Activation of Adenovirus Type 2 Early Region 4 ORF4 Cytoplasmic Death Function by Direct Binding to Src Kinase Domain
2004; Elsevier BV; Volume: 279; Issue: 24 Linguagem: Inglês
10.1074/jbc.m400933200
ISSN1083-351X
AutoresClaudia Champagne, Marie-Claude Landry, Marie‐Claude Gingras, Josée N. Lavoie,
Tópico(s)RNA regulation and disease
ResumoAdenovirus type 2 (Ad2) early region 4 ORF4 (E4orf4) triggers a major death pathway that requires its accumulation in cellular membranes and its tyrosine phosphorylation. This program is regulated by Src family kinases and triggers a potent ZVAD (benzyloxycarbonyl-VAD)- and Bcl2-resistant cell death response in human-transformed cells. How E4orf4 deregulates Src-dependent signaling is unknown. Here we provide strong evidence that a physical interaction requiring the kinase domain of Src and the arginine-rich motif of E4orf4 is involved. The Src binding domain of E4orf4 overlaps with, but is distinct from that of the Bα subunit of protein phosphatase 2A (PP2A-Bα) and some E4orf4 complexes contain both PP2A and Src. Functional assays using mutant E4orf4 revealed that deregulation of Src signaling, activation of the Jun kinase pathway, and cell blebbing were all critically dependent on Src binding. In contrast, PP2A-Bα binding per se was not required to engage the Src-dependent death pathway but was more critical for triggering a distinct death activity. Both E4orf4 death activities were manifested within a given cell population, were typified by distinct morphological features, and contributed to overall cell killing, although to different extents in various cell types. We conclude that E4orf4 binding to the Src kinase domain leads to deregulation of Src signaling and plays a crucial role in induction of the cytoplasmic death pathway. Nonetheless, both Src and PP2A enzymes are critical targets of E4orf4 that likely cooperate to trigger E4orf4-induced tumor cell killing and whose relative contributions may vary in function of the cellular background. Adenovirus type 2 (Ad2) early region 4 ORF4 (E4orf4) triggers a major death pathway that requires its accumulation in cellular membranes and its tyrosine phosphorylation. This program is regulated by Src family kinases and triggers a potent ZVAD (benzyloxycarbonyl-VAD)- and Bcl2-resistant cell death response in human-transformed cells. How E4orf4 deregulates Src-dependent signaling is unknown. Here we provide strong evidence that a physical interaction requiring the kinase domain of Src and the arginine-rich motif of E4orf4 is involved. The Src binding domain of E4orf4 overlaps with, but is distinct from that of the Bα subunit of protein phosphatase 2A (PP2A-Bα) and some E4orf4 complexes contain both PP2A and Src. Functional assays using mutant E4orf4 revealed that deregulation of Src signaling, activation of the Jun kinase pathway, and cell blebbing were all critically dependent on Src binding. In contrast, PP2A-Bα binding per se was not required to engage the Src-dependent death pathway but was more critical for triggering a distinct death activity. Both E4orf4 death activities were manifested within a given cell population, were typified by distinct morphological features, and contributed to overall cell killing, although to different extents in various cell types. We conclude that E4orf4 binding to the Src kinase domain leads to deregulation of Src signaling and plays a crucial role in induction of the cytoplasmic death pathway. Nonetheless, both Src and PP2A enzymes are critical targets of E4orf4 that likely cooperate to trigger E4orf4-induced tumor cell killing and whose relative contributions may vary in function of the cellular background. IntroductionThe adenovirus type 2 (Ad2) 1The abbreviations used are: Ad2, adenovirus type 2; E4orf4, Ad2 early region 4 ORF4; GFP, green fluorescent protein; mT, polyomavirus middle T-antigen; GST, glutathione S-transferase; PBS, phosphate-buffered saline; SH, Src homology domain; ERK, extracellular signal-regulated kinase; MAP, mitogen-activated protein; JNK, Jun N-terminal kinase; FAK, focal adhesion kinase; fmk, fluoromethylketone; Boc, t-butoxycarbonyl; PP2A, protein phosphatase 2A. early region 4 ORF 4 (E4orf4) is a 14-kDa early viral gene product whose function remains ill defined. E4orf4 is not essential for viral growth, although several activities were reported during viral infection that were linked to its ability to interact with cellular PP2A. However, the exact consequence on PP2A activity remains unclear (1Tauber B. Dobner T. Gene (Amst.). 2001; 278: 1-23Crossref PubMed Scopus (57) Google Scholar, 2Muller U. Kleinberger T. Shenk T. J. Virol. 1992; 66: 5867-5878Crossref PubMed Google Scholar, 3Kleinberger T. Shenk T. J. Virol. 1993; 67: 7556-7560Crossref PubMed Google Scholar, 4Bondesson M. Ohman K. Manervik M. Fan S. Akusjarvi G. J. Virol. 1996; 70: 3844-3851Crossref PubMed Google Scholar, 5Kanopka A. Muhlemann O. Petersen-Mahrt S. Estmer C. Ohrmalm C. Akusjarvi G. Nature. 1998; 393: 185-187Crossref PubMed Scopus (166) Google Scholar, 6Estmer Nilsson C. Petersen-Mahrt S. Durot C. Shtrichman R. Krainer A.R. Kleinberger T. Akusjarvi G. EMBO J. 2001; 20: 864-871Crossref PubMed Scopus (75) Google Scholar). It is believed but not proven that E4orf4 cooperates with other viral products to trigger cell death at the end of the infectious cycle to propagate viral progeny (7Marcellus R.C. Lavoie J.N. Boivin D. Shore G.C. Ketner G. Branton P.E. J. Virol. 1998; 72: 7144-7153Crossref PubMed Google Scholar). In mammalian cells, over-expression of E4orf4 triggers p53-independent cell death, and evidence accumulates that E4orf4 killing is much higher in transformed and cancer cells (7Marcellus R.C. Lavoie J.N. Boivin D. Shore G.C. Ketner G. Branton P.E. J. Virol. 1998; 72: 7144-7153Crossref PubMed Google Scholar, 8Lavoie J.N. Nguyen M. Marcellus R.C. Branton P.E. Shore G.C. J. Cell Biol. 1998; 140: 637-645Crossref PubMed Scopus (188) Google Scholar, 9Shtrichman R. Kleinberger T. J. Virol. 1998; 72: 2975-2982Crossref PubMed Google Scholar, 10Shtrichman R. Sharf R. Barr H. Dobner T. Kleinberger T. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 10080-10085Crossref PubMed Scopus (131) Google Scholar, 11Branton P.E. Roopchand D.E. Oncogene. 2001; 20: 7855-7865Crossref PubMed Google Scholar). Because, adenoviral infection drives a process similar to cell transformation by stimulating proliferation and inhibiting p53-dependent cell death, it is conceivable that E4orf4 has evolved to act in a transformed-like genetic background. Deciphering the mechanisms involved in cell death induction is of great interest, because they may unravel ways to manipulate oncogene signaling to trigger tumor-selective death programs.Emerging evidence suggests that E4orf4 has several activities, some of which are cell type-specific. The specific localization of E4orf4 to the cytoplasm, cytoskeleton, and the nucleus illustrates the complexity of E4orf4 signaling with a number of potentially important cellular targets (12Lavoie J.N. Champagne C. Gingras M.C. Robert A. J. Cell Biol. 2000; 150: 1037-1056Crossref PubMed Scopus (66) Google Scholar, 13Gingras M.-C. Champagne C. Roy M.A. Lavoie J.N. Mol. Cell. Biol. 2002; 22: 41-56Crossref PubMed Scopus (41) Google Scholar). Caspase-dependent apoptosis was observed in specific cell lines, but E4orf4-induced cell death generally proceeds independently of the classical pathways for apoptosis induction (death receptor and mitochondrial pathways) (14Livne A. Shtrichman R. Kleinberger T. J. Virol. 2001; 75: 789-798Crossref PubMed Scopus (61) Google Scholar, 15Robert A. Miron M.J. Champagne C. Gingras M.C. Branton P.E. Lavoie J.N. J. Cell Biol. 2002; 158: 519-528Crossref PubMed Scopus (53) Google Scholar). Indeed in most transformed and cancer cells, E4orf4 triggers a ZVAD (benzyloxycarbonyl-VAD)- and Bcl-2-resistant cell death pathway associated with some features of apoptosis, including externalization of phosphatidylserines, cell and nuclear shrinkage, chromatin condensation, and phagocytosis by neighboring cells (8Lavoie J.N. Nguyen M. Marcellus R.C. Branton P.E. Shore G.C. J. Cell Biol. 1998; 140: 637-645Crossref PubMed Scopus (188) Google Scholar, 12Lavoie J.N. Champagne C. Gingras M.C. Robert A. J. Cell Biol. 2000; 150: 1037-1056Crossref PubMed Scopus (66) Google Scholar, 15Robert A. Miron M.J. Champagne C. Gingras M.C. Branton P.E. Lavoie J.N. J. Cell Biol. 2002; 158: 519-528Crossref PubMed Scopus (53) Google Scholar). 2J. N. Lavoie and C. Champagne, unpublished results. We have shown that this cell death pathway is associated with E4orf4 accumulation in the cell membrane-cytoskeleton and is first manifested by the appearance of dramatic actin changes leading to dramatic cell blebbing (12Lavoie J.N. Champagne C. Gingras M.C. Robert A. J. Cell Biol. 2000; 150: 1037-1056Crossref PubMed Scopus (66) Google Scholar). This so-called cytoplasmic death activity is regulated by Src tyrosine kinases, requires the tyrosine phosphorylation of E4orf4, and involves the calcium-regulated cysteine proteases calpains (13Gingras M.-C. Champagne C. Roy M.A. Lavoie J.N. Mol. Cell. Biol. 2002; 22: 41-56Crossref PubMed Scopus (41) Google Scholar, 15Robert A. Miron M.J. Champagne C. Gingras M.C. Branton P.E. Lavoie J.N. J. Cell Biol. 2002; 158: 519-528Crossref PubMed Scopus (53) Google Scholar). Importantly, we have shown that E4orf4 targeting to cellular membranes recapitulates the Src-dependent death activity (15Robert A. Miron M.J. Champagne C. Gingras M.C. Branton P.E. Lavoie J.N. J. Cell Biol. 2002; 158: 519-528Crossref PubMed Scopus (53) Google Scholar), suggesting that tyrosine-phosphorylated E4orf4 acts by disrupting some critical tyrosine kinase signaling pathway on membranes. A distinct death activity was also observed, in the absence of E4orf4 tyrosine phosphorylation (15Robert A. Miron M.J. Champagne C. Gingras M.C. Branton P.E. Lavoie J.N. J. Cell Biol. 2002; 158: 519-528Crossref PubMed Scopus (53) Google Scholar). This death program was rather associated with E4orf4 accumulation in the cell nucleus and led to a distinct morphological phenotype, characterized by dramatic cell shrinkage in the absence of early blebbing induction. The nuclear targets of E4orf4 remain unknown, but may be related to its ability to trigger an irreversible G2/M growth arrest in yeasts and in specific cancer cell lines (16Kornitzer D. Sharf R. Kleinberger T. J. Cell Biol. 2001; 154: 331-344Crossref PubMed Scopus (97) Google Scholar, 17Roopchand D.E. Lee J.M. Shahinian S. Paquette D. Bussey H. Branton P.E. Oncogene. 2001; 20: 5279-5290Crossref PubMed Scopus (51) Google Scholar). Notably, E4orf4 killing and growth inhibition was shown to depend on its ability to bind to the regulatory subunit Bα of protein phosphatase 2A (PP2A-Bα) and recruit PP2A phosphatase activity (10Shtrichman R. Sharf R. Barr H. Dobner T. Kleinberger T. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 10080-10085Crossref PubMed Scopus (131) Google Scholar, 16Kornitzer D. Sharf R. Kleinberger T. J. Cell Biol. 2001; 154: 331-344Crossref PubMed Scopus (97) Google Scholar, 17Roopchand D.E. Lee J.M. Shahinian S. Paquette D. Bussey H. Branton P.E. Oncogene. 2001; 20: 5279-5290Crossref PubMed Scopus (51) Google Scholar, 18Marcellus R.C. Chan H. Paquette D. Thirlwell S. Boivin D. Branton P.E. J. Virol. 2000; 74: 7869-7877Crossref PubMed Scopus (84) Google Scholar). Whether E4orf4 acts by inhibiting and/or stimulating PP2A activity toward specific substrates in a way that relies on recruitment to PP2A-Bα to specific cell compartments remains unclear. Whatever the case, these studies indicated that extra functions are required for cell killing and that some PP2A-independent killing also exists. Although it is clear that E4orf4 possesses distinct activities in various cell types, the functional relationship between Src and PP2A was not addressed, and the molecular mechanisms involved in engagement of the so-called cytoplasmic death pathway remain elusive.We have delineated the determinants of E4orf4-Src association at the molecular level. E4orf4 mutant proteins were used to address the role of Src and PP2A-Bα binding in induction of the cytoplasmic death pathway typified by early changes in actin dynamics. The results strongly suggest that a physical interaction with the kinase domain of Src-like tyrosine kinases is critical to activate E4orf4 cytoplasmic death functions. This interaction requires a highly basic motif on E4orf4 that overlaps with, but is distinct from, the PP2A-Bα binding site. Based on the available data, E4orf4 binding to PP2A-Bα per se is dispensable for deregulation of Src signaling and activation of the cytoplasmic death pathway and appears more critical for induction of a distinct death program.MATERIALS AND METHODSExpression Vectors and Mutagenesis—The following expression vectors were described previously: Ad2 HA-E4orf4 (8Lavoie J.N. Nguyen M. Marcellus R.C. Branton P.E. Shore G.C. J. Cell Biol. 1998; 140: 637-645Crossref PubMed Scopus (188) Google Scholar); FLAG-E4orf4 (13Gingras M.-C. Champagne C. Roy M.A. Lavoie J.N. Mol. Cell. Biol. 2002; 22: 41-56Crossref PubMed Scopus (41) Google Scholar); Myc-E4orf4 (12Lavoie J.N. Champagne C. Gingras M.C. Robert A. J. Cell Biol. 2000; 150: 1037-1056Crossref PubMed Scopus (66) Google Scholar); FLAG-E4orf4-green-fluorescent protein (GFP) and FLAG-GFP-NLS (15Robert A. Miron M.J. Champagne C. Gingras M.C. Branton P.E. Lavoie J.N. J. Cell Biol. 2002; 158: 519-528Crossref PubMed Scopus (53) Google Scholar); GST-SH2v-Src and GST-SH3v-Src (designated SH2v and SH3v) and Myc-p130Cas from Dr. Michel Tremblay, McGill University, Montreal, Canada (19Angers-Loustau A. Cote J.F. Charest A. Dowbenko D. Spencer S. Lasky L.A. Tremblay M.L. J. Cell Biol. 1999; 144: 1019-1031Crossref PubMed Scopus (247) Google Scholar, 20Cote J.F. Charest A. Wagner J. Tremblay M.L. Biochemistry. 1998; 37: 13128-13137Crossref PubMed Scopus (92) Google Scholar); GST-c-Src, GST-SH2c-Src, and GST-c-SrcΔSH2 (13Gingras M.-C. Champagne C. Roy M.A. Lavoie J.N. Mol. Cell. Biol. 2002; 22: 41-56Crossref PubMed Scopus (41) Google Scholar); polyomavirus middle T-antigen (mT); and MT200Δ10 from Dr. Stephen M. Dilworth, Royal Postgraduate Medical School, Hammersmith Hospital, London, UK (21Glover H.R. Brewster C.E. Dilworth S.M. Oncogene. 1999; 18: 4364-4370Crossref PubMed Scopus (30) Google Scholar); Myc-FAK from Dr. J. Thomas Parson, University of Virginia School of Medicine, Charlottesville (22Xiong W.C. Macklem M. Parsons J.T. J. Cell Sci. 1998; 111: 1981-1991Crossref PubMed Google Scholar); chicken c-Src (Y527F) in pLNCX from Dr. Joan S. Brugge, Harvard Medical School, Boston, MA (23Thomas J.E. Soriano P. Brugge J.S. Science. 1991; 254: 568-571Crossref PubMed Scopus (84) Google Scholar); GFP-actin in pEGFP C2 (24Choidas A. Jungbluth A. Sechi A. Murphy J. Ullrich A. Marriott G. Eur. J. Cell Biol. 1998; 77: 81-90Crossref PubMed Scopus (82) Google Scholar); and FLAG-PP2A-Bα, Ad2 E4orf4 (R69A/R70A), (R73A/R74A/R75A), (R69A/R70A/R72A/R73A/R74A/R75A), (R81A/F84A), and (F84A) in pCDNA3 from Dr. Philip E. Branton, McGill University, Montreal, Quebec, Canada (18Marcellus R.C. Chan H. Paquette D. Thirlwell S. Boivin D. Branton P.E. J. Virol. 2000; 74: 7869-7877Crossref PubMed Scopus (84) Google Scholar). The mutant E4orf4 (R69A/R70A/R72A/R73A/R74A/R75A) was designated (6R-A). E4orf4 (6R-A/R81A/F84A) was generated by PCR with the QuikChange site-directed mutagenesis kit (Stratagene) according to the manufacturer's recommendations using E4orf4 (6R-A) as template with the following primers: 5′-TCT GTT TGT CAC GCC GCC ACC TGG GCT TGC TTC AGG AAA TAT GAC-3′ and 5′-GTA GTC ATA TTT CCT GAA GCA AGC CCA GGT GGC GGC GTG ACA AAC-3′. FLAG-E4orf4 mutant constructs were generated from HA-E4orf4 mutants as described in Ref. 12Lavoie J.N. Champagne C. Gingras M.C. Robert A. J. Cell Biol. 2000; 150: 1037-1056Crossref PubMed Scopus (66) Google Scholar. Untagged E4orf4 proteins were obtained by subcloning the HindIII-XhoI E4orf4 DNA fragments from HA-E4orf4 constructs into pCDNA3 vector. E4orf4-GFP constructs were produced by PCR amplification using HA-tagged versions and the following primers: 5′-AGA GGG CCC AG GTT CTT CCA GCT CTT CCA-3′and 5′-CTC CCC GGG CTG GAA CTG TGG CTC AGT-3′. The resulting DNA fragments were inserted in ApaI-XhoI sites of the FLAG-GFP vector (25Lee S. Neumann M. Stearman R. Stauber R. Pause A. Pavlakis G.N. Klausner R.D. Mol. Cell. Biol. 1999; 19: 1486-1497Crossref PubMed Google Scholar). FLAG-E4orf4 (4YF)-GFP, designated nonphosphorylatable E4orf4 (E4orf4[NP]) was produced by site-directed mutagenesis, using FLAG-E4orf4(3YF)-GFP (13Gingras M.-C. Champagne C. Roy M.A. Lavoie J.N. Mol. Cell. Biol. 2002; 22: 41-56Crossref PubMed Scopus (41) Google Scholar) as template and primers: 5′-GAG TGG ATA TTC TTC AAC TAC TAC ACA GAG-3′ and 5′-GTA GTA GTT GAA GAA TAT CCA CTC TCT CAA-3′. GFPE4orf4 (amino acids 62-79) and (amino acids 62-95) were from Dr. Philip E. Branton, McGill University, Montreal, Quebec, Canada and were generated by adding the sequence corresponding to amino acids 62-79 or 62-95 of E4orf4 to GFP by standard PCR techniques using the following primers: sense, 5′-CGC GGA TCC ATG GTG AGC AAG GGC GAG GAG-3′ and antisense for 62-79, 5′-CGC GAA TTC CTA GTG ACA AAC AGA TCT GCG TCT CCG GTC TCG TCG CTT AGC TCG CTC TGT GTA GTA CTT GTA CAG CTC GTC CAT GCC GAG AG-3′ or antisense for 62-95, 5′-CGC GAA TTC CTA GGA ACG CCG GAC GTA GTC ATA TTT CCT GAA GCA AAA CCA GGT GCG GGC GTG ACA AAC AGA TCT G-3′. The resulting fragments were inserted in pCDNA3. GST-FLAG-E4orf4 was generated by PCR using FLAG-E4orf4 as template and the sense and antisense oligonucleotides 5′-GG GGT ACC ATG GTT CTT CCA GCT CTT CC-3′ and 5′-ATT TAG GTG ACA CTA TAG-3′ (SP6 primer). The resulting DNA fragment was subcloned in SmaI-BamHI sites of pGEX6P-1 (Amersham Biosciences). GST-SH3c was generated by PCR using pCI-neo-c-Src (12Lavoie J.N. Champagne C. Gingras M.C. Robert A. J. Cell Biol. 2000; 150: 1037-1056Crossref PubMed Scopus (66) Google Scholar) as template and the primers: 5′-CCGGAATTCC ACC ACC TTT GTG GCC CTC TA-3′ and 5′-TTGCGGCCGCCC CTA GGA GGG CGC CAC ATA GT-3′. GST-ΔSH3c was generated by PCR extension overlap (to join amino acids Val85 to Glu148 of c-Src sequence) using paired sense and anti-sense primers 5′-CCACAGGTGTCCACT-3′ and 5′-CCA CTC CTC CAC CCC ACC TGC CAG AGG CCC-3′ and 5′-GGT GGG GTG GAG GAG TGG TAC TTT GGC AAG-3′ and 5′-CTAGTTGTGGTTTGTCC-3′. The resulting fragment was subcloned in EcoRI-NotI sites of pGEX4T-3 (Amersham Biosciences) and pCI-neo (Promega) vectors. The kinase (SH1) domain of c-Src was excised by PCR using the sense and anti-sense primers: 5′-CC GGA ATT CG ATG GCG TGG GAG ATC CCC CG-3′ and 5′-CTAGTTGTGGTTTGTCC-3′ and was subcloned in EcoRI-NotI sites of pGEX4T-3 and pCI-neo. To generate c-Src ΔSH1, the pGEX-4T-3/c-Src and pCI-neo-c-Src were treated with XmaI-S1 nuclease, and the resulting DNA fragments were ligated to join Pro265 to Glu533 of c-Src. All mutations and DNA fragments generated by PCR were confirmed by DNA sequence analysis.Cell Culture, Transfections, and Functional Assays—293T cells were derived from human embryonic kidney cells, although this cell line may be of neuronal origin (26Shaw G. Morse S. Ararat M. Graham F.L. FASEB J. 2002; 16: 869-871Crossref PubMed Scopus (569) Google Scholar) and express Ad5 E1A and E1B proteins and large T antigen (27Graham F.L. Smiley J. Russell W.C. Nairn R. J. Gen. Virol. 1977; 36: 59-74Crossref PubMed Scopus (3468) Google Scholar). Human C-33A (American Type Culture Collection, HTB-31) is from cervical carcinoma. 293T and C-33A were maintained in Dulbecco's modified Eagle medium and in α-modified Eagle's medium, respectively, both supplemented with 10% fetal bovine serum and streptomycin sulfate-penicillin (100 units/ml). Transfections of 293T were performed by the calcium phosphate method in culture dishes coated with polylysine as described (12Lavoie J.N. Champagne C. Gingras M.C. Robert A. J. Cell Biol. 2000; 150: 1037-1056Crossref PubMed Scopus (66) Google Scholar, 15Robert A. Miron M.J. Champagne C. Gingras M.C. Branton P.E. Lavoie J.N. J. Cell Biol. 2002; 158: 519-528Crossref PubMed Scopus (53) Google Scholar). C-33A cells were transfected using GeneSHUTTLE-40 (Qbiogene, Inc.), according to the manufacturer's recommendations. In vivo localization of GFP proteins was performed using a Nikon TE-200 inverted microscope equipped with a CO2/thermoregulated chamber and a ×40 0.6 numerical aperture (NA) objective. Images were captured as 16-bit TIFF files with a Photometrix Coolsnap FX cooled CCD camera (-30 °C) (Rooper Scientific, Tucson, AZ) driven by Metaview software version 4.5 (Universal Imaging Corp., Downingtown, PA). Confocal microscopy was performed using a Bio-Rad MRC-1024 imaging system mounted on a Nikon Diaphot-TMD with ×20 (zoom ×3) or ×60 0.85 NA (zoom ×1.84) objective lens. The blebbing inducing activity and chromatin condensation were determined as described (13Gingras M.-C. Champagne C. Roy M.A. Lavoie J.N. Mol. Cell. Biol. 2002; 22: 41-56Crossref PubMed Scopus (41) Google Scholar). Briefly, cells were gently washed in PBS containing 1 mm MgCl2 and 0.5 mm CaCl2, fixed in 3.7% formaldehyde/PBS for 20 min, and permeabilized in 0.2% Triton X-100/PBS for 5 min. Immunodetection of HA-E4orf4, E4orf4-GFP, and untagged E4orf4 was performed using mouse HA.11 anti-HA antibody (Sigma-Aldrich), rabbit anti-GFP (Clontech), or rabbit 2419 anti-E4orf4, respectively, followed by Alexa 594- or Alexa 488-labeled goat anti-rabbit IgG (Molecular Probes), and staining of DNA was performed using 4′-6-diamidino-2-phenylindole dihydrochloride (DAPI, Molecular Probes). Long-term cell killing was determined by colony-forming assays as described (13Gingras M.-C. Champagne C. Roy M.A. Lavoie J.N. Mol. Cell. Biol. 2002; 22: 41-56Crossref PubMed Scopus (41) Google Scholar). When indicated, the broad spectrum caspase inhibitor Boc-Asp-(OMe)-CH2F (BocD-fmk, Calbiochem) was added to the culture medium at 15 μm during transfection and at 30 μm after transfection. Fresh inhibitor was added back every 24 h.GST Fusion Proteins and in Vitro Binding Assays—GST-c-Src constructs were introduced in Escherichia coli BL21 strain expressing GroES and GroEL chaperones encoded by pRep4-GroESL vector (a generous gift from Drs. Kurt Amrein and Martin Stieger, Roche Research Center, Hoffmann-La Roche Inc., Nutley, NJ) (28Amrein K.E. Takacs B. Stieger M. Molnos J. Flint N.A. Burn P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 1048-1052Crossref PubMed Scopus (150) Google Scholar). The fusion proteins were produced by growing bacterial cultures to an OD600 of between 0.6 and 0.8 followed by a treatment with 0.1 to 0.5 mm isopropyl-β-d-galactopyranoside for 2-6 h at 30 °C. Bacteria were recovered by centrifugation and kept at -20 °C overnight. Bacterial pellets were thawed at room temperature and lysed as described (13Gingras M.-C. Champagne C. Roy M.A. Lavoie J.N. Mol. Cell. Biol. 2002; 22: 41-56Crossref PubMed Scopus (41) Google Scholar). The supernatants were incubated with glutathione-Sepharose 4B beads for 2 h at 4 °C, and the beads were washed three times in washing buffer (PBS containing 1% Triton X-100 and 1 mm EDTA). To remove the bound chaperones, beads were incubated in ATP buffer (50 mm Tris-HCl, pH 7.4, 10 mm MgSO4, and 2 mm ATP) for 20 min at 37 °C, washed three times in washing buffer, and used for in vitro binding assays. The amount of purified proteins was evaluated on a Coomassie Blue-stained gel. In vitro binding assays were performed as described (13Gingras M.-C. Champagne C. Roy M.A. Lavoie J.N. Mol. Cell. Biol. 2002; 22: 41-56Crossref PubMed Scopus (41) Google Scholar) by incubating equal amounts of transfected total cell lysates (1 mg) with 5 μg of freshly prepared immobilized GST fusion proteins overnight at 4 °C. The beads were recovered by centrifugation, washed once in lysis buffer, then twice in lysis buffer containing only 0.1% Nonidet P-40, and boiled in sodium dodecyl sulfate (SDS) sample buffer. Bound proteins were analyzed by Western blot analysis with the appropriate antibody. The GST-FLAG-E4orf4 was produced as described (12Lavoie J.N. Champagne C. Gingras M.C. Robert A. J. Cell Biol. 2000; 150: 1037-1056Crossref PubMed Scopus (66) Google Scholar). The GST moiety was removed by incubating the beads in protease buffer (50 mm Tris-HCl, pH 7.4, 150 mm NaCl, 1 mm EDTA, 1 mm dithiothreitol, and 4 units of PreScission Protease™ (Amersham BioSciences)) overnight at 4 °C with rotation. Recombinant FLAG-E4orf4 protein was recovered in the supernatant, and direct binding to c-Src kinase domain was performed using 5 μg of the indicated GST-c-Src proteins and 200 ng of purified FLAG-E4orf4 protein, as described above. In vitro kinase assays were performed using 250 ng of bound GST-c-Src and 2.5 μg of purified FLAG-E4orf4 or GST in kinase buffer (50 mm Hepes pH 7.4, 10 mm MgCl2, 0.1% β-mercaptoethanol, 0.1 mm Na3Vo4) containing 0.1 mm ATP and 10 μCi of [γ-P32]ATP in a 50-μl reaction mixture. The reactions were allowed to proceed for 20 min at 30 °C and stopped by adding 25 μl of 3× SDS sample buffer. Labeled samples were resolved on SDS-PAGE and visualized by autoradiography or analyzed by Western blot after electrotransfer on nitrocellulose.Immunoprecipitation, Western Blotting, and Antibodies—For coprecipitations, cells on 10-cm plates were lysed with 0.5 ml of modified radioimmune precipitation assay buffer (RIPA: 50 mm Hepes pH 7.4, 150 mm NaCl, 1.5 mm MgCl2, 1 mm EGTA, 1% Triton, 1% sodium deoxycholate, 0.1% SDS, 10% glycerol, 50 mm NaF, 10 mm β-glycerophosphate, 1 mm Na3Vo4, 15 μg/ml leupeptin, 5 μg/ml aprotinin, 1 μg/ml pepstatin A), and lysates were immediately diluted with 0.5 ml of HNTG buffer (50 mm Hepes pH 7.4, 150 mm NaCl, 0.1% Triton X-100, and 10% glycerol), as described (29Sieg D.J. Hauck C.R. Ilic D. Klingbeil C.K. Schaefer E. Damsky C.H. Schlaepfer D.D. Nat. Cell Biol. 2000; 2: 249-256Crossref PubMed Scopus (1050) Google Scholar). The lysates were incubated with protein G-Sepharose (Amersham Biosciences) or protein G Plus-agarose (Oncogene Research Products) for 20 min on ice and cleared by centrifugation. Immunoprecipitation was carried out with mouse anti-FLAG M2 (Sigma-Aldrich), rabbit SRC2 anti-Src (Santa Cruz Biotechnology), mouse Ab1 anti-Src (Calbiochem), mAb 327 anti-c-Src (26Shaw G. Morse S. Ararat M. Graham F.L. FASEB J. 2002; 16: 869-871Crossref PubMed Scopus (569) Google Scholar), or rabbit 2419 anti-E4orf4 for 2.5 h at 4 °C. Immune complexes were collected on protein G-Sepharose or protein G Plus-agarose and washed three times in modified RIPA containing only 1% Triton X-100 before analysis on SDS-PAGE. Equal amounts of immune complexes were resolved on 9 or 11% SDS-PAGE, transferred to nitrocellulose, and processed for immunoblotting. To disrupt the antigen-antibody complex before reprobing, immunoblots were incubated in stripping buffer (62.5 mm Tris-HCl pH 6.7, 2% SDS, 100 mm β-mercaptoethanol) for 30 min at 60 °C, washed in PBS-Tween (0.1%) at room temperature and reprocessed for immunoblotting. The following antibodies were used for immunoblotting analyses: mouse anti-cortactin (4F11, Upstate Biotechnology); mouse anti-FLAG (M2, Sigma); mouse anti-HA (HA.11, Babco); mouse anti-Myc (9E10, Sigma); mouse anti-polyomavirus middle T-antigen (Pab 762) (30Dilworth S.M. Horner V.P. J. Virol. 1993; 67: 2235-2244Crossref PubMed Google Scholar); mouse anti-phosphotyrosine (RC20:HRPO, Transduction Laboratories); rabbit anti-Src (SRC2, Santa Cruz Biotechnology); mouse anti-phospho-Src (Tyr416) (Cell Signaling, NEB), mouse anti-PP2A-Cα (clone46, BD Biosciences), rabbit anti-phospho-p-38 (Thr180/Tyr182) (Cell Signaling, NEB), rabbit anti-ERK2 (clone 856) (31Huot J. Lambert H. Lavoie J.N. Guimond A. Houle F. Landry J. Eur. J. Biochem. 1995; 227: 416-427Crossref PubMed Scopus (170) Google Scholar), mouse E10 anti-phospho-p44/42 MAPK (Thr202/Tyr204) (Cell Signaling, NEB), rabbit H-79 anti-c-Jun (Santa Cruz Biotechnology), rabbit anti-phospho-c-Jun (Ser63) II (Cell Signaling, NEB), mouse anti-β-actin (AC-74, Sigma-Aldrich), and rabbit 2419 anti-E4orf4. Rabbit 2419, 2418, and 2420 anti-E4orf4 antibodies were described (12Lavoie J.N. Champagne C. Gingras M.C. Robert A. J. Cell Biol. 2000; 150: 1037-1056Crossref PubMed Scopus (66) Google Scholar). The anti-phospho-E4orf4 (Tyr42) was produced by injecting rabbits with a chemically synthesized peptide comprising the phosphorylated Tyr42 (HEGVY[PO3H2]IEPEARGRLC), which was coupled to mcKLH (Imject Mariculture keyhole limpet hemocyanin, Pierce) following the manufacturer's recommendations. The serum was purified against the immobilized nonphosphorylated peptide (HEGVYIEPEARGRLC), was absorbed on immobilized phosphorylated peptide using the SulfoLink Kit (Pierce), and was finally purified against phosphotyrosines linked to agarose (O-phospho-l-tyrosine-agarose, Sigma-Aldrich) according to the manufacturer's recommendations. The specificity of the resulting anti-phospho-E4orf4 (Tyr42) was tested by Western blot analysis of E4orf4 immune complexes and total cell lysates from 293T cells transfected with wild-type FLAG-E4orf4, compared with mutant FLAG-E4orf4 (Y42F) alone or together with c-Src or v-Src to induce maximum tyrosine phosphorylation of Ad2 E4orf4, as described (13Gingras M.-C. Champagne C. Roy M.A. Lavoie J.N. Mol. Cell. Biol. 2002; 22: 41-56Crossref PubMed Scopus (41) Google Scholar). The antibody reacted specifically with wild-type Ad2 E4orf4 but not with mutant E4orf4 (Y42F), and the specific signal was proportional to the level of tyrosine phosphorylation.RESULTSAd2 E4orf4 Directly Interacts with the Kinase Domain of Src—In several transformed and cancer cell lines expressing Ad2 E4orf4, including 293T, C-33A, and H1299, a membrane-cytoskeleton fraction of E4orf4 is associated with Src family kinases, and inhibition of Src kinases interferes with cell death induction (12Lavoie J.N. Champagne C. Gingras M.C. Robert A. J. Cell Biol. 2000; 150: 1037-1056Crossref PubMed Scopus (66) Google Scholar, 13Gingras M.-C. Champagne C. Roy M.A. Lavoie J.N. Mol. Cell. Biol. 2002; 22: 41-56Crossref Pu
Referência(s)