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Central Regulatory Role for SIN1 in Interferon γ (IFNγ) Signaling and Generation of Biological Responses*

  • Barbara Kroczynska
    Affiliations
    Robert H. Lurie Comprehensive Cancer Center and Division of Hematology-Oncology, Feinberg School of Medicine, Chicago, Illinois 60611

    Department of Radiation Oncology, Northwestern University, Chicago, Illinois 60611
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  • Gavin T. Blyth
    Affiliations
    Robert H. Lurie Comprehensive Cancer Center and Division of Hematology-Oncology, Feinberg School of Medicine, Chicago, Illinois 60611
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  • Robert L. Rafidi
    Affiliations
    Robert H. Lurie Comprehensive Cancer Center and Division of Hematology-Oncology, Feinberg School of Medicine, Chicago, Illinois 60611
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  • Beata Majchrzak-Kita
    Affiliations
    Toronto General Research Institute, University Health Network, and Department of Immunology, University of Toronto, Toronto, Ontario M5G 2M1, Canada
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  • Lucy Xu
    Affiliations
    Robert H. Lurie Comprehensive Cancer Center and Division of Hematology-Oncology, Feinberg School of Medicine, Chicago, Illinois 60611
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  • Diana Saleiro
    Footnotes
    Affiliations
    Robert H. Lurie Comprehensive Cancer Center and Division of Hematology-Oncology, Feinberg School of Medicine, Chicago, Illinois 60611
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  • Ewa M. Kosciuczuk
    Affiliations
    Robert H. Lurie Comprehensive Cancer Center and Division of Hematology-Oncology, Feinberg School of Medicine, Chicago, Illinois 60611

    Division of Hematology-Oncology, Department of Medicine, Jesse Brown Veterans Affairs Medical Center, Chicago, Illinois 60612
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  • Jacek Jemielity
    Footnotes
    Affiliations
    Centre of New Technologies, University of Warsaw, 02-097 Warsaw, Poland
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  • Bing Su
    Footnotes
    Affiliations
    Department of Immunobiology and the Vascular Biology and Therapeutics Program, Yale University, New Haven, Connecticut 06520

    Shanghai Institute of Immunology and Department of Microbiology and Immunology, Shanghai JiaoTong University School of Medicine, Shanghai 200000, China
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  • Jessica K. Altman
    Affiliations
    Robert H. Lurie Comprehensive Cancer Center and Division of Hematology-Oncology, Feinberg School of Medicine, Chicago, Illinois 60611

    Division of Hematology-Oncology, Department of Medicine, Jesse Brown Veterans Affairs Medical Center, Chicago, Illinois 60612
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  • Elizabeth A. Eklund
    Affiliations
    Robert H. Lurie Comprehensive Cancer Center and Division of Hematology-Oncology, Feinberg School of Medicine, Chicago, Illinois 60611

    Division of Hematology-Oncology, Department of Medicine, Jesse Brown Veterans Affairs Medical Center, Chicago, Illinois 60612
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  • Eleanor N. Fish
    Footnotes
    Affiliations
    Toronto General Research Institute, University Health Network, and Department of Immunology, University of Toronto, Toronto, Ontario M5G 2M1, Canada
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  • Leonidas C. Platanias
    Correspondence
    To whom correspondence should be addressed: Robert H. Lurie Comprehensive Cancer Center, 303 E. Superior Ave., Lurie 3-125, Chicago, IL 60611. Tel.: 312-503-4267;
    Affiliations
    Robert H. Lurie Comprehensive Cancer Center and Division of Hematology-Oncology, Feinberg School of Medicine, Chicago, Illinois 60611

    Division of Hematology-Oncology, Department of Medicine, Jesse Brown Veterans Affairs Medical Center, Chicago, Illinois 60612
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  • Author Footnotes
    2 Supported by National Science Centre (Poland) Grant UMO-2013/09/B/ST5/01341.
    * This work was supported by National Institutes of Health Grants CA155566, CA161796, CA77816, and CA121192 and by Department of Veterans Affairs Grant I01CX000916. The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
    1 Supported by NCI, National Institutes of Health, Grant T32 CA080621.
    3 Supported by National Natural Science Foundation of China Grants 31470845 and 81430033 and Shanghai Science and Technology Commission Grant 13JC1404700.
    4 A Tier 1 Canada Research Chair.
Open AccessPublished:February 07, 2017DOI:https://doi.org/10.1074/jbc.M116.757666
      The precise signaling mechanisms by which type II IFN receptors control expression of unique genes to induce biological responses remain to be established. We provide evidence that Sin1, a known element of the mammalian target of rapamycin complex 2 (mTORC2), is required for IFNγ-induced phosphorylation and activation of AKT and that such activation mediates downstream regulation of mTORC1 and its effectors. These events play important roles in the assembly of the eukaryotic translation initiation factor 4F (eIF4F) and mRNA translation of IFN-stimulated genes. Interestingly, IFNγ-induced tyrosine phosphorylation of STAT1 is reduced in cells with targeted disruption of Sin1, leading to decreased transcription of several IFNγ-inducible genes in an mTORC2-independent manner. Additionally, our studies establish that Sin1 is essential for generation of type II IFN-dependent antiviral effects and antiproliferative responses in normal and malignant hematopoiesis. Together, our findings establish an important role for Sin1 in both transcription and translation of IFN-stimulated genes and type II IFN-mediated biological responses, involving both mTORC2-dependent and -independent functions.

      Introduction

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      The abbreviations used are: ISG
      IFN-stimulated gene
      mTORC
      mammalian target of rapamycin complex
      eIF4B
      eukaryotic translation initiation factor
      EMCV
      encephalomyocaditis virus
      CPE
      cytopathic effect(s)
      MEF
      mouse embryo fibroblast
      ANOVA
      analysis of variance.
      (
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      • Su B.
      SIN1/MIP1 maintains rictor-mTOR complex integrity and regulates Akt phosphorylation and substrate specificity.
      ). Because mTORC2 is a critical regulator of AKT, a kinase that is often activated in cancer cells and mediates survival signals, this mTOR complex has become a target for cancer therapy and has led to efforts to develop catalytic mTOR inhibitors that block the mTOR kinase (
      • Beauchamp E.M.
      • Platanias L.C.
      The evolution of the TOR pathway and its role in cancer.
      ,
      • Sabatini D.M.
      mTOR and cancer.
      ,
      • Janes M.R.
      • Limon J.J.
      • So L.
      • Chen J.
      • Lim R.J.
      • Chavez M.A.
      • Vu C.
      • Lilly M.B.
      • Mallya S.
      • Ong S.T.
      • Konopleva M.
      • Martin M.B.
      • Ren P.
      • Liu Y.
      • Rommel C.
      • Fruman D.A.
      Effective and selective targeting of leukemia cells using a TORC1/2 kinase inhibitor.
      ).
      In efforts to map and define the roles of different mTORC2-driven elements in the induction of IFNγ responses, we evaluated the role of Sin1 in the IFNγ system. Our studies demonstrate that Sin1 is required for IFNγ-induced phosphorylation of AKT and that Sin1-mediated AKT activation mediates downstream regulation of mTORC1 effectors to control mRNA translation of ISGs. Remarkably, IFNγ-induced STAT1 activation and subsequent transcription of several ISGs is diminished in cells with targeted disruption of Sin1, demonstrating the existence of an mTORC2-independent function of Sin1 as a regulator of Jak-Stat pathways.

      Results

      In initial studies, we compared type II IFN-dependent mTORC2 phosphorylation of AKT in wild type (Sin1+/+) versus Sin1 knock-out (Sin1−/−) mouse embryonic fibroblasts (MEFs). IFNγ treatment induced phosphorylation of AKT on Thr-450, Ser-473, and Thr-308 in Sin1+/+ MEFs but not in Sin1−/− MEFs (Fig. 1, A–C, respectively), thus establishing a requirement for Sin1 in induction of AKT activity. This result is consistent with previous findings demonstrating that mTORC2 activation is required for phosphorylation of the turn motif in AKT on Thr-450 and of the hydrophobic motif on Ser-473 and that this phosphorylation is required for enabling PDK1 to phosphorylate AKT at Thr-308 (
      • Jacinto E.
      • Facchinetti V.
      • Liu D.
      • Soto N.
      • Wei S.
      • Jung S.Y.
      • Huang Q.
      • Qin J.
      • Su B.
      SIN1/MIP1 maintains rictor-mTOR complex integrity and regulates Akt phosphorylation and substrate specificity.
      ,
      • 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.
      Ablation in mice of the mTORC components raptor, rictor, or mLST8 reveals that mTORC2 is required for signaling to Akt-FOXO and PKCα, but not S6K1.
      ,
      • 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.
      The mammalian target of rapamycin complex 2 controls folding and stability of Akt and protein kinase C.
      ,
      • Oh W.J.
      • Wu C.C.
      • Kim S.J.
      • Facchinetti V.
      • Julien L.A.
      • Finlan M.
      • Roux P.P.
      • Su B.
      • Jacinto E.
      mTORC2 can associate with ribosomes to promote cotranslational phosphorylation and stability of nascent Akt polypeptide.
      ,
      • Hresko R.C.
      • Mueckler M.
      mTOR-RICTOR is the Ser473 kinase for AKT/protein kinase B in 3T3-L1 adipocytes.
      ). Similarly, the absence of Sin1 resulted in decreased IFN-inducible phosphorylation of mTOR at Ser-2481 (Fig. 1D) and Ser-2448 (Fig. 1E), possibly reflecting reduced mTORC2 and mTORC1 catalytic activity, respectively (
      • Copp J.
      • Manning G.
      • Hunter T.
      TORC-specific phosphorylation of mammalian target of rapamycin (mTOR): phospho-Ser2481 is a marker for intact mTOR signaling complex 2.
      ). The defective type II IFN-dependent phosphorylation of AKT and mTOR at Ser-2448, taken together with our previous findings implicating AKT upstream of mTORC1 in IFNγ signaling, led us to examine mTORC1 effectors in the type II IFN system. IFNγ-induced phosphorylation of 4E-BP1 on Thr-37/46 (Fig. 1F) and S6K on Thr-389 (Fig. 1G) was reduced in Sin1−/− MEFs compared with Sin1+/+ MEFs. As expected, IFNγ treatment resulted in induction of phosphorylation of the downstream effectors of S6K, rpS6, and eukaryotic translation initiation factor 4B (eIF4B) (Fig. 1, H and I), but these phosphorylation levels were decreased in Sin1−/− MEFs. We also examined whether transient knockdown of Sin1 by specific siRNA-mediated targeting in malignant hematopoietic cells also results in reduced type II IFN-dependent mTOR activity. IFNγ-induced phosphorylation of AKT, mTOR, 4E-BP1, and S6K were impaired in U937 cells where Sin1 was selectively knocked down (Fig. 1, J–N), consistent with the findings in Sin1−/− MEFs.
      Figure thumbnail gr1
      FIGURE 1IFNγ-dependent engagement of mTOR effectors is Sin1-dependent. A–I, serum-starved Sin1+/+ and Sin1−/− MEFs were treated with mouse IFNγ (5 × 103 IU/ml) for the indicated times. A–I (top panels), cell lysates were prepared, and equal amounts of protein were resolved by SDS-PAGE and then subjected to immunoblotting with the indicated specific anti-phosphoantibodies. The blots in the respective top panels were stripped and probed with anti-AKT (A–C), anti-mTOR (D and E), anti-4E-BP1 (F), anti-p70S6K (G), anti-rpS6 (H), and anti-eIF4B (I) antibodies. J–N, serum-starved U937 cells transfected with control siRNA or Sin1 siRNA were treated with human IFNγ (5 × 103 IU/ml) for the indicated times. Cell lysates were prepared, and proteins were resolved by SDS-PAGE and then processed for immunoblotting with anti- phospho-Ser-473 AKT, AKT, Sin1, and GAPDH antibodies (J), anti-phospho-Thr-308 AKT and anti-AKT antibodies (K), antibodies against phospho-Ser-2481 mTOR, mTOR, Sin1, or GAPDH (L), anti-phospho-Thr-37/46 4E-BP1 and anti-4E-BP1 antibodies (M), and anti-phospho-Thr-389 p70S6K and anti-p70S6K antibodies (N). A–K (bottom panels), bands from three independent experiments (including the blots shown) were quantified by densitometry. Data are expressed as ratios of phosphoprotein over respective total protein values, and bar graphs represent means ± S.E. (error bars) of three independent experiments for each experimental condition.
      We subsequently sought to determine whether engagement of Sin1 in IFNγ signaling regulates expression of IFNγ-inducible proteins. For this purpose, Sin+/+ and Sin1−/− MEFs were incubated in the presence or absence of mouse IFNγ, and cell lysates were resolved by SDS-PAGE and immunoblotted with anti-IP10 and DAPK1 antibodies. The expression of IP10 (CXCL10), an IFN-induced chemokine that has diverse roles in infectious diseases and induces apoptosis and cell growth inhibition (
      • Liu M.
      • Guo S.
      • Stiles J.K.
      The emerging role of CXCL10 in cancer.
      ), was defective in Sin1−/− MEFs (Fig. 2A). We also examined whether Sin1 is required for the expression of DAPK1, a tumor suppressor protein (
      • Shiloh R.
      • Bialik S.
      • Kimchi A.
      The DAPK family: a structure-function analysis.
      ,
      • Gade P.
      • Ramachandran G.
      • Maachani U.B.
      • Rizzo M.A.
      • Okada T.
      • Prywes R.
      • Cross A.S.
      • Mori K.
      • Kalvakolanu D.V.
      An IFN-γ-stimulated ATF6-C/EBP-β-signaling pathway critical for the expression of death associated protein kinase 1 and induction of autophagy.
      ). As anticipated, IFNγ treatment induced the expression of DAPK1 in Sin1+/+, but this did not occur in Sin1−/− MEFs (Fig. 2B). Consistent with these findings, IFNγ-induced expression of IP10, DAPK1, and SLFN5 (
      • Sassano A.
      • Mavrommatis E.
      • Arslan A.D.
      • Kroczynska B.
      • Beauchamp E.M.
      • Khuon S.
      • Chew T.L.
      • Green K.J.
      • Munshi H.G.
      • Verma A.K.
      • Platanias L.C.
      Human Schlafen 5 (SLFN5) is a regulator of motility and invasiveness of renal cell carcinoma cells.
      ) was also reduced in U937 cells in which Sin1 was knocked down using specific siRNAs (Fig. 2, C and D).
      Figure thumbnail gr2
      FIGURE 2Requirement of Sin1 for type II IFN-induced ISG protein expression. A and B, serum-starved Sin1+/+ and Sin1−/− MEFs were treated with mouse IFNγ (1.5 × 103 IU/ml) as indicated. Cell lysates were prepared, and proteins were resolved by SDS-PAGE and immunoblotted with anti-IP10 (A) or anti-DAPK1 (B). C and D, serum-starved U937 cells transfected with control siRNA or Sin1 siRNA were treated with human IFNγ (1.5 × 103 IU/ml) for the indicated times. Cell lysates were prepared, and proteins were resolved by SDS-PAGE and then processed for immunoblotting with anti-IP10 (C) and anti-DAPK1 and anti-SLFN5 (D) antibodies. A–D, anti-GAPDH antibody was used for loading control.
      Next, using the 5′-cap binding assay, we undertook studies to evaluate the effects of Sin1 targeting the assembly of the translation initiation complex during IFNγ signaling (
      • Holz M.K.
      • Ballif B.A.
      • Gygi S.P.
      • Blenis J.
      mTOR and S6K1 mediate assembly of the translation preinitiation complex through dynamic protein interchange and ordered phosphorylation events.
      ). As expected, IFNγ treatment resulted in dissociation of 4E-BP1 from eIF4E and subsequently enhanced binding of eIF4G and eIF4A to the 5′-cap analog in Sin1+/+ cells. This was not the case in Sin1−/− MEFs (Fig. 3A). To determine whether impaired assembly of the eIF4F complex, which consists of eIF4E, eIF4G, and eIF4A, in Sin1−/− MEFs accounts for defective mRNA translation of type II IFN-dependent ISGs, we next evaluated the mRNA levels for Ip10, Dapk1, and Slfn2 (
      • Katsoulidis E.
      • Carayol N.
      • Woodard J.
      • Konieczna I.
      • Majchrzak-Kita B.
      • Jordan A.
      • Sassano A.
      • Eklund E.A.
      • Fish E.N.
      • Platanias L.C.
      Role of Schlafen 2 (SLFN2) in the generation of interferon α-induced growth inhibitory responses.
      ) in polysome fractions. Sin1+/+ and Sin1−/− MEFs were left untreated or were treated with IFNγ, polysomal mRNA was fractionated (Fig. 3B), and quantitative real-time RT-PCR was used to measure mRNA levels of ISGs in the polysome fractions. Ip10, Dapk1, and Slfn2 mRNA levels were decreased in Sin−/− MEFs compared with Sin+/+ MEFs (Fig. 3, C–E), indicating a reduction in translation of those mRNAs when Sin1 is absent. Although these results are consistent with the impaired activation of mTOR effectors, whose functions are required for initiation of mRNA translation, observed in Sin1−/− cells, these studies do not address the effects of Sin1 on the transcription of ISGs. Accordingly, we next examined the mRNA levels of Dapk1, Ip10, and Slfn2 in IFNγ-treated Sin1+/+ and Sin1−/− MEFs by quantitative real-time RT-PCR. Interestingly, Dapk1 mRNA expression was not defective in Sin1−/− cells (Fig. 4A), but both Ip10 and Slfn2 mRNA levels were reduced in the absence of Sin1 (Fig. 4, B and C). Together, these results suggest that Sin1 expression is required for mRNA translation of ISGs, but also for IFNγ-induced transcription of selective ISGs.
      Figure thumbnail gr3
      FIGURE 3Essential role for Sin1 in mRNA translation of type II ISGs. A, serum-starved Sin1+/+ and Sin1−/− MEFs were either left untreated or treated with mouse IFNγ (5 × 103 IU/ml) for the indicated times. Cell lysates were bound to the cap analog m7GTP biotin-labeled and streptavidin beads (
      • Sarbassov D.D.
      • Guertin D.A.
      • Ali S.M.
      • Sabatini D.M.
      Phosphorylation and regulation of AKT/PKB by the rictor-mTOR complex.
      ). After extensive washing, bound proteins were resolved by SDS-PAGE and immunoblotted with antibodies against eIF4G, eIF4A, eIF4E, and 4E-BP1. The cell lysates used for this experiment are from the same experiment shown in F. B, Sin1+/+ and Sin1−/− MEFs were either left untreated or treated with mouse IFNγ (1.5 × 103 IU/ml) in DMEM containing 0.5% FBS for 24 h. Cell lysates were layered on 5–50% sucrose gradients and subjected to density gradient centrifugation, and fractions were collected by continuous monitoring of optical density (O.D.) at 254 nm. Optical density at 254 nm is shown as a function of gradient depth and the 40S, 60S, and 80S peaks, and polysomal fractions are indicated. C–E, polysomal fractions were pooled, and RNA was isolated. Subsequently, quantitative real-time PCR was carried out to determine Ip10 (C) Dapk1 (D), and Slfn2 (E) mRNA expression in the polysomal fractions, using Gapdh for normalization. Data are expressed as -fold change over control untreated cells, and bar graphs represent means ± S.E. (error bars) of three independent experiments, including the ones shown in B. Statistical analyses were performed using Student's t test (*, p < 0.05).
      Figure thumbnail gr4
      FIGURE 4Essential role of Sin1 in transcription of Type II ISGs. Serum-starved Sin1+/+ or Sin1−/− MEFs were either left untreated or were treated with mouse IFNγ (2.5 × 103 IU/ml) for 6 h, and total RNA was isolated as described under “Experimental Procedures.” mRNA expression of Dapk1 (A), Ip10 (B), and Slfn2 (C) was evaluated by quantitative real-time PCR, and Gapdh was used for normalization. Data are expressed as -fold change over control untreated cells, and bar graphs represent means ± S.E. (error bars) of three independent experiments. Statistical analyses were performed using Student's t test (*, p < 0.05).
      Previously, we demonstrated that mTORC2 elements regulate transcription of type I ISGs by controlling activation of STAT-driven pathways, through regulation of STAT2 phosphorylation on Tyr-689 and STAT1 phosphorylation on Ser-727 (
      • Kaur S.
      • Kroczynska B.
      • Sharma B.
      • Sassano A.
      • Arslan A.D.
      • Majchrzak-Kita B.
      • Stein B.L.
      • McMahon B.
      • Altman J.K.
      • Su B.
      • Calogero R.A.
      • Fish E.N.
      • Platanias L.C.
      Critical roles for Rictor/Sin1 complexes in interferon-dependent gene transcription and generation of antiproliferative responses.
      ). In addition, prior data showed that the transcription of Ip10 and Slfn2 is STAT1-dependent, and transcription of DAPK1 is STAT1-independent (
      • Liu M.
      • Guo S.
      • Stiles J.K.
      The emerging role of CXCL10 in cancer.
      ,
      • Shiloh R.
      • Bialik S.
      • Kimchi A.
      The DAPK family: a structure-function analysis.
      ). This led us to consider the possibility that defective transcription of IFNγ-regulated genes in the absence of Sin1 could reflect impaired STAT1 activation. Previous studies have established that phosphorylation of STAT1 on Tyr-701 is required for STAT1 dimerization (
      • Darnell Jr, J.E.
      • Kerr I.M.
      • Stark G.R.
      Jak-STAT pathways and transcriptional activation in response to IFNs and other extracellular signaling proteins.
      ,
      • Levy D.E.
      • Darnell Jr., J.E.
      Stats: transcriptional control and biological impact.
      ), whereas phosphorylation on Ser-727 is essential for full transcriptional function of the protein (
      • Kotenko S.V.
      • Izotova L.S.
      • Pollack B.P.
      • Mariano T.M.
      • Donnelly R.J.
      • Muthukumaran G.
      • Cook J.R.
      • Garotta G.
      • Silvennoinen O.
      • Ihle J.N.
      Interaction between the components of the interferon gamma receptor complex.
      ,
      • Darnell Jr, J.E.
      • Kerr I.M.
      • Stark G.R.
      Jak-STAT pathways and transcriptional activation in response to IFNs and other extracellular signaling proteins.
      ,
      • Levy D.E.
      • Darnell Jr., J.E.
      Stats: transcriptional control and biological impact.
      ). We performed studies to examine the effects of targeted disruption of the Sin1 gene on phosphorylation of STAT1. As shown in Fig. 5 (A and B), IFNγ treatment led to greater phosphorylation of STAT1 on Tyr-701 and on Ser-727 in Sin1+/+ cells compared with Sin−/− MEFs, suggesting that Sin1 is essential for optimal STAT1 transcriptional activity. In different settings, Sin1 has been shown to interact with IFNAR2 and TGF receptors (
      • Wang S.Z.
      • Roberts R.M.
      Interaction of stress-activated protein kinase-interacting protein-1 with the interferon receptor subunit IFNAR2 in uterine endometrium.
      ,
      • Li C.
      • Wang Q.
      • Wang J.F.
      Transforming growth factor-β (TGF-β) induces the expression of chondrogenesis-related genes through TGF-β receptor II (TGFRII)-AKT-mTOR signaling in primary cultured mouse precartilaginous stem cells.
      ), which led us to examine whether Sin1 binds IFNGR1. As shown in Fig. 5C, Sin1 co-immunoprecipitates with IFNGR1, suggesting that Sin1 may be required for transmission of IFNGR downstream signaling events. Notably, STAT1 was found to bind IFNGR1 only in the presence of Sin1, suggesting that IFNGR1 requires Sin1 to create a docking site for the STAT1 protein. Binding of IFNγ to IFNGR2 and IFNGR1 induces phosphorylation of JAKs (
      • Kotenko S.V.
      • Izotova L.S.
      • Pollack B.P.
      • Mariano T.M.
      • Donnelly R.J.
      • Muthukumaran G.
      • Cook J.R.
      • Garotta G.
      • Silvennoinen O.
      • Ihle J.N.
      Interaction between the components of the interferon gamma receptor complex.
      ,
      • Darnell Jr, J.E.
      • Kerr I.M.
      • Stark G.R.
      Jak-STAT pathways and transcriptional activation in response to IFNs and other extracellular signaling proteins.
      ). Active JAK kinases phosphorylate the cytoplasmic domain of IFNGR1, which allows for the recruitment of STAT1 and its phosphorylation at Tyr-701 (
      • Kotenko S.V.
      • Izotova L.S.
      • Pollack B.P.
      • Mariano T.M.
      • Donnelly R.J.
      • Muthukumaran G.
      • Cook J.R.
      • Garotta G.
      • Silvennoinen O.
      • Ihle J.N.
      Interaction between the components of the interferon gamma receptor complex.
      ,
      • Darnell Jr, J.E.
      • Kerr I.M.
      • Stark G.R.
      Jak-STAT pathways and transcriptional activation in response to IFNs and other extracellular signaling proteins.
      ). In subsequent studies, we sought to determine whether Sin1 knock-out affects IFNγ-dependent JAK1 and JAK2 phosphorylation. IFNγ treatment induced phosphorylation of JAK1 and JAK2 in Sin1+/+ but not in Sin−/− MEFs (Fig. 5, D and E), suggesting defective IFNγ-dependent activation of JAKs in the absence of Sin1. To further examine the regulation of IFNγ-dependent STAT1 activation, we immunoprecipitated IFNGR1 from mLST8+/− and mLST8−/− MEFs and evaluated binding to STAT1 and Jak1 (Fig. 5F). Importantly, the interaction between Sin1, JAK1, STAT1, and IFNGR1 is independent of mLST8 expression (Fig. 5F), a key component of both mTORC1 and mTORC2, suggesting that Sin1 controls IFNγ-mediated STAT1 activation independently of its role in mTORC2. In agreement, mLST8 expression is not required for IFNγ-induced phosphorylation of STAT1 on Tyr-701 (Fig. 5G).
      Figure thumbnail gr5
      FIGURE 5IFNγ-induced tyrosine phosphorylation of STAT1 is Sin1-dependent. A and B, serum-starved Sin1+/+ and Sin1−/− MEFs were treated with mouse IFNγ (5 × 103 IU/ml) for the indicated times. Cell lysates were prepared, and equal amounts of protein were resolved by SDS-PAGE and then subjected to immunoblotting analyses with the indicated antibodies. A and B (bottom panels), bands from three independent experiments (including the blots shown) were quantified by densitometry. Data are expressed as ratios of phospho-Stat1 over total Stat1, and bar graphs represent means ± S.E. (error bars) of three independent experiments for each experimental condition. C–E, serum-starved Sin1+/+ and Sin1−/− MEFs were treated with mouse IFNγ (5 × 103 IU/ml) for the indicated times. C, equal amounts of protein were processed for immunoprecipitation (IP) with anti-IFNGR1 antibody or control IgG, as indicated. The immunoprecipitated proteins were resolved by SDS-PAGE and then subjected to immunoblot analyses with anti-IFNGR1, anti-Sin1, and anti-Stat1 antibodies. D and E, equal amounts of protein were processed for immunoprecipitation with anti-Jak1 (D) or anti-Jak2 (E) and control IgG, as indicated. The immunoprecipitated proteins were resolved by SDS-PAGE and then subjected to immunoblot analyses with anti-phospho-Tyr-1022/1023 Jak1, Jak1, and Sin1 antibodies (D) or with anti-phospho-Tyr-1007/1008 Jak2, Jak2, and Sin1 antibodies (E). F, serum-starved mLST8+/− and mLST8−/− MEFs were treated with mouse IFNγ (5 × 103 IU/ml) for the indicated times. Equal amounts of protein were processed for immunoprecipitation with anti-IFNGR1 antibody or control IgG as indicated. The immunoprecipitated proteins were resolved by SDS-PAGE and then subjected to immunoblot analyses with anti-IFNGR1, Sin1, Jak1, and Stat1 antibodies. G, the same cell lysates used in H were resolved by SDS-PAGE and then subjected to immunoblot analyses with anti-phospho-Tyr-701 Stat1, Stat1, mLST8, Sin1, and GAPDH antibodies.
      Previously, AKT was found to be required for Sin1 phosphorylation at Thr-86 (
      • Yang G.
      • Murashige D.S.
      • Humphrey S.J.
      • James D.E.
      A Positive Feedback Loop between Akt and mTORC2 via SIN1 Phosphorylation.
      ). Thus, in subsequent studies, we sought to determine whether IFNγ-induced Sin1 phosphorylation at this site affects IFNγ-induced STAT1 phosphorylation. For this, we assessed the effects of rapamycin, an mTORC1 inhibitor, and MK-2206, an AKT inhibitor, in MEFs (Fig. 6). IFNγ-induced phosphorylation of AKT and Sin1 was blocked by co-treatment with MK-2206 (Fig. 6, A and B). However, the AKT inhibitor did not affect STAT1 phosphorylation at Tyr-701 and Ser-727 (Fig. 6C). As expected, rapamycin treatment had no effect on IFNγ-mediated phosphorylation of Sin1, AKT, and STAT1 (Fig. 6, A–C). These results confirm that IFNγ-mediated Sin1 phosphorylation at Thr-86 is AKT-dependent. However, Sin1 phosphorylation at this site does not affect IFNγ-dependent activation of JAK-STAT signaling, further supporting the requirement of Sin1 activity, independently of both mTORC1 and mTORC2 activation.
      Figure thumbnail gr6
      FIGURE 6IFNγ-induced phosphorylation of Sin1 at Thr-86 is AKT-dependent but not required for STAT1 phosphorylation. A–C, serum-starved Sin1+/+ MEFs were treated with rapamycin (20 nm) or MK-2206 (10 μm) for 30 min, followed by co-treatment with mouse IFNγ (5 × 103 IU/ml) for the indicated times. Cell lysates were prepared, and equal amounts of protein were resolved by SDS-PAGE and then subjected to immunoblotting with the indicated specific anti-phosphoantibodies. These blots were stripped and probed with the respective anti-total antibodies.
      To assess the biological relevance and functional significance of Sin1 in the type II IFN system, we next examined the role of Sin1 in the generation of IFNγ-dependent anti-leukemic activity. The inhibitory effects of IFNγ on primitive leukemic progenitors (CFU-L) were partially reversed in U937 cells transfected with Sin1 siRNA (Fig. 7A), suggesting that Sin1 mediates IFNγ-induced anti-leukemic responses. Studies were also carried out to determine the potential role of Sin1 as a mediator of the suppressive effects of IFNγ on normal hematopoiesis. Normal human bone marrow-derived CD34+ cells transduced with control siRNA or Sin1-specific siRNA were treated with IFNγ, and normal myeloid (CFU-GM) or early erythroid (BFU-E) colony formation was determined by clonogenic assays in methylcellulose. As expected, treatment with IFNγ resulted in suppression of hematopoietic progenitor colony formation, but these effects were partially reversed by Sin1 knockdown (Fig. 7B), directly establishing a role for Sin1 in the process.
      Figure thumbnail gr7
      FIGURE 7Requirement of Sin1 for generation of IFNγ biological effects. A, U937 cells were transfected with either control siRNA or siRNA specifically targeting Sin1, as indicated. The cells were subsequently plated in methylcellulose, in the absence or presence of human IFNγ, and leukemic CFU-L colony formation was assessed. Data are expressed as percentage of colony formation of untreated control siRNA-transfected cells, and bar graphs represent means ± S.E. (error bars) of three independent experiments. Statistical significance was calculated using one-way ANOVA followed by Tukey's test: *, p < 0.05. B, normal human CD34+ bone marrow-derived cells were transfected with control siRNA or Sin1 siRNA and incubated in methylcellulose in the presence or absence of human IFNγ, as indicated. CFU-GM and BFU-E progenitor colonies were scored after 14 days in culture. Data are expressed as percentage of colony formation of untreated control siRNA-transfected cells, and bar graphs represent means ± S.E. of three independent experiments. Statistical significance was calculated using one-way ANOVA followed by Tukey's test: *, p < 0.05. C, Sin1+/+ and Sin1−/− MEFs were seeded in wells of a 96-well plate in DMEM. 24 h later, the cells were either left untreated or treated with the indicated doses of mouse IFNγ for 16 h and subsequently challenged with EMCV (0.02 MOI). Virus-induced CPE were quantified 24 h later, as described under “Experimental Procedures.” Data are expressed as percentage protection from EMCV CPE and are representative of three independent experiments. Values are means ± S.E. of quadruplicate assays.
      Because our data demonstrated defective IFNγ signaling events in Sin1−/− cells, we compared induction of IFNγ-dependent antiviral responses in Sin1+/+ and Sin1−/− cells. Notably, Sin1−/− cells are more sensitive to encephalomyocaditis virus (EMCV) infection (data not shown). We found that Sin1+/+ MEFs were responsive to the antiviral effects of IFNγ, as reflected by protection from the cytopathic effects (CPE) of EMCV (Fig. 7C). Using the same infective dose, we found that identical IFNγ doses did not provide protection in the Sin1−/− MEFs, suggesting a role for Sin1 in the generation of IFNγ-induced antiviral effects.

      Discussion

      Recent advances in cancer immunotherapy underscore the critical relevance of the type II IFN system in immune surveillance against cancer (
      • Borden E.C.
      • Sen G.C.
      • Uze G.
      • Silverman R.H.
      • Ransohoff R.M.
      • Foster G.R.
      • Stark G.R.
      Interferons at age 50: past, current and future impact on biomedicine.
      ,
      • Platanias L.C.
      Interferons and their antitumor properties.
      ,
      • George P.M.
      • Badiger R.
      • Alazawi W.
      • Foster G.R.
      • Mitchell J.A.
      Pharmacology and therapeutic potential of interferons.
      ,
      • Young N.S.
      • Calado R.T.
      • Scheinberg P.
      Current concepts in the pathophysiology and treatment of aplastic anemia.
      ). The emerging understanding of the central role of IFNγ in the network of immune regulatory mechanisms that control malignant cell survival underscores the importance of precisely defining type II IFN signaling events. Recently, there has been an increased interest in identifying the mechanisms by which IFNs regulate transcription and mRNA translation of ISGs to develop unique approaches that could modulate the IFN response and lead to ways to enhance the generation of antiviral or antiproliferative responses by these cytokines. In earlier studies, we provided evidence implicating mTOR complexes in the expression of type I and II ISG products (
      • Kaur S.
      • Sassano A.
      • Joseph A.M.
      • Majchrzak-Kita B.
      • Eklund E.A.
      • Verma A.
      • Brachmann S.M.
      • Fish E.N.
      • Platanias L.C.
      Dual regulatory roles of phosphatidylinositol 3-kinase in IFN signaling.
      ,
      • Kaur S.
      • Sassano A.
      • Majchrzak-Kita B.
      • Baker D.P.
      • Su B.
      • Fish E.N.
      • Platanias L.C.
      Regulatory effects of mTORC2 complexes in type I IFN signaling and in the generation of IFN responses.
      ,
      • Kaur S.
      • Kroczynska B.
      • Sharma B.
      • Sassano A.
      • Arslan A.D.
      • Majchrzak-Kita B.
      • Stein B.L.
      • McMahon B.
      • Altman J.K.
      • Su B.
      • Calogero R.A.
      • Fish E.N.
      • Platanias L.C.
      Critical roles for Rictor/Sin1 complexes in interferon-dependent gene transcription and generation of antiproliferative responses.
      ,
      • Kroczynska B.
      • Rafidi R.L.
      • Majchrzak-Kita B.
      • Kosciuczuk E.M.
      • Blyth G.T.
      • Jemielity J.
      • Warminska Z.
      • Saleiro D.
      • Mehrotra S.
      • Arslan A.D.
      • Fish E.N.
      • Platanias L.C.
      Interferon γ (IFNγ) signaling via mechanistic target of rapamycin complex 2 (mTORC2) and regulatory effects in the generation of type II interferon biological responses.
      ,
      • Kroczynska B.
      • Mehrotra S.
      • Arslan A.D.
      • Kaur S.
      • Platanias L.C.
      Regulation of interferon-dependent mRNA translation of target genes.
      ,
      • Kroczynska B.
      • Kaur S.
      • Katsoulidis E.
      • Majchrzak-Kita B.
      • Sassano A.
      • Kozma S.C.
      • Fish E.N.
      • Platanias L.C.
      Interferon-dependent engagement of eukaryotic initiation factor 4B via S6 kinase (S6K)- and ribosomal protein S6K-mediated signals.
      ,
      • Kroczynska B.
      • Joshi S.
      • Eklund E.A.
      • Verma A.
      • Kotenko S.V.
      • Fish E.N.
      • Platanias L.C.
      Regulatory effects of ribosomal S6 kinase 1 (RSK1) in IFNλ signaling.
      ). Other earlier work from our group raised the possibility that mTORC2 is engaged in a unique way in IFN signaling, as reflected by IFN-specific effects on the AKT/mTORC1 axis (
      • Kaur S.
      • Sassano A.
      • Majchrzak-Kita B.
      • Baker D.P.
      • Su B.
      • Fish E.N.
      • Platanias L.C.
      Regulatory effects of mTORC2 complexes in type I IFN signaling and in the generation of IFN responses.
      ,
      • Kroczynska B.
      • Rafidi R.L.
      • Majchrzak-Kita B.
      • Kosciuczuk E.M.
      • Blyth G.T.
      • Jemielity J.
      • Warminska Z.
      • Saleiro D.
      • Mehrotra S.
      • Arslan A.D.
      • Fish E.N.
      • Platanias L.C.
      Interferon γ (IFNγ) signaling via mechanistic target of rapamycin complex 2 (mTORC2) and regulatory effects in the generation of type II interferon biological responses.
      ). Notably, in a recent study, we established that Rictor and Sin1 are essential for type I IFN-mediated activation of STAT-driven signaling pathways required for optimal type I ISG transcription (
      • Kaur S.
      • Kroczynska B.
      • Sharma B.
      • Sassano A.
      • Arslan A.D.
      • Majchrzak-Kita B.
      • Stein B.L.
      • McMahon B.
      • Altman J.K.
      • Su B.
      • Calogero R.A.
      • Fish E.N.
      • Platanias L.C.
      Critical roles for Rictor/Sin1 complexes in interferon-dependent gene transcription and generation of antiproliferative responses.
      ), suggesting cross-talk between mTOR and STAT signaling cascades upon engagement of the type I IFN receptor (
      • Saleiro D.
      • Platanias L.C.
      Intersection of mTOR and STAT signaling in immunity.
      ). Overall, the emerging evidence has been consistent, with a key role for mTOR pathways in the generation of IFN responses, but the elements of the mTOR signaling machinery that mediate interactions with other IFN-activated pathways remain to be identified, and their roles have yet to be precisely defined.
      In the current study, we sought to determine the role of Sin1 in the generation of IFNγ responses. We used cells with targeted disruption of the Sin1 gene to define the role of Sin1 in IFNγ signaling and the control of IFNγ responses. IFNγ-induced phosphorylation of AKT was defective in Sin1−/− cells, establishing that intact mTORC2 is required for induction of AKT activity during its engagement by the type II IFN receptor. Moreover, we found decreased or defective IFNγ-induced phosphorylation of downstream mTORC1 effectors in the absence of Sin1, indicating Sin1-dependent sequential activation of mTORC2 and mTORC1. Our data also demonstrate that Sin1 expression is required for protein expression of ISGs, such as IP10 and DAPK1. This could be explained, in part, by the fact that in the absence of Sin1, there appears to be defective mTORC1-dependent formation of eIF4F and consequent decreased mRNA translation (
      • Katsoulidis E.
      • Carayol N.
      • Woodard J.
      • Konieczna I.
      • Majchrzak-Kita B.
      • Jordan A.
      • Sassano A.
      • Eklund E.A.
      • Fish E.N.
      • Platanias L.C.
      Role of Schlafen 2 (SLFN2) in the generation of interferon α-induced growth inhibitory responses.
      ).
      Interestingly, for the first time, we uncovered a unique role for Sin1 in IFNγ signaling that appears to be independent of its function as a component of mTORC2. Upon binding of IFNγ to IFNGR1, these two chains associate with the two IFNGR2 chains, and JAK1 and JAK2 are activated by phosphorylation (
      • Saleiro D.
      • Kosciuczuk E.M.
      • Platanias L.C.
      Beyond autophagy: new roles for ULK1 in immune signaling and interferon responses.
      ). This interaction leads to JAK1 and JAK2 dimerization, activation, and ultimately phosphorylation of Tyr-457 of IFNGR1, which comprises the receptor docking site for STAT1 (
      • Saleiro D.
      • Kosciuczuk E.M.
      • Platanias L.C.
      Beyond autophagy: new roles for ULK1 in immune signaling and interferon responses.
      ). Subsequently, STAT1 binds to IFNGR1 at this site and is phosphorylated at Tyr-701 by JAK1 and JAK2 (
      • Kotenko S.V.
      • Izotova L.S.
      • Pollack B.P.
      • Mariano T.M.
      • Donnelly R.J.
      • Muthukumaran G.
      • Cook J.R.
      • Garotta G.
      • Silvennoinen O.
      • Ihle J.N.
      Interaction between the components of the interferon gamma receptor complex.
      ,
      • Darnell Jr, J.E.
      • Kerr I.M.
      • Stark G.R.
      Jak-STAT pathways and transcriptional activation in response to IFNs and other extracellular signaling proteins.
      ,
      • Levy D.E.
      • Darnell Jr., J.E.
      Stats: transcriptional control and biological impact.
      ,
      • Stark G.R.
      • Darnell Jr., J.E.
      The JAK-STAT pathway at twenty.
      ,
      • Saleiro D.
      • Kosciuczuk E.M.
      • Platanias L.C.
      Beyond autophagy: new roles for ULK1 in immune signaling and interferon responses.
      ,
      • Rodig S.J.
      • Meraz M.A.
      • White J.M.
      • Lampe P.A.
      • Riley J.K.
      • Arthur C.D.
      • King K.L.
      • Sheehan K.C.
      • Yin L.
      • Pennica D.
      • Johnson Jr., E.M.
      • Schreiber R.D.
      Disruption of the Jak1 gene demonstrates obligatory and nonredundant roles of the Jaks in cytokine-induced biologic responses.
      ,
      • Parganas E.
      • Wang D.
      • Stravopodis D.
      • Topham D.J.
      • Marine J.C.
      • Teglund S.
      • Vanin E.F.
      • Bodner S.
      • Colamonici O.R.
      • van Deursen J.M.
      • Grosveld G.
      • Ihle J.N.
      Jak2 is essential for signaling through a variety of cytokine receptors.
      ). Our data revealed that Sin1 binds IFNGR1 and that this interaction is essential for the IFNγ-induced association of JAK1 and subsequent docking of STAT1 to the IFNGR. IFNγ-mediated tyrosine phosphorylation of JAK1, JAK2, and STAT1 was impaired in Sin1−/− cells, leading to defective transcription of STAT-driven ISGs. Taken in context, these studies provide the first evidence implicating Sin1 in the functional activation of IFNGR-mediated signaling. Moreover, we found that AKT-dependent phosphorylation of Sin1 at Thr-86 is not necessary for STAT1 phosphorylation. We infer that Sin1 may act as a scaffold protein between JAK1 and IFNGR. Overall, our studies establish that Sin1-dependent type II IFN signaling events are required for the IFNγ suppressive effects on normal hematopoiesis and the generation of antiproliferative and antiviral responses, via control of both transcription and translation of specific type II ISGs. Because of the key role of the type II IFN system in the pathophysiology of different diseases, a better understanding of the involvement of Sin1 in the functional activation of IFNGR may contribute to the design of novel and improved therapeutic approaches toward several pathologies.

      Experimental Procedures

      Cell Lines and Reagents

      Immortalized Sin1+/+, Sin1−/− (
      • Kaur S.
      • Sassano A.
      • Majchrzak-Kita B.
      • Baker D.P.
      • Su B.
      • Fish E.N.
      • Platanias L.C.
      Regulatory effects of mTORC2 complexes in type I IFN signaling and in the generation of IFN responses.
      ,
      • Jacinto E.
      • Facchinetti V.
      • Liu D.
      • Soto N.
      • Wei S.
      • Jung S.Y.
      • Huang Q.
      • Qin J.
      • Su B.
      SIN1/MIP1 maintains rictor-mTOR complex integrity and regulates Akt phosphorylation and substrate specificity.
      ), mLST8+/−, and mLST8−/− MEFs provided by David Sabatini (Whitehead Institute, Cambridge, MA) (
      • Kaur S.
      • Sassano A.
      • Majchrzak-Kita B.
      • Baker D.P.
      • Su B.
      • Fish E.N.
      • Platanias L.C.
      Regulatory effects of mTORC2 complexes in type I IFN signaling and in the generation of IFN responses.
      ,
      • 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.
      Ablation in mice of the mTORC components raptor, rictor, or mLST8 reveals that mTORC2 is required for signaling to Akt-FOXO and PKCα, but not S6K1.
      ) were grown in DMEM supplemented with 10% fetal bovine serum (FBS) and antibiotics. Bone marrow-derived CD34+ cells were from Stemcell Technologies (Vancouver, Canada). U937 cells were grown in RPMI 1640 medium supplemented with 10% FBS and antibiotics. Phospho-specific antibodies against mTOR, AKT, Sin1, p70S6K, rpS6, 4E-BP1, eIF4B, JAK1, JAK2, and Stat1 and antibodies against mTOR, AKT, p70S6K, rpS6, 4E-BP1, eIF4B, JAK1, JAK2, mLST8, eIF4G, eIF4A, and eIF4E were from Cell Signaling (Boston, MA). The antibody against CXCL10 (IP10) was purchased from Abcam (Cambridge, MA). Antibodies against Sin1 and GAPDH were from Millipore (Billerica, MA). Anti-DAPK1 and anti-IFNGR1 antibodies were from Proteintech Group, Inc. (Chicago, IL). Antibody against STAT1 was from Santa Cruz Biotechnology, Inc. (Dallas, TX). Anti-SLFN5 was from Sigma-Aldrich (St. Louis, MO). Recombinant human and mouse IFNγ were from Life Technologies, Inc. (Waltham, MA). Rapamycin was obtained from Calbiochem (Billerica, MA). The Akt inhibitor MK-2206 was purchased from Santa Cruz Biotechnology.

      Cap Binding Assays

      These studies were performed as described previously (
      • Kroczynska B.
      • Rafidi R.L.
      • Majchrzak-Kita B.
      • Kosciuczuk E.M.
      • Blyth G.T.
      • Jemielity J.
      • Warminska Z.
      • Saleiro D.
      • Mehrotra S.
      • Arslan A.D.
      • Fish E.N.
      • Platanias L.C.
      Interferon γ (IFNγ) signaling via mechanistic target of rapamycin complex 2 (mTORC2) and regulatory effects in the generation of type II interferon biological responses.
      ). Briefly, Sin1+/+ and Sin1−/− MEFs were incubated overnight in serum-free medium and then treated with mouse IFNγ (5 × 103 IU/ml) for the indicated times. Cell lysates were incubated for 24 h with a dinucleotide mRNA 5′-cap analog (m7GpppG) labeled with biotin attached to ribose of the second dinucleotide (
      • Jemielity J.
      • Lukaszewicz M.
      • Kowalska J.
      • Czarnecki J.
      • Zuberek J.
      • Darzynkiewicz E.
      Synthesis of biotin labelled cap analogue-incorporable into mRNA transcripts and promoting cap-dependent translation.
      ,
      • Warminski M.
      • Warminska Z.
      • Kowalska J.
      • Jemielity J.
      mRNA cap modification through carbamate chemistry: synthesis of amino- and carboxy-functionalised cap analogues suitable for labelling and bioconjugation.
      ) and subsequently with streptavidin beads for 4 h. After extensive washing with lysis buffer, the retained proteins were eluted from the beads, boiled and resolved by SDS-PAGE, transferred onto Immobilon-P membranes (Millipore), and probed with the indicated antibodies.

      Immunoprecipitation, Immunoblotting Analysis, and siRNA Knockdown

      Immortalized MEFs were starved overnight in DMEM containing 0.5% FBS and were then treated with mouse IFNγ at the indicated doses in DMEM containing 0.5% FBS for the indicated times. U937 cells were nucleofected with control siRNA or a pool of four different synthetic Sin1 siRNA (Dharmacon) using the Amaxa nucleofector kit C, following the manufacturer's protocol (Lonza). Transfected cells were starved overnight in RPMI containing 0.5% FBS and then treated with human IFNγ at the indicated doses in RPMI containing 0.5% FBS for the indicated times. Following treatment, cells were washed with PBS and lysed in 40 mm HEPES, pH 7.5, containing 0.1% Nonidet P-40, 120 mm sodium chloride, 1 mm EDTA, 10 mm sodium pyrophosphate, 50 mm NaF, 10 mm B-glycerophosphate, and protease and phosphatase inhibitors (Calbiochem). For immunoblotting analyses, lysates were resolved by SDS-PAGE gradient gels (Bio-Rad), transferred to Immobilon-P PVDF membranes (Millipore), which were probed with primary and secondary antibodies, and then detected by enhanced chemiluminescence as in previous studies (
      • Kaur S.
      • Lal L.
      • Sassano A.
      • Majchrzak-Kita B.
      • Srikanth M.
      • Baker D.P.
      • Petroulakis E.
      • Hay N.
      • Sonenberg N.
      • Fish E.N.
      • Platanias L.C.
      Regulatory effects of mammalian target of rapamycin-activated pathways in type I and II interferon signaling.
      ,
      • Kroczynska B.
      • Rafidi R.L.
      • Majchrzak-Kita B.
      • Kosciuczuk E.M.
      • Blyth G.T.
      • Jemielity J.
      • Warminska Z.
      • Saleiro D.
      • Mehrotra S.
      • Arslan A.D.
      • Fish E.N.
      • Platanias L.C.
      Interferon γ (IFNγ) signaling via mechanistic target of rapamycin complex 2 (mTORC2) and regulatory effects in the generation of type II interferon biological responses.
      ). For co-immunoprecipitation analyses, total cell lysates were incubated overnight at 4 °C with rotation with rabbit antibody against either IFNGR1, JAK1, or JAK2 (Cell Signaling), as indicated, followed by incubation at 4 °C with protein G-Sepharose 4 Fast Flow beads (GE Healthcare). As a control, the same procedure was followed, but using Rabbit IgG. After immunoprecipitation, the beads were washed with Nonidet P-40 buffer. Protein complexes were eluted from the beads, and eluates were resolved by SDS-PAGE and processed for immunoblotting analyses.

      Polysomal Isolation

      Immortalized Sin1+/+ and Sin1−/− MEFs were either left untreated or were treated with 1.5 × 103 IU/ml of mouse IFNγ for 24 h in DMEM supplemented with 0.5% FBS. Cells were washed twice with Dulbecco's PBS with 100 μg/ml cycloheximide and then lysed in lysis buffer (0.5% Triton X-100, 0.5% sodium deoxycholate, 5 mm Tris, pH 7.5, 2.5 mm MgCl2, 1.5 mm KCl, 100 μg/ml cycloheximide, 2 mm DTT, protease inhibitor, and 1 unit/μl RNase inhibitor). Lysates were then centrifuged at 12,000 × g for 5 min at 4 °C, and supernatants were collected and snap-frozen in liquid nitrogen. Equal amounts of cell lysates of each sample were layered on a sucrose gradient of 5–50% prepared by using the BioComp Gradient Master 108 (Biocomp Instruments, Fredericton, Canada), according to the manufacturer's instructions. Samples were centrifuged at 4 °C for 110 min at 35,000 rpm using a Beckman SW41-Ti rotor. After centrifugation, samples were loaded on the Density Gradient Fractionation System (Brandel), and absorbance was measured continuously at 254 nm with the following settings: pump speed, 0.80 ml/min; 10 drops/fraction; chart speed, 300 cm/h; sensitivity, 1; peak separator, off; noise filter, 0.5 s. Assignments of the 40S, 60S, and 80S peaks and polysomes were determined based on the absorbance profile. RNA from polysomal fractions was isolated using an RNA all-prep kit from Qiagen. 1 μg of polysomal RNA was reverse transcribed using oligo(dT) primers (Life Technologies) and the Omniscript RT kit (Qiagen), as described previously (
      • Kroczynska B.
      • Rafidi R.L.
      • Majchrzak-Kita B.
      • Kosciuczuk E.M.
      • Blyth G.T.
      • Jemielity J.
      • Warminska Z.
      • Saleiro D.
      • Mehrotra S.
      • Arslan A.D.
      • Fish E.N.
      • Platanias L.C.
      Interferon γ (IFNγ) signaling via mechanistic target of rapamycin complex 2 (mTORC2) and regulatory effects in the generation of type II interferon biological responses.
      ).

      Hematopoietic Progenitor Assays

      Malignant leukemic progenitor (CFU-L) colony formation in methylcellulose from U937 cells transfected with control siRNA or Sin1 siRNA was assessed as described previously (
      • Kaur S.
      • Kroczynska B.
      • Sharma B.
      • Sassano A.
      • Arslan A.D.
      • Majchrzak-Kita B.
      • Stein B.L.
      • McMahon B.
      • Altman J.K.
      • Su B.
      • Calogero R.A.
      • Fish E.N.
      • Platanias L.C.
      Critical roles for Rictor/Sin1 complexes in interferon-dependent gene transcription and generation of antiproliferative responses.
      ). Cells were plated in methylcellulose, in the absence or presence of human IFNγ (5 × 102 IU/ml), and leukemic CFU-L colony formation was assessed as in our previous studies (
      • Kaur S.
      • Kroczynska B.
      • Sharma B.
      • Sassano A.
      • Arslan A.D.
      • Majchrzak-Kita B.
      • Stein B.L.
      • McMahon B.
      • Altman J.K.
      • Su B.
      • Calogero R.A.
      • Fish E.N.
      • Platanias L.C.
      Critical roles for Rictor/Sin1 complexes in interferon-dependent gene transcription and generation of antiproliferative responses.
      ). Normal hematopoietic progenitor colony formation for late erythroid progenitors (BFU-E), or myeloid progenitors (CFU-GM) from normal CD34+ bone marrow-derived cells transfected with control siRNA or Sin1 siRNA was determined in clonogenic assays in methylcellulose, in the absence or presence of human IFNγ (50–100 IU/ml), as in our previous studies (
      • Kroczynska B.
      • Rafidi R.L.
      • Majchrzak-Kita B.
      • Kosciuczuk E.M.
      • Blyth G.T.
      • Jemielity J.
      • Warminska Z.
      • Saleiro D.
      • Mehrotra S.
      • Arslan A.D.
      • Fish E.N.
      • Platanias L.C.
      Interferon γ (IFNγ) signaling via mechanistic target of rapamycin complex 2 (mTORC2) and regulatory effects in the generation of type II interferon biological responses.
      ). The transfections were performed using the siRNA transfection reagent TransIT-TKO (Mirus).

      Quantitative RT-PCR

      Sin1+/+ and Sin1−/− MEFs were starved overnight in DMEM containing 0.5% FBS and then treated for 6 h with 2.5 × 103 IU/ml mouse IFNγ. Total RNA was isolated using the RNeasy minikit (Qiagen) per the manufacturer's instructions. 1 μg of total cellular mRNA was reverse transcribed into cDNA using the Omniscript RT kit (Qiagen) and oligo(dT)12–18 primers (Life Technologies). Real-time PCR was carried out using an ABI7500 sequence detection system (Applied Biosystems) using commercially available FAM-labeled probes and primers (Applied Biosystems) to determine mRNA expression of Ip10, Dapk1, Slfn2, and Gapdh. Gapdh was used for normalization as described in our previous studies (
      • Kaur S.
      • Sassano A.
      • Majchrzak-Kita B.
      • Baker D.P.
      • Su B.
      • Fish E.N.
      • Platanias L.C.
      Regulatory effects of mTORC2 complexes in type I IFN signaling and in the generation of IFN responses.
      ,
      • Kroczynska B.
      • Rafidi R.L.
      • Majchrzak-Kita B.
      • Kosciuczuk E.M.
      • Blyth G.T.
      • Jemielity J.
      • Warminska Z.
      • Saleiro D.
      • Mehrotra S.
      • Arslan A.D.
      • Fish E.N.
      • Platanias L.C.
      Interferon γ (IFNγ) signaling via mechanistic target of rapamycin complex 2 (mTORC2) and regulatory effects in the generation of type II interferon biological responses.
      ).

      Antiviral Assays

      Sin1+/+ and Sin1−/− MEFs were seeded in individual wells of 96-well plates. 24 h later, the cells were either left untreated or treated with mouse IFNγ at the indicated doses for 16 h, before infection with EMCV. The infective dose of EMCV was predetermined as the minimum viral dilution that leads to 100% CPE in the Sin1+/+ cells at 24 h (0.02 MOI). Virus-induced CPE was quantified 24 h postinfection using a colorimetric assay as described earlier (
      • Kaur S.
      • Sassano A.
      • Majchrzak-Kita B.
      • Baker D.P.
      • Su B.
      • Fish E.N.
      • Platanias L.C.
      Regulatory effects of mTORC2 complexes in type I IFN signaling and in the generation of IFN responses.
      ,
      • Kroczynska B.
      • Rafidi R.L.
      • Majchrzak-Kita B.
      • Kosciuczuk E.M.
      • Blyth G.T.
      • Jemielity J.
      • Warminska Z.
      • Saleiro D.
      • Mehrotra S.
      • Arslan A.D.
      • Fish E.N.
      • Platanias L.C.
      Interferon γ (IFNγ) signaling via mechanistic target of rapamycin complex 2 (mTORC2) and regulatory effects in the generation of type II interferon biological responses.
      ). Data are expressed as percentage protection from EMCV CPE and are representative of three independent experiments. Values are means ± S.E. of quadruplicate assays.

      Statistical Analyses

      Statistical significance was analyzed using Student's t test or one-way ANOVA followed by Tukey's test as indicated. Differences were considered statistically significant when p values were <0.05.

      Author Contributions

      B. K., G. T. B., R. L. R., L. X., E. M. K., B. M.-K., D. S., S. M., and G. T. B. performed research and analyzed data; B. S. and J. J. developed, produced and provided key experimental reagents for the study; B. K., E. N. F., J. K. A., E. A. E., and L. C. P. conceived and analyzed data; D. S., L. X., G. T. B., R. L. F., and E. N. F., contributed to manuscript drafting. B. K. and L. C. P. designed research and wrote the manuscript.

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