contents from all of these independent experiments are shown in the bar graphs. Data are presented as means ± S.D. (Student'st test). Representative blots are also shown.
The PH domain of AKT binds to PtdIns(3,4,5)P3 and is involved in PtdIns(3,4,5)P3-mediated AKT translocation. In addition to this translocation role, the PH domain, upon interaction with PtdIns(3,4,5)P3, can induce AKT conformational change (31), which may make the AKT a better substrate for phosphorylation by mTORC2. To eliminate any possible effect of the PH domain, we generated the Myr-ΔPH-AKT(KT) mutant that lacks the PH domain (Fig. 1A). When this mutant was tested, the phospho-Thr473 AKT antibody detected weaker HM phosphorylation of this mutant than that of Myr-AKT(KT) (Fig. 1B). These results suggest that the AKT PH domain may render AKT a better substrate for HM phosphorylation. Nonetheless, insulin could still induce HM phosphorylation on Myr-ΔPH-AKT(KT), which was inhibited by LY294002 (Fig. 1B). We also examined the effects of insulin and LY294002 treatment on Ser450 phosphorylation of both AKT substrates. Consistent with previous findings (19,21), phospho-Ser450 contents were not affected by either treatment (Fig. 1B). Thus, these results together suggest that insulin can regulate AKT HM phosphorylation independently of the PH domain, but in a PI3K-dependent manner.
The Ras protein is one of the signaling molecules for growth factors, including insulin, and has been implicated in the regulation of mTORC2 (32,33). We examined the role of Ras in the HM phosphorylation of Myr-ΔPH-AKT(KT). Expression of a constitutive active mutant of H-Ras (G12V) strongly induced HM phosphorylation of Myr-ΔPH-AKT(KT) (Fig. 1C), whereas expression of a dominant negative mutant of H-Ras (S17N) markedly blocked insulin-induced HM phosphorylation (Fig. 1C). Because Ras can activate PI3K (34–36), we tested the effect of LY294002 on active Ras-mediated HM phosphorylation. LY294002 almost completely blocked Ras-G12V-induced HM phosphorylation (Fig. 1D). To more directly demonstrate that PI3K can induce HM phosphorylation of Myr-ΔPH-AKT(KT), a constitutive active PI3K (PI3K-CAAX) (37) was expressed, and it increased the HM phosphorylation (Fig. 1D). These results together support a conclusion that insulin and its downstream signaling molecule Ras may stimulate AKT HM phosphorylation via PI3K.
Given that mTORC2 is an upstream kinase for phosphorylation of AKT HM, we tested whether LY294002 could affect mTOR-Ser2481 phosphorylation. This phosphorylation is stimulated by insulin, and its phosphorylation in mTORC2 may require the intact complex (29), even though the phosphorylation also occurs in mTORC1 (30). We found that insulin could induce the phosphorylation in HEK293T cells, which could be inhibited by LY294002 (Fig. 2A). In addition, expression of the constitutive active PI3K or H-Ras in HEK293T cells increased mTOR-Ser2481 phosphorylation, which could also be blocked by LY294002 (Fig. 2B). These results are consistent with the conclusion that Ras and PI3K may be upstream regulators of mTOR in response to insulin.
FIGURE 2.
Insulin, Ras, and PI3K increase mTORC2-Ser2481 phosphorylation.A, HEK293 cells were treated as described in the legend toFig. 1B followed by Western analysis.B, HEK293 cells were transfected with plasmids encoding LacZ, PI3K-CAAX or Ras-G12V, followed by LY294002 (LY) treatment and Western analysis. Data analysis and presentation are as described in the legend toFig. 1. Data are presented as means ± S.D. (Student'st test).
To more directly assess the roles of PI3K in insulin-mediated regulation of mTORC2 kinase activity, we immunoprecipitated mTORC2 using a RICTOR antibody from serum/nutrient-starved cells stimulated with insulin and treated with/without LY294002 and carried out anin vitro kinase assay using recombinant GSK-AKT(KT) prepared fromE. coli as the substrate. The immunocomplexes pulled down from cells treated with insulin caused significantly greater HM phosphorylation of GST-AKT(KT) than those from nontreated cells (Fig. 3A). In addition, treatment of cells with LY294002 prior to insulin stimulation could block insulin-mediated increases in HM phosphorylation by immunoprecipitated mTOR (Fig. 3A). We also tested another known mTORC2 substrate, PKCα. Immunoprecipitated mTORC2 stimulated phosphorylation of recombinant GST-PKCα at Ser638, which was blocked by LY294002 treatment (Fig. 3A). Because LY294002 inhibits PI3K activity by competitively inhibiting ATP binding to the catalytic subunit of PI3K, there is a possibility that LY294002 might directly inhibit ATP binding to mTOR. To exclude this possibility, we treated the immunocomplexes pulled down by the mTOR antibody with LY294002 before the substrate and ATP were added to initiate the kinase assay. Direct treatment of immunoprecipitated mTOR by LY294002 did not inhibit its phosphorylation of GST-AKT(KT) at HM (Fig. 3B). Therefore, these results indicate that insulin can stimulate mTORC2 kinase activity via PI3K.
FIGURE 3.
Insulin stimulates mTORC2 kinase activity via PI3K.A, HEK293 cells were treated as in described in the legend toFig. 1B and lysed. The cell lysates were used for immunoprecipitation (IP) with a control antibody (IgG) or Rictor antibody. Immunocomplexes were used to phosphorylate GST-AKT(KT) or GST-PKCα in anin vitro kinase assay. The kinase assay mixtures were subjected to Western analysis by an antibody to phospho-Ser473 AKT, phospho-pan PKC (which recognizes the phospho-HM site of PKCα), Rictor, AKT, or mTOR.B, cells were stimulated with insulin, and their lysates were subjected to immunoprecipitation with an mTOR antibody. The immunocomplexes were then treated with LY294002 (LY) for 15 min before they were used in thein vitro kinase assay to phosphorylate GST-AKT(KT). Data analysis and presentation are as described in the legend toFig. 1. Data are presented as means ± S.D. (Student'st test).
To determine whether the PI3K product PtdIns(3,4,5)P3 can directly regulate mTOR-mediated HM phosphorylationin vitro, we tested the effects of lipid vesicles containing PtdIns(3,4,5)P3 in thein vitro kinase assays with immunoprecipitated mTORC2. In addition to recombinant GST-AKT(KT) and GST-PKCα, GST-ΔPH-AKT(KT) was also used as a substrate. The immunocomplexes pulled down by the RICTOR antibody from serum/nutrient-starved HEK293T cells were incubated with or without phosphatidylethanolamine (PE) vesicles containing 0, 5, and 25 ng/ml of PtdIns(3,4,5)P3 before the kinase assay was initiated by adding the substrates and ATP. HM phosphorylation of GST-ΔPH-AKT(KT), GST-AKT(KT) (Fig. 4A), and GST-PKCa (Fig. 4B) was increased significantly in the samples treated with PtdIns(3,4,5)P3-containing PE vesicles compared with those treated with the PE vesicle alone. The PE vesicle itself, or the one containing PtdIns(4,5)P2, had little effect on the phosphorylation (Fig. 4A). In addition, the increase in HM phosphorylation was dependent on the PtdIns(3,4,5)P3 dose. Moreover, PtdIns(3,4,5)P3-containing PE vesicles induced significantly stronger HM phosphorylation of GST-AKT(KT) than that of GST-ΔPH-AKT(KT), whereas HM phosphorylation of GST-AKT(KT) and GST-ΔPH-AKT(KT) remained the same in the absence of PtdIns(3,4,5)P3 regardless of the presence of PE vesicles (Fig. 4A). These results indicate that PtdIns(3,4,5)P3 can directly activate the kinase activity of immunoprecipitated mTORC2. In addition, they confirm that PtdIns(3,4,5)P3 has additional roles in mTORC2-mediated HM phosphorylation probably via its interaction with the PH domain of the substrate.
FIGURE 4.
PIP3 can directly activate mTORC2 kinase activity.A, HEK293 cells were lysed, and their lysates were subjected to immunoprecipitation (IP) with the Rictor antibody. The immunocomplexes were incubated with PE (100 ng/ul) vesicles containing 0 or 25 ng/ml of PtdIns(4,5)P2, 5 or 25 ng/ml of PtdIns(3,4,5)P3 and GST-ΔPH-AKT(KT), GST-AKT(KT), or GST-PKCα in anin vitro kinase assay. Data analysis and presentation are as described in the legend toFig. 1.B, a model for the regulation of AKT HM phosphorylation by insulin/Ras/PI3K signaling. PtdIns(3,4,5)P3, whose contents are elevated upon insulin stimulation, may regulate the HM phosphorylation via at least three mechanisms: direct stimulation of the mTORC2 kinase activity, recruitment of AKT to the plasma membrane, and making AKT a better substrate. The latter two mechanisms appear to be mediated by the N-terminal PH domain.
In this report, we used two AKT mutants as probes to investigate the role of PI3K and PtdIns(3,4,5)P3 in their regulation of mTORC2-mediated HM phosphorylation and found that PtdIns(3,4,5)P3 regulates the phosphorylation via at least three different mechanisms as illustrated inFig. 4B. Previous studies have demonstrated that the PH domain at the AKT N terminus has an important role in membrane translocation of the AKT molecule and is thus required for ligand-stimulated HM phosphorylation. Our studies indicate that the PH domain may have an additional role,i.e. its presence may make a better substrate for HM phosphorylation. Although we postulate that this may be the result of a conformational change induced upon the interaction of the PH domain with PtdIns(3,4,5)P3, other possible explanations may exist. Although Ras has been implicated in the regulation of mTORC2, our results suggest that it may primarily act through PI3K and PtdIns(3,4,5)P3. In addition to the effects mediated by the PH domain, ourin vitro kinase assay results clearly demonstrated that PtdIns(3,4,5)P3 directly regulates the kinase activity of mTORC2. However, we do not know the precise mechanisms by which PtdIns(3,4,5)P3 regulates mTORC2 kinase activity. PtdIns(3,4,5)P3, may directly regulate the kinase activity of mTOR or one of the components of the complex. Several of the mTORC2 components contain PH domains and may subject to regulation by PtdIns(3,4,5)P3. Further work is needed to characterize these detailed mechanisms.
This work was supported, in whole or in part, by National Institutes of Health Grants HL070694, CA132317, and CA139395 (to D. W.).
- HM
- hydrophobic motif
- PtdIns(3,4,5)P3
- phosphatidylinositol 3,4,5-trisphosphate
- PH
- pleckstrin homology
- PE
- phosphatidylethanolamine
- KT
- K179M and T308A.
REFERENCES
- 1.Polak P., Hall M. N. (2009) Curr. Opin. Cell. Biol. 21, 209–218 [DOI] [PubMed] [Google Scholar]
- 2.Wullschleger S., Loewith R., Hall M. N. (2006) Cell 124, 471–484 [DOI] [PubMed] [Google Scholar]
- 3.Fonseca B. D., Smith E. M., Lee V. H., MacKintosh C., Proud C. G. (2007) J. Biol. Chem. 282, 24514–24524 [DOI] [PubMed] [Google Scholar]
- 4.Hara K., Maruki Y., Long X., Yoshino K., Oshiro N., Hidayat S., Tokunaga C., Avruch J., Yonezawa K. (2002) Cell 110, 177–189 [DOI] [PubMed] [Google Scholar]
- 5.Kim D. H., Sarbassov D. D., Ali S. M., King J. E., Latek R. R., Erdjument-Bromage H., Tempst P., Sabatini D. M. (2002) Cell 110, 163–175 [DOI] [PubMed] [Google Scholar]
- 6.Kim D. H., Sarbassov D. D., Ali S. M., Latek R. R., Guntur K. V., Erdjument-Bromage H., Tempst P., Sabatini D. M. (2003) Mol. Cell 11, 895–904 [DOI] [PubMed] [Google Scholar]
- 7.Kovacina K. S., Park G. Y., Bae S. S., Guzzetta A. W., Schaefer E., Birnbaum M. J., Roth R. A. (2003) J. Biol. Chem. 278, 10189–10194 [DOI] [PubMed] [Google Scholar]
- 8.Jacinto E., Facchinetti V., Liu D., Soto N., Wei S., Jung S. Y., Huang Q., Qin J., Su B. (2006) Cell 127, 125–137 [DOI] [PubMed] [Google Scholar]
- 9.Pearce L. R., Huang X., Boudeau J., Pawlowski R., Wullschleger S., Deak M., Ibrahim A. F., Gourlay R., Magnuson M. A., Alessi D. R. (2007) Biochem. J. 405, 513–522 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10.Sarbassov D. D., Ali S. M., Kim D. H., Guertin D. A., Latek R. R., Erdjument-Bromage H., Tempst P., Sabatini D. M. (2004) Curr. Biol. 14, 1296–1302 [DOI] [PubMed] [Google Scholar]
- 11.Woo S. Y., Kim D. H., Jun C. B., Kim Y. M., Haar E. V., Lee S. I., Hegg J. W., Bandhakavi S., Griffin T. J. (2007) J. Biol. Chem. 282, 25604–25612 [DOI] [PubMed] [Google Scholar]
- 12.Yang Q., Inoki K., Ikenoue T., Guan K. L. (2006) Genes Dev. 20, 2820–2832 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 13.Jacinto E., Lorberg A. (2008) Biochem. J. 410, 19–37 [DOI] [PubMed] [Google Scholar]
- 14.Mamane Y., Petroulakis E., LeBacquer O., Sonenberg N. (2006) Oncogene 25, 6416–6422 [DOI] [PubMed] [Google Scholar]
- 15.Tremblay F., Marette A. (2001) J. Biol. Chem. 276, 38052–38060 [DOI] [PubMed] [Google Scholar]
- 16.Um S. H., Frigerio F., Watanabe M., Picard F., Joaquin M., Sticker M., Fumagalli S., Allegrini P. R., Kozma S. C., Auwerx J., Thomas G. (2004) Nature 431, 200–205 [DOI] [PubMed] [Google Scholar]
- 17.Hayashi T., Hatanaka M., Nagao K., Nakaseko Y., Kanoh J., Kokubu A., Ebe M., Yanagida M. (2007) Genes Cells 12, 1357–1370 [DOI] [PubMed] [Google Scholar]
- 18.Sarbassov D. D., Ali S. M., Sengupta S., Sheen J. H., Hsu P. P., Bagley A. F., Markhard A. L., Sabatini D. M. (2006) Mol. Cell 22, 159–168 [DOI] [PubMed] [Google Scholar]
- 19.Facchinetti V., Ouyang W., Wei H., Soto N., Lazorchak A., Gould C., Lowry C., Newton A. C., Mao Y., Miao R. Q., Sessa W. C., Qin J., Zhang P., Su B., Jacinto E. (2008) EMBO J. 27, 1932–1943 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 20.García-Martínez J. M., Alessi D. R. (2008) Biochem. J. 416, 375–385 [DOI] [PubMed] [Google Scholar]
- 21.Ikenoue T., Inoki K., Yang Q., Zhou X., Guan K. L. (2008) EMBO J. 27, 1919–1931 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 22.Loewith R., Jacinto E., Wullschleger S., Lorberg A., Crespo J. L., Bonenfant D., Oppliger W., Jenoe P., Hall M. N. (2002) Mol. Cell 10, 457–468 [DOI] [PubMed] [Google Scholar]
- 23.Sarbassov D. D., Guertin D. A., Ali S. M., Sabatini D. M. (2005) Science 307, 1098–1101 [DOI] [PubMed] [Google Scholar]
- 24.Frias M. A., Thoreen C. C., Jaffe J. D., Schroder W., Sculley T., Carr S. A., Sabatini D. M. (2006) Curr. Biol. 16, 1865–1870 [DOI] [PubMed] [Google Scholar]
- 25.Guertin D. A., Stevens D. M., Thoreen C. C., Burds A. A., Kalaany N. Y., Moffat J., Brown M., Fitzgerald K. J., Sabatini D. M. (2006) Dev. Cell 11, 859–871 [DOI] [PubMed] [Google Scholar]
- 26.Shiota C., Woo J. T., Lindner J., Shelton K. D., Magnuson M. A. (2006) Dev. Cell 11, 583–589 [DOI] [PubMed] [Google Scholar]
- 27.Jacinto E., Loewith R., Schmidt A., Lin S., Rüegg M. A., Hall A., Hall M. N. (2004) Nat. Cell Biol. 6, 1122–1128 [DOI] [PubMed] [Google Scholar]
- 28.Schmidt A., Kunz J., Hall M. N. (1996) Proc. Natl. Acad. Sci. U.S.A. 93, 13780–13785 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 29.Copp J., Manning G., Hunter T. (2009) Cancer Res. 69, 1821–1827 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 30.Soliman G. A., Acosta-Jaquez H. A., Dunlop E. A., Ekim B., Maj N. E., Tee A. R., Fingar D. C. (2010) J. Biol. Chem. 285, 7866–7879 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 31.Milburn C. C., Deak M., Kelly S. M., Price N. C., Alessi D. R., Van Aalten D. M. (2003) Biochem. J. 375, 531–538 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 32.Lee S., Comer F. I., Sasaki A., McLeod I. X., Duong Y., Okumura K., Yates J. R., 3rd, Parent C. A., Firtel R. A. (2005) Mol. Biol. Cell 16, 4572–4583 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 33.Lee S., Parent C. A., Insall R., Firtel R. A. (1999) Mol. Biol. Cell 10, 2829–2845 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 34.Li W., Zhu T., Guan K. L. (2004) J. Biol. Chem. 279, 37398–37406 [DOI] [PubMed] [Google Scholar]
- 35.Pollock C. B., Shirasawa S., Sasazuki T., Kolch W., Dhillon A. S. (2005) Cancer Res. 65, 1244–1250 [DOI] [PubMed] [Google Scholar]
- 36.Wabnitz G. H., Nebl G., Klemke M., Schröder A. J., Samstag Y. (2006) J. Immunol. 176, 1668–1674 [DOI] [PubMed] [Google Scholar]
- 37.Bondeva T., Pirola L., Bulgarelli-Leva G., Rubio I., Wetzker R., Wymann M. P. (1998) Science 282, 293–296 [DOI] [PubMed] [Google Scholar]