Activation of Protein Kinase Cη by Type I Interferons
2009; Elsevier BV; Volume: 284; Issue: 16 Linguagem: Inglês
10.1074/jbc.m807254200
ISSN1083-351X
AutoresAmanda J. Redig, Antonella Sassano, Beata Majchrzak-Kita, Efstratios Katsoulidis, Hui Liu, Jessica K. Altman, Eleanor N. Fish, Amittha Wickrema, Leonidas C. Platanias,
Tópico(s)Protein Kinase Regulation and GTPase Signaling
ResumoType I interferons (IFNs) are cytokines with diverse biological properties, including antiviral, growth inhibitory, and immunomodulatory effects. Although several signaling pathways are activated during engagement of the type I IFN receptor and participate in the induction of IFN responses, the mechanisms of generation of specific signals for distinct biological effects remain to be elucidated. We provide evidence that a novel member of the protein kinase C (PKC) family of proteins is rapidly phosphorylated and activated during engagement of the type I IFN receptor. In contrast to other members of the PKC family that are also regulated by IFN receptors, PKCη does not regulate IFN-inducible transcription of interferon-stimulated genes or generation of antiviral responses. However, its function promotes cell cycle arrest and is essential for the generation of the suppressive effects of IFNα on normal and leukemic human myeloid (colony-forming unit-granulocyte macrophage) bone marrow progenitors. Altogether, our studies establish PKCη as a unique element in IFN signaling that plays a key and essential role in the generation of the regulatory effects of type I IFNs on normal and leukemic hematopoiesis. Type I interferons (IFNs) are cytokines with diverse biological properties, including antiviral, growth inhibitory, and immunomodulatory effects. Although several signaling pathways are activated during engagement of the type I IFN receptor and participate in the induction of IFN responses, the mechanisms of generation of specific signals for distinct biological effects remain to be elucidated. We provide evidence that a novel member of the protein kinase C (PKC) family of proteins is rapidly phosphorylated and activated during engagement of the type I IFN receptor. In contrast to other members of the PKC family that are also regulated by IFN receptors, PKCη does not regulate IFN-inducible transcription of interferon-stimulated genes or generation of antiviral responses. However, its function promotes cell cycle arrest and is essential for the generation of the suppressive effects of IFNα on normal and leukemic human myeloid (colony-forming unit-granulocyte macrophage) bone marrow progenitors. Altogether, our studies establish PKCη as a unique element in IFN signaling that plays a key and essential role in the generation of the regulatory effects of type I IFNs on normal and leukemic hematopoiesis. Type I interferons (IFNs) 2The abbreviations used are: IFN, interferon; PKC, protein kinase C; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; siRNA, short interfering RNA; RT, reverse transcription; ISRE, interferon-stimulated response element; CFU-GM, colony-forming unit-granulocyte macrophage; BFU-E, burst-forming unit-erythroid; CML, chronic myelogenous leukemia; EMCV, encephalomyocarditis virus. exhibit important biological effects, including antiviral properties and regulation of normal and malignant cell growth (1Pestka S. Langer J.A. Zoon K.C. Samuel C.E. Annu. Rev. Biochem... 1987; 56: 727-777Google Scholar, 2Stark G.R. Kerr I.M. Williams B.R. Silverman R.H. Schreiber R.D. Annu. Rev. Biochem... 1998; 67: 227-264Google Scholar, 3Platanias L.C. Fish E.N. Exp. Hematol... 1999; 27: 1583-1592Google Scholar, 4Parmar S. Platanias L.C. Curr. Opin. Oncol... 2003; 15: 431-439Google Scholar-5Borden E.C. Sen G.C. Uze G. Silverman R.H. Ransohoff R.M. Foster G.R. Stark G.R. Nat. Rev. Drug Discov... 2007; 6: 975-990Google Scholar). Inducible or constitutive production of IFNs appears to be a key component of cellular defense mechanisms against viral infections and the immunosurveillance against cancer (1Pestka S. Langer J.A. Zoon K.C. Samuel C.E. Annu. Rev. Biochem... 1987; 56: 727-777Google Scholar, 2Stark G.R. Kerr I.M. Williams B.R. Silverman R.H. Schreiber R.D. Annu. Rev. Biochem... 1998; 67: 227-264Google Scholar, 3Platanias L.C. Fish E.N. Exp. Hematol... 1999; 27: 1583-1592Google Scholar, 4Parmar S. Platanias L.C. Curr. Opin. Oncol... 2003; 15: 431-439Google Scholar-5Borden E.C. Sen G.C. Uze G. Silverman R.H. Ransohoff R.M. Foster G.R. Stark G.R. Nat. Rev. Drug Discov... 2007; 6: 975-990Google Scholar). These cytokines exhibit important regulatory effects on cell cycle progression, gene transcription, and mRNA translation (6Darnell Jr. J.E. Kerr I.M. Stark G.R. Science.. 1994; 264: 1415-1421Google Scholar, 7Darnell Jr. J.E. 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One of the most important biological activities of IFNs is their ability to act as regulators of normal hematopoietic progenitor cell growth and to control normal hematopoiesis. It has been known for the last 3 decades that type I IFNs are potent suppressors of hematopoietic progenitor cell growth in vitro (14van't Hull E. Schellekens H. Löwenberg B. de Vries M.J. Cancer Res... 1978; 38: 911-914Google Scholar, 15Broxmeyer H.E. Lu L. Platzer E. Feit C. Juliano L. Rubin B.Y. J. Immunol... 1983; 131: 1300-1305Google Scholar, 16Raefsky E.L. Platanias L.C. Zoumbos N.C. Young N.S. J. Immunol... 1985; 135: 2507-2512Google Scholar, 17Broxmeyer H.E. Cooper S. Rubin B.S. Taylor M.W. J. Immunol... 1985; 135: 2502-2506Google Scholar-18Means Jr. R.T. Krantz S.B. J. Clin. Investig... 1993; 91: 416-419Google Scholar), and such effects may account for the development of pancytopenias that patients receiving IFN treatment frequently develop. IFNs inhibit the growth of all different classes of normal bone marrow-derived hematopoietic precursors, including progenitors of myeloid (CFU-GM), erythroid (CFU-E and BFU-E) and megakaryocytic (CFU-MK) lineages (14van't Hull E. Schellekens H. Löwenberg B. de Vries M.J. Cancer Res... 1978; 38: 911-914Google Scholar, 15Broxmeyer H.E. Lu L. Platzer E. Feit C. Juliano L. Rubin B.Y. J. Immunol... 1983; 131: 1300-1305Google Scholar, 16Raefsky E.L. Platanias L.C. Zoumbos N.C. Young N.S. J. Immunol... 1985; 135: 2507-2512Google Scholar, 17Broxmeyer H.E. Cooper S. Rubin B.S. Taylor M.W. J. Immunol... 1985; 135: 2502-2506Google Scholar, 18Means Jr. R.T. Krantz S.B. J. Clin. Investig... 1993; 91: 416-419Google Scholar, 19Verma A. Deb D.K. Sassano A. Uddin S. Varga J. Wickrema A. Platanias L.C. J. Biol. Chem... 2002; 277: 7726-7735Google Scholar-20Weekx S.F. Van Bockstaele D.R. Plum J. Moulijn A. Rodrigus I. Lardon F. De Smedt M. Nijs G. Lenjou M. Loquet P. Berneman Z.N. Snoeck H.W. Exp. Hematol... 1998; 26: 1034-1039Google Scholar). The effects of IFNs have been also shown to occur on cell populations that contain bone marrow cells at an early precursor stage (CD34+CD38–) (20Weekx S.F. Van Bockstaele D.R. Plum J. Moulijn A. Rodrigus I. Lardon F. De Smedt M. Nijs G. Lenjou M. Loquet P. Berneman Z.N. Snoeck H.W. Exp. Hematol... 1998; 26: 1034-1039Google Scholar), underscoring the ability of IFNs to regulate both early and late stages of hematopoietic development. Over the years, the mechanisms of type I IFN signaling have been extensively studied and defined in a variety of cellular systems and backgrounds. Clearly, engagements of Jak kinases and Stat proteins are events of critical importance in the generation of the biological properties of IFNs (2Stark G.R. Kerr I.M. Williams B.R. Silverman R.H. Schreiber R.D. Annu. Rev. Biochem... 1998; 67: 227-264Google Scholar, 3Platanias L.C. Fish E.N. Exp. Hematol... 1999; 27: 1583-1592Google Scholar, 4Parmar S. Platanias L.C. Curr. Opin. Oncol... 2003; 15: 431-439Google Scholar, 5Borden E.C. Sen G.C. Uze G. Silverman R.H. Ransohoff R.M. Foster G.R. Stark G.R. Nat. Rev. Drug Discov... 2007; 6: 975-990Google Scholar, 6Darnell Jr. J.E. Kerr I.M. Stark G.R. Science.. 1994; 264: 1415-1421Google Scholar, 7Darnell Jr. J.E. Science.. 1997; 277: 1630-1635Google Scholar-8Platanias L.C. Nat. Rev. Immunol... 2005; 5: 375-386Google Scholar). The activation of Jaks occurs at the receptor level, followed by direct activation of interacting Stats, providing a mechanism of rapid turnover of signals from the cell surface to the nucleus (2Stark G.R. Kerr I.M. Williams B.R. Silverman R.H. Schreiber R.D. Annu. Rev. Biochem... 1998; 67: 227-264Google Scholar, 3Platanias L.C. Fish E.N. Exp. Hematol... 1999; 27: 1583-1592Google Scholar, 4Parmar S. Platanias L.C. Curr. Opin. Oncol... 2003; 15: 431-439Google Scholar, 5Borden E.C. Sen G.C. Uze G. Silverman R.H. Ransohoff R.M. Foster G.R. Stark G.R. Nat. Rev. Drug Discov... 2007; 6: 975-990Google Scholar, 6Darnell Jr. J.E. Kerr I.M. Stark G.R. Science.. 1994; 264: 1415-1421Google Scholar, 7Darnell Jr. J.E. Science.. 1997; 277: 1630-1635Google Scholar-8Platanias L.C. Nat. Rev. Immunol... 2005; 5: 375-386Google Scholar). Type I IFNs also activate p38 mitogen-activated protein kinase signaling pathways that complement the function of Jak-Stat pathways and are required for optimal transcriptional activation of IFN-regulated genes (21Uddin 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: 27634-27640Google Scholar, 22Uddin 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: 30127-30131Google Scholar, 23Goh K.C. Haque S.J. Williams B.R. EMBO J... 1999; 18: 5601-5608Google Scholar-24Platanias L.C. Pharmacol. Ther... 2003; 98: 129-142Google Scholar). In addition, there is accumulating evidence that type I IFNs activate the Akt/mammalian target of rapamycin signaling pathway and its downstream effectors (25Lekmine F. Uddin S. Sassano A. Parmar S. Brachmann S.M. Majchrzak B. Sonenberg N. Hay N. Fish E.N. Platanias L.C. J. Biol. Chem... 2003; 278: 27772-27780Google Scholar, 26Thyrell L. Hjortsberg L. Arulampalam V. Panaretakis T. Uhles S. Dagnell M. Zhivotovsky B. Leibiger I. Grandér D. Pokrovskaja K. J. Biol. Chem... 2004; 279: 24152-24162Google Scholar, 27Kaur 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. J. Biol. Chem... 2007; 282: 1757-1768Google Scholar-28Kaur S. Sassano A. Dolniak B. Joshi S. Majchrzak-Kita B. Baker D.P. Hay N. Fish E.N. Platanias L.C. Proc. Natl. Acad. Sci. U. S. A... 2008; 105: 4808-4813Google Scholar) and that such activation is required for the initiation of mRNA translation of interferon-stimulated genes (28Kaur S. Sassano A. Dolniak B. Joshi S. Majchrzak-Kita B. Baker D.P. Hay N. Fish E.N. Platanias L.C. Proc. Natl. Acad. Sci. U. S. A... 2008; 105: 4808-4813Google Scholar). Members of the PKC family (δ, ϵ, and θ) have been shown previously to be activated and play roles in the generation of type I and/or type II IFN responses (29Uddin 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: 14408-14416Google Scholar, 30Srivastava K.K. Batra S. Sassano A. Li Y. Majchrzak B. Kiyokawa H. Altman A. Fish E.N. Platanias L.C. J. Biol. Chem... 2004; 279: 29911-29920Google Scholar, 31Zhao K.W. Li D. Zhao Q. Huang Y. Silverman R.H. Sims P.J. Chen G.Q. J. Biol. Chem... 2005; 280: 42707-42714Google Scholar, 32Ivaska J. Bosca L. Parker P.J. Nat. Cell Biol... 2003; 5: 363-369Google Scholar-33Venkatesan B.A. Mahimainathan L. Ghosh-Choudhury N. Gorin Y. Bhandari B. Valente A.J. Abboud H.E. Choudhury G.G. Cell. Signal... 2006; 18: 508-518Google Scholar). However, much remains to be defined regarding the overall contribution of the PKC family to the generation of IFN responses as well as the specific roles of distinct isoforms in IFN signaling. In this study we provide the first evidence for engagement of PKCη, a member of the novel subgroup of PKC isotypes in type I IFN signaling. Our data establish that this PKC isoform is phosphorylated/activated by IFNα or IFNβ treatment of sensitive cells. We also show that engagement of PKCη by the type I IFN receptor regulates IFNα-dependent G0/G1 cell cycle arrest and plays an essential role in the generation of the suppressive effects of type I IFNs on normal and leukemic myeloid (CFU-GM) progenitors. Altogether, our findings implicate PKCη as a novel member of the PKC family with an important role in the generation of IFN responses and define a unique and specific role for this PKC isoform in IFN-mediated control of myelopoiesis. Cells and Reagents—The CML-derived lymphoblastoid crisis KT1 cell line, the multiple myeloma U266 cell line, and the acute myelomonocytic U937 cell line were grown in RPMI 1640 media supplemented with 10% fetal bovine serum and antibiotics. The osteosarcoma U20S cell line was grown in McCoy's media supplemented with 10% fetal bovine serum and antibiotics. Primary human CD34+ progenitor cells were either purchased from Stem Cell Technologies (Vancouver, British Columbia, Canada) or obtained from the bone marrow of normal donors after obtaining informed consent approved by the Institutional Review Board of Northwestern University. Bone marrow mononuclear cells were isolated using Histopaque (Sigma), and CD34+ cells were further purified using indirect positive selection (Miltenyi, Bergisch Gladbach, Germany), as in our previous studies (19Verma A. Deb D.K. Sassano A. Uddin S. Varga J. Wickrema A. Platanias L.C. J. Biol. Chem... 2002; 277: 7726-7735Google Scholar, 34Katsoulidis E. Li Y. Yoon P. Sassano A. Altman J. Kannan-Thulasiraman P. Balasubramanian L. Parmar S. Varga J. Tallman M.S. Verma A. Platanias L.C. Cancer Res... 2005; 65: 9029-9037Google Scholar). Recombinant human IFNα was obtained from Hoffmann-La Roche. Recombinant IFNβ was obtained from Biogen Idec. An antibody against the phosphorylated form of PKCη on Ser-674 was purchased from Upstate Biotechnology, Inc. (Billerica, MA); an antibody against PKCη was obtained from Santa Cruz Biotechnology (Santa Cruz, CA), and an antibody against GAPDH was obtained from Chemicon (Billerica, MA). PKCη and PKCζ peptide inhibitors were purchased from Calbiochem. U937 cells were transfected by nucleofection following the manufacturer's protocol (Amaxa AG, Cologne, Germany). A constitutively active PKCη mutant (36Brandlin I. Hubner S. Eiseler T. Martinez-Moya M. Horschinek A. Hausser A. Link G. Rupp S. Storz P. Pfizenmaier K. Johannes F.J. J. Biol. Chem... 2002; 277: 6490-6496Google Scholar) was provided by Dr. Gottfried Baier (Innsbruck Medical University, Innsbruck, Austria) and was used in overexpression experiments. Cell Lysis and Immunoblotting—Cells were serum-starved, stimulated with 1 × 104 IU/ml of the indicated IFN for the indicated times, and subsequently lysed in phosphorylation buffer as described previously (19Verma A. Deb D.K. Sassano A. Uddin S. Varga J. Wickrema A. Platanias L.C. J. Biol. Chem... 2002; 277: 7726-7735Google Scholar, 37Uddin S. Fish E.N. Sher D. Gardziola C. Colamonici O.R. Kellum M. Pitha P.M. White M.F. Platanias L.C. Blood.. 1997; 90: 2574-2582Google Scholar). Immunoprecipitation and immunoblotting using an enhanced chemiluminescence (ECL) method were performed as in previous studies (19Verma A. Deb D.K. Sassano A. Uddin S. Varga J. Wickrema A. Platanias L.C. J. Biol. Chem... 2002; 277: 7726-7735Google Scholar, 37Uddin S. Fish E.N. Sher D. Gardziola C. Colamonici O.R. Kellum M. Pitha P.M. White M.F. Platanias L.C. Blood.. 1997; 90: 2574-2582Google Scholar). Evaluation of Erythroid Differentiation—Human primary erythroid progenitor cells were enriched by in vitro culture of CD34+ cells isolated from normal bone marrows or obtained commercially from Stem Cell Technologies. After CD34+ cell isolation, differentiating erythroid progenitors were obtained by culturing cells for 4–14 days in medium with 15% fetal bovine serum, 15% human AB serum, 10 ng/ml interleukin-3, 2 units/ml erythropoietin, and 50 ng/ml stem cell factor (19Verma A. Deb D.K. Sassano A. Uddin S. Varga J. Wickrema A. Platanias L.C. J. Biol. Chem... 2002; 277: 7726-7735Google Scholar, 35Uddin S. Ah-Kang J. Ulaszek J. Mahmud D. Wickrema A. Proc. Natl. Acad. Sci. U. S. A... 2004; 101: 147-152Google Scholar). At the indicated time points, an aliquot of cells was removed from culture, washed with phosphate-buffered saline, and stained with glycophorin A and CD71 or the appropriate antibody controls (BD Biosciences) prior to flow cytometric analysis. RNA Isolation and PCR—Real time RT-PCR was performed as in our previous studies (27Kaur 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. J. Biol. Chem... 2007; 282: 1757-1768Google Scholar, 28Kaur S. Sassano A. Dolniak B. Joshi S. Majchrzak-Kita B. Baker D.P. Hay N. Fish E.N. Platanias L.C. Proc. Natl. Acad. Sci. U. S. A... 2008; 105: 4808-4813Google Scholar). RNA was isolated using a standard methodology (Qiagen, Hilden, Germany) and used as substrate for reverse transcription reactions. Quantitative real time PCR (Applied Biosystems, Foster City, CA) was then used to measure the relative expression of indicated mRNA transcripts with normalization to GAPDH. PCR was performed under the following conditions: 1 cycle at 50 °C for 2 min, 1 cycle at 95 °C for 10 min, and 40 cycles at 95 °C for 15 s followed by 1 min at 60 °C. Hematopoietic Cell Progenitor Assays—Bone marrow from normal donors and bone marrow or peripheral blood from CML patients was collected after obtaining consent approved by the Institutional Review Board of Northwestern University. Mononuclear cells were isolated using Histopaque (Sigma) separation, and CD34+ cells were further purified using indirect positive selection (Miltenyi, Bergisch Gladbach, Germany). Primary human CD34+ progenitor cells were also purchased from Stem Cell Technologies (Vancouver, British Columbia, Canada). CD34+ cells were then transfected using transfection reagent purchased from Mirus (Madison, WI) with control siRNA or siRNAs targeting specific PKC isoforms. Two different siRNA targeting PKCη (Ambion ID777 and ID778), as well as siRNA targeting PKCα siRNA, PKCβ siRNA, PKCι siRNA, and control siRNA were purchased from Ambion (Foster City, CA). Growth of erythroid or myeloid progenitors was subsequently determined in clonogenic assays in methylcellulose, as in our previous studies (34Katsoulidis E. Li Y. Yoon P. Sassano A. Altman J. Kannan-Thulasiraman P. Balasubramanian L. Parmar S. Varga J. Tallman M.S. Verma A. Platanias L.C. Cancer Res... 2005; 65: 9029-9037Google Scholar, 38Parmar S. Katsoulidis E. Verma A. Li Y. Sassano A. Lal L. Majchrzak B. Ravandi F. Tallman M.S. Fish E.N. Platanias L.C. J. Biol. Chem... 2004; 279: 25345-25352Google Scholar, 39Parmar S. Smith J. Sassano A. Uddin S. Katsoulidis E. Majchrzak B. Kambhampati S. Eklund E.A. Tallman M.S. Fish E.N. Platanias L.C. Blood.. 2005; 106: 2436-2443Google Scholar). In some experiments progenitor colonies from methylcellulose cultures were plucked and used for RT-PCR analysis. In Vitro Kinase Assays—Immune complex assays to detect the kinase activity of specified proteins were performed as in our previous studies (29Uddin 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: 14408-14416Google Scholar). Briefly, cells were serum-starved, stimulated with 1 × 104 IU/ml of the indicated IFNs and then immunoprecipitated overnight with an anti-PKCη antibody or rabbit IgG as a control. Immunoprecipitates were then washed three times with phosphorylation lysis buffer and twice with kinase buffer (25 mm Tris-HCl (pH 7.4), 5 mm MgCl2, 0.5 mm EDTA, 1 mm dithiothreitol, and 20 μm ATP). Immunoprecipitated proteins were resuspended in 30 μl of kinase buffer to which 5 μg of histone H1 and 10 μCi of [γ-32P]ATP were added. The reaction was allowed to proceed for 20 min at room temperature prior to termination following the addition of SDS-sample buffer. Proteins were then analyzed by SDS-PAGE, and phosphorylated histone H1 was detected by autoradiography. The blot was subsequently immunoblotted with an anti-PKCη antibody. Luciferase Assays—Cells were transfected with a β-galactosidase expression vector and either an ISRE luciferase construct (22Uddin 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: 30127-30131Google Scholar) or a luciferase reporter gene containing eight GAS elements linked to a minimal prolactin promoter (8×-GAS) (40Horvai 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: 1074-1079Google Scholar), using the Superfect transfection reagent in accordance with the manufacturer's recommended procedure (Qiagen). The ISRE-luciferase construct was previously provided by Dr. Richard Pine (Public Health Research Institute, New York). The 8×-GAS construct was previously provided by Dr. Christopher Glass (University of California, San Diego). Forty eight hours after transfection, triplicate cultures were either left untreated or treated with PKCη or PKCζ peptide inhibitors (Calbiochem) for 60 min. Following inhibitor incubation, triplicate cultures were then either left untreated or treated with 5 × 103 units/ml IFNα or IFNβ as indicated, and luciferase activity was measured as in our previous studies (22Uddin 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: 30127-30131Google Scholar, 29Uddin 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: 14408-14416Google Scholar). In addition, in some experiments U2OS cells were also transfected with a β-galactosidase expression vector, an ISRE luciferase construct (22Uddin 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: 30127-30131Google Scholar), and either an empty vector plasmid or a constitutively active PKCη mutant provided by Dr. Gottfried Baier (Innsbruck Medical University, Innsbruck, Austria) (36Brandlin I. Hubner S. Eiseler T. Martinez-Moya M. Horschinek A. Hausser A. Link G. Rupp S. Storz P. Pfizenmaier K. Johannes F.J. J. Biol. Chem... 2002; 277: 6490-6496Google Scholar). Evaluation of Apoptosis and Cell Cycle—Cells were transfected with the indicated plasmid constructs via nucleofection (Amaxa, Cologne, Germany), according to the manufacturer's instructions. The evaluation of apoptosis was assessed by annexin V-propidium iodide staining using an apoptosis detection kit (Pharmingen), as in previous studies (41Kannan-Thulasiraman P. Katsoulidis E. Tallman M.S. Arthur J.S. Platanias L.C. J. Biol. Chem... 2006; 281: 22446-22452Google Scholar, 42Giafis N. Katsoulidis E. Sassano A. Tallman M.S. Higgins L.S. Nebreda A.R. Davis R.J. Platanias L.C. Cancer Res... 2006; 66: 6763-6771Google Scholar). The evaluation of cell cycle was assessed by propidium iodide staining and flow cytometric analysis. Briefly, the cells were synchronized by serum starvation for 24 h, and then re-plated in media containing serum, pretreated with PKCη or PKCζ peptide inhibitor (Calbiochem) for 60 min, followed by treatment with 2500–3000 units/ml of IFNα for 24 h. Cells were then harvested, washed in cold phosphate-buffered saline, fixed with ice-cold ethanol, and incubated for 20 min with propidium iodide (Sigma) prior to flow cytometric analysis. Antiviral Assays—The antiviral effects of human IFNα and IFNβ were determined as in our previous studies (27Kaur 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. J. Biol. Chem... 2007; 282: 1757-1768Google Scholar), using EMCV as the challenge virus. In initial studies, we sought to determine whether PKCη is phosphorylated in response to treatment of cells with different type I IFNs. We examined the effects of IFNα on the phosphorylation of PKCη in IFN-sensitive hematopoietic cell lines. KT1 or U266 cells were incubated in the presence or absence of IFNα for different times, and cell lysates were resolved by SDS-PAGE and immunoblotted with an antibody against the phosphorylated form of PKCη against Ser-674. IFNα induced strong phosphorylation of PKCη in both cell lines studied (Fig. 1, A and B). Similarly, treatment of KT1 or U266 cells with another type I IFN, IFNβ, also resulted in strong phosphorylation of PKCη (Fig. 1, C and D). To directly determine whether the kinase domain of PKCη is activated in a type I IFN-dependent manner, experiments were performed in which lysates from IFNα- or IFNβ-treated cells were immunoprecipitated with an anti-PKCη antibody and subjected to in vitro kinase assays using histone H1 as an exogenous substrate. As shown in Fig. 2, treatment of cells with either IFNα (Fig. 2A) or IFNβ (Fig. 2B) resulted in PKCη kinase activity, indicating that the kinase domain of this PKC isoform is activated during its engagement by the type I IFN receptor.FIGURE 2Type I IFN-dependent activation of PKCη. A, KT1 cells were serum-starved overnight, treated with IFNα for 20 min, and lysed in phosphorylation lysis buffer. Cell lysates were immunoprecipitated (IP) with an anti-PKCη antibody or control nonimmune rabbit immunoglobulin (RIgG) as indicated and subjected to in vitro kinase assays, using histone H1 as an exogenous substrate. Immunoprecipitated proteins were resolved by SDS-PAGE, and phosphorylated histone H1 was detected by autoradiography. The blot from the kinase assay was subsequently immunoblotted with an anti-PKCη antibody to control for loading. B, KT1 cells were serum-starved overnight, treated with IFNβ for 20 min, and lysed in phosphorylation lysis buffer. Cell lysates were immunoprecipitated with an anti-PKCη antibody or control nonimmune rabbit immunoglobulin as indicated and subjected to in vitro kinase assays, using histone H1 as an exogenous substrate. Immunoprecipitated proteins were resolved by SDS-PAGE, and phosphorylated histone H1 was detected by autoradiography. The blot from the kinase assay was subsequently immunoblotted with an anti-PKCη antibody to control for loading.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Previous work has shown that another member of the PKC family of isoforms, PKCδ, plays an important role in IFNα-dependent transcriptional regulation (20Weekx S.F. Van Bockstaele D.R. Plum J. Moulijn A. Rodrigus I. Lardon F. De Smedt M. Nijs G. Lenjou M. Loquet P. Berneman Z.N. Snoeck H.W. Exp. Hematol... 1998; 26: 1034-1039Google Scholar). To determine whether PKCη also regulates type IFN-dependent transcription, luciferase promoter assays were performed to determine the effects of PKCη inhibition on IFN-dependent transcriptional activity via ISRE or GAS elements. U2OS cells were transfected with either ISRE or 8×-GAS luciferase constructs and pretreated with a PKCη-specific peptide inhibitor prior to treatment with either IFNα or IFNβ. Luciferase activity was then measured and normalized to β-galactosidase activity. Inhibition of PKCη activity had no significant effects on IFNα-inducible luciferase activity for ISRE elements (Fig. 3A). Similarly, although there was some minimal decrease in IFNβ-inducible luciferase activity in the presence of the PKCη peptide inhibited, there was still clear inducible IFNβ-dependent transcription (Fig. 3B), suggesting that PKCη activity is not essential for type I IFN-dependent transcriptional activation via ISRE elements. Consistent with this, overexpression of a constitutively active PKCη mutant did not result in enhanced transcription via ISRE elements (Fig. 3C). Pretreatment of cells with an inhibitor against an atypical PKC isoform, PKCζ (used as a control), had also no significant effects on type I IFN-dependent transcriptional activation via ISRE elements (Fig. 3D). Similarly, inhibition of either PKCη (Fig. 3, E and F) or PKCζ (Fig. 3G) activities had no significant effects on type I IFN-dependent transcription via GAS elements. Because IFN-induced transcription is strongly associated with generation of IFN-dependent antiviral responses, we also assessed the effects of PKCη inhibition on antiviral activity. U2OS cells were pretreated with PKCη pseudo-substrate inhibitor and then challenged with EMCV. As shown in Fig. 4, both IFNα and IFNβ protected U2OS cells from the cytopathic effects of EMCV in a dose-dependent manner, but inhibition of PKCη activity did not reverse such IFN-induced antiviral protection (Fig. 4, A and B). Thus, in contrast to two other members of the group of novel PKC isoforms (δ and θ) whose activities are required for type I IFN-dependent gene transcription (29Uddin 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: 14408-14416Google Scholar, 30Srivastava K.K. Batra S. Sassano A. Li Y. Majchrzak B. Kiyokawa H. Altman A. Fish E.N. Platanias L.C. J. Biol. Chem... 2004; 279: 29911-29920Google Scholar), PKCη does not regulate transcriptional activation of inter
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