Sin1 phosphorylation impairs mTORC2 complex integrity and inhibits downstream Akt signalling to suppress tumorigenesis - PubMed (original) (raw)

. 2013 Nov;15(11):1340-50.

doi: 10.1038/ncb2860. Epub 2013 Oct 27.

Wenjian Gan, Hiroyuki Inuzuka, Adam S Lazorchak, Daming Gao, Omotooke Arojo, Dou Liu, Lixin Wan, Bo Zhai, Yonghao Yu, Min Yuan, Byeong Mo Kim, Shavali Shaik, Suchithra Menon, Steven P Gygi, Tae Ho Lee, John M Asara, Brendan D Manning, John Blenis, Bing Su, Wenyi Wei

Affiliations

Sin1 phosphorylation impairs mTORC2 complex integrity and inhibits downstream Akt signalling to suppress tumorigenesis

Pengda Liu et al. Nat Cell Biol. 2013 Nov.

Erratum in

Abstract

The mechanistic target of rapamycin (mTOR) functions as a critical regulator of cellular growth and metabolism by forming multi-component, yet functionally distinct complexes mTORC1 and mTORC2. Although mTORC2 has been implicated in mTORC1 activation, little is known about how mTORC2 is regulated. Here we report that phosphorylation of Sin1 at Thr 86 and Thr 398 suppresses mTORC2 kinase activity by dissociating Sin1 from mTORC2. Importantly, Sin1 phosphorylation, triggered by S6K or Akt, in a cellular context-dependent manner, inhibits not only insulin- or IGF-1-mediated, but also PDGF- or EGF-induced Akt phosphorylation by mTORC2, demonstrating a negative regulation of mTORC2 independent of IRS-1 and Grb10. Finally, a cancer-patient-derived Sin1-R81T mutation impairs Sin1 phosphorylation, leading to hyper-activation of mTORC2 by bypassing this negative regulation. Together, our results reveal a Sin1-phosphorylation-dependent mTORC2 regulation, providing a potential molecular mechanism by which mutations in the mTORC1-S6K-Sin1 signalling axis might cause aberrant hyper-activation of the mTORC2-Akt pathway, which facilitates tumorigenesis.

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Conflict of interest statement

Competing Financial Interests

The authors declare no competing financial interests.

Figures

Figure 1

Figure 1. S6K phosphorylates Sin1 on both T86 and T398 sites

a. Immunoblot (IB) analysis of whole cell lysates (WCL) and Flag-immunoprecipitates (IP) derived from Flag-Sin1-transfected HeLa cells that were serum-starved for 24 hours and then collected after serum stimulation for 30 minutes. Where indicated, the kinase inhibitors (AktVIII: 10 μM, PP242: 1 μM, Rapamycin: 20 nM, S6K1-I: 10 μM) were added together with insulin (100 nM). DMSO was used as a negative control. b. IB analysis of WCL and IP derived from 293T cells transfected with Flag-Sin1 and the indicated HA-tagged constitutive active AGC family kinases. c. IB analysis of WCL and IP derived from 293T cells transfected with Flag-Sin1 and HA-S6K1 (or empty vector as a negative control). Where indicated, the S6K inhibitor was added. d. Schematic illustration of the two evolutionarily conserved putative S6K phosphorylation sites, T86 and T398 within Sin1. e. IB analysis of WCL and IP derived from 293T cells transfected with constitutive active form of S6K (HA-S6K-R3A) and the indicated Flag-Sin1 constructs. f. In vitro kinase assays depicting major S6K phosphorylation sites in Sin1. Please note that the GST-Rictor fusion protein used here is not the full-length protein but rather the truncated version that contains the S6K phosphorylation site T1135 (GST-Rictor-C-tail [aa1390–1708]). g. IB analysis of WCL and Flag-IP derived from HeLa cells transfected with the indicated Flag-Sin1 constructs. Where indicated, cells were serum starved for 12 hours and stimulated by 100 nM insulin for 30 minutes before harvesting. h. IB analysis of WCL and Flag-IP derived from HeLa cells depleted of Raptor transfected with Flag-Sin1 (shGFP as a negative control). Where indicated, cells were serum starved for 12 hours and stimulated by the indicated stimuli before harvesting.

Figure 2

Figure 2. S6K-dependent phosphorylation of Sin1 dissociates Sin1 from the mTORC2 complex

a. Immunoblot (IB) analysis of whole cell lysates (WCL) and Flag immunoprecipitates (IP) derived from 293T cells transfected with the indicated Flag-Sin1 constructs (EV: empty vector control; WT: Sin1-WT; AA: Sin1-T86A/T398A; EE: Sin1-T86E/T398E). b–d. GST pull down assays to demonstrate that S6K phosphorylation of GST-Sin1-WT-FL (full-length) but not GST-Sin1-T86A/T398A led to impaired interaction with Rictor (b), mTOR-kinase domain (KD) (c) or GβL (d). As indicated, GST-Sin1 proteins were phosphorylated by active recombinant S6K in vitro for 1 hour before using as a bait to pull down HA-Rictor (b), mTOR-kinase domain (KD) (c) or GβL (d) expressed in 293T cells. e. Gel filtration experiments to illustrate that comparing with WT-Sin1, Sin1-EE lost interaction with the functional mTORC2 complex components in vivo. IB analysis of the indicated fractionations derived from the gel filtration experiment with HeLa cells co-transfected with HA-Sin1-WT and Flag-Sin1-EE constructs. Prior to running cell lysates, the molecular weight resolution of the column was first estimated by running native molecular weight markers (Thyroglobulin ~669KD, Ferritin ~440KD, Aldolase ~158KD, Conalbumin ~75KD and Ovalbumin ~44KD) to determine their retention times on coomassie-stained SDS-PAGE protein gels. f. Deletion of endogenous TSC2, which led to increased S6K kinase activity, resulted in a reduction of Rictor association with Sin1. IB analysis of WCL and anti-Sin1-IP derived from TSC2+/+ or TSC2−/− MEFs. g. Schematic representation of the indicated domains of Sin1 as well as the locations of the two Sin1 phosphorylation sites: T86 is in the N-terminal domain while T398 is located in the PH domain. h. GST pull down assays to depict the Sin1 domains that interact with Rictor, mTOR-KD or GβL, respectively (* indicates the sizes of GST-Sin1 proteins). I–j. GST pull down assays to demonstrate that Sin1 T86E or T398E mutation led to reduced interaction with Rictor (i) or mTOR-KD (j), respectively.

Figure 3

Figure 3. Sin1 phosphorylation induced by various stimuli impairs mTORC2 integrity

a. Immunoblot (IB) analysis of whole cell lysates (WCL) and endogenous Sin1 immunoprecipitates (IP) derived from Sin1-WT MEFs that were serum-starved for 24 hours and then collected after EGF (100 ng/ml) stimulation for the indicated time periods. b. IB analysis of WCL and Flag-IP derived from Flag-Sin1-transfected HeLa cells that were serum-starved for 24 hours and then collected after insulin stimulation for the indicated time periods. c. Either Sin1-T86A or T398A mutation impaired the dynamic interaction between Sin1 and other essential mTORC2 components. IB analysis of WCL and Flag-IP derived from HeLa cells transfected with the indicated Flag-Sin1 constructs that were serum starved for 12 hours and then treated with the EGF (100 ng/ml) for the indicated time periods before harvesting for IB analysis. d. Either Sin1-T86A or T398A mutation led to sustained Akt activation upon EGF stimulation. IB analysis of WCL derived from HeLa cells transfected with the indicated Flag-Sin1 constructs that were serum starved for 12 hours and then treated with the EGF (100 ng/ml) for the indicated time periods before harvesting for IB analysis. e. Rapamycin or S6K1-I treatment led to a relatively sustained Akt-pS473 upon insulin stimulation. IB analysis of WCL derived from HeLa cells serum starved for 12 hours and stimulated with 100 ng/ml insulin before harvesting at the indicated time points. Where indicated, 20 nM rapamycin or 10 μM S6K1-I was added.

Figure 4

Figure 4. Sin1 phosphomimetic mutation is deficient in interacting with the mTORC2 substrate Akt1, but not SGK1

a–b. Immunoblot (IB) analysis of whole cell lysates (WCL) and HA (a) or Flag (b) immunoprecipitates (IP) derived from 293T cells that were transfected with the indicated Flag-Sin1 constructs with HA-Akt1. c. Sin1-T86E or Sin1-T398E disrupts Sin1-N-terminus or Sin1-PH domain interaction with Akt1. Indicated GST-Sin1 proteins were used as a bait to pull down HA-Akt1 expressed in 293T cells. d–e. IB analysis of WCL and HA-IP derived from 293T cells that were transfected with the indicated Flag-Sin1 constructs with HA-SGK-Δ60. f–g. IB analysis of WCL and HA-IP or Flag-IP derived from 293T cells that were transfected with the indicated Flag-Sin1 constructs with HA-S6K1.

Figure 5

Figure 5. Sin1 phosphorylation suppresses mTORC2 kinase activity towards phosphorylating Akt in vitro

a–b. 293T cells were transfected with the indicated Flag-tagged Sin1 constructs. 36 hours post-transfection, whole cell lysates (WCL) were collected and the mTORC2 complex was purified by Flag-immunoprecipitation (IP). The Flag-IPs were incubated in vitro with purified GST-Akt1 in the presence of ATP and the kinase reaction buffer. Thirty minutes later, the reaction was stopped by the addition of the loading buffer. Akt1 phosphorylation status was examined by immunoblot (IB) analysis. c–d. Prolonged insulin treatment (45 min) induces Sin1 phosphorylation, leading to dissociation of mTORC2 complex and abolished Akt activation. Flag-Sin1-WT or R81T mutant was transfected into HeLa cells and 48 hours later the transfected cells were harvested upon insulin (100 nM) stimulation for 45 min after 12 hours of serum starvation in CHAPS buffer. The whole cell lysates were filtered and run through FPLC superdex 200 column. 500 μL elute was collected for each fraction and 1/20 volume of each fraction was incubated with 2 μg GST-Akt-tail (aa 408–480) at 30°C for 30 min. Afterwards, the resulting samples were resolved on SDS-PAGE and subjected to IB analysis. e–f. Rapamycin treatment restored Sin1 phosphorylation resulted from EGF treatment, leading to reassembly of mTORC2 complex and Akt activation. Flag-Sin1-WT was transfected into TSC2-depleted HeLa cells and 24 hours later, the transfected cells were treated with 20 nM rapamycin for another 12 hours prior to EGF stimulation (100 nM) before harvested and analyzed as in (c–d).

Figure 6

Figure 6. Sin1 phosphorylation attenuates mTORC2 kinase activity towards phosphorylating Akt in vivo

a. Sin1−/− MEFs were transfected with the indicated Flag-Sin1 constructs. 30 hours post-transfection, the resulting cells were serum-starved for 24 hours and then collected after stimulation with insulin for 30 minutes for immunoblot (IB) analysis. b. Reconstitution of Sin1−/− MEFs with WT-, but not EE-Sin1, could restore Akt-Ser473 phosphorylation under various stimulation conditions. Sin1−/− MEFs were transfected with the indicated Flag-Sin1 constructs and serum-starved for 24 hours before harvesting after treatment with indicated stimuli for IB analysis. c. The indicated Flag-Sin1 constructs were transfected into Sin1−/− MEFs and Flag immunoprecipitation (IP) was recovered as the kinase source to phosphorylate GST-Akt1-tail (aa 408–480) in vitro. d. The indicated Flag-Sin1 constructs were transfected into Sin1−/− MEFs and endogenous Akt IP was performed as the kinase source to phosphorylate crosstide in vitro. Data was shown as mean ± SD for n= 3 independent experiments. e–g. Sin1−/− MEFs were transfected with the indicated Flag-Sin1 constructs (with empty vector as a negative control). 24 hours post-transfection, the resulting cells were cultured in 10% FBS-containing medium with the indicated concentrations of etoposide (e) or cisplatin (f) for 48 hours before performing the cell viability assays (e,f) or IB analysis (g). Data was shown as mean ± SD from n=3 independent experiments. * indicates p < 0.05 (Student’s t-test).

Figure 7

Figure 7. The pathological Sin1-R81T mutation led to attenuated Sin1-T86 phosphorylation and sustained Akt phosphorylation upon physiological stimulations

a. Schematic illustrations of the ovarian cancer patient-derived Sin1-R81T mutation and the skin cancer patient-derived S84L mutation. b. Immunoblot (IB) analysis of whole cell lysate (WCL) and Flag-immunoprecipitates (IP) derived from 293T cells transfected with the indicated Flag-Sin1 constructs and HA-S6K1. c. Sin1-R81T does not interfere with the Sin1-pT86 antibody to recognize Sin1-pT86. Indicated Sin1 synthetic peptides were dotted on nitrocellulose membrane for IB analysis. d. HeLa cells were transfected with the indicated Flag-Sin1 constructs and serum-starved for 24 hours and then collected after stimulation with the indicated stimuli for 30 minutes for IB analysis and Flag-IP. e. Sin1−/− MEFs were transfected with the indicated Flag-Sin1 constructs and serum-starved overnight followed by IB analysis upon 100 ng/ml EGF stimulation for the indicated time points. f–g. Sin1 depleted OVCAR5 cells stably expressing Sin1-WT or -R81T were serum starved overnight followed by IB analysis after treatment with 100 ng/ml EGF (f) or 100 nM insulin (g) for the indicated time points.

Figure 8

Figure 8. The pathological Sin1-R81T mutation displayed elevated oncogenic activity in part by bypassing Sin1 phosphorylation-mediated negative regulation of Akt-pS473

a–c. Sin1−/− MEFs were transfected with the indicated Flag-Sin1 constructs and were treated with the indicated concentrations of etoposide (a) or cisplatin (b,c) for 48 hours before performing the cell viability assays (a,b) or immunoblot (IB) analysis (c). Data was shown as mean ± SD from n=3 independent experiments. * indicates p < 0.05 (t-test). d. Soft agar assays for Sin1-depleted OVCAR5 cells stably expressing EV, WT or R81T. Data was presented as mean ± SD from n=3 independent experiments. e–f. Growth curves (e) and mass of the dissected tumors (f) from xenograft experiments with the indicated cells injected subcutaneously into n=10 mice for each cell line. The visible tumors were measured at the indicated days. Error bars, ±SEM and * indicates p < 0.05 (t-test). g. Representative images of the dissected tumors presented in Figure 8e,f. h. Eight-week-old mice were fasted overnight and then refed for 6 hours following a 30 min pretreatment with vehicle or rapamycin (10 mg/kg). N=4 mice per condition. Livers were dissected and liver lysates were subjected to IB analysis. i. Four representative images of IHC with indicated Sin1 and Akt phosphorylation status out of 58 ovarian patient samples under 400x magnification. Scale bar represents 100 μm.

Comment in

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