Rictor is a novel target of p70 S6 kinase-1

Abstract

The rapamycin-insensitive companion of mammalian target of rapamycin (mTOR) (Rictor) is a key member of mTOR complex-2 (mTORC2), which phosphorylates the AGC kinases Akt/PKB, PKC and SGK1 at a C-terminal hydrophobic motif. We identified several novel sites on Rictor that are phosphorylated, including Thr1135, which is conserved across all vertebrates. Phosphorylation of this site on Rictor is stimulated by amino acids and growth factors through a rapamycin-sensitive signaling cascade. We demonstrate here that Rictor is a direct target of the ribosomal protein S6 kinase-1 (S6K1). Rictor phosphorylation at Thr1135 does not lead to major changes in mTORC2-kinase activity. However, phosphorylation of this site turns over rapidly and mediates 14-3-3 binding to Rictor and mTORC2, providing possibility for altered interactions of the complex. These findings reveal an unexpected signaling input into mTORC2, which is regulated by amino acids, growth factors and rapamycin.

Introduction

Mammalian target of rapamycin (mTOR) complex-1 (mTORC1) and mTOR complex 2 (mTORC2) are two structurally and functionally distinct multi-protein complexes. They share the same catalytic subunit, the highly conserved Ser/Thr kinase mTOR (mammalian target of rapamycin, also known as mechanistic target of rapamycin), which is a major regulator of cell growth, proliferation and survival (Guertin and Sabatini, 2007). mTORC1 consists of mTOR, Raptor (Regulatory-associated protein of mTOR), mLST8/GβL, PRAS40 (proline-rich Akt substrate of 40KD) and DEPTOR (Hara et al., 2002; Kim et al., 2002, 2003; Loewith et al., 2002; Sancak et al., 2007; Vander Haar et al., 2007; Peterson et al., 2009). This complex plays a major role in the regulation of cell growth in response to amino acids, energy sufficiency and growth factors by regulating several cellular processes, including translation, transcription, ribosome biogenesis, nutrients transport and autophagy. The ribosomal S6 kinases (S6Ks) and the eukaryotic initiation factor 4E-binding protein-1, both of which regulate protein synthesis, are the best-characterized mTORC1 substrates and play a critical role in the control of cell growth by mTORC1 (Inoki et al., 2005; Wullschleger et al., 2006; Ma and Blenis, 2009). The second complex, mTORC2, also contains mTOR, mLST8/GβL and DEPTOR associated with Rictor (the rapamycin-insensitive companion of mTOR), Sin1 (stress-activated, protein kinase-interacting protein-1) and Protor (protein associated with Rictor) (Jacinto et al., 2004, 2006; Sarbassov et al., 2004; Frias et al., 2006; Yang et al., 2006; Pearce et al., 2007; Peterson et al., 2009). mTOR assembled into mTORC2 modulates cell proliferation and survival in response to growth factors by promoting the phosphorylation and activation of Akt/PKB, PKC (protein kinase-C) and SGK1 (serum-glucocorticoid-induced protein kinase-1) (Sarbassov et al., 2004, 2005; Guertin et al., 2006; Facchinetti et al., 2008; Garcia-Martinez and Alessi, 2008; Ikenoue et al., 2008).

Rapamycin, first discovered as a product of the bacterium Streptomyces hygroscopicus, is a potent inhibitor of mTORC1, and hence S6Ks. By contrast, mTORC2 is referred to as the rapamycin-insensitive complex. Despite the fact that prolonged rapamycin treatment has been described to disrupt mTORC2 assembly in a subset of cells, rapamycin does not appear to be a general inhibitor of mTORC2 (Sarbassov et al., 2006). In contrast, rapamycin inhibits a negative feedback loop mechanism downstream of mTORC1 that targets the Akt/PKB pathway, leading to its activation (Radimerski et al., 2002; Zhang et al., 2003; Harrington et al., 2004; Shah et al., 2004; Um et al., 2004). This reveals the close interconnections that exist between the two mTOR complexes, with the activity of each complex being able directly or indirectly to modulate the activity of the other. Both complexes are activated in response to growth factors through a phosphatidyl inositol-3 (PI-3) kinase-dependent signaling cascade (Bhaskar and Hay, 2007). The mTORC2 target, Akt/PKB, plays an important role in the control of mTORC1 activity in response to growth factors, by releasing the inhibitory effect of TSC (tuberous sclerosis complex) and PRAS40, positioning mTOR both upstream and downstream of Akt/PKB (Inoki et al., 2002; Sancak et al., 2007). One of the striking differences between the two complexes is their relative sensitivity to nutrients. Indeed, mTOR activity is regulated by amino acids only when part of mTORC1.

There is increasing evidence that the mTOR pathway is deregulated in common diseases, including cancer and diabetes, emphasizing the importance of defining and understanding the mechanisms involved in the control of mTOR signaling. Recently, mTORC2, and more particularly Rictor, has been report to be required for cancer progression in a prostate cancer model driven by PTEN loss (Guertin et al., 2009). Little is known about the signaling cascade leading to activation of mTORC2, or any regulation that could directly target this complex. It has been suggested that Rictor is prone to phosphorylation, although, neither the specific sites involved nor the responsible kinases have been described (Jacinto et al., 2004; Sarbassov et al., 2004; Frias et al., 2006; Akcakanat et al., 2007). Nevertheless, these phosphorylation events may be involved in the regulation of mTORC2 activity and could lead to identification of new components of the mTORC2 signaling pathway. We set out to characterize phosphorylation sites within Rictor using a proteomic approach, which led us to the identification of 15 phosphorylated residues. We concentrated our study on the characterization of the residue Thr1135, which is conserved across all vertebrates. Rictor phosphorylation at Thr1135 is induced by amino acids and growth factors, and is rapamycin-sensitive. Indeed, we demonstrate that Thr1135 is a direct target of S6K1. Phosphorylation on this site is rapidly turned over, but does not cause major changes in mTORC2-kinase activity. It does, however, create a docking site for 14-3-3 proteins. This work reveals a novel amino-acid and rapamycin-sensitive input into Rictor and mTORC2.

Results

Identification of phosphorylation sites within Rictor

We used mass spectrometry (MS) to identify phosphorylation sites within Rictor. Endogenous Rictor was immunoprecipitated from human embryonic kidney-293 (HEK-293) cells growing in serum. After separation by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and Coomassie blue staining, the band containing Rictor was excised from the gel and analysed by MS. Two independent samples were analysed (74 and 60% sequence coverage respectively; data not shown) leading to the identification of 25 phosphopeptides corresponding to 14 distinct sequences, with a total of 15 phosphorylation events identified (Figure 1a). Ser21, Ser1035, Ser1177, Ser1235, Ser1302 and Ser1479 were confidently identified as phosphorylated residues (Figure 1a, sites marked in red). In some cases, definitive localization of the phosphorylation site could not be obtained (Figure 1a, sites marked in green). Some of these sites have previously been reported, including Ser21 and Ser1177, which have been identified in different large-scale analyses of phosphoproteins (Beausoleil et al., 2004; Dai et al., 2007; Cantin et al., 2008; Daub et al., 2008; Dephoure et al., 2008; Zanivan et al., 2008). In addition, the phosphopeptides containing Ser1174, Ser1219, Thr1222, Thr1295, Ser1385, Tyr1386 and Ser1388, which have recently reported been, were also detected (Dephoure et al., 2008; Zanivan et al., 2008). The majority of the residues that we identified, eight out of 15, are novel unreported phosphorylation sites.

Figure 1
figure1

Identification of the phosphorylation site Thr1135 within Rictor. (a) MS was performed on Rictor protein purified from HEK-293 cells. Coomassie-stained Rictor protein was isolated from an SDS–polyacrylamide gel and analysed by LC/MS/MS. In red are pictured the phosphorylation sites confidently assigned. The phosphorylation sites pictured in green are not confidently assigned. (b) Sequence alignment showing conservation of Rictor Thr1135 among vertebrates. (c) HEK-293 cells were transfected with empty vector or wild-type myc-tagged Rictor. Immunoprecipitated Rictor was analysed by immunoblotting using a phospho(Ser/Thr) PKB substrate antibody that recognizes the R/KxR/KxxpS/T consensus motif and an myc tag antibody. Whole-cell lysates were analysed by immunoblotting using the indicated antibodies. (d) A549 cells were transfected with 25 nM scrambled or Rictor siRNAs. Seventy-two hours later, immunoprecipitated Rictor was analysed by immunoblotting using phospho(Ser/Thr) PKB substrate and Rictor antibodies. Whole-cell lysates were analysed by immunoblotting using indicated antibodies. (e) Rictor was immunoprecipitated from A549 cells and incubated with or without CIP. Immunoprecipitated Rictor was analysed by immunoblotting using phospho(Ser/Thr) PKB substrate and Rictor antibodies. Whole-cell lysates were analysed by immunoblotting using the indicated antibodies. (f) HEK-293 cells were transfected with empty vector, wild-type, T1135A, T1135D or S1138A myc-tagged Rictor. Immunoprecipitated Rictor was analysed by immunoblotting using phospho-Rictor (Thr1135), phospho(Ser/Thr) PKB substrate and myc tag antibodies. Whole-cell lysates were analysed by immunoblotting using the indicated antibodies. HEK, human embryonic kidney; LC, liquid chromatography; MS, mass spectrometry; PKB, protein kinase-B; Rictor, rapamycin-insensitive companion of mammalian target of rapamycin; SDS, sodium dodecyl sulfate; siRNA, small interfering RNA.

An analysis of human Rictor protein sequence with the Scansite algorithm (http://scansite.mit.edu) indicates that residue Thr1135, which we identified as a potential phosphorylation site, is a putative phosphorylation site for Akt/PKB at high stringency (Obenauer et al., 2003). The sequence of this site is NRRIRTLpTEPSV, which conforms to the Akt/PKB consensus motif, Arg-x-Arg-x-x-pSer/Thr (RxRxxpS/T, where x is any amino acid). Alignment of Rictor homologues of different species shows that the Thr1135 and the consensus motif are conserved across all vertebrates (Figure 1b). To confirm the phosphorylation of this site in vivo, we examined whether we could detect phosphorylation of Rictor using an antibody that recognizes the phosphorylated consensus ‘Akt/PKB-substrate’ motif Arg/Lys-x-Arg/Lys-x-x-pSer/Thr (R/KxR/KxxpS/T). Interestingly, myc-tagged Rictor immunoprecipitated from HEK-293 (Figure 1c) as well as endogenous Rictor immunoprecipitated from A549 cells (Figure 1d) were recognized by this phospho-motif antibody. This antibody specifically recognized the phosphorylated form of Rictor, as demonstrated by the fact that knockdown of Rictor expression using small interfering RNA (siRNA) (Figure 1d) or phosphatase treatment of immunoprecipitated Rictor (Figure 1e) resulted in complete loss of immunoreactivity. These results show that Rictor is phosphorylated in vivo on a (R/KxR/KxxpS/T) motif.

In order to determine whether the phospho-motif antibody detects the phosphorylation of Rictor at Thr1135, we expressed myc-tagged, wild-type Rictor or different mutants of Rictor at Thr1135 or at Ser1138 in HEK-293 cells. The phospho-motif antibody detects wild-type Rictor and an S1138A mutant form. In contrast, there is no detection of Rictor mutants where Thr1135 has been mutated into either alanine (T1135A) or aspartic acid (T1135D) (Figure 1f). These results show that the phospho-motif antibody specifically detects Rictor phosphorylation at Thr1135. Finally, an antibody was raised against the phosphopeptide RIRTLpTEPSVDC, corresponding to the Thr1135 site in the human Rictor sequence. This specifically recognized Rictor phosphorylated at Thr1135, but not the T1135A or T1135D mutants (Figure 1f).

Thus, a MS approach led us to the identification of the phosphorylation site Thr1135 within Rictor, which is part of an RxRxxpS/T consensus motif.

Amino acids and growth factors stimulate Rictor phosphorylation at Thr1135 via mTORC1

The RxRxxpS/T consensus motif can be phosphorylated by Akt/PKB and also other AGC-family kinases, including S6K, SGK and RSK (Alessi et al., 1996).To determine which kinase was responsible for Rictor phosphorylation, we analysed Thr1135 phosphorylation of myc-tagged Rictor in HEK-293 cells after treatment with PI-3-kinase inhibitors (PI-103 and LY294002), an mTORC1 inhibitor (rapamycin) or an MEK inhibitor (U0126). Thr1135 phospho-specific antibody (Figure 2a) and Akt/PKB substrate phospho-motif antibody (Supplementary Figure S1a) were used. Both PI-3-kinase inhibitors prevent Rictor phosphorylation at Thr1135, while the MEK inhibitor does not. mTORC1 inhibition by rapamycin treatment is also sufficient to completely block Rictor phosphorylation at Thr1135. These results were confirmed on endogenous Rictor with the phospho-motif antibody, as shown in Figure 2b, and with the Thr1135 phospho-specific antibody as shown in Supplementary Figure S1b. Consistent with these observations, we found that potent activators of mTORC1, such as amino acids, insulin and growth factors, robustly stimulated Rictor phosphorylation at Thr1135 (Figure 2c and Supplementary Figure S1c). As expected, the phospho-motif antibody, characterized in Figure 1, specifically detects the phosphorylation of Rictor at Thr1135, which is induced by insulin treatment (Supplementary Figure S1d). In addition, pretreatment with rapamycin totally abrogated Rictor phosphorylation at Thr1135 induced by amino acids and insulin (Figure 2d and Supplementary Figure S1e).

Figure 2
figure2

Amino acids and growth factors induce rapamycin-sensitive Rictor phosphorylation at Thr1135. (a) HEK-293 cells were transfected with empty vector or wild-type myc-tagged Rictor. Cells were treated for 30 min with dimethyl sulfoxide (DMSO), PI-103(1 μM), LY294002(50 μM), rapamycin (100 nM) or U0126(10 μM), where indicated. Immunoprecipitated Rictor was analysed by immunoblotting using phospho-Rictor (Thr1135) and Myc tag antibodies. Whole-cell lysates were analysed by immunoblotting using the indicated antibodies. (b) A549 cells were treated for 30 min with DMSO, PI-103(1 μM), LY294002(50 μM), rapamycin(100 nM) or U0126(10 μM), where indicated. Immunoprecipitated Rictor was analysed by immunoblotting using phospho(Ser/Thr) PKB substrate and Rictor antibodies. Whole-cell lysates were analysed by immunoblotting using the indicated antibodies. (c) A549 cells were deprived of serum for 16 h and of amino acids for 2 h. Cells were stimulated with amino acids, insulin(10−7M), EGF(100 ng/ml) or serum(10%) for 15 min, where indicated. Immunoprecipitated Rictor was analysed by immunoblotting using phospho(Ser/Thr) PKB substrate and Rictor antibodies. Whole-cell lysates were analysed by immunoblotting the using indicated antibodies. (d) A549 cells were deprived of serum for 16 h and of amino acids for 2 h. Cells were treated with rapamycin (100 nM) for 30 min, followed by 15-min stimulation with amino acids or insulin (10−7M), where indicated. Immunoprecipitated Rictor was analysed by immunoblotting using phospho-Rictor (Thr1135) and Rictor antibodies. Whole-cell lysates were analysed by immunoblotting using the indicated antibodies. DMSO, dimethylsulfoxide; HEK, human embryonic kidney; PI, phosphatidyl inositol; PKB, protein kinase-B; Rictor, rapamycin-insensitive companion of mammalian target of rapamycin.

Thus, we show that amino acids and growth factors stimulate Rictor phosphorylation at Thr1135 through an mTORC1-dependent signaling pathway.

Rictor is a direct target of S6K1

The results shown in Figure 2 suggest that a kinase located downstream of mTORC1 phosphorylates Rictor. Of the known mTORC1-target proteins, the ribosomal protein S6Ks, S6K1 and S6K2, phosphorylate substrates with the consensus motif RxRxxS/T and thus represent prime candidates for this role. To test whether S6Ks are involved in Rictor phosphorylation, we analysed its phosphorylation in mouse embryo fibroblasts (MEFs) in which the genes encoding S6K1 and S6K2 had been deleted. As shown in HEK-293 and A549 cells in Figure 2, PI-3-kinase inhibitor and rapamycin treatment inhibit Rictor phosphorylation at Thr1135 in wild-type MEFs, but Rictor phosphorylation at this site is undetectable in S6K1/2−/− MEFs (Figure 3a), indicating that S6Ks play a major role in the control of Rictor phosphorylation. Indeed, knockdown of TSC2, a negative regulator of mTORC1, activates S6K, leading to increase in Rictor phosphorylation at Thr1135 (Supplementary Figure S2a).

Figure 3
figure3

S6K1 phosphorylates Rictor at Thr1135. (a) S6K1/2+/+ and S6K1/2−/− MEFs were treated for 30 min with DMSO, LY294002 (50 μM), rapamycin (100 nM) or U0126 (10 μM), where indicated. Sin1 was immunoprecipitated and associated Rictor phosphorylation was analysed by immunoblotting using phospho-Rictor (Thr1135), Rictor and Sin1 antibodies. Whole-cell lysates were analysed by immunoblotting using the indicated antibodies. (b) An in vitro kinase assay was performed by incubating Rictor, immunoprecipitated from HEK-293 cells treated for 30 min with PI-103 (1 μM), with a recombinant active S6K1. Rictor phosphorylation was analysed by immunoblotting using phospho-Rictor (Thr1135), Rictor and S6K1 antibodies. (c) A549 cells were transfected with 25 nM scrambled, mTOR, Rictor, S6K1 or S6K2 siRNAs alone or in combination. Seventy-hours later, immunoprecipitated Rictor was analysed by immunoblotting using phospho-Rictor (Thr1135), Rictor, mTOR and Sin1 antibodies. Whole-cell lysates were analysed by immunoblotting using the indicated antibodies. (d) HEK-293 cells were transfected with empty vector, wild-type or E389ΔCT HA-tagged S6K1. Cells were treated for 30 min with rapamycin (100 nM), where indicated. Immunoprecipitated Rictor was analysed by immunoblotting using phospho(Ser/Thr) PKB substrate and Rictor antibodies. Whole-cell lysates were analysed by immunoblotting using the indicated antibodies. (e) HEK-293 cells were transfected with empty vector, wild-type or E388ΔCT HA-tagged S6K2. Cells were treated for 30 min with rapamycin (100 nM), where indicated. Immunoprecipitated Rictor was analysed by immunoblotting using phospho(Ser/Thr) PKB substrate and Rictor antibodies. Whole-cell lysates were analysed by immunoblotting using the indicated antibodies. DMSO, dimethylsulfoxide; HA, hemagglutinin; HEK, human embryonic kidney; MEF, mouse embryo fibroblast; mTOR ,mammalian target of rapamycin; PI, phosphatidyl inositol; PKB, protein kinase-B; Rictor, rapamycin-insensitive companion of mammalian target of rapamycin; S6K, S6 kinase; siRNA, small interfering RNA; Sin1, stress-activated, protein kinase-interacting protein-1.

To test whether Rictor is a direct target of S6Ks, we performed S6K in vitro kinase assays using Rictor as a substrate. Rictor was immunoprecipitated from HEK-293 cells treated with PI-103 to downregulate the phosphorylation of Thr1135 and recombinant active S6Ks were then added. As revealed with the Thr1135 phospho-specific antibody (Figure 3b) and with the phospho-motif antibody (Supplementary Figure S2b), S6K1 phosphorylates Rictor at Thr1135 in vitro. Similar results were seen with purified S6K2 (data not shown). In addition, as shown Supplementary Figure S2c, S6K1 phosphorylates Rictor specifically at Thr1135, but not the mutant forms of Rictor, which lack this site.

In order to examine the individual contributions of S6K1 and S6K2 in vivo, we used siRNA specifically targeting each isoform. Both siRNAs decrease the phosphorylation level of the ribosomal protein S6, a well-described substrate of S6K1 and S6K2. Consistent with the results obtained using S6K1/2−/− MEFs, Rictor phosphorylation at Thr1135 is totally abrogated when S6K1 and S6K2 siRNA are used in combination. Knockdown of S6K1 expression alone blocks Rictor phosphorylation at Thr1135. In contrast, siRNA targeting S6K2 induces a sharp decrease in S6 phosphorylation at both Ser235/236 and Ser240/244, but does not affect Rictor phosphorylation level at Thr1135 (Figure 3c and Supplementary Figure S2d). To confirm these results, we analysed Rictor phosphorylation status in HEK-293 cells expressing wild-type S6K1 and S6K2, or the constitutively active and rapamycin-resistant mutants of S6K1 and S6K2 (respectively, S6K1E389ΔCT and S6K2E388ΔCT; Schalm and Blenis, 2002). As expected, Rictor phosphorylation at Thr1135 remained sensitive to rapamycin in cells transfected with the wild-type forms of S6K1 and S6K2 (Figures 3d and e), but rapamycin failed to suppress Rictor phosphorylation in cells expressing constitutively active and rapamycin-resistant S6K1, but not in cells expressing the equivalent mutant of S6K2 (Figures 3d and e, and Supplementary Figure S2e).

These results suggest that, in cells, Rictor is a direct target of S6K and that S6K1, rather than S6K2, is likely to be the major kinase responsible for Rictor phosphorylation at Thr1135.

Rictor phosphorylation at Thr1135 has minor effects on mTORC2-kinase activity

To determine whether phosphorylation at Thr1135 plays a role in the control of mTORC2 activity, we expressed wild-type Rictor or mutants T1135A, T1135D and S1138A in Rictor-deficient MEFs. Cells were cultured in the presence of serum and the phosphorylation level of different mTORC2 substrates in the cells was analysed. In agreement with previous studies, in the Rictor-knockout MEFs, Akt/PKB and PKCα exhibited only a very low level of phosphorylation at their hydrophobic motifs (respectively, Ser473 and Ser657) and turn motifs (respectively, Thr450 and Thr641), while normal amount of phosphorylation was detected in the activation loop of Akt/PKB (Thr308) (Figures 4a and b). However, Rictor-knockout MEFs still possessed mTORC1 function, as S6K1 was still phosphorylated at its hydrophobic motif Thr389 (Figure 4a; Shiota et al., 2006; Garcia-Martinez and Alessi, 2008). Despite impaired phosphorylation of Ser473 and Thr450 in Akt/PKB, the phosphorylation levels of Ser21/9 in glycogen synthase kinase-3α (GSK3α)/β, and of Thr1462 in TSC2, which are well-defined substrates for Akt/PKB, were not altered, consistent with published work (Guertin et al., 2006; Jacinto et al., 2006). In contrast with other reports, we did not detect any modification of the phosphorylation level of Thr32 in FOXO3A (Figures 4a and b). We were not able to study the phosphorylation level of SGK1 at the hydrophobic motif (Ser422), due to the absence of signal with all the antibodies tested. However, the phosphorylation level of NDRG1, a physiological substrate of SGK1, is undetectable in Rictor-deficient MEFs, but clear in wild-type MEFs (Figures 4a and b). As expected, re-introduction of wild-type Rictor, in Rictor−/− MEFs, restored the phosphorylation of the hydrophobic and turn motifs of Akt/PKB and PKCα, and the phosphorylation of Thr346 of NDRG1. Roughly the same level of phosphorylation was induced by expression of mutants T1135A, T1135D or S1138A of Rictor (Figures 4b and c). This result suggests that phosphorylation of Rictor at Thr1135 is not a prerequisite for mTORC2 protein-kinase activity and is at best a minor regulatory influence, at least toward the substrates studied here.

Figure 4
figure4

Rictor phosphorylation at Thr1135 does not have a major impact on mTORC2's activity. (a) Rictor+/+ and Rictor−/− MEFs deprived of serum for 16 h, were stimulated with serum (10%) for 10 min, where indicated. Whole-cell lysates were analysed by immunoblotting using the indicated antibodies. (b) Rictor−/− MEFs were transfected with empty vector, wild-type, T1135A, T1135D or S1138A myc-tagged Rictor. Whole-cell lysates were analysed by immunoblotting using the indicated antibodies. (c) Rictor−/− MEFs were transfected with empty vector, wild-type, T1135A or T1135D myc-tagged Rictor. After 6 h of serum starvation, cells were treated with rapamycin (100 nM) for 30 min, followed by 10-min stimulation with serum(10%), where indicated. Whole-cell lysates were analysed by immunoblotting using the indicated antibodies. MEF, mouse embryo fibroblast; mTORC, mammalian target of rapamycin complex; Rictor, rapamycin-insensitive companion of mammalian target of rapamycin.

mTORC1 inhibitors such as rapamycin have been shown to activate Akt/PKB while suppressing mTORC1 signaling in various different cancer cell lines and clinical human tumor samples, due to inhibition of a negative feedback loop involving insulin receptor substrate-1 (Zhang et al., 2003; Harrington et al., 2004; Shah et al., 2004). To determine whether the phosphorylation of Rictor at Thr1135 could be another part of the feedback mechanism involved in this control of Akt/PKB activity, we analysed Akt/PKB phosphorylation after rapamycin treatment in Rictor-deficient MEFs where wild-type Rictor or mutants T1135A or T1135D had been re-introduced. Rapamycin treatment stimulates Akt/PKB phosphorylation at Ser473 and Thr308 in Rictor−/− MEFs where wild-type Rictor has been re-expressed, but we did not detect any modification of the phosphorylation level of the turn motif of Akt/PKB (Thr450) or of the hydrophobic or turn motifs of PKCα. The effect of rapamycin treatment on the phosphorylation of Ser473 and Thr308 of Akt/PKB is the same whether the wild type, or T1135A or T1135D Rictor mutants are re-expressed in Rictor−/− MEFs (Figure 4c and Supplementary Figure S3).

Thus, phosphorylation of Thr1135 of Rictor by S6K1 does not appear to be an obvious part of the negative feedback loop by which mTORC1 represses Akt/PKB signaling.

Phosphorylation of Rictor at Thr1135 regulates 14-3-3 binding

To examine the role of this phosphorylation on the integrity of mTORC2, we isolated mTORC2 complex associated with Rictor and then determined its composition. As shown Figure 5a, the same amount of mTOR, Sin1 and GβL is associated with wild-type Rictor and with mutants T1135A or T1135D. In addition, inhibition of Thr1135 phosphorylation by rapamycin treatment (Figure 5b) or by knockdown of S6K1 expression using siRNA (Figure 3b), or increase of Thr1135 phosphorylation by treatment with the phosphatase inhibitor calyculin-A (Figure 5c), does not modulate the amount of mTOR, Sin1 and GβL associated with Rictor. These results suggest that Rictor phosphorylation levels at Thr1135 do not modify the composition of mTORC2, at least regarding Rictor, Sin1 and mTOR. However, the rapid increase in Rictor Thr1135 phosphorylation following treatment of cells with calyculin-A for 10 min (Figure 5c) suggests that phosphorylation of this site turns over very rapidly and is subject to stringent regulation by phosphatases.

Figure 5
figure5

Rictor phosphorylation at Thr1135 does not modify mTORC2 integrity. (a) HEK-293 cells were transfected with empty vector, wild-type, T1135A or T1135D myc-tagged Rictor. Myc immunoprecipitates were analysed by immunoblotting using myc tag, mTOR, Sin1 and GβL antibodies. Whole-cell lysates were analysed by immunoblotting using the indicated antibodies. (b) HEK-293 cells were treated with rapamycin (100 nM) for 30 min. Rictor immunoprecipitates were analysed by immunoblotting using phospho-Rictor (Thr1135), Rictor, mTOR, Sin1 and GβL antibodies. Whole-cell lysates were analysed by immunoblotting using the indicated antibodies. (c) HEK-293 cells were treated with calyculin-A (50 nM) for 10 min. Rictor immunoprecipitates were analysed by immunoblotting using phospho-Rictor (Thr1135), Rictor, mTOR, Sin1 and GβL antibodies. Whole-cell lysates were analysed by immunoblotting using the indicated antibodies. HEK, human embryonic kidney; mTOR, mammalian target of rapamycin; mTORC, mammalian target of rapamycin complex; Rictor, rapamycin-insensitive companion of mammalian target of rapamycin; Sin1, stress-activated, protein kinase-interacting protein-1.

The sequence around Thr1135 of Rictor conforms to the Akt/PKB consensus motif (RxRxxpS/T) and is also predicted to be a putative 14-3-3-binding motif (14-3-3 mode-1-binding motif: RS/TxpS/TxP; Yaffe et al., 1997). Alignment of Rictor homologues of different species shows that the consensus 14-3-3-binding motif is conserved across all vertebrates (Figure 6a). In addition, three isoforms of 14-3-3 have been detected in preparations of Rictor purified by tandem affinity purification (Pearce et al., 2007). To determine whether 14-3-3 proteins might bind to Thr1135 of Rictor in a phospho-specific manner, peptides with Rictor sequences surrounding either non-phosphorylated or phosphorylated Thr1135 were coupled to beads and incubated with lysate from HEK-293 cells; then bound proteins were analysed by SDS–PAGE. Only the peptide phosphorylated at Thr1135, but not the non-phosphorylated form, interacts with 14-3-3 in this assay (Figure 6b). In addition, co-immunoprecipitation experiments revealed that 14-3-3 interacts with wild-type Rictor but not with Rictor mutated at Thr1135 (Figure 6c). Consistent with these observations, the interaction between 14-3-3 and Rictor is inhibited by rapamycin treatment, an effect seen both with overexpressed (Figure 6d) and endogenous Rictor (Figure 6e). In agreement with these results, combined treatment with amino acids and growth factors stimulate the interaction between Rictor and 14-3-3 (Figure 6f).

Figure 6
figure6

Rictor phosphorylation at Thr1135 is required for binding to 14-3-3. (a) Sequence alignment showing conservation of the 14-3-3 mode-1 binding motif surrounding Rictor Thr1135 among vertebrates. (b) Non-phosphorylated or phosphorylated peptides corresponding to the Rictor sequence surrounding Thr1135 were coupled to beads and incubated with the lysate from HEK-293 cells. Samples were analysed by immunoblotting using a 14-3-3ɛ antibody. (c) HEK-293 cells were transfected with empty vector, wild-type, T1135A or T1135D myc-tagged Rictor. Myc immunoprecipitates were analysed by immunoblotting using Myc tag, and 14-3-3ɛ antibodies. Whole-cell lysates were analysed by immunoblotting using the indicated antibodies. (d) HEK-293 cells were transfected with empty vector or wild-type myc-tagged Rictor. Cells were treated for 30 min with DMSO or rapamycin (100 nM), where indicated. Immunoprecipitated Rictor was analysed by immunoblotting using 14-3-3ɛ and Rictor antibodies. Whole-cell lysates were analysed by immunoblotting using the indicated antibodies. (e) A549 cells were treated for 30 min with DMSO or rapamycin (100 nM), where indicated. Rictor and Sin1 immunoprecipitates were analysed by immunoblotting using 14-3-3ɛ, Sin1 and Rictor antibodies. Whole-cell lysates were analysed by immunoblotting using the indicated antibodies. (f) A549 cells were deprived of serum for 16 h and amino acids for 2 h. Cells were stimulated with amino acids and serum (10%) for 15 min, where indicated. Rictor immunoprecipitates were analysed by immunoblotting using 14-3-3ɛ and Rictor antibodies. Whole-cell lysates were analysed by immunoblotting using the indicated antibodies. DMSO, dimethylsulfoxide; HEK, human embryonic kidney; Rictor, rapamycin-insensitive companion of mammalian target of rapamycin; Sin1, stress-activated, protein kinase-interacting protein-1.

Altogether, these results indicate that Rictor interacts with 14-3-3, dependent on its phosphorylation at Thr1135.

Discussion

Our investigation of phosphorylation sites within Rictor revealed that Rictor is highly phosphorylated. Most of the sites that we identified are in the C-terminal part of the protein, which does not contain any conserved region (Sarbassov et al., 2004). The numerous sites identified suggest that Rictor may be modified by different kinases, which could integrate different signals. These phosphorylation events might play an important role in the control of mTORC2 signaling. We report here the phosphorylation of Rictor at Thr1135, which is part of an AGC-kinase consensus sequence (RxRxxpS/T) and conserved among vertebrate Rictors. Other groups have reported that Rictor may be phosphorylated by observing its electrophoretic mobility and have suggested that these phosphorylations could be regulated (Sarbassov et al., 2004; Yang et al., 2006; Akcakanat et al., 2007). Although no effect of short-term rapamycin treatment has been reported on the electrophoretic mobility of Rictor, possibly reflecting the high molecular weight of Rictor, we show here using phospho-specific antibodies that amino acid and growth factor stimulation of cells induces Rictor phosphorylation at Thr1135 in a rapamycin-sensitive manner (Figure 2 and Supplementary Figure S1). These results reveal that Rictor, described as a nutrient-insensitive partner of mTOR, is phosphorylated in response to amino acids through an mTORC1-dependent signaling cascade, suggesting that Rictor could also play a role in the amino-acid response. The fact that phosphorylation at Thr1135 does not modify the association between Rictor, mTOR, Sin1 and GβL, shows that amino acids stimulate the phosphorylation of Rictor as part of mTORC2.

The effect of rapamycin on mTORC2 is still poorly understood. Long-term rapamycin treatment has been shown to modify the integrity, phosphorylation state and activity of mTORC2, suggesting that rapamycin could indirectly act on this complex (Sarbassov et al., 2006; Akcakanat et al., 2007). Rapamycin is known to be a potent inhibitor of mTORC1, and hence of its direct target S6K1. We demonstrated here that Rictor is a direct target of S6K1, revealing a direct link between mTORC2 and rapamycin. This result shows that Rictor's phosphorylation state is directly controlled by mTORC1's activity and places Rictor/mTORC2 downstream of mTORC1, adding a new level of complexity in the functional relationship between the two mTOR complexes.

Mammalian cells express two forms of S6 kinases, S6K1 and S6K2, encoded by two distinct genes. Despite a high level of overall sequence homology, murine S6K1 and S6K2-knockout models and RNA interference studies reveal both common and isoform-specific functions. S6K1-deficient mice are smaller at birth because of a specific defect in cell size, with adipose tissues, skeletal muscle and pancreatic β-cells most affected (Shima et al., 1998; Pende et al., 2000). By contrast, S6K2-deficient mice do not display any defect in size (Pende et al., 2004). On a growth level, double-knockout mice are similar to S6K1-deficient mice, suggesting that S6K1 has specific substrates promoting cell growth. The ribosomal protein S6, which is a common target of S6K1 and S6K2, was the first and for several years the only S6K substrate described. Despite the description of several new substrates, little is known about isoform specificity (Ruvinsky and Meyuhas, 2006). Although animal models suggest the existence of specific targets for each kinase, SKAR is the only isoforms-specific target described so far—for S6K1 (Richardson et al., 2004). We report here that Rictor is a direct target of S6K1 and that S6K1 rather than S6K2 is likely to be the major kinase responsible for Rictor phosphorylation at Thr1135, suggesting that Rictor may play a role in the non-redundant functional specificity of S6K1.

We found here that Rictor plays a critical role in the control of the phosphorylation level of the hydrophobic and turn motifs of Akt/PKB and PKC, but is not associated with alteration of the phosphorylation of the Akt/PKB targets GSK3 and TSC2, as previously shown by others (Guertin et al., 2006; Jacinto et al., 2006; Shiota et al., 2006; Facchinetti et al., 2008; Ikenoue et al., 2008). However, in contrast with other reports, we did not detect any impact of Rictor deletion on the phosphorylation of the transcription factor FOXO3A at Thr32 (Guertin et al., 2006; Jacinto et al., 2006). We were unable to determine the phosphorylation state of SGK1, but, as reported by others, we notice a strong decrease in the phosphorylation level of one of its substrates, NDRG1 (N-myc downstream-regulated gene-1), in Rictor-null MEFs (Garcia-Martinez and Alessi, 2008). This finding indicates that Rictor plays a major role in the regulation of the SGK1 signaling cascade, which has been suggested to be a primary effector of mTORC2's function in yeast (Kamada et al., 2005; Aronova et al., 2008) and Caenorhabditis elegans (Jones et al., 2009; Soukas et al., 2009), but suggests that it may be somewhat less critical in the regulation of the activity of Akt/PKB. It is noteworthy that in muscle-specific, Rictor-knockout mice, Akt/PKB phosphorylation at S473 is decreased, while phosphorylation of FOXO3A at T32 and FOXO1 at T24 is maintained (Bentzinger et al., 2008; Kumar et al., 2008). As reported here (Figure 4), mutation of Rictor at Thr1135 does not block the ability of Rictor to restore the phosphorylation of Akt/PKB, PKC and NDRG1 in Rictor-null MEFs, revealing that this phosphorylation is not required for mTORC2 activity toward these substrates. This is consistent with the fact that amino acids are not required for Akt/PKB, PKC or SGK1 signaling.

In a subset of cancer cell lines and patient tumors, inhibition of mTORC1 induces Akt/PKB phosphorylation at Ser473 (O’Reilly et al., 2006). This is due to the inhibition of a negative feedback loop, whereby mTORC1/S6K activation inhibits PI3-kinase signaling by phosphorylating and inhibiting insulin receptor substrate proteins and regulating platelet-derived growth factor receptor expression (Zhang et al., 2003; Harrington et al., 2004; Manning, 2004; Shah et al., 2004). The existence of other feedback loops is still a distinct possibility, and a rapamycin-sensitive phosphorylation event within Rictor could have been an example of this. But, as shown in Figure 4, expression of the Rictor T1135A mutant does not block the effect of rapamycin on Akt/PKB. Rapamycin treatment increases the phosphorylation level of Akt/PKB at Thr308 in Rictor-null MEFs, suggesting that Rictor is not required for rapamycin's effect on Akt/PKB signaling (Figure 4 and Supplementary Figure S3). Indeed, it has been described recently that inhibition of mTORC1 initiates Akt/PKB activation independent of Rictor (Wang et al., 2008).

Whereas mutation of Rictor at the S6K1-dependent phosphorylation site did not affect its interaction with mTOR, GβL or Sin1, we found that this phosphorylation event modulates Rictor's association with 14-3-3. Previously, 14-3-3α, β and ɛ were reported to co-purify with tandem affinity purification-tagged Rictor, although they were not detected in complex with the endogenous protein (Pearce et al., 2007). 14-3-3 proteins regulate numerous cellular signaling events by binding to proteins following their phosphorylation at a defined motif (Hermeking, 2003). Thr1135 is part of a mode-1-binding motif (RS/TxpS/TxP), which is conserved across vertebrates (Yaffe et al., 1997). 14-3-3 binding regulates the activity of its target by a number of different mechanisms, including inter- and intra-compartmental sequestration or promotion/inhibition of protein interaction, which could be applicable to Rictor. The importance of mTOR localization has recently been highlighted (Sancak et al., 2008), although so far details of the localization of endogenous Rictor have not been established. We note that the mTORC1 component Raptor has been shown previously to interact with 14-3-3 when phosphorylated by AMPK, in this case leading to inhibition of the activity of the mTORC1 complex (Gwinn et al., 2008). Another mTORC1 component, PRAS40, associates with 14-3-3 when phosphorylated by Akt/PKB, although in that case the effect on mTORC1 kinase activity is still subject to debate.

The full functional significance of the phosphorylation of Rictor by S6K1 at T1135 remains to be determined. This is clearly a tightly regulated event, responding rapidly to upstream activators of S6K1, but also being rapidly reversed by the action of phosphatases. While the effects on mTORC2-kinase activity toward previously defined substrates are marginal in our experiments, it is possible that other as yet unidentified substrates are regulated more impressively, or that the effects on subcellular localization within the cell are important. Rictor is also known to have effects on the actin cytoskeleton, which have not yet been elucidated fully (Jacinto et al., 2004); phosphorylation at Thr1135 might influence these in some way. The phosphorylation of Rictor by S6K1 might play a role under certain pathological conditions; indeed, Rictor/mTORC2 activity seems to be important in malignant transformation (Guertin et al., 2009) and S6K1 plays a central role under nutritional conditions in which mTOR is chronically activated, such as high-fat diet (Um et al., 2004). The role of Rictor phosphorylation by S6K1 in cancer or diet-induced obesity could be addressed by creation of mice expressing the T1135A mutation in Rictor.

In conclusion, in this study we have uncovered a rapamycin-sensitive modification of mTORC2, mediated by phosphorylation of Rictor at T1135 by S6K1.

Materials and methods

Materials

Protein-A–sepharose, protein-G–sepharose and insulin were purchased from Sigma (St Louis, MO, USA). EGF, rapamycin, LY294002, U0126, PI-103 and calyculin-A were purchased from Calbiochem (San Diego, CA, USA). Amino acids were purchased from Gibco (Langley, OK, USA). siRNA Smart Pools were purchased from Dharmacon. Purified recombinant, full-length human p70S6K was purchased from Cell Signaling Technology (Danvers, MA, USA).

Antibodies

Anti-Rictor and anti-Sin1 antibodies were obtained from Novus Biologicals (Littleton, CO, USA). Anti-phospho-PKB substrate, anti-phospho-PKB (Ser308), anti-phospho-PKB (Ser450), anti-phospho-PKB (Ser473), pan PKB, anti-phospho-GSK3α/β (Ser21/9), anti-phospho TSC2 (Thr1462), anti-TSC2, anti-phospho-PKCα/β (Thr638/641), anti-phospho-PKC pan (βII Ser660), anti-phospho-p70S6K (Thr389), anti-p70S6K, anti-phospho-S6 (Ser235/Ser236), anti-phosho-S6 (Ser240/Ser244), anti-S6, anti-phospho-Erk1/2 (Thr202/Tyr204), anti-phospho-NDRG1 (Thr346), anti-14-3-3ɛ, anti-mTOR and anti-GβL antibodies were obtained from Cell Signaling Technology. Anti-α-tubulin antibody was obtained from Sigma. Anti-lamin-B and anti-phospho-PKCα (Ser657) antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-phospho-FKHRL1/FOXO3A (Thr32) antibody was obtained from Upstate Biotechnology (Waltham, MA, USA). Anti-Myc and anti-hemagglutinin (HA) antibodies were produced in-house. Anti-mouse and anti-rabbit antibodies coupled to horseradish peroxidase were obtained from GE Healthcare (Munich, Germany). The rabbit polyclonal phospho-specific antibody against Rictor Thr1135 was generated against the peptide RIRTLpTEPSVDC, on the basis of the sequence surrounding the residue Thr1135 in human Rictor.

Constructs

pRK5-myc-Rictor, pRK7-HA-S6K1wt, pRK7-HA-S6K1-E389ΔCT and pcDNA3-HA-S6K2wt constructs were obtained from Addgene.org (plasmids 11367(Sarbassov et al., 2004), 8984, 8993 and 17729 (Schalm and Blenis, 2002), respectively). Mutagenesis of residue Thr1135 and residue Ser1138, within Rictor, was performed using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA, USA). The Rictor Δ mutant (deletion of amino acids 1030–1323) was generated by XbaI digestion of pRK5-myc-Rictor. pcDNA3-S6K2-E388ΔCT was generated by introducing a stop codon at amino acid 399 into pcDNA3-S6K2-E388-D3E obtained from Addgene.org (plasmid 17731), using the QuikChange site-directed mutagenesis kit (Stratagene). These mutations were confirmed by sequencing.

Cell culture and transfections

The wild-type control S6K1/2+/+ and S6K1/2−/− MEFs were described previously (Pende et al., 2004). The wild-type control Rictor+/+ and Rictor−/− MEFs were described previously (Shiota et al., 2006). S6K1/2+/+, S6K1/2−/−, Rictor+/+ and Rictor−/− MEFs were maintained in Dulbecco's modified Eagle's medium, supplemented with 10% fetal bovine serum, 100 μg/ml streptomycin and 100 U/ml penicillin at 37 °C and 5% CO2. HEK-293 cells (human embryonic kidney) and A549 cells (non-small-cell lung carcinoma) were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 100 μg/ml streptomycin and 100 U/ml penicillin at 37 °C and 10% CO2.

Plasmid transfections were performed with the Effectene reagent (Qiagen, Hilden, Germany) in HEK-293 or with Lipofectamin Plus (Invitrogen, Carlsbad, CA, USA) in Rictor−/− MEFs according to the manufacturer's protocol. Transfections of siRNA were performed with Oligofectamine (Invitrogen) according to the manufacturer's protocol.

Cell lysis, immunoprecipitations and immunoblots

Cells were rinsed once with phosphate-buffered saline and lysed in ice-cold lysis buffer (40 mM Hepes (pH 7.5), 120 mM NaCl, 50 mM NaF, 1 mM EDTA, 10 mM pyrophosphate, 10 mM glycerophosphate, 0.3% CHAPS, EDTA-free protease inhibitors (Roche Diagnostics, Basel, Switzerland) and phosphatase inhibitor cocktails (Sigma)). Clear cell lysates were incubated with the precipitating antibody for 2 h followed by a 1-h incubation with protein-A/G–sepharose (Sigma). Immunoprecipitates were washed three times with lysis buffer and 4 × Laemmli sample buffer (Invitrogen) was added. Samples were resolved on 4–12% gradient gel from Invitrogen and proteins were transferred to a polyvinylidene difluoride membrane (Millipore, Bedford, MA, USA). The membranes were blocked in TBS-T (TBS containing 0.1% Tween-20) with 5% milk, incubated with the indicated antibody, washed in TBS-T, incubated with horseradish peroxidase-conjugated secondary antibody, washed again in TBS-T and revealed by enhanced chemiluminiscence (GE Healthcare).

Mass spectrometry

MS analysis was performed by microcapillary liquid chromatography (LC)/MS/MS techniques by the Taplin Biological Mass Spectrometry Facility (Harvard Medical School, Boston, MA, USA; http://taplin.med.harvard.edu/?q=node/2).

In vitro kinase assays

S6K in vitro kinase assays were performed according to the manufacturer's protocol (Cell Signaling). Endogenous Rictor, the wild-type form of Rictor or T1135A, Δ and S1138A mutant forms of Rictor were immunoprecipitated from HEK-293 cells treated for 30 min with PI-103(1 μM) and then used as substrate. Beads from immunoprecipitations were washed thrice in lysis buffer and once in kinase buffer (4 mM MOPS (pH 7.2), 2.5 mM β-glycerophosphate, 1 mM EGTA, 0.4 mM EDTA, 30 mM MgCl2, 0.05 mm dithiothreitol and 250 μM ATP (Cell Signaling)). A 200-ng weight of p70S6K active recombinant protein (Cell Signaling) was added to the reaction for 30 min at 30 °C in 15 μl kinase buffer. Reactions were stopped by the addition of 4 × Laemmli sample buffer (Invitrogen) and the reaction products were subject to SDS–PAGE.

Peptide pull-down assays

Peptides corresponding to the sequence surrounding residue Thr1135 in human Rictor either non-phosphorylated (T1135 (RIRTLTEPSVDC)) or phosphorylated ((p)T1135 (RIRTLpTEPSVDC)), were used. One milligram of peptide was coupled to 500 μl of packed Affi-Gel-15 beads (Bio-Rad, Hercules, CA, USA) according to the manufacturer's instructions, and then beads were washed once in lysis buffer. Clear cell lysates were incubated with 30 μl of peptide-coupled beads overnight. Beads were washed three times with lysis buffer and 4 × Laemmli sample buffer (Invitrogen) was added. Samples were analysed by western blotting.

Conflict of interest

The authors declare no conflict of interest.

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Acknowledgements

We thank Julie Bastien, Megan Cully, David Hancock and Oliver Pardo for helpful discussions and technical advice. This work was funded by Cancer Research UK.

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Correspondence to J Downward.

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Supplementary Information accompanies the paper on the Oncogene website (http://www.nature.com/onc)

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Treins, C., Warne, P., Magnuson, M. et al. Rictor is a novel target of p70 S6 kinase-1. Oncogene 29, 1003–1016 (2010). https://doi.org/10.1038/onc.2009.401

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Keywords

  • Rictor
  • S6K
  • feedback
  • amino acids
  • Akt/PKB
  • mTORC2

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