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The TORC1-activated Proteins, p70S6K and GRB10, Regulate IL-4 Signaling and M2 Macrophage Polarization by Modulating Phosphorylation of Insulin Receptor Substrate-2*

  • Kristi J. Warren
    Affiliations
    From Johns Hopkins University, School of Medicine, Department of Anesthesiology and Critical Care Medicine, Baltimore, Maryland 21205
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  • Xi Fang
    Affiliations
    From Johns Hopkins University, School of Medicine, Department of Anesthesiology and Critical Care Medicine, Baltimore, Maryland 21205
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  • Nagaraj M. Gowda
    Affiliations
    From Johns Hopkins University, School of Medicine, Department of Anesthesiology and Critical Care Medicine, Baltimore, Maryland 21205
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  • Joshua J. Thompson
    Affiliations
    From Johns Hopkins University, School of Medicine, Department of Anesthesiology and Critical Care Medicine, Baltimore, Maryland 21205
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  • Nicola M. Heller
    Correspondence
    To whom correspondence should be addressed: Johns Hopkins University School of Medicine, Dept. of ACCM, Division of Allergy and Clinical Immunology, 720 Rutland Ave., Ross 367, Baltimore, MD 21205. Tel.: 410-955-1743; Fax: 410-614-0083;
    Affiliations
    From Johns Hopkins University, School of Medicine, Department of Anesthesiology and Critical Care Medicine, Baltimore, Maryland 21205
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  • Author Footnotes
    * This work was supported, in whole or in part, by National Institutes of Health Grant K99/R00 HL096897 (NHLBI; to N. M. H.). The authors declare that they have no conflict 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.
      Lung M2 macrophages are regulators of airway inflammation, associated with poor lung function in allergic asthma. Previously, we demonstrated that IL-4-induced M2 gene expression correlated with tyrosine phosphorylation of the insulin receptor substrate-2 (IRS-2) in macrophages. We hypothesized that negative regulation of IRS-2 activity after IL-4 stimulation is dependent upon serine phosphorylation of IRS-2. Herein, we describe an inverse relationship between tyrosine phosphorylation (Tyr(P)) and serine phosphorylation (Ser(P)) of IRS-2 after IL-4 stimulation. Inhibiting serine phosphatase activity increased Ser(P)-IRS-2 and decreased Tyr(P)-IRS-2 leading to reduced M2 gene expression (CD200R, CCL22, MMP12, and TGM2). We found that inhibition of p70S6K, downstream of TORC1, resulted in diminished Ser(P)-IRS-2 and prolonged Tyr(P)-IRS-2 as well. Inhibition of p70S6K increased expression of CD200R and CCL22 indicating that p70S6K negatively regulates some, but not all, human M2 genes. Knocking down GRB10, another negative regulatory protein downstream of TORC1, enhanced both Tyr(P)-IRS-2 and increased expression of all four M2 genes. Furthermore, GRB10 associated with IRS-2, NEDD4.2 (an E3-ubiquitin ligase), IL-4Rα, and γC after IL-4 stimulation. Both IL-4Rα and γC were ubiquitinated after 30 min of IL-4 treatment, suggesting that GRB10 may regulate degradation of the IL-4 receptor-signaling complex through interactions with NEDD4.2. Taken together, these data highlight two novel regulatory proteins that could be therapeutically manipulated to limit IL-4-induced IRS-2 signaling and polarization of M2 macrophages in allergic inflammation.

      Introduction

      Both genetic and environmental factors influence the onset and exacerbation of allergic asthma, which remains one of the most costly, non-communicable diseases of the human population. Allergic inflammation is characterized by airway hyper-reactivity, type 2 cytokines (IL-4, -5, and -13), IgE, and the overproduction of mucus in the lung (
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      ). Macrophages present at the site of allergic inflammation can be polarized by Th2-cytokines to become “alternatively activated” or M2 macrophages. In addition, the abundance of M2 macrophages in the lungs of asthmatics correlates with symptom severity (
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      ). As M2 macrophage polarization is dependent upon IL-4 stimulation, this cytokine and its signaling pathways represent excellent targets for asthma therapies.
      Our previous work has identified IL-4 signaling through the type I IL-4 receptor complex and activation of insulin receptor substrate 2 (IRS-2)
      The abbreviations used are: IRS-2, insulin receptor substrate 2; IP, immunoprecipitation; qPCR, quantitative PCR; ANOVA, analysis of variance.
      as important for the degree of polarization of M2 macrophages (
      • Heller N.M.
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      • Junttila I.S.
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      Type I IL-4Rs selectively activate IRS-2 to induce target gene expression in macrophages.
      ). IL-4 binds first to the IL-4Rα chain with high affinity followed by recruitment of the common γ chain (IL-2Rγ or γC) to create a functional type I receptor. Assembly of this receptor complex leads to recruitment and phosphorylation of the JAK1 and -3 proteins followed by robust, yet transient, tyrosine phosphorylation of IRS-2 and STAT6. Phosphorylated STAT6 homodimerizes and translocates to the nucleus, where it activates transcription of M2 macrophage-specific genes (ArgI, Retnla, and Chi3l3 in the mouse and TGM2, MMP12, CCL22, CD200R in humans). Activation of the IRS-2 pathway by IL-4 binding the type I IL-4 receptor, but not the type II IL-4 receptor, further enhances the degree of M2 macrophage gene expression (
      • Heller N.M.
      • Qi X.
      • Junttila I.S.
      • Shirey K.A.
      • Vogel S.N.
      • Paul W.E.
      • Keegan A.D.
      Type I IL-4Rs selectively activate IRS-2 to induce target gene expression in macrophages.
      ). Although Shp-1 and SHIP-1 have been implicated in negative regulation of IL-4-induced STAT6 signaling (
      • Haque S.J.
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      • Tabrizi M.
      • Yi T.
      • Williams B.R.
      Protein-tyrosine phosphatase Shp-1 is a negative regulator of IL-4- and IL-13-dependent signal transduction.
      ), nothing is known of the negative regulatory processes that suppress the IL-4-induced IRS-2 signaling pathway.
      Serine phosphorylation of the IRS proteins is one mechanism by which insulin-induced IRS signaling is terminated (
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      Insulin-induced serine phosphorylation of IRS-2 via ERK1/2 and mTOR: studies on the function of Ser-675 and Ser-907.
      ). Serine phosphorylation of IRS-1 and IRS-2 prevents p85 binding and PI3K activation (
      • Hançer N.J.
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      • Copps K.D.
      • White M.F.
      Insulin and metabolic stress stimulate multisite serine/threonine phosphorylation of insulin receptor substrate 1 and inhibit tyrosine phosphorylation.
      ), promotes IRS degradation, and promotes dissociation of IRS molecules from the insulin receptor (
      • Monami G.
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      Grb10/Nedd4-mediated multiubiquitination of the insulin-like growth factor receptor regulates receptor internalization.
      ,
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      Modulation of insulin-stimulated degradation of human insulin receptor substrate-1 by serine 312 phosphorylation.
      ). A number of different serine/threonine kinases (e.g. ERK1/2, TORC1/2, p70S6K, GSK-3α/β, JNK) have been shown to phosphorylate-specific serine residues of IRS-1 to inhibit insulin signaling (
      • Copps K.D.
      • White M.F.
      Regulation of insulin sensitivity by serine/threonine phosphorylation of insulin receptor substrate proteins IRS1 and IRS2.
      ,
      • Fritsche L.
      • Neukamm S.S.
      • Lehmann R.
      • Kremmer E.
      • Hennige A.M.
      • Hunder-Gugel A.
      • Schenk M.
      • Häring H.U.
      • Schleicher E.D.
      • Weigert C.
      Insulin-induced serine phosphorylation of IRS-2 via ERK1/2 and mTOR: studies on the function of Ser-675 and Ser-907.
      • Hançer N.J.
      • Qiu W.
      • Cherella C.
      • Li Y.
      • Copps K.D.
      • White M.F.
      Insulin and metabolic stress stimulate multisite serine/threonine phosphorylation of insulin receptor substrate 1 and inhibit tyrosine phosphorylation.
      ). Recent publications, however, highlight the importance of the TOR complexes and TOR-activated proteins in regulating M2 polarization in mouse macrophages in response to IL-4 (
      • Byles V.
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      The TSC-mTOR pathway regulates macrophage polarization.
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      TSC1 controls macrophage polarization to prevent inflammatory disease.
      ,
      • Festuccia W.T.
      • Pouliot P.
      • Bakan I.
      • Sabatini D.M.
      • Laplante M.
      Myeloid-specific Rictor deletion induces M1 macrophage polarization and potentiates in vivo pro-inflammatory response to lipopolysaccharide.
      ,
      • Hallowell R.W.
      • Collins S.
      • Zhang Y.
      • Chan-Li Y.
      • Powell J.
      • Horton M.R.
      Mtor signaling pathways regulate macrophage differentiation and function.
      • Mercalli A.
      • Calavita I.
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      • Citro A.
      • Cantarelli E.
      • Nano R.
      • Melzi R.
      • Maffi P.
      • Secchi A.
      • Sordi V.
      • Piemonti L.
      Rapamycin unbalances the polarization of human macrophages to M1.
      ). Because our previous work showed that IRS-2 tyrosine phosphorylation correlated with M2 polarization, we sought to determine whether serine phosphorylation of IRS-2 and TOR-activated regulatory pathways were responsible for controlling IL-4 signaling through IRS-2 in human macrophages.

      Results

       IL-4 Had Opposite Effects on Tyrosine and Serine Phosphorylation of IRS-2

      Previously we described IL-4-induced tyrosine phosphorylation of IRS-2 correlating with M2 macrophage polarization in both mouse macrophages and human monocytes (
      • Heller N.M.
      • Qi X.
      • Junttila I.S.
      • Shirey K.A.
      • Vogel S.N.
      • Paul W.E.
      • Keegan A.D.
      Type I IL-4Rs selectively activate IRS-2 to induce target gene expression in macrophages.
      ,
      • Keegan A.D.
      • Nelms K.
      • White M.
      • Wang L.M.
      • Pierce J.H.
      • Paul W.E.
      An IL-4 receptor region containing an insulin receptor motif is important for IL-4-mediated IRS-1 phosphorylation and cell growth.
      ,
      • Wang H.Y.
      • Zamorano J.
      • Keegan A.D.
      A role for the insulin-interleukin (IL)-4 receptor motif of the IL-4 receptor α-chain in regulating activation of the insulin receptor substrate 2 and signal transducer and activator of transcription 6 pathways: analysis by mutagenesis.
      ). We hypothesized that IL-4-induced tyrosine phosphorylation of IRS-2 is subject to down-regulation by serine phosphorylation of IRS-2. To test this hypothesis, we simultaneously evaluated tyrosine-phosphorylated (Tyr(P)-) IRS-2 and serine-phosphorylated (Ser(P)-) IRS-2 in human monocytic cells stimulated with IL-4 (10 ng/ml). Maximum tyrosine phosphorylation of IRS-2 occurs between 15 and 30 min of IL-4 stimulation; therefore, we evaluated later time points (time = 60, 90, 120, 150 min) after the peak of IRS-2 activation (
      • Heller N.M.
      • Qi X.
      • Junttila I.S.
      • Shirey K.A.
      • Vogel S.N.
      • Paul W.E.
      • Keegan A.D.
      Type I IL-4Rs selectively activate IRS-2 to induce target gene expression in macrophages.
      ). Accordingly, the amount of Tyr(P)-IRS-2 peaked at 15–30 min of IL-4 stimulation then returned to the level found in unstimulated cells by 90 min (Fig. 1A). In contrast, IRS-2 was highly phosphorylated on serine residues before stimulation (Fig. 1A, lower panel). After IL-4 stimulation, the amount of Ser(P)-IRS-2 decreased dramatically. Ser(P)-IRS-2 returned to the amount seen in unstimulated cells by 150 min of IL-4 stimulation.
      Figure thumbnail gr1
      FIGURE 1.IL-4 has opposite effects on tyrosine and serine phosphorylation of IRS-2. Human monocytic (U937) cells were serum-starved for 2 h then stimulated with IL-4 (10 ng/ml) for the indicated times. A, IRS-2 protein was immunoprecipitated from cell lysates, and Western blot (IB) membranes were probed with anti-phosphotyrosine (pY), anti-phosphoserine (pS), and anti-IRS-2 antibodies. Representative images of the films (left) are shown. Films were scanned, and densitometry was performed on the bands. The amount of Tyr(P)-IRS-2 normalized to the total IRS-2 is graphed from three separate experiments (right). Mean ± S.E. are represented. *, p < 0.05; A.U., arbitrary units. B, U937 cells stimulated with IL-4 for 30 min then treated with 25 nm calyculin A or DMSO vehicle control. Tyr(P)- and Ser(P)-IRS-2 was assessed as in A. A representative Western blot film and densitometry for three separate experiments are shown. Mean ± S.E. is represented. *, p < 0.05; **, p < 0.01; ***, p < 0.001. C, phospho-TOR (Ser-2448), -AKT (Ser-473), and -STAT6 (Tyr-641) were assessed at the indicated time points in the DMSO- or calyculin A-treated cells. A representative Western blot image is shown. The white vertical bar indicates that the bands were from non-adjacent lanes. The representative bands are from the same gel/membrane. Densitometry is representative of two separate experiments, and statistical significance was determined using multiple Student's t tests. **, p < 0.01. D, human M2 gene expression was determined by quantitative PCR at 6 and 24 h of IL-4 stimulation with or without calyculin A, as described in B. Relative -fold induction of mRNA is expressed using the standard 2ΔΔCt calculation, compared with unstimulated cells. Mean ± S.E. is graphed (n = 3; statistical analysis was performed using two-way ANOVAs with Bonferroni post-tests. *, p < 0.05; **, p < 0.01; +, p < 0.1).
      We further interrogated the role of Ser(P)-IRS-2 by inhibiting total serine phosphatase activity with calyculin A. IL-4 signaling was initiated, and calyculin A was added after 30 min (Fig. 1B). Tyrosine phosphorylation of IRS-2 was significantly reduced after the addition of calyculin A (Fig. 1B, top panel). Concomitantly, we detected a significant increase in Ser(P)-IRS-2 (Fig. 1B, middle panel) with a corresponding increase in the molecular weight of IRS-2 as a result (Fig. 1B, bottom panel). Calyculin A did not increase STAT6 phosphorylation; however, phosphorylation of AKT (Ser-473) and mTOR (Ser-2448) were modulated downstream of IRS-2 (Fig. 1C) (
      • Pozuelo-Rubio M.
      • Leslie N.R.
      • Murphy J.
      • Mackintosh C.
      Mechanism of activation of PKB/Akt by the protein phosphatase inhibitor Calyculin A.
      ,
      • Chen D.
      • Fucini R.V.
      • Olson A.L.
      • Hemmings B.A.
      • Pessin J.E.
      Osmotic shock inhibits insulin signaling by maintaining Akt/protein kinase B in an inactive dephosphorylated state.
      ). As anticipated after the calyculin A-induced ablation of Tyr(P)-IRS-2, we detected significant decreases in M2 gene expression of CD200R (*, p < 0.05) and TGM-2 and CCL22 (**, p < 0.01, Fig. 1D). These results confirmed an inverse correlation between serine and tyrosine phosphorylation of IRS-2 following IL-4 stimulation.

       TORC1-specific Inhibitors Altered IRS-2 Phosphorylation and M2 Gene Expression

      Activation of 44 serine/threonine kinases, including serine/threonine kinases of the AKT/TOR pathway, were screened and evaluated using a phosphokinase array (Fig. 2A). As we observed in the previous experiment (Fig. 1C), IL-4 induced phosphorylation of TOR (Ser-2448) and AKT (Ser-473) (Fig. 2A, left and middle panel). P70S6K, downstream of TOR, also showed changes in phosphorylation with IL-4 stimulation. Phosphorylation of AKT and p70S6K in response to IL-4 was confirmed by Western blot (Fig. 2A, right panel, DMSO-treated cells).
      Figure thumbnail gr2
      FIGURE 2.TORC1-specific inhibitors altered IL-4 signaling and M2 gene expression. A, left, cell lysates from IL-4-stimulated U937 were incubated with kinase array membranes. A representative Western blot image of the membranes (top left), a 10× image of the area on the array membrane corresponding to phosphorylated STAT6 (Tyr-641), and quantitative densitometry (bottom left) are shown. Quantitative densitometry for phosphorylated AKT (Ser-473, and Thr-308), TOR (Ser-2448), p70S6K (Thr-389), and p70S6K (Thr-421/Ser-424) are shown (right). Mean pixel intensity (1000×) for each phosphoprotein is shown. A, right, validation of serine kinase inhibition with rapamycin (5 nm), PP242 (500 nm), and PF4708671 (20 μm). Cells were pretreated for 1 h with the different inhibitors and stimulated with IL-4 for the indicated times. Phosphorylation of AKT (Ser-473), p70S6K (Thr-389), S6 (Ser-235/236), and the total target protein was determined by Western blot (IB). B, IRS-2 was immunoprecipitated as in A after serine kinase inhibition. Tyr(P)-IRS-2 and Ser(P)-IRS-2 were determined in three separate experiments. Tyr(P)-STAT6 was determined and quantitated relative to total STAT 6 protein. C, human M2 gene expression was determined by quantitative PCR at 6 and 24t h of IL-4 stimulation with or without TORC1, TORC1/2 and p70S6K inhibition, as described in B. Relative -fold induction of mRNA was determined. Results were normalized across experiments using the maximum expression vehicle control to determine the % of maximum with indicated inhibitor. Mean ± S.E. are graphed (n = 3; statistical analysis was performed using two-way ANOVA with Bonferroni post-tests where appropriate and simple Student's t test. **, p < 0.01; *, p < 0.05; +, p < 0.1).
      Next, we used inhibitors of TORC1/2 (PP242), TORC1 (rapamycin), and p70S6K (PF4708671) to determine their role in serine phosphorylation of IRS-2 after IL-4 stimulation. We first validated the specificity of each inhibitor in the U937 cells (Fig. 2A, right side) (
      • Pearce L.R.
      • Alton G.R.
      • Richter D.T.
      • Kath J.C.
      • Lingardo L.
      • Chapman J.
      • Hwang C.
      • Alessi D.R.
      Characterization of PF-4708671, a novel and highly specific inhibitor of p70 ribosomal S6 kinase (S6K1).
      ,
      • Feldman M.E.
      • Apsel B.
      • Uotila A.
      • Loewith R.
      • Knight Z.A.
      • Ruggero D.
      • Shokat K.M.
      Active-site inhibitors of mTOR target rapamycin-resistant outputs of mTORC1 and mTORC2.
      • Abraham R.T.
      • Wiederrecht G.J.
      Immunopharmacology of rapamycin.
      ). Phosphorylation of serine 473 of AKT, a TORC2 phosphorylation site, was inhibited by PP242 but not rapamycin (5 nm) (Fig. 2A, right top panel). Rapamycin and PP242 both inhibited TORC1 activity, as demonstrated by diminished phosphorylation of the TORC1 substrate, p70S6K (Thr-389) (Fig. 2A, right middle panel). PF-4708671 inhibited p70S6K, as shown by diminished phosphorylation of its substrate, S6 (Fig. 2A, right bottom panel).
      We pretreated U937 cells for 1 h with each serine kinase inhibitor to determine the duration of IRS-2 signaling and phosphorylation of AKT, p70S6K, and TOR as before (Fig. 1, A and B). Rapamycin and p70S6K inhibitor treatments prolonged Tyr(P)-IRS-2 compared with DMSO treatment (Fig. 2B, top panel of blots and upper panel of graphs). The amount of serine phosphorylation of IRS-2 significantly decreased in cells treated with the p70S6K inhibitor (Fig. 3A, middle panel of blots and lower panel of graphs). Treatment with PP242 did not change the amount of Tyr(P)- or Ser(P)-IRS-2. STAT6 phosphorylation at Tyr-641 was not changed by any of the inhibitors (Fig. 2B, right side of panel).
      Figure thumbnail gr3
      FIGURE 3.GRB10 was activated and transiently associated with IL-4 receptor subunits after IL-4 stimulation. A, GRB10. B, γC. C, IL-4Rα. D, IRS-2 were immunoprecipitated from U937 cells stimulated with IL-4. Immunoprecipitation was confirmed by Western blot for the indicated proteins (GRB10, γC, IL-4Rα, and IRS-2). We detected SHP-1 with IL-4Rα and JAK3 with γC as the positive control. Representative blotting images are shown (γC IP with GRB10 (n = 4); GRB10 IP with pGRB10 (n = 5); GRB10 with NEDD4.2 (n = 3); γC with NEDD4.2 (n = 2); γC and IL-4Rα with ubiquitin (n = 2); IRS-2 with all targets (n = 2)).
      Next, we assessed the induction of M2 macrophage-specific genes in U937 cells treated with the various inhibitors. IL-4 induced expression of TGM-2, MMP12, CCL22, and CD200R in DMSO control-treated cells. Inhibition of TORC1 with rapamycin (Fig. 2C, open bars) modestly increased expression of CCL22 at 24 h (+, p < 0.1) but not the other M2 genes. PP242 treatment did not significantly alter expression of any of the M2 genes. However, inhibition of p70S6K significantly increased expression of CD200R and CCL22 above that of DMSO control at 24 h (**, p < 0.01; *, p < 0.05; Fig. 2C, black bars). This finding matched the prolonged Tyr(P)-IRS-2 and diminished Ser(P)-IRS-2 signaling we observed with this inhibitor. Expression of MMP12 and TGM2 was not increased by p70S6K inhibition. These data suggest that p70S6K is involved in negatively regulating IRS-2 signaling and expression of some, but not all, human M2 genes.

       GRB10 Was Activated and Transiently Associated with IL-4 Receptor Subunits after IL-4 Stimulation

      Because not all M2 genes were augmented by p70S6K inhibition, we evaluated other negative regulatory proteins downstream of TORC1. Other studies have shown that the small adaptor protein, GRB10, is also activated by TORC1 (
      • Hsu P.P.
      • Kang S.A.
      • Rameseder J.
      • Zhang Y.
      • Ottina K.A.
      • Lim D.
      • Peterson T.R.
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      • Gray N.S.
      • Yaffe M.B.
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      The mTOR-regulated phosphoproteome reveals a mechanism of mTORC1-mediated inhibition of growth factor signaling.
      ,
      • Yu Y.
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      • Yang Q.
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      • Gygi S.P.
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      Phosphoproteomic analysis identifies Grb10 as an mTORC1 substrate that negatively regulates insulin signaling.
      ) and negatively regulates IRS-1 signaling after insulin and IGF-I stimulation (
      • Hsu P.P.
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      The mTOR-regulated phosphoproteome reveals a mechanism of mTORC1-mediated inhibition of growth factor signaling.
      • Yu Y.
      • Yoon S.O.
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      • Yang Q.
      • Ma X.M.
      • Villén J.
      • Kubica N.
      • Hoffman G.R.
      • Cantley L.C.
      • Gygi S.P.
      • Blenis J.
      Phosphoproteomic analysis identifies Grb10 as an mTORC1 substrate that negatively regulates insulin signaling.
      ,
      • Vecchione A.
      • Marchese A.
      • Henry P.
      • Rotin D.
      • Morrione A.
      The Grb10/Nedd4 complex regulates ligand-induced ubiquitination and stability of the insulin-like growth factor i receptor.
      • Wick K.R.
      • Werner E.D.
      • Langlais P.
      • Ramos F.J.
      • Dong L.Q.
      • Shoelson S.E.
      • Liu F.
      Grb10 inhibits insulin-stimulated insulin receptor substrate (IRS)-phosphatidylinositol 3-kinase/Akt signaling pathway by disrupting the association of IRS-1/IRS-2 with the insulin receptor.
      ). We hypothesized that GRB10 would regulate IL-4-activated IRS-2 signaling. First, we confirmed that GRB10 was activated in response to IL-4 by evaluating the phosphorylation of target residues of TORC1 activity. We observed GRB10 (Ser-501/503) phosphorylation at 30 min of IL-4 stimulation. Phosphorylation of GRB10 declined at 90 and 120 min of IL-4 stimulation (Fig. 3A). This kinetic of phosphorylation of GRB10 (Ser-501/503) correlated with tyrosine and serine phosphorylation of IRS-2.
      Next we showed that GRB10 associated with subunits of the IL-4 receptor complex. We observed a transient association of phosphorylated GRB10 with the IL-4Rα chain and γC by co-IP after IL-4 stimulation (Fig. 3, B and C). We also detected GRB10 and the IL-4Rα and γC together, leading us to hypothesize that GRB10 may regulate IL-4-induced responses by mediating internalization and degradation the type I IL-4 receptor signaling complex. We detected NEDD4.2 and ubiquitination of γC and IL-4Rα with IL-4 stimulation (Fig. 3, A and B). These findings are consistent with reports of NEDD4.2 binding to γC, and γC ubiquitination and degradation in T-cells (
      • Malardé V.
      • Proust R.
      • Dautry-Varsat A.
      • Gesbert F.
      NEDD4–2 associates with γc and regulates its degradation rate.
      ). To our knowledge we are the first to show GRB10 binding to subunits of the type I IL-4 receptor complex after IL-4-induced activation. We also demonstrated that GRB10, phosphorylated TOR, and phospho-p70S6K could be detected in the IRS-2 immunoprecipitates after IL-4 stimulation (Fig. 3D).

       GRB10 Knockdown Increased Tyrosine Phosphorylation of IRS-2 and M2 Macrophage Gene Expression

      To determine whether GRB10 was involved in modulating Tyr(P)-IRS-2 and M2 gene expression, we used a small interfering RNA approach to specifically target GRB10 for knockdown. U937 cells were nucleofected with siGRB10 or control siRNA and stimulated with IL-4. We confirmed GRB10 knockdown by Western blot and PCR (65–95% knockdown) (Fig. 4A). Tyrosine phosphorylation of IRS-2 and STAT6 were determined after IL-4 treatment (Fig. 4B). We detected a significant increase in Tyr(P)-IRS-2 in cells transfected with siGRB10 compared with siRNA-control cells (*, p < 0.05; Fig. 4B, upper two panels). Phosphorylation of STAT6 was not different between siGRB10 and control siRNA (Fig. 4B, lower two panels).
      Figure thumbnail gr4
      FIGURE 4.GRB10 knockdown increased tyrosine phosphorylation of IRS-2 and M2 macrophage gene expression. A, U937 cells were nucleofected with siRNA to GRB10 or negative control siRNA (50 nm). 24 h later, siRNA-transfected cells were stimulated with 10 ng/ml IL-4. Phosphorylation of GRB10 and total GRB10 was determined by Western blot (left) and by qPCR in unstimulated cells (right). B, tyrosine phosphorylation of IRS-2 and STAT6 were analyzed as in A and . IB, Western blot. A.U., absorbance units. C, human M2 gene expression was determined by quantitative PCR after 24 and 48 h of IL-4 stimulation as described in C. Mean ± S.E. are graphed (n = 3 independent experiments). *, p < 0.05. D, cell surface CD200R and CD206 were detected by flow cytometry in U937 cells stimulated with IL-4 for 24 h. Data are representative of two-three separate experiments. Statistical significance was determined by Student's t test; *, indicates p < 0.05.
      We next examined the effect of GRB10 knockdown on IL-4-induced M2 gene expression; cells lacking GRB10 expressed significantly more mRNA for TGM-2, CCL22, and MMP12 at 24 and 48 h of IL-4 stimulation (*, p < 0.05, Fig. 4C). CD200R expression was also modestly increased in siGRB10-treated U937 cells. Expression of another hallmark human M2 protein, CD206, was significantly increased on the surface of cells transfected with siGRB10 and stimulated with IL-4 as compared with control siRNA cells (*, p < 0.05; Fig. 4D). There was also a trend toward elevated cell surface CD200R in the GRB10 siRNA-transfected cells after IL-4 stimulation. Taken together with our co-IP and FACS data, these results demonstrate that GRB10 negatively regulates IL-4-induced IRS-2 signaling and M2 macrophage gene expression.

      Discussion

      IRS-2 has been described as a key regulator of M2 macrophage polarization (
      • Heller N.M.
      • Qi X.
      • Junttila I.S.
      • Shirey K.A.
      • Vogel S.N.
      • Paul W.E.
      • Keegan A.D.
      Type I IL-4Rs selectively activate IRS-2 to induce target gene expression in macrophages.
      ,
      • Heller N.M.
      • Qi X.
      • Gesbert F.
      • Keegan A.D.
      The extracellular and transmembrane domains of the γC and interleukin (IL)-13 receptor α1 chains, not their cytoplasmic domains, dictate the nature of signaling responses to IL-4 and IL-13.
      ). Given the correlation between the presence of M2 macrophages in the asthmatic lung and asthma severity in humans (
      • Melgert B.N.
      • ten Hacken N.H.
      • Rutgers B.
      • Timens W.
      • Postma D.S.
      • Hylkema M.N.
      More alternative activation of macrophages in lungs of asthmatic patients.
      ,
      • Viksman M.Y.
      • Bochner B.S.
      • Peebles R.S.
      • Schleimer R.P.
      • Liu M.C.
      Expression of activation markers on alveolar macrophages in allergic asthmatics after endobronchial or whole-lung allergen challenge.
      • St-Laurent J.
      • Turmel V.
      • Boulet L.P.
      • Bissonnette E.
      Alveolar macrophage subpopulations in bronchoalveolar lavage and induced sputum of asthmatic and control subjects.
      ), understanding the regulation of IL-4-activated signaling pathways that lead to M2 polarization is critical for interrupting asthma progression.
      The goal of this study was to determine mechanisms that negatively regulate the activity of IRS-2 in response to IL-4. Our current results show that down-regulation of IRS-2 signaling by serine phosphorylation follows a paradigm previously established for insulin responses (
      • Sun X.J.
      • Miralpeix M.
      • Myers Jr, M.G.
      • Glasheen E.M.
      • Backer J.M.
      • Kahn C.R.
      • White M.F.
      Expression and function of IRS-1 in insulin signal transmission.
      • Paz K.
      • Hemi R.
      • LeRoith D.
      • Karasik A.
      • Elhanany E.
      • Kanety H.
      • Zick Y.
      A molecular basis for insulin resistance. Elevated serine/threonine phosphorylation of IRS-1 and IRS-2 inhibits their binding to the juxtamembrane region of the insulin receptor and impairs their ability to undergo insulin-induced tyrosine phosphorylation.
      ,
      • Greene M.W.
      • Garofalo R.S.
      Positive and negative regulatory role of insulin receptor substrate 1 and 2 (IRS-1 and IRS-2) serine/threonine phosphorylation.
      • Zick Y.
      Ser/Thr phosphorylation of IRS proteins: a molecular basis for insulin resistance.
      ). Various well defined mechanisms, including serine/threonine kinase activation, steric hindrance, and ubiquitin-mediated degradation of IRS proteins (
      • Rui L.
      • Yuan M.
      • Frantz D.
      • Shoelson S.
      • White M.F.
      SOCS-1 and SOCS-3 block insulin signaling by ubiquitin-mediated degradation of IRS1 and IRS2.
      ), are known to limit insulin-induced IRS-1 activation. Our findings similarly show an on-to-off tyrosine/serine phosphorylation switch for IRS-2 in response to IL-4 (summarized in Fig. 5). IL-4 activates tyrosine phosphorylation and M2 macrophage polarization, whereas serine phosphorylation inhibits IL-4-activated IRS-2 signaling. Using pharmacological and siRNA knockdown approaches, we found that two TORC1-activated proteins, p70S6K and GRB10, were important negative regulators of IL-4-induced IRS-2 signaling and M2 gene expression in human monocytes.
      Figure thumbnail gr5
      FIGURE 5.Regulatory pathways of IL-4 signaling and M2 macrophage differentiation. IL-4 stimulation is a potent inducer of M2 macrophage polarization. 1, IL-4 can bind two kinds of IL-4 receptor complexes on monocyte-macrophages, type I (shown here) and type II. 2, both STAT6 and IRS-2 are recruited to the IL-4 receptor α chain after JAK phosphorylation of key tyrosine residues in the cytoplasmic domain. 3, phosphorylation of tyrosine residues of IRS-2 occurs, presumably by proximity to the activated JAKs. Tyrosine phosphorylation of IRS-2 occurs most potently as a result of engagement of IL-4 with type I receptor complexes. Engagement of type II receptor complexes by either IL-4 or IL-13 does not elicit robust tyrosine phosphorylation of IRS-2. 4, PI3K is activated through docking and activation of p85α with tyrosine phosphorylated IRS-2. PIP3 activation of PDK1 leads to phosphorylation of AKT Thr308. 5a, AKT activates mTORC1 as well as possibly other pathways downstream of AKT that directly contribute to M2 gene expression. 5b. PIP3-activated mTORC2 fully activates AKT function by phosphorylation of Ser-473. 6a, mTORC1 phosphorylates p70S6K, which interacts with IRS-2 to phosphorylate serine residues and thereby limit IL-4 signaling. 6b, GRB10 is directly activated and stabilized by mTORC1. 7, GRB10 interacts with IRS-2, common γ chain (γC), IL-4Rα and NEDD4.2 to target these proteins for ubiquitination (Ub) and, most likely, proteasomal degradation. Our current study establishes that the mTORC-1 activated proteins p70S6K and GRB10 limit signaling of tyrosine-phosphorylated IRS-2 through serine phosphorylation of IRS-2 and ubiquitination, respectively. The effect of these two negative regulatory mechanisms is a reduction in M2 polarization of human macrophages.
      Based upon the results of the Ser(P)- and Tyr(P)-IRS-2 studies, we expected induction of the human M2 gene panel after amplifying Tyr(P)-IRS-2 tyrosine with p70S6K inhibition. Indeed, two of four M2 genes were enhanced with p70S6K inhibition, indicating a greater complexity to regulation of M2 gene expression than anticipated. Many M2 genes share common activating and inhibitory pathways (
      • Covarrubias A.J.
      • Aksoylar H.I.
      • Horng T.
      Control of macrophage metabolism and activation by mTOR and Akt signaling.
      ,
      • Dasgupta P.
      • Dorsey N.J.
      • Li J.
      • Qi X.
      • Smith E.P.
      • Yamaji-Kegan K.
      • Keegan A.D.
      The adaptor protein insulin receptor substrate 2 inhibits alternative macrophage activation and allergic lung inflammation.
      ) and can be AKT-dependent or -independent (
      • Covarrubias A.J.
      • Aksoylar H.I.
      • Yu J.
      • Snyder N.W.
      • Worth A.J.
      • Iyer S.S.
      • Wang J.
      • Ben-Sahra I.
      • Byles V.
      • Polynne-Stapornkul T.
      • Espinosa E.C.
      • Lamming D.
      • Manning B.D.
      • Zhang Y.
      • Blair I.A.
      • Horng T.
      Akt-mTORC1 signaling regulates Acly to integrate metabolic input to control of macrophage activation.
      ). For example, studies of macrophage polarization using macrophages derived from Tsc1 (
      • Byles V.
      • Covarrubias A.J.
      • Ben-Sahra I.
      • Lamming D.W.
      • Sabatini D.M.
      • Manning B.D.
      • Horng T.
      The TSC-mTOR pathway regulates macrophage polarization.
      ,
      • Zhu L.
      • Yang T.
      • Li L.
      • Sun L.
      • Hou Y.
      • Hu X.
      • Zhang L.
      • Tian H.
      • Zhao Q.
      • Peng J.
      • Zhang H.
      • Wang R.
      • Yang Z.
      • Zhang L.
      • Zhao Y.
      TSC1 controls macrophage polarization to prevent inflammatory disease.
      )-, Raptor (

      Robert, H., Samuel, C., Yee, C.-L., Jonathan, P., and Maureen, R. H., (May 18–23, 2012) MTOR regulates the differentiation of classically and alternatively activated macrophages. In C92. Control of Macrophage Polarization and Effector Functions, American Thoracic Society, pp. A5061–A5061, 2012 International Conference, San Francisco,.

      )-, and Rictor (
      • Byles V.
      • Covarrubias A.J.
      • Ben-Sahra I.
      • Lamming D.W.
      • Sabatini D.M.
      • Manning B.D.
      • Horng T.
      The TSC-mTOR pathway regulates macrophage polarization.
      ,

      Robert, H., Samuel, C., Yee, C.-L., Jonathan, P., and Maureen, R. H., (May 18–23, 2012) MTOR regulates the differentiation of classically and alternatively activated macrophages. In C92. Control of Macrophage Polarization and Effector Functions, American Thoracic Society, pp. A5061–A5061, 2012 International Conference, San Francisco,.

      )-deficient mice have concluded that TORC1 down-regulates IL-4 signaling/M2 polarization (
      • Byles V.
      • Covarrubias A.J.
      • Ben-Sahra I.
      • Lamming D.W.
      • Sabatini D.M.
      • Manning B.D.
      • Horng T.
      The TSC-mTOR pathway regulates macrophage polarization.
      ,
      • Zhu L.
      • Yang T.
      • Li L.
      • Sun L.
      • Hou Y.
      • Hu X.
      • Zhang L.
      • Tian H.
      • Zhao Q.
      • Peng J.
      • Zhang H.
      • Wang R.
      • Yang Z.
      • Zhang L.
      • Zhao Y.
      TSC1 controls macrophage polarization to prevent inflammatory disease.
      ) and TORC2 promotes M2 polarization (

      Robert, H., Samuel, C., Yee, C.-L., Jonathan, P., and Maureen, R. H., (May 18–23, 2012) MTOR regulates the differentiation of classically and alternatively activated macrophages. In C92. Control of Macrophage Polarization and Effector Functions, American Thoracic Society, pp. A5061–A5061, 2012 International Conference, San Francisco,.

      ). However, TORC1 serves as a signaling “node” that can activate multiple downstream signaling pathways (e.g. p70S6K, GRB10, 4E-BP, and others) that may also influence macrophage polarization (
      • Laplante M.
      • Sabatini D.M.
      mTOR signaling at a glance.
      ,
      • Weichhart T.
      Mammalian target of rapamycin: a signaling kinase for every aspect of cellular life.
      ). It is clear from our study that control of IL-4 signaling and M2 gene expression downstream of TORC1 is not all mediated via p70S6K. To that end, we demonstrated that another TORC1-activated protein, GRB10, could regulate IL-4-induced IRS-2 signaling to promote human M2 gene expression. From our co-immunoprecipitation experiments we suggested a second mechanism by which GRB10 bound to receptor subunits that make up the type I IL-4 receptor complex (IL-4Rα, γC) mediates interaction with NEDD4.2. In this way IL-4 signaling could be suppressed by GRB10 by reducing cell surface availability of the type I IL-4 receptor. Future work will address the precise role of GRB10 in regulating this pathway.
      In summary, our work shows for the first time that serine phosphorylation of IRS-2 is an important mechanism for down-regulating activation of IRS-2 and M2 gene expression in response to IL-4. We show that tyrosine phosphorylation of IRS-2 and M2 gene expression is controlled by p70S6K and by GRB10, two proteins activated by TORC1 after IL-4 stimulation. Specifically, p70S6K regulated serine phosphorylation of IRS-2 in response to IL-4 and expression of some, but not all, human M2 macrophage genes. GRB10 also negatively regulated the IL-4-activated IRS-2 pathway and M2 gene expression in human monocytes, likely by regulating cell surface expression of the type I IL-4 receptor. To our knowledge we are the first to show GRB10 binding to the components of the IL-4 receptor complex and its effect on IL-4-induced signaling responses and human M2 macrophage gene expression. Our work may provide new therapeutic avenues where p70S6K or GRB10 activity could be enhanced to prevent M2 macrophage polarization in diseases like asthma.

      Experimental Procedures

       Reagents and Antibodies

      The serine kinase inhibitors used in this study were as follows: rapamycin (LC Laboratories, Woburn, MA), PP242 (Millipore, Billerica, MA), PF4708671 (Sigma). The antibodies used for immunoprecipitation and Western blotting were as follows: rabbit anti-human NEDD4.2 (Cell Signaling Technologies, Boston, MA), rabbit anti-human phospho-NEDD4.2 (Ser-448, clone EPR8269) (
      • Vercelli D.
      Advances in asthma and allergy genetics in 2007.
      ), Abcam, Cambridge, MA), rabbit anti-mouse GRB10 (Millipore), rabbit anti-human phospho-GRB10 (Ser-501/Ser-503, Millipore), mouse anti-phosphotyrosine-HRP (clone 4G10, Millipore), goat anti-human IRS-2 (M-19, Santa Cruz Biotechnology, Dallas, TX), rabbit anti-human STAT6 (Santa Cruz Biotechnology), rabbit anti-human IL-4Rα (clone C-20), rabbit anti-human γC (clone C-20), goat anti-human JAK3 (clone: N15), and mouse anti-human β-actin (clone C4, Santa Cruz Technologies), rabbit anti-human phospho-TOR (Ser-2448), rabbit anti-human p70S6K, rabbit anti-human phospho-p70S6K (Thr-389), rabbit anti-human AKT, rabbit anti-human phospho-AKT (Ser-473), rabbit anti-human S6, and rabbit anti-human phospho-S6 (Ser-235/236, Cell Signaling Technologies), anti-ubiquitin-HRP (Enzo, Farmingdale, NY), mouse anti-human phospho-serine (BD Biosciences), and mouse anti-human SHP-1 (clone 52/PTP1C/SHP1) and mouse anti-human phospho-STAT6 (Tyr-641) (Cell Signaling Technologies). The antibodies for flow cytometry were as follows: mouse anti-human CD200R-PE (clone OX-108, Biolegend, San Diego, CA), mouse anti-human IL-4Rα-APC (clone 25463, R&D Systems, Minneapolis, MN), and mouse anti-human CD206-APC (clone 19.2), rat anti-human γC-PE (clone TUGh4), and mouse anti-human phospho-STAT6 (Tyr-641, clone 18/P-Stat6, BD Pharmingen).

       Cell Culture

      Human monocytic cells, U937, were maintained in RPMI (Life Technologies) media supplemented with 10% FBS, 2 mm l-glutamine, penicillin (100 units/ml), and streptomycin (100 μg/ml).

       Gene Knockdown, Overexpression, and Nucleofection

      siRNA against GRB10, GAPDH, and HPRT (hypoxanthine-guanine phosphoribosyltransferase) (Sigma) were transfected using an Amaxa Nucleofector Kit C (Lonza, Allendale, NJ) at concentrations up to 500 nm according to the manufacturer's protocol. Protein knockdown was confirmed by Western blotting for the target protein, and gene knockdown was confirmed using quantitative PCR.

       Cell Signaling Experiments

      Cell signaling was carried out as previously described (
      • Heller N.M.
      • Qi X.
      • Junttila I.S.
      • Shirey K.A.
      • Vogel S.N.
      • Paul W.E.
      • Keegan A.D.
      Type I IL-4Rs selectively activate IRS-2 to induce target gene expression in macrophages.
      ,
      • Heller N.M.
      • Qi X.
      • Gesbert F.
      • Keegan A.D.
      The extracellular and transmembrane domains of the γC and interleukin (IL)-13 receptor α1 chains, not their cytoplasmic domains, dictate the nature of signaling responses to IL-4 and IL-13.
      ). Briefly, cells were serum-starved for a minimum of 2 h, up to 12 h, then pretreated for 1 h with serine kinase inhibitors at the concentrations indicated in the figure legend before stimulation with IL-4 (10 ng/ml). For the calyculin A experiments, cells were stimulated with IL-4 (10 ng/ml) for 30 min, then calyculin A (25–100 nm) was added for the duration of the experiment. A separate signaling experiment with DMSO treatment was included in all experiments as a vehicle control. Cells were collected and lysed using 1% Nonidet P-40 lysis buffer (described below) at different time points as indicated.

       Immunoprecipitation and Western Blotting

      Cells were lysed with buffer containing 1% Nonidet P-40 detergent, protease inhibitors, 1 mm PMSF, 1 mm sodium orthovanadate, and 25 nm calyculin A (previously described Refs.
      • Heller N.M.
      • Qi X.
      • Junttila I.S.
      • Shirey K.A.
      • Vogel S.N.
      • Paul W.E.
      • Keegan A.D.
      Type I IL-4Rs selectively activate IRS-2 to induce target gene expression in macrophages.
      and
      • Heller N.M.
      • Qi X.
      • Gesbert F.
      • Keegan A.D.
      The extracellular and transmembrane domains of the γC and interleukin (IL)-13 receptor α1 chains, not their cytoplasmic domains, dictate the nature of signaling responses to IL-4 and IL-13.
      ). Immunoprecipitating antibodies were incubated with cell lysates for a minimum of 2 h up to 12 h. Antibodies were rotated with protein G-agarose beads, collected by centrifugation, and washed three times before Western blotting. A Proteome Profiler Human Phospho-Kinase Array (R&D Systems) was used to detect serine kinases phosphorylated in response to IL-4 stimulation. Briefly, 1 × 107 cells were stimulated with 10 ng/ml of IL-4 for 0–120 min and lysed in 1 ml of lysis buffer as described above. Detection membranes were incubated with 375 μg of protein from cell lysates. Detection of bound protein was determined according to the manufacturer's protocol. All films were developed and digitally scanned, then analyzed using the NIH ImageJ software to determine pixel density of each spot or band. All protein data is normalized to the loading control (GAPDH or β-actin) or unstimulated control as indicated.

       Quantitative PCR

      U937 cells were pretreated with calyculin A or serine kinase inhibitors (concentrations described under “Results”) for 1 h then stimulated with IL-4 (10 ng/ml) for up to 48 h. RNA isolation was carried out using an RNeasy Mini kit (Qiagen, Valencia, CA) following the manufacturer's instructions. cDNA was synthesized from 500 ng of purified RNA, and qPCR was performed. The qPCR primer sets used are as described previously (
      • Heller N.M.
      • Qi X.
      • Junttila I.S.
      • Shirey K.A.
      • Vogel S.N.
      • Paul W.E.
      • Keegan A.D.
      Type I IL-4Rs selectively activate IRS-2 to induce target gene expression in macrophages.
      ,
      • Heller N.M.
      • Qi X.
      • Gesbert F.
      • Keegan A.D.
      The extracellular and transmembrane domains of the γC and interleukin (IL)-13 receptor α1 chains, not their cytoplasmic domains, dictate the nature of signaling responses to IL-4 and IL-13.
      ) and in Table 1.
      TABLE 1Primer sequences for mRNA detection by PCR
      Target geneSequence (5′ → 3′)
      GRB10 forwardCAA ACA GGA CGC GTG ATA GA
      GRB10 reverseGAG GAC ATC TGC GGT CAT AAG
      CD200R forwardCAT CGT GGA TAT CAC CTC CAA G
      CD200R reverseCTT GCT TAG TGG CAC AAT CGC
      MMP12 forwardAAC CAA CGC TTG CCA AAT CC
      MMP12 reverseCCT TCA GCC AGA AGA ACC TGT
      CCL22 forwardGCG TGG TGT TGC TAA CCT TC
      CCL22 reverseGAG AGT TGG CAC AGG CTT CT
      TGM2 forwardCAG GAG AAG AGC GAA GGG AC
      TGM2 reverseGAG GTT GGA CTC CGT AAG GC

       Statistical Analyses

      All statistical analysis was carried out using GraphPad Prism Version 6. One-way analysis of variance (ANOVA) was used to establish differences among treatment groups in each experiment. A two-way ANOVA with repeated measures was used where appropriate to determine interaction between time and treatment (i.e. serine kinase inhibition, phosphatase inhibition) on cell signaling outcomes over time. Bonferroni post-tests were completed for every experiment to determine statistical differences between each treatment group compared with non-stimulated control. Statistical trend levels were set at p < 0.1, and statistical significance levels for all tests were set at p < 0.05.

      Author Contributions

      K. J. W. conceptualized the experiments presented in FIGURE 2., FIGURE 3., FIGURE 4. and provided data acquisition in FIGURE 1., FIGURE 2., FIGURE 3., FIGURE 4., statistical analysis throughout, and manuscript preparation. X. F. provided data acquisition and statistical analysis for FIGURE 2., FIGURE 3.. N. M. G. conceptualized the experiments and provided data acquisition in FIGURE 1., FIGURE 2.. J. J. T. provided data acquisition of Fig. 2. N. M. G. provided funding support, conceptualization of experiments throughout, figure design, and manuscript preparation.

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