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Tyrphostin AG 555 Inhibits Bovine Papillomavirus Transcription by Changing the Ratio between E2 Transactivator/Repressor Function

2003; Elsevier BV; Volume: 278; Issue: 39 Linguagem: Inglês

10.1074/jbc.m304449200

1083-351X

Sabine Baars, Anastasia Bachmann, Alexander Levitzki, Frank Rösl,

Infectious Diseases and Mycology

The tyrosine kinase inhibitor (tyrphostin) AG 555 selectively interferes with viral transcription in bovine papillomavirus type 1 (BPV-1)-transformed fibroblasts and induces suppression of cyclin-dependent kinase activity and cell cycle arrest. Concomitant with inhibition of viral transcription, c-Jun was strongly up-regulated, which was consistent with the observation that AG 555 treatment also led to an activation of the mitogen-activated protein kinase pathway by enhancing phosphorylation of JNK and p38. Increased JNK and p38 activity resulted in higher phosphorylation of the AP-1 family members c-Jun and activating transcription factor 2. Scanning the BPV-1 genome for potential binding sequences, an intragenic AP-1 site (BAP-1) within the E7 open reading frame was detected. Enhanced dimerization of phosphorylated activating transcription factor 2 together with c-Jun and binding to BAP-1 seem to be responsible for viral dysregulation because both suppression of BPV-1 and induction of c-Jun mRNA could be almost entirely abrogated by simultaneous treatment with SB 203580, an inhibitor of p38 mitogen-activated protein kinase activity. Moreover, dissecting the complex transcriptional pattern of episomal BPV-1 with specific primer sets for reverse transcription-PCR analysis, the repressive effect could be attributed to a selective down-regulation of the mRNA encoding the E2 transactivator function in favor of the E2 repressor, whose mRNA level remained constant during AG 555 treatment. These data indicate that tyrphostin AG 555 disturbs the balance of negative and positive regulatory factors necessary to maintain the homeostasis of a virus-transformed phenotype. The tyrosine kinase inhibitor (tyrphostin) AG 555 selectively interferes with viral transcription in bovine papillomavirus type 1 (BPV-1)-transformed fibroblasts and induces suppression of cyclin-dependent kinase activity and cell cycle arrest. Concomitant with inhibition of viral transcription, c-Jun was strongly up-regulated, which was consistent with the observation that AG 555 treatment also led to an activation of the mitogen-activated protein kinase pathway by enhancing phosphorylation of JNK and p38. Increased JNK and p38 activity resulted in higher phosphorylation of the AP-1 family members c-Jun and activating transcription factor 2. Scanning the BPV-1 genome for potential binding sequences, an intragenic AP-1 site (BAP-1) within the E7 open reading frame was detected. Enhanced dimerization of phosphorylated activating transcription factor 2 together with c-Jun and binding to BAP-1 seem to be responsible for viral dysregulation because both suppression of BPV-1 and induction of c-Jun mRNA could be almost entirely abrogated by simultaneous treatment with SB 203580, an inhibitor of p38 mitogen-activated protein kinase activity. Moreover, dissecting the complex transcriptional pattern of episomal BPV-1 with specific primer sets for reverse transcription-PCR analysis, the repressive effect could be attributed to a selective down-regulation of the mRNA encoding the E2 transactivator function in favor of the E2 repressor, whose mRNA level remained constant during AG 555 treatment. These data indicate that tyrphostin AG 555 disturbs the balance of negative and positive regulatory factors necessary to maintain the homeostasis of a virus-transformed phenotype. Increased tyrosine phosphorylation is a common feature of many cancers (1Ullrich A. Schlessinger J. Cell. 1990; 61: 203-212Abstract Full Text PDF PubMed Scopus (4619) Google Scholar, 2Levitzki A. Pharmacol. Ther. 1999; 82: 231-239Crossref PubMed Scopus (202) Google Scholar). Up-regulation of specific receptors or/and enhanced tyrosine kinase activity concomitantly elevate intracellular phosphorylation of many downstream regulatory proteins, which guarantees the maintenance of unscheduled DNA synthesis and cell proliferation (for review, see Ref. 3Levitzki A. Gazit A. Science. 1995; 267: 1782-1788Crossref PubMed Scopus (1628) Google Scholar). In recent years, defined synthetic compounds have been designed which can efficiently block tyrosine kinase activity (tyrphostins) (4Lipson K.E. Pang L. Huber L.J. Chen H. Tsai J.M. Hirth P. Gazit A. Levitzki A. McMahon G. J. Pharmacol. Exp. Ther. 1998; 285: 844-852PubMed Google Scholar) and in turn the proliferative phenotype. In principle, two types of tyrphostin have been developed: one is acting at the substrate binding site of the enzyme, whereas the other functions by competing with the ATP binding domain, which is highly conserved among tyrosine kinases (1Ullrich A. Schlessinger J. Cell. 1990; 61: 203-212Abstract Full Text PDF PubMed Scopus (4619) Google Scholar). Tyrphostins structurally resemble tyrosine and erbstatin moieties and carry hydrophobic residues that enable crossing the cell membrane. The potential application of tyrphostins is not only considered for treatment of malignant disorders but also in other diseases such as atherosclerosis, psoriasis, and septic shock where increased tyrosine kinase activity could be discerned (5Ben-Bassat H. Vardi D.V. Gazit A. Klaus S.N. Chaouat M. Hartzstark Z. Levitzki A. Exp. Dermatol. 1995; 4: 82-88Crossref PubMed Scopus (37) Google Scholar, 6Brenner T. Poradosu E. Soffer D. Sicsic C. Gazit A. Levitzki A. Exp. Neurol. 1998; 154: 489-498Crossref PubMed Scopus (22) Google Scholar). Furthermore, tyrphostins are also useful tools to study regulatory mechanisms where enhanced tyrosine phosphorylation is involved (2Levitzki A. Pharmacol. Ther. 1999; 82: 231-239Crossref PubMed Scopus (202) Google Scholar). In the case of papillomavirus-linked diseases, alterations of epidermal growth factor (EGF) 1The abbreviations used are: EGF, epidermal growth factor; ATF-2, activating transcription factor 2; BPV-1, bovine papillomavirus type 1; EMSA, electrophoretic mobility shift assay; ERK, extracellular signal-regulated kinase; HPV, human papillomavirus; IGF, insulin-like growth factor; JNK, c-Jun NH2-terminal kinase; MAP, mitogen-activated protein; Me2SO, dimethyl sulfoxide; ORF, open reading frame; RT, reverse transcription./insulin-like growth factor I (IGF-I) signal transduction and in turn increased tyrosine phosphorylation seem to play a pivotal role during multistep progression toward malignancy (7Maruo T. Yamasaki M. Ladines-Llave C.A. Mochizuki M. Cancer. 1992; 69: 1182-1187Crossref PubMed Scopus (50) Google Scholar, 8Morrione A. DeAngelis T. Baserga R. J. Virol. 1995; 69: 5300-5303Crossref PubMed Google Scholar, 9Crusius K. Auvinen E. Alonso A. Oncogene. 1997; 15: 1437-1444Crossref PubMed Scopus (87) Google Scholar). For example, the bovine papillomavirus type 1 (BPV-1) E5 oncoprotein exerts its transforming function through constitutive activation of growth stimulatory pathways via interaction with platelet-derived growth factor and EGF receptors (10Petti L. DiMaio D. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6736-6740Crossref PubMed Scopus (112) Google Scholar, 11Klein O. Kegler-Ebo D. Su J. Smith S. DiMaio D. J. Virol. 1999; 73: 3264-3272Crossref PubMed Google Scholar). Furthermore, both BPV-1 E5 and the corresponding homolog of the human papillomavirus (HPV) type 16 can cooperate with ectopic EGF receptor expression in cell transformation assays (12Martin P. Vass W.C. Schiller J.T. Lowy D.R. Velu T.J. Cell. 1989; 59: 21-32Abstract Full Text PDF PubMed Scopus (123) Google Scholar, 13Leechanachai P. Banks L. Moreau F. Matlashewski G. Oncogene. 1992; 7: 19-25PubMed Google Scholar). Although the E5 open reading frame (ORF) of HPV-16 is often found to be deleted in cervical carcinoma cells after integration (14Jeon S. Allen-Hoffmann B.L. Lambert P.F. J. Virol. 1995; 69: 2989-2997Crossref PubMed Google Scholar), the protein may have a function in premalignant lesions (15Kabsch K. Alonso A. J. Virol. 2002; 76: 12162-12172Crossref PubMed Scopus (95) Google Scholar), in which the DNA is still episomal (16Alazawi W. Pett M. Arch B. Scott L. Freeman T. Stanley M.A. Colem N. Cancer Res. 2002; 62: 6959-6965PubMed Google Scholar). Intriguing also is the observation that rodent cells harboring a targeted disruption of the IGF receptor gene (IGF-R1) cannot be transformed by BPV-1 unless a functional IGF-R1 cDNA is provided (8Morrione A. DeAngelis T. Baserga R. J. Virol. 1995; 69: 5300-5303Crossref PubMed Google Scholar). This supports the notion that enhanced tyrosine kinase activity is apparently a general feature in viral carcinogenesis because elevated expression of EGF/IGF receptors has been reported in a wide proportion of papillomavirus-induced malignancies (17Nakamura K. Hongo A. Kodama J. Miyagi Y. Yoshinouchi M. Kudo T. Cancer Res. 2000; 60: 760-765PubMed Google Scholar, 18Mathur S.P. Mathur R.S. Rust P.F. Young R.C. Am. J. Reprod. Immunol. 2000; 46: 280-287Crossref Scopus (41) Google Scholar). A suitable model system, in which enhanced intracellular tyrosine phosphorylation signaling can be studied in the context of viral transcription and transformation, is provided by BPV-1-transformed mouse fibroblasts (ID13 cells) (19Law M.F. Lowy D.R. Dvoretzky I. Howley P.M. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 2727-2731Crossref PubMed Scopus (230) Google Scholar). Here, E5 is considered the main oncoprotein (20DiMaio D. Guralski D. Schiller J.T. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 1797-1801Crossref PubMed Scopus (109) Google Scholar) because mutations within the ORFs of E6 and E7 (encoding the major HPV-16/18-transforming proteins) (for review, see Ref. 21zur Hausen H. Nature Rev. Cancer. 2002; 5: 342-350Crossref Scopus (3148) Google Scholar) have only marginal effects in focus formation assays using rodent cells as recipients (22Rabson M.S. Yee C. Yang Y.C. Howley P.M. J. Virol. 1986; 60: 626-634Crossref PubMed Google Scholar). However, E6 can also affect tyrosine phosphorylation by interacting with the focal adhesion protein paxillin, known to be involved in coordination of cell spreading and motility (23Tong X. Howley P.M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4412-4417Crossref PubMed Scopus (224) Google Scholar). Similar to premalignant HPV-positive keratinocytes (24Dürst M. Kleinheinz A. Hotz M. Gissmann L. J. Gen. Virol. 1985; 66: 1515-1522Crossref PubMed Scopus (374) Google Scholar), BPV-1 persists as multicopy episomal nucleoprotein complexes in transformed cells (25Rösl F. Waldeck W. Zentgraf H. Sauer G. J. Virol. 1986; 58: 500-507Crossref PubMed Google Scholar, 26Rösl F. Waldeck W. Mol. Carcinogenesis. 1991; 4: 248-256Crossref Scopus (2) Google Scholar). This has the advantage that effects on the multipromoter-driven transcriptional activity (27Baker C.C. Howley P.M. EMBO J. 1987; 6: 1027-1035Crossref PubMed Scopus (110) Google Scholar, 28Szymanski P. Stenlund A. J. Virol. 1991; 65: 5710-5720Crossref PubMed Google Scholar) can be investigated independently from any position effects (25Rösl F. Waldeck W. Zentgraf H. Sauer G. J. Virol. 1986; 58: 500-507Crossref PubMed Google Scholar) often occurring after viral integration into the host genome (29Jeon S. Lambert P.F. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 1654-1658Crossref PubMed Scopus (426) Google Scholar). In the present study, we show that the tyrphostin AG 555 can selectively suppress BPV-1 transcription through MAP kinase pathway activation and binding of phosphorylated Jun/ATF-2 at a novel intragenic regulatory sequence. We also demonstrate that AG 555 affects the transcription of the major regulatory viral protein E2 by shifting the ratio between E2 transactivator in favor to the repressor function. These data indicate that fine tuning of BPV-1 gene expression in transformed cells is regulated by tyrosine phosphorylation. Cell Culture and Treatment—ID13 mouse fibroblasts (19Law M.F. Lowy D.R. Dvoretzky I. Howley P.M. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 2727-2731Crossref PubMed Scopus (230) Google Scholar) were maintained in Dulbecco's modified Eagle's medium (Sigma) supplemented with 5% (v/v) fetal calf serum (Invitrogen), 100 units/ml penicillin, and 100 μg/ml streptomycin (Sigma). Cells were seeded at 2.5 × 104 cells/cm2 to ensure logarithmic growth. The final concentration of the tyrphostin AG 555 (cyano-3-(3,4-dihydroxyphenyl)-N-(3-phenylpropyl)-2-propenamide) was 30 μm in all experiments. Stock solutions of 10 mm in Me2SO were stored at –80 °C. To exclude potential Me2SO effects, the same final concentrations (0.3%) of Me2SO were added to nontreated controls. Actinomycin D (Calbiochem) was dissolved in water and added to cells in a final concentration of 5 μg/ml. SB 203580 (Calbiochem) was dissolved in Me2SO (stock concentration: 50 mm) and used in a final concentration of 10 μm. Northern Blot Analysis—RNA was extracted according to the guanidinium-thiocyanate protocol described by Chomczynski and Sacchi (30Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (63232) Google Scholar). Approximately 5 μg of total cellular RNA was separated on 1% agarose gels (31Khandjian E.W. Meric C. Anal. Biochem. 1986; 159: 227-232Crossref PubMed Scopus (47) Google Scholar) and transferred to GeneScreen Plus membranes (PerkinElmer Life Sciences). The filters subsequently were hybridized with specific DNA probes that were labeled with [32P]dCTP by random priming (32Feinberg A.P. Vogelstein B. Anal. Biol. 1983; 132: 6-13Crossref PubMed Scopus (16651) Google Scholar). DNA Probes—pBPV-1 represents a unit length of BPV-1 DNA (33Chen E.Y. Howley P.M. Levinson A.D. Seeburg P.H. Nature. 1982; 299: 529-534Crossref PubMed Scopus (194) Google Scholar) cloned in pBR322. c-Jun, encoding the mouse cDNA in a Rous sarcoma virus-driven vector, was kindly obtained from Peter Angel, Deutsches Krebsforschungszentrum Heidelberg (34Angel P. Karin M. Biochim. Biophys. Acta. 1991; 1072: 129-157Crossref PubMed Scopus (3279) Google Scholar). pHF-β A1 (35Gunning P. Ponte P. Okayama H. Engel J. Blau H. Kedes L. Mol. Cell. Biol. 1983; 3: 787-795Crossref PubMed Scopus (958) Google Scholar), harboring an approximately full-length insert of the fibroblast β-actin gene, was a generous gift from L. Kedes (Medical Center, Palo Alto, CA). Electrophoretic Mobility Shift Assays (EMSAs)—For gel retardation and competition assays, the following oligonucleotides were used: AP-1 consensus sequence, 5′-CGCTTGATGACTCAGCCGGAA-3′; TRE consensus sequence, 5′-AGCTAAAGTGGTGACTCATCACTAT-3′ derived from the collagenase 3-/matrix metalloprotease 13 promoter) (34Angel P. Karin M. Biochim. Biophys. Acta. 1991; 1072: 129-157Crossref PubMed Scopus (3279) Google Scholar); SP-1 binding sequence: 5′-ATTCGATCGGGGCGGGGCGAGC-3′, derived from the SV 40 promoter (36Dynan W.S Tjian R. Cell. 1983; 35: 79-87Abstract Full Text PDF PubMed Scopus (912) Google Scholar); and the BPV-1-specific AP-1 oligonucleotide 5′-AAACTTGGATGATTCACCTGC-3′, position 505–525 in the BPV-1 genome, detected by scanning the BPV-1 genomic sequence with a 7-bp consensus oligonucleotide of AP-1 (5′-TGACTCA-3′) using computer program Gap. The oligonucleotides were synthesized in an Applied Biosystems synthesizer using phosphoramitide chemistry and purified further by high performance liquid chromatography. For EMSAs, the annealed oligonucleotides were labeled with [γ-32P]ATP (Amersham Biosciences, 3,000 Ci/mmol) with T4 polynucleotide kinase and gel purified from a 15% polyacrylamide gel. Cellular extracts were prepared according to Schreiber et al. (37Schreiber E. Matthias P. Muller M.M. Schaffner W. Nucleic Acids Res. 1989; 17: 6419Crossref PubMed Scopus (3918) Google Scholar) with the only modification that N-N-(l-3-trans-carboxyoxirane-2-carbonyl)-l-leucyl-agmatine (E64) and 4-(2-aminoethyl)-benzolsulfonylfluoride (Pefabloc SC) were included as protease inhibitors in concentrations suggested by the manufacturer (Roche Applied Science). The binding was performed exactly as described (38Soto U. Das B.C. Lengert M. Finzer P. zur Hausen H. Rösl F. Oncogene. 1999; 18: 3187-3198Crossref PubMed Scopus (88) Google Scholar). The sequence specificity of the binding was controlled in competition experiments by the addition of a 100-fold molar excess of either unlabeled homologous or heterologous oligonucleotides. For monitoring c-Jun/ATF-2 composition in supershift assays, 2 μg of polyclonal antibodies directed against c-Jun or ATF-2 was added, and the reaction was incubated further for 1 h at 4 °C. In detail, the following antibodies (all obtained from Santa Cruz, Biotechnology as TransCRuz™ supershift reagents) were used: c-Jun-Ab, which recognizes both the unphosphorylated and phosphorylated form of c-Jun (epitope corresponding to amino acids 56–69 mapping within the amino-terminal domain of the mouse c-Jun protein); ATF-2 antibody, specific for Thr71-phosphorylated ATF-2). The DNA-protein complexes were resolved on 5.5% non-denaturating polyacrylamide gels (29:1 cross-linking ratio), dried, and exposed overnight to Kodak X-Omat films. Western Blot Analyses—Total cellular protein extracts were prepared using ice cold RIPA buffer (0.5% deoxycholate, 0.5% Nonidet P-40, 0.5% SDS, 50 mm Tris, pH 7.4, and 100 mm NaCl) supplemented with freshly added protease inhibitor mixture (Complete™, Roche Applied Science). The protein concentration was determined by the method of Bradford (39Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (217529) Google Scholar) (Bio-Rad), and 50 μg of total protein was electrophoresed on 10% SDS-polyacrylamide gels under reducing conditions. After electrotransfer to Immobilon™-P polyvinylidene difluoride membranes (Millipore Corporation), the membranes were incubated for 1 h at room temperature with blocking solution: TBS (20 mm Tris-HCl, pH 7.6, 0.14 m NaCl) containing 0.1% Tween 20 (Sigma) and 5% skim milk powder (Merck). After blocking, the membranes were incubated with the first antibody (diluted in blocking solution) overnight at 4 °C. The membranes were washed three times with TBS containing 0.1% Tween 20 for 30 min and then incubated for 2 h at room temperature with the second antibody (anti-rabbit or anti-mouse IgG conjugated with horseradish peroxidase) diluted 1:5,000 in blocking solution. Finally the membranes were washed again for 30 min, incubated 2 min with the ECL reagents (Amersham Biosciences), and exposed on Kodak X-Omat films. For stripping, the membranes were incubated with 200 mm NaOH for 5 min and washed with water prior to incubation with an additional antibody. The following polyclonal antibodies were used (all purchased from New England Biolabs): phospho-p38, p38, phospho-JNK, JNK, phospho-ERK1/2, phospho-c-Jun, phospho-ATF-2, and ATF-2. c-Jun antibody, and Cdk2 antibody were from Santa Cruz Biotechnology. The BPV E5 monoclonal antibody was a kind gift from R. Schlegel (Georgetown University, Washington, D. C.). Equal protein transfer and loading were routinely controlled by reincubating the filters with a monoclonal actin-specific antibody (ICN Biomedicals). p38 MAP Kinase Assay—All steps of the nonradioactive kinase assay system were performed exactly according to the manufacturer's instructions (New England Biolabs). Cells were lysed in buffer containing 20 mm Tris, pH 7.5, 150 mm NaCl, 1 mm EDTA, 1 mm EGTA, 1% Triton, 2.5 mm sodium pyrophosphate, 1 mm-glycerophosphate, 1 mm Na3VO4, and 1 μg/ml leupeptin. The first step includes an immunoprecipitation of p38 MAP kinase from a 100-μg protein extract via immobilized phospho-p38 MAP kinase (Thr180/Tyr182) monoclonal antibody. In vitro kinase assay was performed using purified ATF-2 protein as substrate. ATF-2 phosphorylation was detected by Western blot using phospho-ATF-2 (Thr71) antibody. Fluorescence-activated Cell Sorter Analysis—For fluorescence analysis, cells were washed twice with phosphate-buffered saline and stained 1 h with 50 μg/ml propidiumiodide + 40 μg/ml RNaseA in phosphate-buffered saline in the dark at 4 °C. Analysis was performed using a BD Biosciences flow cytometer (40Larsen J.K. Munch-Petersen B. Christiansen J. Jorgensen K. Cytometry. 1986; 1: 54-63Crossref Scopus (80) Google Scholar). Cell cycle distribution and quantification of flow cytometric data were performed according to Dean and Jett (41Dean P.N. Jett J.H. J. Cell Biol. 1974; 60: 523-527Crossref PubMed Scopus (666) Google Scholar). For each measurement, 10,000 cells were counted. RT-PCR—Total RNA was isolated using the RNeasy kit from Qiagen according to the instructions of the provider. 30 μg of RNA was treated with 1 unit of DNase for 20 min at 37 °C to remove residual genomic DNA. The RT reaction was performed using the Omniscript RT kit from Qiagen; 2 μg of RNA was used for a 20-μl reaction volume to generate cDNA. E2 sequence-specific PCR was performed with 0.5 μl of RT Mix, now containing cDNA, 5 mmol of dNTPs, 5 pmol of primers, and 0.7 unit of Expand High Fidelity Polymerase (Roche Applied Science) in a 20-μl reaction volume using primers for full-length, amino-terminal, and carboxyl-terminal E2 DNA sequences: forward (F) primer, 5′-AGACAGCATGCGAACGTTTA-3′; reverse (R) primer, 5′-TGCTTGGCTCTCTCTTGACA-3′; amino-terminal Rn primer, 5′-TCCTCTTCTTCCTGCCTTGA-3′; carboxyl-terminal Fc primer: 5′-AGCCCAGCCTGTCTCTTCT-3. The PCR program (MJ Research PCR Cycler) was as follows: step 1, denaturation at 95 °C for 1 min; step 2, denaturation at 95 °C for 30 s; step 3, annealing with touch down 65–1 °C/cycle; step 4, elongation at 72 °C for 4 min; step 5, repeat steps 2–4, 19 times; step 6, denaturation at 95 °C for 30 s; step 7, annealing at 55 °C for 30 s; step 8, elongation at 72 °C for 4 min; step 9, repeat steps 6–8, 19 times; step 10, elongation at 72 °C for 12 min and cooling at 4 °C. PCR products were separated on 1% agarose gels and detected after ethidium bromide staining under UV. Immunoprecipitation and Cdk2 Assay—Cdk2 activity was measured after immunoprecipitation of the protein from cell extracts and phosphorylation of histone H1 as substrate (42Blomberg I. Hoffmann I. Mol. Cell. Biol. 1999; 19: 6183-6194Crossref PubMed Scopus (255) Google Scholar). 100 μg of each cell extract (treated and controls) was supplemented with 1 μl of anti-Cdk2 antibody (Santa Cruz) and incubated on ice for 2 h. The following step was the binding of protein-antibody aggregates by A/G-agarose beads and centrifugation to separate the bound complexes from free protein. The beads were washed using lysis buffer (50 mm Tris, 250 mm NaCl, 1% Triton X-100, 5 mm EDTA, 50 mm NaF, 1 mm dithiothreitol, 0,1 mm phenylmethylsulfonyl fluoride, 100 μm natriumvanadate, 1 μg/ml aprotinin, 10 μg/ml soybean trypsin inhibitor, 10 μg/ml tosylphenylalanyl chloromethyl ketone, 5 μg/ml 1-chloro-3-tosylamido-7-amino-2-heptanone) and kinase reaction buffer (50 mm Tris, pH 7.5, 10 mm MgCl2, 1 mm dithiothreitol), and afterward a premix consisting of histone H1 (calf thymus, Calbiochem-Novabiochem), ATP (Roche Applied Science), and radioactive γ-ATP (Amersham Biosciences) was added. The kinase reaction was incubated for 15 min at 30 °C, and the detection of phosphorylated histone H1 was performed after 10% SDS-PAGE, Coomassie staining, and autoradiography of dried gels. AG 555 Selectively Down-regulates BPV-1 Transcription— Because of their structural similarity with tyrosine (Fig. 1, panel A) tyrphostins can block protein-tyrosine kinase activity by binding to the substrate binding site (2Levitzki A. Pharmacol. Ther. 1999; 82: 231-239Crossref PubMed Scopus (202) Google Scholar). To test the effect of AG 555 on viral transformed cells, BPV-1-transformed mouse fibroblasts (ID13 cells) were treated for different periods of time, and total RNA was examined by Northern blot analysis. As shown in Fig. 1, panel B, cell incubation in the presence of 30 μm AG 555 resulted in a selective down-regulation of the most abundant viral transcripts (27Baker C.C. Howley P.M. EMBO J. 1987; 6: 1027-1035Crossref PubMed Scopus (110) Google Scholar, 43Barksdale S.K. Baker C.C. J. Virol. 1993; 67: 5605-5616Crossref PubMed Google Scholar) already 4 h after drug application. To demonstrate that AG 555-mediated BPV-1 suppression was a selective process and not the consequence of a general transcriptional block per se, the RNA filters were rehybridized with endogenous reference genes. Notably, under conditions in which BPV-1 transcription was suppressed, an even costimulatory effect on c-Jun gene expression could be discerned. c-Jun induction was detectable already 2 h after AG 555 addition and remained elevated up to 8–10 h. Subsequent hybridization of the same filters with a housekeeping gene probe (β-actin) demonstrated that suppression was specifically directed against the virus-specific transcription cassette and did not represent the result of a nonspecific transcriptional impairment of the cells by the tyrphostin. Down-regulation of BPV-1 Transcription and Cell Cycle Arrest: Suppression of Cdk2 Activity—Measuring the cell cycle profile by flow cytometric analysis under conditions in which BPV-1 expression was found to be down-regulated, the majority of ID13 cells were growth-arrested with an accumulation of the cells at the late S/early G2 boundary, whereas the G1 phase was diminished (Fig. 2, panel A). For example, considering the fluorescence-activated cell sorter profile 8 h after AG 555 treatment, an increase of the S/G2 phase from 7.7 to 18.5% and from 12 to 25% and a decrease of G1 from 75 to 43%, respectively, could be noted (see Fig. 2, panel A). To analyze whether the inhibition of BPV-1 transcription and cell cycle arrest correlated with cyclin-dependent kinase activity suppression, cyclin-Cdk2 complexes were first immunoprecipitated with a Cdk2-specific antibody and subsequently functionally tested in an in vitro phosphorylation assay using histone H1 as substrate (Fig. 2, panel B, upper part). Compared with the untreated controls, Cdk2 remained active up to 2 h but declined significantly between 6 and 8 h after the addition of AG 555. To exclude that Cdk2 activity was reduced because of quantitative changes in Cdk2 itself, total cellular extracts were monitored by Western blot analysis. Consecutive incubation of the filter with specific antibodies against Cdk2 and actin as internal loading control confirmed equal Cdk2 expression (Fig. 2, panel C). In addition, consistent with the transcriptional data presented above (Fig. 1, panel B), the level of the E5 oncoprotein was down-regulated in the same time range, whereas the expression of Ha-ras and actin as internal reference was not affected (Fig. 2, panel D). These results suggest that AG 555 was efficiently inducing cell cycle arrest by negatively interfering with BPV-1 transcription and Cdk2 function. MAP Kinase Pathway Activation by AG 555—Because c-Jun expression was induced upon AG 555 treatment, we reasoned that tyrphostin treatment may act as a cellular stress signal (for review, see Ref. 44Ichijo H. Oncogene. 1999; 18: 6087-6093Crossref PubMed Scopus (474) Google Scholar) that activates the MAP kinase pathway and in turn downstream target proteins. Mammalian MAP kinases consist of three groups: ERK, JNK, and the p38 MAP kinases (45Schaeffer J.J. Weber M.J. Mol. Cell. Biol. 1999; 19: 2435-2444Crossref PubMed Scopus (1407) Google Scholar). To prove whether AG 555 was affecting MAP kinase activity, Western blot analyses using phosphorylation-specific antibodies raised against ERK, JNK, and p38 were performed. Fig. 3, panel A, shows that only the stress kinases JNK and p38 were phosphorylated, reaching a maximum between 4 and 6 h after the addition of AG 555. In contrast, ERK1 and ERK2 were unaltered under the same experimental conditions. Phosphorylation could be not attributed to an enhanced protein synthesis because control incubations of the filters with nonphosphorylation-specific antibodies revealed that the net amount of the corresponding proteins was the same. p38 MAP kinases are normally activated by dual phosphorylation on Thr and Tyr within a Thr-Gly-Tyr motif (46Raingeaud J. Gupta S. Rogers J.S. Dickens M. Han J. Ulevitch R.J. Davis R.J. J. Biol. Chem. 1995; 270: 7420-7426Abstract Full Text Full Text PDF PubMed Scopus (2046) Google Scholar). It was therefore unexpected that a tyrphostin, which can potentially interfere with tyrosine phosphorylation (2Levitzki A. Pharmacol. Ther. 1999; 82: 231-239Crossref PubMed Scopus (202) Google Scholar), can activate p38. Because the p38 antibody used cannot distinguish between these two phosphorylation sites, it was mandatory to test whether phosphorylation was in fact accompanied with an increased enzymatic activity. p38 was therefore first immunoprecipitated from nuclear extracts and subsequently assayed in vitro using purified ATF-2 (in the presence of ATP) as a substrate. Phosphorylation of ATF-2 was detectable already 30 min after the addition of AG 555, which coincides temporarily with p38 activation (Fig. 3, panel B) but precedes viral suppression (see below). JNK activity was measured in vivo by probing nuclear extracts with c-Jun phosphorylation-specific antibodies directed against the serine residues at positions 63 and 73 within the amino-terminal transactivating domain. As depicted in Fig. 3, panel B, even though c-Jun was induced on a transcriptional level (Fig. 1, panel B), no enhanced accumulation of the corresponding protein could be noted. These data indicate that the biological outcome of AG 555-mediated MAP kinase activation was restricted mainly to post-translational phosphorylation events on c-Jun and ATF-2. Identification of a Potential Intragenic AP-1 site within the BPV-1 Genome—To determine whether the repression of viral transcription is linked causally with MAP kinase activation, we anticipated that the activation of the AP-1 family members c-Jun and ATF-2 may either directly or indirectly influence BPV-1 expression. Because no AP-1 binding site within the BPV-1 genome has been described until now, we scanned the nucleotide sequence for putative bind