Activation of the p70 S6 Kinase and Phosphorylation of the 4E-BP1 Repressor of mRNA Translation by Type I Interferons
2003; Elsevier BV; Volume: 278; Issue: 30 Linguagem: Inglês
10.1074/jbc.m301364200
ISSN1083-351X
AutoresFatima Lekmine, Shahab Uddin, Antonella Sassano, Simrit Parmar, Saskia M. Brachmann, Beata Majchrzak, Nahum Sonenberg, Nissim Hay, Eleanor N. Fish, Leonidas C. Platanias,
Tópico(s)Protein Kinase Regulation and GTPase Signaling
ResumoThe Type I IFN receptor-generated signals required for initiation of mRNA translation and, ultimately, induction of protein products that mediate IFN responses, remain unknown. We have previously shown that IFNα and IFNβ induce phosphorylation of insulin receptor substrate proteins and downstream engagement of the phosphatidylinositol (PI) 3′-kinase pathway. In the present study we provide evidence for the existence of a Type I IFN-dependent signaling cascade activated downstream of PI 3′-kinase, involving p70 S6 kinase. Our data demonstrate that p70 S6K is rapidly phosphorylated on threonine 421 and serine 424 and is activated during treatment of cells with IFNα or IFNβ. Such activation of p70 S6K is blocked by pharmacological inhibitors of the PI 3′-kinase or the FKBP 12-rapamycin-associated protein/mammalian target of rapamycin (FRAP/mTOR). Consistent with this, the Type I IFN-dependent phosphorylation/activation of p70 S6K is defective in embryonic fibroblasts from mice with targeted disruption of the p85α and p85β subunits of the PI 3′-kinase (p85α–/–β–/–). Treatment of sensitive cell lines with IFNα or IFNβ also results in phosphorylation/inactivation of the 4E-BP-1 repressor of mRNA translation. Such 4E-BP1 phosphorylation is also PI3′-kinase-dependent and rapamycin-sensitive, indicating that the Type I IFN-inducible activation of PI3′-kinase and FRAP/mTOR results in dissociation of 4E-BP1 from the eukaryotic initiation factor-4E (eIF4E) complex. Altogether, our data establish that the Type I IFN receptor-activated PI 3′-kinase pathway mediates activation of the p70 S6 kinase and inactivation of 4E-BP1, to regulate mRNA translation and induction of Type I IFN responses. The Type I IFN receptor-generated signals required for initiation of mRNA translation and, ultimately, induction of protein products that mediate IFN responses, remain unknown. We have previously shown that IFNα and IFNβ induce phosphorylation of insulin receptor substrate proteins and downstream engagement of the phosphatidylinositol (PI) 3′-kinase pathway. In the present study we provide evidence for the existence of a Type I IFN-dependent signaling cascade activated downstream of PI 3′-kinase, involving p70 S6 kinase. Our data demonstrate that p70 S6K is rapidly phosphorylated on threonine 421 and serine 424 and is activated during treatment of cells with IFNα or IFNβ. Such activation of p70 S6K is blocked by pharmacological inhibitors of the PI 3′-kinase or the FKBP 12-rapamycin-associated protein/mammalian target of rapamycin (FRAP/mTOR). Consistent with this, the Type I IFN-dependent phosphorylation/activation of p70 S6K is defective in embryonic fibroblasts from mice with targeted disruption of the p85α and p85β subunits of the PI 3′-kinase (p85α–/–β–/–). Treatment of sensitive cell lines with IFNα or IFNβ also results in phosphorylation/inactivation of the 4E-BP-1 repressor of mRNA translation. Such 4E-BP1 phosphorylation is also PI3′-kinase-dependent and rapamycin-sensitive, indicating that the Type I IFN-inducible activation of PI3′-kinase and FRAP/mTOR results in dissociation of 4E-BP1 from the eukaryotic initiation factor-4E (eIF4E) complex. Altogether, our data establish that the Type I IFN receptor-activated PI 3′-kinase pathway mediates activation of the p70 S6 kinase and inactivation of 4E-BP1, to regulate mRNA translation and induction of Type I IFN responses. Type I interferons (IFNs) 1The abbreviations used are: IFN, interferon; STAT, signal transducer and activator of transcription; PI, phosphatidylinositol; ISRE, interferon-stimulated response element; SIE, sis-inducible element; GAS, IFNγ-activated site; p70 S6K, p70 S6 kinase; FRAP, FKBP12 rapamycin-associated protein; mTOR, mammalian target of rapamycin; eIF4E, eukaryotic initiation factor-4E; 4E-BP1, 4E-binding protein 1; MAPK, mitogen-activated protein kinase; IRS, insulin receptor substrate; MEF, mouse embryonic fibroblast; RIgG, rabbit immunoglobulin; SIF, sis-inducible factor.1The abbreviations used are: IFN, interferon; STAT, signal transducer and activator of transcription; PI, phosphatidylinositol; ISRE, interferon-stimulated response element; SIE, sis-inducible element; GAS, IFNγ-activated site; p70 S6K, p70 S6 kinase; FRAP, FKBP12 rapamycin-associated protein; mTOR, mammalian target of rapamycin; eIF4E, eukaryotic initiation factor-4E; 4E-BP1, 4E-binding protein 1; MAPK, mitogen-activated protein kinase; IRS, insulin receptor substrate; MEF, mouse embryonic fibroblast; RIgG, rabbit immunoglobulin; SIF, sis-inducible factor. are pleiotropic cytokines that exhibit multiple biological effects on cells and tissues, including inhibition of cell proliferation of normal and malignant cells, induction of antiviral responses, as well as immunomodulatory activities (1Pestka S. Langer J.A. Zoon K.C. Samuel C.E. Annu. Rev. Biochem. 1987; 56: 727-777Crossref PubMed Scopus (1599) Google Scholar, 2Darnell Jr., J.E. Kerr I.M. Stark G.R. Science. 1994; 264: 1415-1420Crossref PubMed Scopus (4974) Google Scholar, 3Darnell Jr., J.E. Science. 1997; 277: 1630-1635Crossref PubMed Scopus (3356) Google Scholar, 4Stark G.R. Kerr I.M. Williams B.R. Silverman R.H. Schreiber R.D. Annu. Rev. Biochem. 1998; 67: 227-264Crossref PubMed Scopus (3366) Google Scholar, 5Platanias L.C. Fish E.N. Exp. Hematol. 1999; 27: 1583-1592Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar, 6Verma A. Platanias L.C. Leuk. Lymphoma. 2002; 43: 703-709Crossref PubMed Scopus (31) Google Scholar, 7Uddin S. Alsayed Y. Grumbach I. Woodson J. Platanias L.C. Haematology. 1999; 2: 192-199Google Scholar). Several signaling pathways are activated following binding of Type I IFNs to the multichain Type I interferon receptor complex, whose IFNaR1 and IFNaR2 subunits are constitutively associated with protein members of the Jak family of kinases (reviewed in Refs. 1Pestka S. Langer J.A. Zoon K.C. Samuel C.E. Annu. Rev. Biochem. 1987; 56: 727-777Crossref PubMed Scopus (1599) Google Scholar, 2Darnell Jr., J.E. Kerr I.M. Stark G.R. Science. 1994; 264: 1415-1420Crossref PubMed Scopus (4974) Google Scholar, 3Darnell Jr., J.E. Science. 1997; 277: 1630-1635Crossref PubMed Scopus (3356) Google Scholar, 4Stark G.R. Kerr I.M. Williams B.R. Silverman R.H. Schreiber R.D. Annu. Rev. Biochem. 1998; 67: 227-264Crossref PubMed Scopus (3366) Google Scholar, 5Platanias L.C. Fish E.N. Exp. Hematol. 1999; 27: 1583-1592Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar, 6Verma A. Platanias L.C. Leuk. Lymphoma. 2002; 43: 703-709Crossref PubMed Scopus (31) Google Scholar, 7Uddin S. Alsayed Y. Grumbach I. Woodson J. Platanias L.C. Haematology. 1999; 2: 192-199Google Scholar). A major Type I IFN-activated cellular pathway is the Jak-STAT signaling cascade (1Pestka S. Langer J.A. Zoon K.C. Samuel C.E. Annu. Rev. Biochem. 1987; 56: 727-777Crossref PubMed Scopus (1599) Google Scholar, 2Darnell Jr., J.E. Kerr I.M. Stark G.R. Science. 1994; 264: 1415-1420Crossref PubMed Scopus (4974) Google Scholar, 3Darnell Jr., J.E. Science. 1997; 277: 1630-1635Crossref PubMed Scopus (3356) Google Scholar, 4Stark G.R. Kerr I.M. Williams B.R. Silverman R.H. Schreiber R.D. Annu. Rev. Biochem. 1998; 67: 227-264Crossref PubMed Scopus (3366) Google Scholar, 5Platanias L.C. Fish E.N. Exp. Hematol. 1999; 27: 1583-1592Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar, 6Verma A. Platanias L.C. Leuk. Lymphoma. 2002; 43: 703-709Crossref PubMed Scopus (31) Google Scholar, 7Uddin S. Alsayed Y. Grumbach I. Woodson J. Platanias L.C. Haematology. 1999; 2: 192-199Google Scholar). Engagement of the Type I IFN receptor results in activation of the Tyk-2 and Jak-1 kinases, which in turn regulate downstream phosphorylation/activation of the STAT1 and STAT2 transcriptional activators. The phosphorylated forms of STAT1 and STAT2 associate with IRF-9 (p48) to form the mature ISGF3 DNA-binding complex that translocates to the nucleus and regulates gene transcription via binding to ISRE elements in the promoters of IFN-stimulated genes (ISGs) (1Pestka S. Langer J.A. Zoon K.C. Samuel C.E. Annu. Rev. Biochem. 1987; 56: 727-777Crossref PubMed Scopus (1599) Google Scholar, 2Darnell Jr., J.E. Kerr I.M. Stark G.R. Science. 1994; 264: 1415-1420Crossref PubMed Scopus (4974) Google Scholar, 3Darnell Jr., J.E. Science. 1997; 277: 1630-1635Crossref PubMed Scopus (3356) Google Scholar, 4Stark G.R. Kerr I.M. Williams B.R. Silverman R.H. Schreiber R.D. Annu. Rev. Biochem. 1998; 67: 227-264Crossref PubMed Scopus (3366) Google Scholar, 5Platanias L.C. Fish E.N. Exp. Hematol. 1999; 27: 1583-1592Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar). Several other STAT complexes are also induced during engagement of the Type I interferon receptor. STAT 1:1 homodimers, STAT 3:3 homodimers, STAT 1:3 heterodimers, STAT 5:5 homodimers, and CrkL:STAT5 heterodimers are formed in a Type I IFN-dependent manner and translocate to the nucleus where they bind to GAS regulatory elements in the promoters of IFN-activated genes to regulate gene transcription (2Darnell Jr., J.E. Kerr I.M. Stark G.R. Science. 1994; 264: 1415-1420Crossref PubMed Scopus (4974) Google Scholar, 3Darnell Jr., J.E. Science. 1997; 277: 1630-1635Crossref PubMed Scopus (3356) Google Scholar, 4Stark G.R. Kerr I.M. Williams B.R. Silverman R.H. Schreiber R.D. Annu. Rev. Biochem. 1998; 67: 227-264Crossref PubMed Scopus (3366) Google Scholar, 5Platanias L.C. Fish E.N. Exp. Hematol. 1999; 27: 1583-1592Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar, 8Fish E.N. Uddin S. Korkmaz M. Majchrzak B. Druker B.J. Platanias L.C. J. Biol. Chem. 1999; 274: 571-573Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar, 9Meinke A. Barahmand-Pour F. Wohrl S. Stoiber D. Decker T. Mol. Cell. Biol. 1996; 16: 6937-6944Crossref PubMed Scopus (155) Google Scholar). In addition to tyrosine phosphorylation of STAT proteins by interferon-activated Jak kinases, phosphorylation on serine residues is required for their full transcriptional activation (10Decker T. Kovarik P. Oncogene. 2000; 19: 2628-2637Crossref PubMed Scopus (707) Google Scholar, 11Wen Z. Zhong Z. Darnell J.E. Cell. 1995; 28: 241-250Abstract Full Text PDF Scopus (1735) Google Scholar, 12Zhu X. Wen Z. Xu L.Z. Darnell Jr., J.E. Mol. Cell. Biol. 1997; 17: 6618-6623Crossref PubMed Scopus (141) Google Scholar, 13Zhang J.J. Zhao Y. Chait B.T. Lathem W.W. Ritzi M. Knippers R. Darnell Jr., J.E. EMBO J. 1998; 17: 6963-6971Crossref PubMed Scopus (189) Google Scholar, 14Wen Z. Darnell Jr., J.E. Nucleic Acids Res. 1997; 25: 2062-2067Crossref PubMed Scopus (266) Google Scholar). It appears that, at least in the case of STAT1, phosphorylation on serine 727 is regulated by a member of the protein kinase C family of proteins, protein kinase C δ (15Uddin S. Sassano A. Deb D.K. Verma A. Majchrzak B. Rahman A. Malik A.B. Fish E.N. Platanias L.C. J. Biol. Chem. 2002; 277: 14408-14416Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar). There is also accumulating evidence that the p38 MAPK pathway is activated in a Type I IFN-dependent manner (16Uddin S. Majchrzak B. Woodson J Arunkumar P. Alsayed Y. Pine R Young P.R. Fish E.N. Platanias L.C. J. Biol. Chem. 1999; 274: 30127-30131Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar, 17Goh K.C. Haque S.J. Williams B.R.G. EMBO J. 1999; 18: 5601-5608Crossref PubMed Scopus (325) Google Scholar) and that its function is essential for gene transcription via ISRE (16Uddin S. Majchrzak B. Woodson J Arunkumar P. Alsayed Y. Pine R Young P.R. Fish E.N. Platanias L.C. J. Biol. Chem. 1999; 274: 30127-30131Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar, 17Goh K.C. Haque S.J. Williams B.R.G. EMBO J. 1999; 18: 5601-5608Crossref PubMed Scopus (325) Google Scholar) or GAS elements (18Uddin S. Lekmine F. Sharma N. Majchrzak B. Mayer I. Young P.R. Bokoch G.M. Fish E.N. Platanias L.C. J. Biol. Chem. 2000; 275: 27634-27640Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar). Such regulatory effects of this pathway play critical roles in IFN signaling, because p38 activation is essential for generation of Type I IFN-dependent antiproliferative responses (19Verma A. Deb D.K. Sassano A. Uddin S. Varga J. Wickrema A. Platanias L.C. J. Biol. Chem. 2002; 277: 7726-7735Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar, 20Mayer I.A. Verma A. Grumbach I.M. Uddin S. Lekmine F. Ravandi F. Majchrzak B. Fujita S. Fish E.N. Platanias L.C. J. Biol. Chem. 2001; 276: 28570-28577Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar, 21Platanias L.C. Pharmacol. Ther. 2003; 98: 129-142Crossref PubMed Scopus (131) Google Scholar). The p70 S6 kinase was originally identified as a kinase that regulates serine phosphorylation of the 40 S ribosomal S6 protein (22Ferrari S. Thomas G. Crit. Rev. Biochem. Mol. Biol. 1994; 29: 385-413Crossref PubMed Scopus (133) Google Scholar, 23Chou M.M. Blenis J. Curr. Opin. Cell Biol. 1995; 7: 806-814Crossref PubMed Scopus (245) Google Scholar, 24Grammer T.C. Cheatham L. Chou M.M. Blenis J. Cancer Surv. 1996; 27: 271-292PubMed Google Scholar, 25Belcham C. Wu S. Avruch J. Curr. Biol. 1999; 9: 93-96Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar, 26Dufner A. Thomas G. Exp. Cell Res. 1999; 253: 100-109Crossref PubMed Scopus (601) Google Scholar, 27Shah O.J. Anthony J.C. Kimball S.R. Jefferson L.S. Am. J. Physiol. Endocrinol. Metab. 2000; 279: 715-729Crossref PubMed Google Scholar, 28Blume-Jensen P. Hunter T. Nature. 2001; 411: 355-365Crossref PubMed Scopus (3109) Google Scholar, 29Kozma S.C. Thomas G. Bioessays. 2002; 24: 65-71Crossref PubMed Scopus (256) Google Scholar). This kinase plays important roles in the regulation of cell-cycle progression, cell survival, as well as regulation of mRNA translation via phosphorylation of the 40 S ribosomal S6 protein (22Ferrari S. Thomas G. Crit. Rev. Biochem. Mol. Biol. 1994; 29: 385-413Crossref PubMed Scopus (133) Google Scholar, 23Chou M.M. Blenis J. Curr. Opin. Cell Biol. 1995; 7: 806-814Crossref PubMed Scopus (245) Google Scholar, 24Grammer T.C. Cheatham L. Chou M.M. Blenis J. Cancer Surv. 1996; 27: 271-292PubMed Google Scholar, 25Belcham C. Wu S. Avruch J. Curr. Biol. 1999; 9: 93-96Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar, 26Dufner A. Thomas G. Exp. Cell Res. 1999; 253: 100-109Crossref PubMed Scopus (601) Google Scholar, 27Shah O.J. Anthony J.C. Kimball S.R. Jefferson L.S. Am. J. Physiol. Endocrinol. Metab. 2000; 279: 715-729Crossref PubMed Google Scholar, 28Blume-Jensen P. Hunter T. Nature. 2001; 411: 355-365Crossref PubMed Scopus (3109) Google Scholar, 29Kozma S.C. Thomas G. Bioessays. 2002; 24: 65-71Crossref PubMed Scopus (256) Google Scholar, 30Chung J. Kuo C.J. Crabtree G.R. Blenis J. Cell. 1992; 69: 1227-1236Abstract Full Text PDF PubMed Scopus (1021) Google Scholar, 31Kuo C.J. Chung J. Fiorentino D.F. Flanagan W.M. Blenis J. Crabtree G.R. Nature. 1992; 370: 71-75Google Scholar, 32Jefferies H.B. Fumagali S. Dennis P.B. Reinhard C. Pearson R.B. Thomas G. EMBO J. 1997; 16: 3693-3704Crossref PubMed Scopus (806) Google Scholar, 33Volarevic S. Stewart M.J. Ledermann B. Zilberman F. Terracciano L. Montini E. Grompe M. Kozma S.C. Thomas G. Science. 2000; 288: 2045-2047Crossref PubMed Scopus (317) Google Scholar, 34Petritsch C. Beug H. Balmain A. Oft M. Genes Dev. 2000; 14: 3093-3101Crossref PubMed Scopus (189) Google Scholar, 35Harada H. Andersen J.S. Mann M. Terada N. Korsmeyer S.J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 9666-9670Crossref PubMed Scopus (465) Google Scholar). Previous studies have established that the activation of this kinase is regulated by the FKBP 12-rapamycin-associated protein (FRAP/mTOR), whose activation is in turn regulated by the upstream activation of the phosphatidylinositol 3′-kinase pathway (36Alessi D. Kozlowski M.T. Weng Q.P. Morrice N. Avruch J. Curr. Biol. 1998; 8: 69-81Abstract Full Text Full Text PDF PubMed Scopus (512) Google Scholar, 37Pullen N. Dennis P.B. Andjelkovic M. Dufner A. Kozma S.C. Hemmings B.A. Thomas G. Science. 1998; 279: 707-710Crossref PubMed Scopus (723) Google Scholar, 38Brennan P. Babbage J.W. Thomas G. Cantrell D. Mol. Cell. Biol. 1999; 19: 4729-4738Crossref PubMed Scopus (115) Google Scholar, 39Romanelli A. Martin K.A. Toker A. Blenis J. Mol. Cell. Biol. 1999; 19: 2921-2928Crossref PubMed Google Scholar, 40Balendran A. Currie R. Armstrong C.G. Avruch J. Alessi D.R. J. Biol. Chem. 1999; 274: 37400-37406Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar, 41Jensen C.J. Buch M.B. Krag T.O. Hemmings B.A. Gammeltoft S. Frodin M. J. Biol. Chem. 1999; 274: 27168-27176Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar). The signals generated by the Type I interferon receptor to ultimately regulate mRNA translation are not known. We have previously demonstrated that Type I IFNs activate the insulin receptor substrate (IRS)-PI 3′-kinase pathway in human and mouse cells (42Uddin S. Yenush L. Sun X. Sweet M.E. White M.F. Platanias L.C. J. Biol. Chem. 1995; 270: 15938-15941Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar, 43Platanias L.C. Uddin S.C. Yetter A. Sun X. White M.F. J. Biol. Chem. 1996; 271: 278-282Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar, 44Uddin S. Fish E.N. Sher D. Gardziola C. Colamonici O.R. Kellum M. Pitha P.M. White M.E. Platanias L.C. Blood. 1997; 90: 2574-2582PubMed Google Scholar, 45Uddin S. Fish E.N. Sher D. Gardziola C. White M.F. Platanias L.C. J. Immunol. 1997; 158: 2390-2397PubMed Google Scholar) and that both the lipid (42Uddin S. Yenush L. Sun X. Sweet M.E. White M.F. Platanias L.C. J. Biol. Chem. 1995; 270: 15938-15941Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar) and serine (45Uddin S. Fish E.N. Sher D. Gardziola C. White M.F. Platanias L.C. J. Immunol. 1997; 158: 2390-2397PubMed Google Scholar) kinase activities of the p110 catalytic subunit of the PI 3′-kinase are activated during engagement of the Type I interferon receptor. In the present study we sought to determine whether the p70 S6 kinase is activated downstream of the PI 3′-kinase to mediate induction of Type I IFN responses. Our data demonstrate that the p70 S6 kinase is rapidly phosphorylated and activated during treatment of sensitive cell lines with IFNα or IFNβ. They also show that the IFNα-dependent phosphorylation/activation of the p70 S6 kinase is defective in mouse embryonic fibroblasts (MEFs) from p85α–/– p85β–/– double knock-out mice. In other studies we establish that the translational mRNA repressor 4E-BP1 is phosphorylated in a Type I IFN-dependent manner, and such phosphorylation is also PI 3′-kinase-dependent, further demonstrating that activation of PI 3-kinase/mTOR by Type I IFNs ultimately induces signals important for mRNA translation. Cells Lines and Reagents—Human recombinant IFNα2 was provided by Hoffmann-La Roche. Human recombinant consensus IFNα was provided by Amgen Inc. Human recombinant IFNβ was provided by Biogen Inc. Antibodies against the phosphorylated forms of p70 S6 kinase, mTOR, and 4EBP-1 were obtained from Cell Signaling Technology Inc. An antibody against 4EB-P1 has been previously described (46Gingras A.-C. Kennedy S.G. O'Leary M.A. Sonenberg N. Hay N. Genes Dev. 1998; 12: 502-513Crossref PubMed Scopus (725) Google Scholar). The FRAP/mTOR inhibitor, rapamycin, and the PI 3′-kinase inhibitors LY294002 and wortmannin were obtained from Calbiochem Inc. (La Jolla, CA). U266 cells were grown in RPMI 1640 supplemented with fetal bovine serum and antibiotics. U2OS and T98G cells were grown in McCoy's and Dulbecco's modified Eagle's media, respectively, supplemented with fetal bovine serum and antibiotics. The generation of p85α–/–β–/– mice, by crossing p85β+/– (48Ueki K. Yballe C.M. Brachmann S.M. Vicent D. Watt J.M. Kahn C.R. Cantley L.C. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 419-424Crossref PubMed Scopus (197) Google Scholar) mice with p85α+/– mice (47Fruman D.A. Mauvais-Jarvis F. Pollard D.A. Yballe C.M. Brazil D. Bronson R.T. Kahn C.R. Cantley L.C. Nat. Genet. 2000; 26: 379-382Crossref PubMed Scopus (251) Google Scholar) will be described elsewhere. 2S. Brachmann and L. C. Cantley, manuscript in preparation. The p85α–/–β–/– mouse embryonic fibroblasts were obtained from p85α–/–β–/– double knock-out mice. Briefly, mouse embryos were harvested at day 14; the limbs, head, and liver removed, and the resultant torso was finely minced. Following trypsinization, the single cell suspension was transferred onto gelatinized tissue culture dishes, and the mouse embryonic fibroblasts were immortalized after a few passages using SV40 large T antigen, expressed by a retroviral vector. The genotypes of the cells were determined by polymerase chain reaction. All transfections were performed using FuGENE 6, according to the manufacturer's instructions (Roche Applied Science). Cell Lysis and Immunoblotting—Cells were stimulated with 1 × 104 units/ml of the indicated IFNs for the indicated times, then lysed in phosphorylation lysis buffer as previously described (49Yetter A. Uddin S. Krolewski J.J. Jiao H. Yi T. Platanias L.C. J. Biol. Chem. 1995; 270: 18179-18182Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). Immunoprecipitations and immunoblotting, using an enhanced chemiluminescence (ECL) method, were performed as previously described (49Yetter A. Uddin S. Krolewski J.J. Jiao H. Yi T. Platanias L.C. J. Biol. Chem. 1995; 270: 18179-18182Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). In the experiments in which pharmacological inhibitors of FRAP/mTOR or the PI 3′-kinase were used, the cells were pretreated for 60 min with the indicated concentrations of the inhibitors and subsequently treated for the indicated times with IFNs, prior to lysis in phosphorylation lysis buffer. In some of the experiments to determine the phosphorylation of 4E-BP1, cell extracts were obtained by three freeze-thaw cycles, as previously described (46Gingras A.-C. Kennedy S.G. O'Leary M.A. Sonenberg N. Hay N. Genes Dev. 1998; 12: 502-513Crossref PubMed Scopus (725) Google Scholar). Chromatography on m7GDP-Agarose—Chromatography of cell extracts from IFN-treated cells on m7GDP-agarose was performed essentially as previously described (46Gingras A.-C. Kennedy S.G. O'Leary M.A. Sonenberg N. Hay N. Genes Dev. 1998; 12: 502-513Crossref PubMed Scopus (725) Google Scholar, 51Miron M. Verdu J. Lachanse P.E.D. Birnbaum M.J. Lasko P.F. Sonenberg N. Nat. Cell Biol. 2001; 3: 596-601Crossref PubMed Scopus (175) Google Scholar). Briefly, cell extracts were obtained by four freeze-thaw cycles, in cold cap-binding buffer, containing 100 mm KCl, 20 mm HEPES, pH 7.6, 7 mm β-mercaptoethanol, 0.2 mm EDTA, 10% glycerol, 50 mm β-glycerol phosphate, 50 mm NaF, 100 μm sodium orthovanadate, and 1 mm phenylmethylsulfonyl fluoride. Protein extracts were subsequently incubated for 60 min with m7GDP-agarose resin (46Gingras A.-C. Kennedy S.G. O'Leary M.A. Sonenberg N. Hay N. Genes Dev. 1998; 12: 502-513Crossref PubMed Scopus (725) Google Scholar, 51Miron M. Verdu J. Lachanse P.E.D. Birnbaum M.J. Lasko P.F. Sonenberg N. Nat. Cell Biol. 2001; 3: 596-601Crossref PubMed Scopus (175) Google Scholar) at 4 °C. The resin was then washed with cap-binding buffer, once with 500 ml and twice with 1 ml, resuspended in Laemmli sample buffer, and boiled. p70 S6 Kinase Assays—Assays to detect the Type I IFN-dependent activation of the p70 S6 kinase were performed as previously described (52Cichy S.B. Uddin S. Danilkovich A. Guo S. Klippel A. Unterman T.G. J. Biol. Chem. 1998; 273: 6482-6487Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar). Briefly, cells were lysed in phosphorylation lysis buffer, and cell lysates were immunoprecipitated with an antibody against p70 S6 kinase or control non-immune rabbit immunoglobulin (RIgG). In vitro kinase assays were performed using a synthetic peptide substrate (AKRRRLSSLRA), and p70 S6 kinase activity was measured using an S6 kinase assay kit (Upstate Biotechnology Inc.) according to the manufacturer's instructions (52Cichy S.B. Uddin S. Danilkovich A. Guo S. Klippel A. Unterman T.G. J. Biol. Chem. 1998; 273: 6482-6487Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar). Values were calculated by subtracting nonspecific activity, detected in RIgG immunoprecipitates, from kinase activity detected in anti-p70 S6K immunoprecipitates. Isolation of Normal Peripheral Blood Granulocytes—Informed consent was obtained from healthy volunteers, according to the guidelines established by the Institutional Review Board of Northwestern University Medical School. Polymorphonuclear leukocytes were separated from peripheral venous blood using the Mono-Poly resolving medium (M-PRM, ICN Biomedicals, Aurora, OH), as previously described (20Mayer I.A. Verma A. Grumbach I.M. Uddin S. Lekmine F. Ravandi F. Majchrzak B. Fujita S. Fish E.N. Platanias L.C. J. Biol. Chem. 2001; 276: 28570-28577Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar). Briefly, after centrifugation at 300 × g for 30 min at room temperature, the plasma and the mononuclear leukocyte band were discarded, and the polymorphonuclear band was transferred into an individual tube. Cells were washed with culture medium and were subsequently resuspended in culture medium, prior to interferon treatment. Mobility Shift Assays—Actively growing cells were treated with IFNα for the indicated times, in the presence or absence of rapamycin, as indicated. 10 μg of nuclear extracts, from untreated or IFNα-treated cells, was analyzed using electrophoretic mobility shift assays, as described previously (19Verma A. Deb D.K. Sassano A. Uddin S. Varga J. Wickrema A. Platanias L.C. J. Biol. Chem. 2002; 277: 7726-7735Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar, 20Mayer I.A. Verma A. Grumbach I.M. Uddin S. Lekmine F. Ravandi F. Majchrzak B. Fujita S. Fish E.N. Platanias L.C. J. Biol. Chem. 2001; 276: 28570-28577Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar). A double-stranded oligodeoxynucleotide (ATTTCCCGTAAATCCC), representing a sis-inducing element (SIE) of the c-fos promoter, was synthesized and used in the gel shift assays. A double-stranded oligodeoxynucleotide (CTGTTGGTTTCGTTTCCTCAGA), representing an ISRE element from the ISG-15 gene, was also synthesized and used to detect ISGF3 complexes. Luciferase Reporter Assays—Cells were transfected with a β-galactosidase expression vector and either an ISRE luciferase construct or a luciferase reporter gene containing eight GAS elements linked to a minimal prolactin promoter (8X-GAS), using the SuperFect transfection reagent as per the manufacturer's recommended procedure (Qiagen). The ISRE-luciferase construct (16Uddin S. Majchrzak B. Woodson J Arunkumar P. Alsayed Y. Pine R Young P.R. Fish E.N. Platanias L.C. J. Biol. Chem. 1999; 274: 30127-30131Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar) included an ISG15 ISRE (TCGGGAAAGGGAAACCGAAACTGAAGCC) cloned via cohesive ends into the BamHI site of the pZtkLuc vector and was provided by Dr. Richard Pine (Public Health Research Institute, New York, NY). The 8X-GAS construct (53Horvai A.E. Xu L. Korzus E. Brard G. Kalafus D. Mullen T.-M. Rose D.W. Rosenfeld M.G. Glass C.K. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1074-1079Crossref PubMed Scopus (388) Google Scholar) was kindly provided by Dr. Christofer Glass (University of California San Diego, San Diego, CA). Forty-eight hours after transfection, triplicate cultures were either left untreated or treated with 5 × 103 units/ml IFNα, and luciferase activity was subsequently measured using the manufacturer's protocol (Promega). The measured luciferase activities were normalized for β-galactosidase activity for each sample. The p70 S6 Kinase Is Activated by Type I Interferons Downstream of the Phosphatidylinositol 3′-Kinase—We initially sought to determine whether the p70 S6 kinase is phosphorylated/activated during treatment of Type I IFN-sensitive cell lines with IFNα or IFNβ. Molt-4 cells were incubated for 30 min in the presence or absence of IFNα. Cells were lysed in phosphorylation lysis buffer, and total cell lysates were analyzed by SDS-PAGE and immunoblotted with an antibody against the phosphorylated form of p70 S6 kinase on threonine 421 and serine 424. IFNα treatment resulted in strong phosphorylation of the p70 S6 kinase, whereas there was no change in the amounts of p70 S6 kinase protein detected after IFNα stimulation (Fig. 1, A and B). Similarly, phosphorylation of p70 S6K was detectable when another Type I IFN, IFNβ, was used (Fig. 1, C and D). In experiments, in which the phosphorylation of the p70 S6 kinase by Type I IFNs was determined, we found that both IFNα (Fig. 2, A and B), as well as IFNβ (Fig. 2, C and D) are capable of inducing phosphorylation of the protein when used at doses as low as 100 IU/ml, further emphasizing the specificity of the process. Furthermore, the Type I interferon-dependent phosphorylation of the p70 S6 kinase was inducible in various cell types, including U2OS osteosarcoma cells (Fig. 3, A and B) or U-266 multiple myeloma cells (Figs. 2A, 2B, 3C, and 3D).Fig. 2Interferon-dependent phosphorylation of the p70 S6 kinase. A, U266 cells were treated with the indicated doses of IFNα for 30 min, as indicated. Equal amounts of total cell lysates were analyzed by SDS-PAGE and immunoblotted with an antibody against the phosphorylated/activated form of the p70 S6-kinase. B, the blot shown in A was stripped and re-probed with an anti-p70 S6K antibody, to control for protein loading. C, Molt-4 cells were treated with the indicated doses of IFNβ for 60 min, as indicated. Equal amounts of total cell lysates were analyzed by SDS-PAGE and
Referência(s)