mTORC1-activated S6K1 phosphorylates Rictor on threonine 1135 and regulates mTORC2 signaling

Institute for Research in Immunology and Cancer, Department of Pathology and Cell Biology, Faculty of Medicine, Université de Montréal, Montreal, Quebec, Canada.
Molecular and Cellular Biology (Impact Factor: 5.04). 12/2009; 30(4):908-21. DOI: 10.1128/MCB.00601-09
Source: PubMed

ABSTRACT The mammalian target of rapamycin (mTOR) is a conserved Ser/Thr kinase that forms two functionally distinct complexes important for nutrient and growth factor signaling. While mTOR complex 1 (mTORC1) regulates mRNA translation and ribosome biogenesis, mTORC2 plays an important role in the phosphorylation and subsequent activation of Akt. Interestingly, mTORC1 negatively regulates Akt activation, but whether mTORC1 signaling directly targets mTORC2 remains unknown. Here we show that growth factors promote the phosphorylation of Rictor (rapamycin-insensitive companion of mTOR), an essential subunit of mTORC2. We found that Rictor phosphorylation requires mTORC1 activity and, more specifically, the p70 ribosomal S6 kinase 1 (S6K1). We identified several phosphorylation sites in Rictor and found that Thr1135 is directly phosphorylated by S6K1 in vitro and in vivo, in a rapamycin-sensitive manner. Phosphorylation of Rictor on Thr1135 did not affect mTORC2 assembly, kinase activity, or cellular localization. However, cells expressing a Rictor T1135A mutant were found to have increased mTORC2-dependent phosphorylation of Akt. In addition, phosphorylation of the Akt substrates FoxO1/3a and glycogen synthase kinase 3 alpha/beta (GSK3 alpha/beta) was found to be increased in these cells, indicating that S6K1-mediated phosphorylation of Rictor inhibits mTORC2 and Akt signaling. Together, our results uncover a new regulatory link between the two mTOR complexes, whereby Rictor integrates mTORC1-dependent signaling.

Download full-text


Available from: Philippe P. Roux, Sep 05, 2014
  • Source
    • "pAKT recovery was reached earlier than p4E-BP1 phosphorylation, suggesting a preferential inhibition of mTOR complex 1 (mTORC1) by BEZ-235, compared to mTORC2. Indeed, it has been shown that mTORC1 could exert an inhibitory effect on mTORC2 (through phospho-p70S6K) [22] [23], which is able to activate AKT (Ser 473 phosphorylation). It could be hypothesized that the maintained inhibition of mTORC1 after BEZ-235 withdrawal, but not of mTORC2, could lead to an mTORC2-mediated AKT phosphorylation, whereas the targets of mTORC1 such as 4E-BP1 remain unphosphorylated. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Targeting the phosphoinositide 3-kinase (PI3K)/mammalian target of rapamycin (mTOR) pathway is a potential means of overcoming chemoresistance in ovarian cancer. We investigated the capability of (18)F-fluororodeoxyglucose ((18)F-FDG) small-animal positron emission tomography (SA-PET) to predict the effects of a dual PI3K/mTOR inhibitor (BEZ-235) in a cisplatin-resistant ovarian cancer model. In a first experiment, nude rats bearing subcutaneous SKOV3 tumors received BEZ-235 for 3 days given alone or after paclitaxel and were compared to controls (either untreated or that were given the excipients of paclitaxel and BEZ-235). SA-PET was performed at baseline, on day 3, and day 7. In a second experiment aiming at further exploring the kinetics of (18)F-FDG tumor uptake during the first 48 hours following drug cessation, untreated controls were compared to rats receiving BEZ-235, which were imaged at baseline, on day 3, on day 4, and on day 5. SA-PET results were compared to cell proliferation assessment (Ki-67), PI3K/mTOR downstream target expression studies (pAKT and phospho-eukaryotic translation initiation factor 4E-binding protein 1), and apoptosis evaluation (cleaved caspase-3). In the first experiment, BEZ-235, compared to untreated controls, induced a marked decrease in (18)F-FDG uptake on day 3, which was correlated to a significant decrease in cell proliferation and to a significant PI3K/mTOR pathway inhibition. No tumor necrosis or apoptosis occurred. Four days following treatment cessation, tumor recovery (in terms of PI3K/mTOR inhibition and cell proliferation) occurred and was identified by (18)F-FDG SA-PET. Paclitaxel plus BEZ-235 showed results similar to BEZ-235 alone. In the second experiment, PI3K/mTOR pathways exhibited partial recovery as early as 24 hours following treatment cessation, but both (18)F-FDG SA-PET and cell proliferation remained unchanged. (18)F-FDG SA-PET is a surrogate marker of target inhibition during treatment with BEZ-235 and predicts tumor recovery 4 days after drug withdrawal, but not during the first 48 hours following drug cessation, when a lag between PI3K/mTOR pathway recovery and metabolic recovery is observed. (18)F-FDG SA-PET could be used for therapy monitoring of PI3K/mTOR inhibitors, but our results also raise questions regarding the potential impact of the delay between PET imaging and the last drug intake on the accuracy of FDG imaging.
    Translational oncology 10/2013; 6(5):586-95. DOI:10.1593/tlo.13100 · 3.40 Impact Factor
  • Source
    • "rearrangement, proliferation, splicing, feedback regulation, and cell survival have been identified (Fenton and Gout, 2011). Many functional aspects of S6K1 have been characterized-mTOR-mediated phosphorylation of p70S6K at T389 also regulates its nucleocytoplasmic localization (Rosner et al., 2012), crosstalk exists between the mTOR/S6K1 and Hedgehog (HH) pathways (Wang et al., 2012), and mTORC1-activated p70S6K phosphorylates Rictor on T1135 and regulates mTORC2 signaling (Dibble et al., 2009; Julien et al., 2010). These findings provide significant insights into the regulation of p70S6K and have increased our understanding of its function. "
    [Show abstract] [Hide abstract]
    ABSTRACT: p70 ribosomal S6 kinase (p70S6K) can integrate nutrient and growth factor signals to promote cell growth and survival. We report our molecular characterization of the complementary DNA (cDNA) that encodes the goat p70S6K gene 40S ribosomal S6 kinase 1 (S6K1) (GenBank accession GU144017) and its 3' noncoding sequence in Inner Mongolia Cashmere goats (Capra hircus). Goat S6K1 cDNA was 2,272 bp and include an open reading frame (ORF) of 1,578 bp, corresponding to a polypeptide of 525 amino acids, and a 694-residue 3' noncoding sequence with a polyadenylation signal at nucleotides 2,218 to 2,223. The relative abundance of S6K1 mRNA was measured by real-time PCR in 6 tissues, and p70S6K expression was examined by immunohistochemistry in heart and testis. The phosphorylation of p70S6K is regulated by mitogen-activated protein kinase (MAPK) signaling in fetal fibroblasts.
    Asian Australasian Journal of Animal Sciences 08/2013; 26(8):1057-64. DOI:10.5713/ajas.2012.12710 · 0.56 Impact Factor
  • Source
    • "Translational Oncology Vol. 6, No. 2, 2013 NVP-BEZ235 and Radiosensitization Kuger et al. 177 the interruption of the negative feedback loops that downregulate PI3K signaling, which in turn can paradoxically promote cell survival, as reported in case of rapalogs elsewhere [43] [44] [45]. This mechanism would explain the lack of radiosensitization by NVP-BEZ235 used in schedule I (i.e., long-term pretreatment). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Previous studies have shown that the dual phosphatidylinositide 3-kinase/mammalian target of rapamycin (PI3K/mTOR) inhibitor NVP-BEZ235 radiosensitizes tumor cells if added shortly before ionizing radiation (IR) and kept in culture medium thereafter. The present study explores the impact of inhibitor and IR schedule on the radiosensitizing ability of NVP-BEZ235 in four human glioblastoma cell lines. Two different drug-IR treatment schedules were compared. In schedule I, cells were treated with NVP-BEZ235 for 24 hours before IR and the drug was removed before IR. In schedule II, the cells were exposed to NVP-BEZ235 1 hour before, during, and up to 48 hours after IR. The cellular response was analyzed by colony counts, expression of marker proteins of the PI3K/AKT/mTOR pathway, cell cycle, and DNA damage. We found that under schedule I, NVP-BEZ235 did not radiosensitize cells, which were mostly arrested in G1 phase during IR exposure. In addition, the drug-pretreated and irradiated cells exhibited less DNA damage but increased expressions of phospho-AKT and phospho-mTOR, compared to controls. In contrast, NVP-BEZ235 strongly enhanced the radiosensitivity of cells treated according to schedule II. Possible reasons of radiosensitization by NVP-BEZ235 under schedule II might be the protracted DNA repair, prolonged G2/M arrest, and, to some extent, apoptosis. In addition, the PI3K pathway was downregulated by the NVP-BEZ235 at the time of irradiation under schedule II, as contrasted with its activation in schedule I. We found that, depending on the drug-IR schedule, the NVP-BEZ235 can act either as a strong radiosensitizer or as a cytostatic agent in glioblastoma cells.
    Translational oncology 04/2013; 6(2):169-79. DOI:10.1593/tlo.12364 · 3.40 Impact Factor
Show more