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Roles of Phosphatidylinositol 3-Kinase in Interferon-γ-dependent Phosphorylation of STAT1 on Serine 727 and Activation of Gene Expression*

  • Hannah Nguyen
    Footnotes
    Affiliations
    Department of Molecular Biology, Lerner Research Institute, The Cleveland Clinic Foundation, Cleveland, Ohio 44195
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  • Chilakamarti V. Ramana
    Affiliations
    Department of Molecular Biology, Lerner Research Institute, The Cleveland Clinic Foundation, Cleveland, Ohio 44195
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  • Joshua Bayes
    Affiliations
    Department of Molecular Biology, Lerner Research Institute, The Cleveland Clinic Foundation, Cleveland, Ohio 44195
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  • George R. Stark
    Correspondence
    To whom correspondence should be addressed: Dept. of Molecular Biology, Lerner Research Inst., The Cleveland Clinic Foundation, 9500 Euclid Ave., Cleveland, OH 44195. Tel.: 216-444-3900; Fax: 216-444-3279;
    Affiliations
    Department of Molecular Biology, Lerner Research Institute, The Cleveland Clinic Foundation, Cleveland, Ohio 44195
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  • Author Footnotes
    * This work was supported in part by Grant P01 CA 62220 from the National Institutes of Health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
    ‡ Supported by a Cancer Research Institute Fellowship.
Open AccessPublished:September 07, 2001DOI:https://doi.org/10.1074/jbc.M105070200
      STAT1 must be phosphorylated on serine 727 to be fully active in transcription. We show that phosphatidylinositol 3-kinase (PI3K) and its effector kinase Akt play an important role in the serine phosphorylation of STAT1 and in the activation of gene expression in response to interferon-γ (IFNγ). IFNγ activates PI3K as well as Akt in a variety of cell lines. Specific inhibition of PI3K abrogates IFNγ-induced, but not interleukin-1- or tumor necrosis factor-α-induced, phosphorylation of STAT1 on serine and reduces STAT1-dependent transcription and gene expression by ∼7-fold. Constitutively active forms of PI3K or Akt activate and their dominant-negative derivatives inhibit STAT1-driven transactivation in response to IFNγ. In addition to PI3K and Akt, JAK1, JAK2, and the tyrosine 440 STAT1 docking residue of IFNGR1 are required for STAT1 to be phosphorylated on serine. Taken together, these results suggest that the following events lead to the activation of STAT1 upon IFNγ stimulation: 1) PI3K and Akt are activated by the occupied receptor and Tyr-440 is phosphorylated by the activated JAKs; 2) STAT1 docks to Tyr-440; and 3) Tyr-701 is phosphorylated by the JAKs and Ser-727 is phosphorylated by a kinase downstream of Akt.
      STAT
      signal transducer and activator of transcription
      GAS
      γ activated sequence
      PI3K
      phosphatidylinositol 3-kinase
      JAK
      Janus kinase
      IFNγ
      interferon γ
      TNF
      tumor necrosis factor-α
      IL
      interleukin
      MAPK
      mitogen-activated protein kinase
      MEFs
      mouse embryo fibroblasts
      PAGE
      polyacrylamide gel electrophoresis
      GBP-1
      guanylate-binding protein-1
      SH2
      Src homology domain 2
      ERK
      extracellular signal-regulated kinase
      Full activation of STAT11 by IFNγ requires two distinct phosphorylation events. Receptor-mediated phosphorylation of STAT1 on tyrosine 701 is required for STAT1 homodimers to form and subsequently to bind to the promoters of IFNγ-responsive genes through γ-activated sequence (GAS) elements (reviewed in Refs.
      • Bach E.A.
      • Aguet M.
      • Schreiber R.D.
      and
      • Stark G.R.
      • Kerr I.M.
      • Williams B.R.
      • Silverman R.H.
      • Schreiber R.D.
      ). The mechanistic basis for STAT1 tyrosine phosphorylation has been established for some time. JAK1 and JAK2, constitutively bound to specific cytoplasmic domains of the IFNGR1 and IFNGR2 subunits, phosphorylate each other when IFNγ binds and the receptor subunits aggregate. The activated JAKs then phosphorylate tyrosine 440 of IFNGR1, creating a docking site that recruits STAT1 to the receptor. STAT1 binds to phosphorylated Tyr-440 through its SH2 domain, allowing its Tyr-701 residue to be phosphorylated by the JAKs. STAT1 then dissociates from the receptor, dimerizes through reciprocal SH2-phosphotyrosine interactions, and binds to IFNγ-inducible promoters.
      At some point, STAT1 is also phosphorylated on serine 727 independently of tyrosine phosphorylation (
      • Wen Z.
      • Zhong Z.
      • Darnell Jr., J.E.
      ,
      • Zhu X.
      • Wen Z.
      • Xu L.Z.
      • Darnell Jr., J.E.
      ,
      • Kovarik P.
      • Stoiber D.
      • Novy M.
      • Decker T.
      ,
      • Goh K.C.
      • Haque S.J.
      • Williams B.R.
      ,
      • Kovarik P.
      • Stoiber D.
      • Eyers P.A.
      • Menghini R.
      • Neininger A.
      • Gaestel M.
      • Cohen P.
      • Decker T.
      ). Serine phosphorylation substantially enhances the transcriptional activity of STAT1; the transactivation potential of the S727A mutant of STAT1 is reduced by ∼80% (
      • Wen Z.
      • Zhong Z.
      • Darnell Jr., J.E.
      ). STAT1 can also be phosphorylated on serine in response to IFNα (
      • Kovarik P.
      • Stoiber D.
      • Novy M.
      • Decker T.
      ,
      • Goh K.C.
      • Haque S.J.
      • Williams B.R.
      ), UV irradiation (
      • Kovarik P.
      • Stoiber D.
      • Eyers P.A.
      • Menghini R.
      • Neininger A.
      • Gaestel M.
      • Cohen P.
      • Decker T.
      ), bacterial lipopolysaccharide (
      • Kovarik P.
      • Stoiber D.
      • Novy M.
      • Decker T.
      ,
      • Kovarik P.
      • Stoiber D.
      • Eyers P.A.
      • Menghini R.
      • Neininger A.
      • Gaestel M.
      • Cohen P.
      • Decker T.
      ), platelet-derived growth factor (
      • Wen Z.
      • Zhong Z.
      • Darnell Jr., J.E.
      ), phorbol esters, interleukins-2 and -12 (
      • Gollob J.A.
      • Schnipper C.P.
      • Murphy E.A.
      • Ritz J.
      • Frank D.A.
      ), cross-linking of B-cell or T-cell receptors (
      • Su L.
      • Rickert R.C.
      • David M.
      ,
      • Gamero A.M.
      • Larner A.C.
      ), or tumor necrosis factor-α (
      • Kovarik P.
      • Stoiber D.
      • Eyers P.A.
      • Menghini R.
      • Neininger A.
      • Gaestel M.
      • Cohen P.
      • Decker T.
      ) (reviewed in Ref.
      • Decker T.
      • Kovarik P.
      ).
      The mechanisms through which STAT1 is phosphorylated on serine are not yet well understood. Serine 727 lies within the mitogen-activated protein kinase (MAPK) consensus motif PXn(S/T)P, where S/T is serine or threonine and n is 1 or 2, suggesting that MAPK family members may be STAT1 serine kinases. Through the use of knockout cells and dominant-negative and chemical inhibitors, several kinases have been implicated directly or indirectly (reviewed in Ref.
      • Decker T.
      • Kovarik P.
      ). Phosphorylation of STAT1 on serine in response to IFNγ has been shown to involve JAK2 (
      • Zhu X.
      • Wen Z.
      • Xu L.Z.
      • Darnell Jr., J.E.
      ), proline-rich tyrosine kinase 2 (Pyk2), potentially through its activation of ERK2 (
      • Takaoka A.
      • Tanaka N.
      • Mitani Y.
      • Miyazaki T.
      • Fujii H.
      • Sato M.
      • Kovarik P.
      • Decker T.
      • Schlessinger J.
      • Taniguchi T.
      ), p38 MAPK (
      • Goh K.C.
      • Haque S.J.
      • Williams B.R.
      ), and double-stranded RNA-activated protein kinase (PKR)(13).
      Phosphatidylinositol 3-kinase (PI3K) is a member of a subfamily of lipid kinases implicated in many physiological processes, including regulation of cell growth, proliferation, survival and differentiation, vesicle trafficking, glucose transport, platelet function, and cytoskeletal remodeling (reviewed in Refs.
      • Fruman D.A.
      • Meyers R.E.
      • Cantley L.C.
      and
      • Wymann M.P.
      • Pirola L.
      ). PI3K is activated by phosphorylation on tyrosine residues in response to many growth factors and cytokines by receptors with intrinsic tyrosine kinase activity or by receptor-associated tyrosine kinases. A heterodimer comprised of one of three catalytic isoforms and one of seven adaptor/regulatory proteins, PI3K, once active, catalyzes the addition of a phosphate moiety specifically to the 3′-OH position of the inositol ring of phosphatidylinositols (reviewed in Refs.
      • Fruman D.A.
      • Meyers R.E.
      • Cantley L.C.
      and
      • Wymann M.P.
      • Pirola L.
      ). The resulting 3-phosphorylated phosphatidylinositols serve as secondary messengers to activate many downstream signaling targets, initiating the physiological effects of PI3K. One of the best characterized PI3K effectors is the serine-threonine protein kinase Akt(PKB), whose activation following growth factor or cytokine stimulation is directly dependent on PI3K-derived phosphorylated phosphatidylinositols (reviewed in Refs.
      • Vanhaesebroeck B.
      • Leevers S.J.
      • Panayotou G.
      • Waterfield M.D.
      ,
      • Coffer P.J.
      • Jin J.
      • Woodgett J.R.
      ,
      • Chan T.O.
      • Rittenhouse S.E.
      • Tsichlis P.N.
      ).
      We now find that the phosphorylation of STAT1 on serine in response to IFNγ requires the activation of PI3K and Akt. Our results reveal that PI3K functions in a novel pathway that plays an important role in signaling and in the activation of gene expression in response to IFNγ and defines receptor-associated functions that are additionally required for the phosphorylation of STAT1 on Ser-727.

      ACKNOWLEDGEMENTS

      We thank Nywana Sizemore for helpful discussions and Nicholas Grammatikakis for the PI3K constructs.

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