ORF36 Protein Kinase of Kaposi's Sarcoma Herpesvirus Activates the c-Jun N-terminal Kinase Signaling Pathway
2004; Elsevier BV; Volume: 279; Issue: 37 Linguagem: Inglês
10.1074/jbc.m400964200
ISSN1083-351X
AutoresMohamed Sabry Hamza, Richard A. Reyes, Yoshihiro Izumiya, R Wisdom, Hsing-Jien Kung, Paul A. Luciw,
Tópico(s)Cytomegalovirus and herpesvirus research
Resumoα-, β-, and γ-Herpesviruses encode putative viral protein kinases. The herpes simplex virus UL13, varicella-zoster virus ORF47, and Epstein-Barr virus BGLF4 genes all show protein kinase domains in their protein sequences. Mutational analysis of these herpesviruses demonstrated that the viral kinase is important for optimal virus growth. Previous studies have shown that ORF36 of Kaposi's sarcoma herpesvirus (KSHV) has protein kinase activity and is autophosphorylated on serine. The gene for ORF36 is expressed during lytic growth of the virus and has been classified as a late gene. Inspection of the ORF36 sequence indicated potential motifs that could be involved in activation of cellular transcription factors. To analyze the function of ORF36, the cDNA for this viral gene was tagged with the FLAG epitope and inserted into an expression vector for mammalian cells. Transfection experiments in 293T and SLK cells demonstrated that expression of ORF36 resulted in phosphorylation of the c-Jun N-terminal kinase. Autophosphorylation of ORF36 is important for JNK activation because a mutation in the predicted catalytic domain of ORF36 blocked its ability to phosphorylate JNK. Western blot analysis, using phosphospecific antibodies, revealed that mitogen-activated kinases MKK4 and MKK7 were phosphorylated by ORF36 but not by the kinase-negative mutant. Binding experiments in transfected cells also demonstrated that both the wild type and kinase-negative mutant of ORF36 form a complex with JNK, MKK4, and MKK7. In addition, using a tetracycline-inducible Rta BCBL-1 cell line (TREx BCBL1-Rta), JNK was phosphorylated during lytic replication, and inhibition of JNK activation blocked late viral gene expression but not early viral gene expression. In summary, these studies demonstrate that KSHV ORF36 activates the JNK pathway; thus this cell signaling pathway may function in the KSHV life cycle by regulating viral and/or cellular transcription. α-, β-, and γ-Herpesviruses encode putative viral protein kinases. The herpes simplex virus UL13, varicella-zoster virus ORF47, and Epstein-Barr virus BGLF4 genes all show protein kinase domains in their protein sequences. Mutational analysis of these herpesviruses demonstrated that the viral kinase is important for optimal virus growth. Previous studies have shown that ORF36 of Kaposi's sarcoma herpesvirus (KSHV) has protein kinase activity and is autophosphorylated on serine. The gene for ORF36 is expressed during lytic growth of the virus and has been classified as a late gene. Inspection of the ORF36 sequence indicated potential motifs that could be involved in activation of cellular transcription factors. To analyze the function of ORF36, the cDNA for this viral gene was tagged with the FLAG epitope and inserted into an expression vector for mammalian cells. Transfection experiments in 293T and SLK cells demonstrated that expression of ORF36 resulted in phosphorylation of the c-Jun N-terminal kinase. Autophosphorylation of ORF36 is important for JNK activation because a mutation in the predicted catalytic domain of ORF36 blocked its ability to phosphorylate JNK. Western blot analysis, using phosphospecific antibodies, revealed that mitogen-activated kinases MKK4 and MKK7 were phosphorylated by ORF36 but not by the kinase-negative mutant. Binding experiments in transfected cells also demonstrated that both the wild type and kinase-negative mutant of ORF36 form a complex with JNK, MKK4, and MKK7. In addition, using a tetracycline-inducible Rta BCBL-1 cell line (TREx BCBL1-Rta), JNK was phosphorylated during lytic replication, and inhibition of JNK activation blocked late viral gene expression but not early viral gene expression. In summary, these studies demonstrate that KSHV ORF36 activates the JNK pathway; thus this cell signaling pathway may function in the KSHV life cycle by regulating viral and/or cellular transcription. Based on molecular and biological properties, the Herpesviridae family has been subdivided into the α-herpesvirus (herpes simplex virus and varicella-zoster virus), β-herpesvirus (human cytomegalovirus), and γ-herpesvirus (Epstein-Barr virus (EBV) 1The abbreviations used are: EBV, Epstein-Barr virus; HSV, herpes simplex virus; VZV, varicella-zoster virus; KSHV, Kaposi's sarcoma herpesvirus; JNK, c-Jun N-terminal kinase; MAP, mitogen-activated protein; MEKK, MAP kinase/extracellular signal-regulated kinase kinase; MKK, mitogen-activated protein kinase kinase; bZIP, basic region-leucine zipper; JIP, JNK-interacting protein; JSAP-1, JNK/stress-activated protein kinase-associated protein-1; JNKi, JNK inhibitor. and Kaposi's sarcoma herpesvirus (KSHV)) subfamilies (1Roizman B. Pellet P.E. Knipe D.M. Howley P.M. Fields Virology. 2. Lippincott Williams and Wilkins, Philadelphia, PA2001: 2381-2397Google Scholar). These double-stranded DNA viruses can remain latent and persist as an episome, expressing a limited number of viral genes, and thereby establish a lifelong infection in the natural host. Immunosuppression or stress can lead to reactivation of latent virus to produce pathogenic manifestations; examples include Burkitt's lymphoma, nasopharyngeal carcinoma, and Hodgkin's disease associated with EBV (for a review, see Ref. 2Kieff E. Rickinson A. Knipe D. Howley P. 4th Ed. Fields Virology. 2. Lippincott Williams and Wilkins, Philadelphia, PA2001: 2576-2673Google Scholar) and Kaposi's sarcoma, primary effusion lymphomas, and multicentric Castleman's disease associated with KSHV (for reviews, see Refs. 3Bubman D. Cesarman E. Hematol. Oncol. Clin. N. Am. 2003; 17: 717-745Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar and 4Cotter II, M.A. Robertson E.S. Front. Biosci. 2002; 7: d358-d375Crossref PubMed Scopus (17) Google Scholar). Reactivation of these latent herpesviruses leads to sequentially regulated expression of viral genes that play an essential role in the replication and assembly of infectious virions (2Kieff E. Rickinson A. Knipe D. Howley P. 4th Ed. Fields Virology. 2. Lippincott Williams and Wilkins, Philadelphia, PA2001: 2576-2673Google Scholar, 3Bubman D. Cesarman E. Hematol. Oncol. Clin. N. Am. 2003; 17: 717-745Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 4Cotter II, M.A. Robertson E.S. Front. Biosci. 2002; 7: d358-d375Crossref PubMed Scopus (17) Google Scholar). During the lytic cycle of infection, herpesviruses express genes that are predicted to encode protein kinases (for a review, see Ref. 5Kawaguchi Y. Kato K. Rev. Med. Virol. 2003; 13: 331-340Crossref PubMed Scopus (74) Google Scholar). These protein kinases are conserved among the Herpesviridae family, and analysis of the amino acid sequence reveals motifs that are shared by mammalian serine/threonine protein kinases. Located within the catalytic region of these viral proteins are 11 conserved subdomains that are common to cellular protein kinases. Recent studies have revealed that mutation of a conserved lysine residue within subdomain II abolishes phosphorylation; this finding shows that this region is necessary for the catalytic activity of these protein kinases (6Johnson L.N. Lowe E.D. Noble M.E. Owen D.J. FEBS Lett. 1998; 430: 1-11Crossref PubMed Scopus (185) Google Scholar, 7Smith R.F. Smith T.F. J. Virol. 1989; 63: 450-455Crossref PubMed Google Scholar). The protein kinases of herpesviruses appear to mimic the function of cellular protein kinases, including Cdc2 and cellular translational factor EF-1δ, by phosphorylating the same amino acid residues on their cognate protein targets (5Kawaguchi Y. Kato K. Rev. Med. Virol. 2003; 13: 331-340Crossref PubMed Scopus (74) Google Scholar, 8Kawaguchi Y. Matsumura T. Roizman B. Hirai K. J. Virol. 1999; 73: 4456-4460Crossref PubMed Google Scholar). Furthermore the viral protein kinases are associated with the virion and may promote dissociation and entry of the virus particle by phosphorylating the viral tegument proteins (9Morrison E.E. Wang Y.F. Meredith D.M. J. Virol. 1998; 72: 7108-7114Crossref PubMed Google Scholar, 10Overton H.A. McMillan D.J. Klavinskis L.S. Hope L. Ritchie A.J. Wong-kai-in P. Virology. 1992; 190: 184-192Crossref PubMed Scopus (72) Google Scholar, 11Wolf D.G. Honigman A. Lazarovits J. Tavor E. Panet A. Arch. Virol. 1998; 143: 1223-1232Crossref PubMed Scopus (26) Google Scholar, 12van Zeijl M. Fairhurst J. Baum E.Z. Sun L. Jones T.R. Virology. 1997; 231: 72-80Crossref PubMed Scopus (66) Google Scholar). The importance of herpesvirus protein kinases for viral replication and disease has been investigated. Kinase-null mutants generated in α-, β-, and γ-herpesviruses demonstrate decreased replication in tissue culture (13Prichard M.N. Gao N. Jairath S. Mulamba G. Krosky P. Coen D.M. Parker B.O. Pari G.S. J. Virol. 1999; 73: 5663-5670Crossref PubMed Google Scholar, 14Sato H. Pesnicak L. Cohen J.I. J. Virol. 2003; 77: 11180-11185Crossref PubMed Scopus (24) Google Scholar, 15Besser J. Sommer M.H. Zerboni L. Bagowski C.P. Ito H. Moffat J. Ku C.C. Arvin A.M. J. Virol. 2003; 77: 5964-5974Crossref PubMed Scopus (53) Google Scholar). Decreased virulence of HSV and VZV kinase-null mutants has been shown in the mouse model (16Coulter L.J. Moss H.W. Lang J. McGeoch D.J. J. Gen. Virol. 1993; 74: 387-395Crossref PubMed Scopus (110) Google Scholar, 17Heineman T.C. Cohen J.I. J. Virol. 1995; 69: 7367-7370Crossref PubMed Google Scholar, 18Moffat J.F. Zerboni L. Sommer M.H. Heineman T.C. Cohen J.I. Kaneshima H. Arvin A.M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 11969-11974Crossref PubMed Scopus (139) Google Scholar). However, the protein kinase of VZV appears to be dispensable for the establishment of latency in rodent models (14Sato H. Pesnicak L. Cohen J.I. J. Virol. 2003; 77: 11180-11185Crossref PubMed Scopus (24) Google Scholar, 18Moffat J.F. Zerboni L. Sommer M.H. Heineman T.C. Cohen J.I. Kaneshima H. Arvin A.M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 11969-11974Crossref PubMed Scopus (139) Google Scholar). Studies on cells productively infected with α-, β-, and γ-herpesviruses have demonstrated that various members of the stress kinase pathway are activated. HSV activates JNK, p38, and AP1 (19McLean T.I. Bachenheimer S.L. J. Virol. 1999; 73: 8415-8426Crossref PubMed Google Scholar, 20Zachos G. Clements B. Conner J. J. Biol. Chem. 1999; 274: 5097-5103Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar, 21Kim D.B. DeLuca N.A. J. Virol. 2002; 76: 6473-6479Crossref PubMed Scopus (35) Google Scholar); cytomegalovirus activates p38 and JNK (22Bruening W. Giasson B. Mushynski W. Durham H.D. Nucleic Acids Res. 1998; 26: 486-489Crossref PubMed Scopus (82) Google Scholar); and VZV activates AP1, Jun, Fos, and ATF-2 (23Rahaus M. Wolff M.H. Virus Res. 2003; 92: 9-21Crossref PubMed Scopus (26) Google Scholar). For EBV, the latent membrane protein LMP2A regulates c-Jun through an extracellular signal-regulated kinase (24Chen S.Y. Lu J. Shih Y.C. Tsai C.H. J. Virol. 2002; 76: 9556-9561Crossref PubMed Scopus (69) Google Scholar), and the immediate early proteins of EBV, BZLF1 and BRLF1, activate ATF-2, p38, and JNK (25Adamson A.L. Darr D. Holley-Guthrie E. Johnson R.A. Mauser A. Swenson J. Kenney S. J. Virol. 2000; 74: 1224-1233Crossref PubMed Scopus (163) Google Scholar). Recent studies have demonstrated the K15 membrane protein of KSHV activates the mitogen-activated protein kinase and NF-κB pathways (26Brinkmann M.M. Glenn M. Rainbow L. Kieser A. Henke-Gendo C. Schulz T.F. J. Virol. 2003; 77: 9346-9358Crossref PubMed Scopus (121) Google Scholar). However, the role of the viral protein kinase in activating the stress kinase pathway for these herpesviruses remains to be determined. In KSHV, ORF36 encodes a serine protein kinase, which is localized in the nucleus of infected cells. In vitro kinase assays have indicated that this viral protein is autophosphorylated, and the lysine residue in the kinase subdomain II is essential for protein kinase activity (27Park J. Lee D. Seo T. Chung J. Choe J. J. Gen. Virol. 2000; 81: 1067-1071Crossref PubMed Scopus (45) Google Scholar). Conservation of the viral protein kinases among the Herpesviridae family implies that these enzymes are indispensable for herpesvirus survival. Based on transcriptional kinetics and sensitivity to DNA replication inhibitors, ORF36 has been classified as a late gene (27Park J. Lee D. Seo T. Chung J. Choe J. J. Gen. Virol. 2000; 81: 1067-1071Crossref PubMed Scopus (45) Google Scholar, 28Sun R. Lin S.F. Staskus K. Gradoville L. Grogan E. Haase A. Miller G. J. Virol. 1999; 73: 2232-2242Crossref PubMed Google Scholar). Reactivation of KSHV in BCBL-1 cells (B-lymphocytes latently infected with KSHV) induces transcription of the viral protein kinase (27Park J. Lee D. Seo T. Chung J. Choe J. J. Gen. Virol. 2000; 81: 1067-1071Crossref PubMed Scopus (45) Google Scholar). Understanding the cellular events that are regulated by the KSHV protein kinase may provide a basis for defining molecular mechanisms that control viral gene expression. Our studies demonstrate the ability of KSHV ORF36 to activate the JNK pathway and the cellular transcription factor c-Jun, and inhibition of JNK activation resulted in the inhibition of late KSHV viral gene expression. Antibodies—Phosphospecific antibodies for SEK1/MKK4 (Thr-261), MKK7 (Ser-271/Thr-275), stress-activated protein kinase/JNK (Thr-183/Tyr-185), ATF-2 (Thr-71), c-Jun (Ser-63), cAMP-response element-binding protein (Ser-133), ELK-1 (Ser-383), and p38 MAP kinase (Thr-180/Tyr-182) were obtained from Cell Signaling Technology (Beverly, MA). Rabbit anti-phosphothreonine and -phosphoserine were obtained from Zymed Laboratories Inc.. MEK-4 (C-20), MEK-7 (H-160), JNK1 (full-length), c-Jun (N terminus), c-Myc, and tubulin (H-300) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-FLAG M2 was obtained from Stratagene (La Jolla, CA). Antibodies to KSHV K8.1 A/B were obtained from Advanced Biotechnologies Inc. (Columbia, MD). Cloning and Plasmids—The pND vector for expression in mammalian cells (a generous gift from Dr. G. Rhodes, University of California, Davis, CA) was used in the cloning of KSHV ORF36. cDNA was synthesized using RNA extracted from BCBL-1 (B-lymphocytes latently infected with KSHV) cells after 48 h of 12-O-tetradecanoylphorbol-13-acetate treatment. KSHV ORF36 was amplified by PCR using appropriate oligonucleotide primers (5′-AAAAGATCTGCCACCATGGATTACAAGGATGACGACGATAAGCGCTGGAAGAGAATGGAGAGGAG-3′ and 5′-AAAGCGGCCGCTCAGAAAACAAGTCCGCGGGT-3′) and cloned into pND. The resulting construct was designated pND-nFLAG-KSHV-ORF36. The forward primer generated a FLAG sequence at the N terminus. The QuikChange XL site-directed mutagenesis kit (Stratagene) was used in the construction of pND-nFLAG-KSHV-ORF36(K108Q). Briefly pND-nFLAG-KSHV-ORF36 was amplified by PCR using oligonucleotide primers 5′-TCCGAGGATCTGTGTGTTGCAGCAGTTTGATAGCCGCCGG-3′ and 5′-CCGGCGGCTATCAAACTTGCTGCACACACAGATCCTCGGA-3′ to generate a kinase-negative mutant of KSHV ORF36 (lysine at position 108 mutated to glutamine). Cells and Transfections—293T and SLK cells (human endothelial cell origin, generous gift from Drs. Sophie Leventon-Kriss, Tel-Aviv, Israel and Jay A. Levy, University of California, San Francisco, CA) were cultured in Dulbecco's modified essential medium supplemented with 10% fetal calf serum, and cultures were maintained at 5% CO2 at 37 °C. Transient expression of KSHV-ORF36 and KSHV-ORF36(K108Q) was performed by transfecting pND-nFLAG-KSHV-ORF36 or pND-nFLAG-KSHV-ORF36(K108Q) using FuGENE 6 transfection reagent according to the manufacturer's protocol (Roche Applied Science). Cells were used 24 h after transfection. Anisomycin-treated cells served as positive controls for JNK activation. The TREx BCBL1-Rta cell line (a generous gift from Dr. Jae U. Jung) was cultured in RPMI 1640 medium supplemented with 20% fetal calf serum, 250 μg/ml blasticidin S HCl (Invitrogen), and 200 μg/ml hygromycin B (Invitrogen). KSHV lytic replication was induced in this cell line with 1 μg/ml doxycycline (Clontech). Cells were harvested 24 h postinduction. JNK Inhibition Assays—TREx BCBL1-Rta cells were treated with 50 nm JNK inhibitor II (SP600125) or 20 μm JNK inhibitor negative control (EMD Biosciences, San Diego, CA) in the presence of 1 μg of doxycycline/ml for 24 h. Cells were harvested for Western blot. Preparation of Cell Extracts—For detection of phosphorylated cellular proteins or viral proteins, cells were treated with SDS sample buffer (62.5 mm Tris-HCl, 2% SDS, 10% glycerol, 50 mm dithiothreitol, 0.01% (w/v) bromphenol blue), sonicated for 15 s, heated for 5 min at 95 °C, and cooled on ice. For immunoprecipitation, cells were treated with cell lysis buffer (20 mm Tris-HCl, 150 mm NaCl, 1 mm EDTA, 1 mm EGTA, 1% Triton X-100, 2.5 mm sodium pyrophosphate, 1 mm glycerol phosphate, 1 mm Na3VO4, 1 μg/ml leupeptin) for 30 min at 4 °C and then centrifuged to remove cell debris. Western Blot Analysis—For detection of phosphoproteins, cells extracts were electrophoresed on a 12% SDS-polyacrylamide gel and transferred onto polyvinylidene difluoride membrane. Blocking buffer (1× Tris-buffered saline, 0.1% Tween 20, and 5% (w/v) nonfat dry milk) was added to the membrane for 30 min at room temperature. Phosphospecific antibodies were diluted in primary antibody dilution buffer (1× Tris-buffered saline, 0.1% Tween 20, 5% (w/v) bovine serum albumin), and membranes were incubated overnight at 4 °C. Membranes were washed three times with wash buffer (1× Tris-buffered saline, 0.1% Tween 20) and incubated with rabbit IgG-horseradish peroxidase in blocking buffer for 30 min at room temperature. Phosphoproteins were detected using SuperSignal West Pico chemiluminescent substrate (Pierce). For detection of total JNK, MKK4, MKK7, K8.1, K-bZIP, and c-Myc (Rta), the respective primary antibodies were diluted in blocking buffer for 30 min at room temperature and then detected with rabbit or mouse IgG-horseradish peroxidase and SuperSignal substrate. ORF36 Viral Protein Kinase Is Phosphorylated on Serine— The conserved lysine residue in the kinase subdomain II has been shown to be essential for kinase activity and autophosphorylation of ORF36 (27Park J. Lee D. Seo T. Chung J. Choe J. J. Gen. Virol. 2000; 81: 1067-1071Crossref PubMed Scopus (45) Google Scholar, 29Smith G.A. Young P.L. Mattick J.S. Arch. Virol. 1991; 119: 199-210Crossref PubMed Scopus (15) Google Scholar). To demonstrate that ORF36 was phosphorylated on serine and that the lysine at position 108 was important for its phosphorylation, we transfected 293T cells with plasmids expressing KSHV ORF36 or the mutant K108Q. Western blot analysis with antibodies that recognized phosphoserine, phosphothreonine, or phosphotyrosine was used to analyze immunoprecipitated FLAG-tagged ORF36 and the mutant K108Q proteins in transfected cells. A band that had the same molecular weight as FLAG-tagged ORF36 was detected with phosphoserine antibody (Fig. 1) but not with phosphothreonine or phosphotyrosine antibody (data not shown). The K108Q mutant protein of ORF36 was not detected with any of these three phosphospecific antibodies (Fig. 1 and data not shown). This finding demonstrates that the lysine at position 108 is important for the autophosphorylation of ORF36. ORF36 Activates c-Jun N-terminal Kinase—To evaluate the ability of KSHV ORF36 to phosphorylate the MAP kinase pathway, SLK cells were transfected with plasmids expressing ORF36 or the kinase-negative mutant K108Q. Twenty-four hours post-transfection, cells were lysed to prepare extracts for Western blot analysis. Cells transfected with the empty pND vector served as negative controls, and cells treated with anisomycin were positive controls for cell activation. As shown in Fig. 2, ORF36 activated the p54 isoform of JNK, which was phosphorylated on threonine 183 and tyrosine 185; the phosphospecific antibody for JNK recognizes JNK only when phosphorylated on threonine 183 and tyrosine 185. The p46 isoform of JNK was not phosphorylated by ORF36. The K108Q mutant was unable to phosphorylate either the p46 or p54 isoform of JNK. The p38 pathway and its downstream targets, ATF-2, cAMP-response element-binding protein, ELK-1, p44/42, and MKK3/MKK6, were analyzed to determine whether ORF36 exclusively activated the stress-activated protein kinase/JNK pathway. Western blot analysis demonstrated that these target proteins were not phosphorylated by either the wild type ORF36 or kinase-negative mutant K108Q (Fig. 2). ORF36 Activates JNK via MKK4 and MKK7—MKK4 and MKK7 function as non-redundant activators of JNK in vivo. Synergistic activation of MKK4 and MKK7 is required for optimal JNK activation. Maximal enzyme activity of JNK is achieved through phosphorylation of threonine 183 and tyrosine 185 with MKK4 having a preference for phosphorylating the tyrosine residue and MKK7 having a preference for the threonine residue (30Lisnock J. Griffin P. Calaycay J. Frantz B. Parsons J. O'Keefe S.J. LoGrasso P. Biochemistry. 2000; 39: 3141-3148Crossref PubMed Scopus (71) Google Scholar, 31Lawler S. Fleming Y. Goedert M. Cohen P. Curr. Biol. 1998; 8: 1387-1390Abstract Full Text Full Text PDF PubMed Google Scholar, 32Fleming Y. Armstrong C.G. Morrice N. Paterson A. Goedert M. Cohen P. Biochem. J. 2000; 352: 145-154Crossref PubMed Scopus (173) Google Scholar, 33Kishimoto H. Nakagawa K. Watanabe T. Kitagawa D. Momose H. Seo J. Nishitai G. Shimizu N. Ohata S. Tanemura S. Asaka S. Goto T. Fukushi H. Yoshida H. Suzuki A. Sasaki T. Wada T. Penninger J.M. Nishina H. Katada T. J. Biol. Chem. 2003; 278: 16595-16601Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). To determine whether the viral kinase utilizes MKK4 and MKK7 in the activation of JNK, lysates from transfected cells expressing ORF36 or the mutant K108Q were analyzed by Western blot with appropriate antibodies. Fig. 3 demonstrates that both MKK4 and MKK7 were activated by ORF36 but not by the kinase-negative mutant K108Q. These observations reveal that the phosphorylation of JNK by ORF36 was dependent on the activation of both MKK4 and MKK7, and autophosphorylation of ORF36 at serine was important for this activity. Phosphorylation of c-Jun by ORF36 via JNK—Activated JNK regulates transcription by phosphorylating c-Jun, ATF-2, and other transcription factors. JNK phosphorylates the serine residues 63 and 73 of the transcription factor c-Jun, which in turn binds to 12-O-tetradecanoylphorbol-13-acetate response elements and thereby increases c-Jun expression (34Hibi M. Lin A. Smeal T. Minden A. Karin M. Genes Dev. 1993; 7: 2135-2148Crossref PubMed Scopus (1724) Google Scholar, 35Galcheva-Gargova Z. Derijard B. Wu I.H. Davis R.J. Science. 1994; 265: 806-808Crossref PubMed Scopus (541) Google Scholar, 36Diehl A.M. Yin M. Fleckenstein J. Yang S.Q. Lin H.Z. Brenner D.A. Westwick J. Bagby G. Nelson S. Am. J. Physiol. 1994; 267: G552-G561PubMed Google Scholar, 37Westwick J.K. Weitzel C. Minden A. Karin M. Brenner D.A. J. Biol. Chem. 1994; 269: 26396-26401Abstract Full Text PDF PubMed Google Scholar). To demonstrate whether KSHV ORF36 activation of JNK results in c-Jun phosphorylation, total cell lysates were analyzed by Western blot analysis for phosphorylation of c-Jun. Fig. 4 demonstrates that activation of JNK by KSHV ORF36 results in c-Jun phosphorylation; the kinase-negative mutant K108Q did not lead to phosphorylation of c-Jun. ORF36 Interacts with MKK4, MKK7, and JNK—Immunoprecipation studies demonstrated that scaffolding proteins, such as JIP-1/2 (JNK-interacting protein 1/2), JSAP-1 (JNK/stress-activated protein kinase-associated protein-1), and β-arrestin-2, interact physically with their downstream targets. JIP-1 and JIP-2 form complexes with JNK, mixed lineage kinase, and MKK7 (38Ikeda A. Hasegawa K. Masaki M. Moriguchi T. Nishida E. Kozutsumi Y. Oka S. Kawasaki T. J. Biochem. (Tokyo). 2001; 130: 773-781Crossref PubMed Scopus (39) Google Scholar, 39Whitmarsh A.J. Cavanagh J. Tournier C. Yasuda J. Davis R.J. Science. 1998; 281: 1671-1674Crossref PubMed Scopus (592) Google Scholar), whereas JSAP-1 forms complexes with MEKK1, MKK4, and JNK (40Ito M. Yoshioka K. Akechi M. Yamashita S. Takamatsu N. Sugiyama K. Hibi M. Nakabeppu Y. Shiba T. Yamamoto K.I. Mol. Cell. Biol. 1999; 19: 7539-7548Crossref PubMed Scopus (231) Google Scholar). These enzyme complexes produce "MAP kinase modules," which facilitate activation and enhance specificity of their respective target proteins (39Whitmarsh A.J. Cavanagh J. Tournier C. Yasuda J. Davis R.J. Science. 1998; 281: 1671-1674Crossref PubMed Scopus (592) Google Scholar). To assess the in vivo association of ORF36 with proteins in the JNK pathway, 293T cells were transfected with ORF36 or the mutant K108Q. Immunoprecipitation of total cell lysates with the FLAG, MKK4, MKK7, or JNK antibody demonstrated physical interaction of ORF36 with these cellular enzymes (Fig. 5). The kinase-negative mutant K108Q was also able to associate with MKK4, MKK7, and JNK. Thus, autophosphorylation of ORF36 was not important for association with MAP kinase modules and proceeded in a phosphorylation-independent manner. Activation of JNK during KSHV Reactivation—The KSHV viral protein Rta encoded by ORF50 is a transcriptional activator and acts as a molecular switch for KSHV reactivation. Expression of Rta efficiently induces lytic replication in latently infected KSHV cell lines. The TREx BCBL1-Rta cell line is a derivative of the BCBL-1 cell line (B-cell lymphoma cell line latently infected with KSHV) in which Rta is under the control of a tetracycline-inducible promoter. This cell line was demonstrated to fully induce lytic replication and produce infectious viral progeny in the presence of tetracycline. In addition, the TREx BCBL1-Rta cell line was demonstrated to induce KSHV gene expression in a more powerful and efficient manner than that induced by 12-O-tetradecanoylphorbol-13-acetate stimulation of BCBL-1 cells (41Nakamura H. Lu M. Gwack Y. Souvlis J. Zeichner S.L. Jung J.U. J. Virol. 2003; 77: 4205-4220Crossref PubMed Scopus (244) Google Scholar). Stimulation of TREx BCBL-1-Rta cells with doxycycline for 24 h stimulated the expression of Rta, and expression levels did not change with the addition of JNK inhibitor (JNKi) II or the JNKi negative inhibitor (Fig. 6). Anisomycin or untreated cells did not express Rta. The same cell lysates were used to determine the activation of JNK and c-Jun. Induction of lytic replication activated JNK and c-Jun, and this activation was abrogated in the presence of JNKi II. Inhibition of JNK also prevented expression of K8.1 (a KSHV glycoprotein that serves as a marker for viral late gene expression) but not K-bZIP (an early lytic gene identified as a basicleucine zipper protein) (Fig. 6). KSHV, a γ-2 herpesvirus, encodes a serine protein kinase (27Park J. Lee D. Seo T. Chung J. Choe J. J. Gen. Virol. 2000; 81: 1067-1071Crossref PubMed Scopus (45) Google Scholar). Our results support previously published data, using phosphoamino acid analysis, that this kinase is phosphorylated on serine (Fig. 1) (27Park J. Lee D. Seo T. Chung J. Choe J. J. Gen. Virol. 2000; 81: 1067-1071Crossref PubMed Scopus (45) Google Scholar). Autophosphorylation was abrogated by mutating the lysine residue in the catalytic subdomain II of ORF36 (Fig. 1). In this study, we established a mechanism by which the viral protein kinase activates the mitogen-activated stress kinase pathway. Using transient transfection experiments, JNK was activated by KSHV ORF36 but not by the kinase-negative mutant K108Q (Fig. 2). This finding indicates that autophosphorylation of ORF36 was important for JNK activation. The p44/42, MKK3/6, and p38 pathways were not activated by ORF36, showing specificity for JNK activation (Fig. 2). Both MKK4 and MKK7 act synergistically to activate JNK (30Lisnock J. Griffin P. Calaycay J. Frantz B. Parsons J. O'Keefe S.J. LoGrasso P. Biochemistry. 2000; 39: 3141-3148Crossref PubMed Scopus (71) Google Scholar, 31Lawler S. Fleming Y. Goedert M. Cohen P. Curr. Biol. 1998; 8: 1387-1390Abstract Full Text Full Text PDF PubMed Google Scholar, 32Fleming Y. Armstrong C.G. Morrice N. Paterson A. Goedert M. Cohen P. Biochem. J. 2000; 352: 145-154Crossref PubMed Scopus (173) Google Scholar). Fig. 3 demonstrates that ORF36 activates MKK4 and MKK7. These findings imply that JNK activation by ORF36 is via phosphorylation of MKK4 and MKK7. Recent data show that scaffolding proteins, such as JIP-1/2 and JSAP-1, associate with MEKK1, mixed lineage kinase, MKK4, MKK7, and JNK, forming enzyme complexes and facilitating the activation of the mitogen/stress-activated protein kinase pathway (38Ikeda A. Hasegawa K. Masaki M. Moriguchi T. Nishida E. Kozutsumi Y. Oka S. Kawasaki T. J. Biochem. (Tokyo). 2001; 130: 773-781Crossref PubMed Scopus (39) Google Scholar, 39Whitmarsh A.J. Cavanagh J. Tournier C. Yasuda J. Davis R.J. 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This demonstrates that initiation of lytic infection and the transcription of early lytic genes may not require the activation of the JNK pathway. Conversely expression levels of K8.1, a structural glycoprotein component of KSHV particles, are dramatically reduced in the presence of SP600125. The physiological role of ORF36 for KSHV replication is not defined, but possible clues to its function have emerged from studies performed on homologous protein kinases found in other herpesviruses. Previous work demonstrated that α-, β-, and γ-herpesviruses encode viral protein kinases, which are represented by HSV-1 ULl3, VZV ORF47, human cytomegalovirus UL97, human herpesvirus-6 U69, EBV EGLF4, KSHV ORF36, and rhesus rhadinovirus ORF36 (5Kawaguchi Y. Kato K. Rev. Med. Virol. 2003; 13: 331-340Crossref PubMed Scopus (74) Google Scholar, 42Kawaguchi Y. Kato K. Tanaka M. Kanamori M. Nishiyama Y. Yamanashi Y. J. Virol. 2003; 77: 2359-2368Crossref PubMed Scopus (124) Google Scholar, 43Kato K. 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Functional studies to demonstrate the significance of these findings will provide clues to the relevance of the mitogen-activated stress kinase pathway in herpesvirus survival and transcription. Such studies could be based on the analysis of kinase-null mutants of the virus, dominant negative JNK pathway mutants, and specific inhibitors for the JNK pathway. Another possible role for ORF36 phosphorylation of JNK may be the activation of transcription factors encoded by KSHV. Marek disease virus, also a γ-herpesvirus, encodes MEQ, which mimics c-Jun. The MEQ protein shares extensive homology with the Jun/Fos family of transcription factors within the basic region-leucine zipper (bZIP) domain. In addition, MEQ dimerizes with itself and other cellular transcription factors and can functionally substitute for c-Jun (55Liu J.L. Kung H.J. Virus Genes. 2000; 21: 51-64Crossref PubMed Google Scholar). KSHV also encodes a gene with a basic region-leucine zipper domain; this protein has been designated K-bZIP and shows homology with BZLF1, an EBV protein essential for viral reactivation and replication (56Lin S.F. Robinson D.R. Miller G. Kung H.J. J. Virol. 1999; 73: 1909-1917Crossref PubMed Google Scholar, 57Izumiya Y. Lin S.F. Ellison T. Chen L.Y. Izumiya C. Luciw P. Kung H.J. J. Virol. 2003; 77: 1441-1451Crossref PubMed Scopus (98) Google Scholar). These observations suggest that ORF36 plays a role in the activation of KSHV transcription by activating viral homologs of cellular c-Jun. Consequently KSHV ORF36 (a viral late protein) might target an immediate early viral protein, such as K-bZIP. This proposed model is supported by the finding that herpesvirus protein kinases are found in purified virions (10Overton H.A. McMillan D.J. Klavinskis L.S. Hope L. Ritchie A.J. Wong-kai-in P. Virology. 1992; 190: 184-192Crossref PubMed Scopus (72) Google Scholar, 12van Zeijl M. Fairhurst J. Baum E.Z. Sun L. Jones T.R. Virology. 1997; 231: 72-80Crossref PubMed Scopus (66) Google Scholar). In fact, using in vitro kinase assays, ORF36 was shown to phosphorylate K-bZIP. 2M. S. Hamza, R. A. Reyes, Y. Izumiya, R. Wisdom, H.-J. Kung, and P. A. Luciw, unpublished results. Future studies to define the role of ORF36 in KSHV replication and transcription will provide clues into the mechanism by which herpesviruses modulate host cell signaling pathways and maintain their survival within the infected host. We thank Dr. Albert Van Geelen for helpful discussions.
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