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• W1974967315 abstract "The MHV-68 latent protein, M2, does not have homology to any known viral or cellular proteins, and its function is unclear. To define the role played by M2 during MHV-68 latency as well as the molecular mechanism involved, we used M2 as bait to screen a yeast two-hybrid mouse B-cell cDNA library. Vav1 was identified as an M2-interacting protein in two independent screenings. Subsequent yeast two-hybrid interaction studies showed that M2 also binds to Vav2, but not Vav3, and that three “PXXP” motifs located at the C terminus of M2 are important for this interaction. The interactions between M2 and Vav proteins were also confirmed in vivo in 293T and WEHI-231 B-cells by co-immunoprecipitation assays. Rac1/GST-PAK “pull-down” experiments and Western blot analysis using a phospho-Vav antibody demonstrated that expression of M2 in WEHI-231 cells enhances Vav activity. We further showed in WEHI-231 cells that M2 expression promotes proliferation and survival and is associated with enhanced cyclin D2 and repressed p27Kip1, p130, and Bim expression. Taken together, these experiments suggest that M2 might have an important role in disseminating the latent virus during the establishment and maintenance of latency by modulating B-cell receptor-mediated signaling events through Vav to promote B-cell activation, proliferation, and survival. The MHV-68 latent protein, M2, does not have homology to any known viral or cellular proteins, and its function is unclear. To define the role played by M2 during MHV-68 latency as well as the molecular mechanism involved, we used M2 as bait to screen a yeast two-hybrid mouse B-cell cDNA library. Vav1 was identified as an M2-interacting protein in two independent screenings. Subsequent yeast two-hybrid interaction studies showed that M2 also binds to Vav2, but not Vav3, and that three “PXXP” motifs located at the C terminus of M2 are important for this interaction. The interactions between M2 and Vav proteins were also confirmed in vivo in 293T and WEHI-231 B-cells by co-immunoprecipitation assays. Rac1/GST-PAK “pull-down” experiments and Western blot analysis using a phospho-Vav antibody demonstrated that expression of M2 in WEHI-231 cells enhances Vav activity. We further showed in WEHI-231 cells that M2 expression promotes proliferation and survival and is associated with enhanced cyclin D2 and repressed p27Kip1, p130, and Bim expression. Taken together, these experiments suggest that M2 might have an important role in disseminating the latent virus during the establishment and maintenance of latency by modulating B-cell receptor-mediated signaling events through Vav to promote B-cell activation, proliferation, and survival. γ-Herpesviruses comprise a subfamily of animal double-stranded DNA viruses widespread in nature. There are two known human γ-herpesviruses, namely Epstein-Barr virus (EBV) 5The abbreviations used are: EBVEpstein-Barr virusHHV-8human herpesvirus-8CDKcyclin-dependent kinasepRbretinoblastoma proteinMHV-68murine γ-herpesvirus 68CMVcytomegalovirusHAhemagglutininGSTglutathione S-transferaseMCSmultiple cloning sitesPAKp21-activated kinaseCKIcyclin-dependent kinase inhibitor 5The abbreviations used are: EBVEpstein-Barr virusHHV-8human herpesvirus-8CDKcyclin-dependent kinasepRbretinoblastoma proteinMHV-68murine γ-herpesvirus 68CMVcytomegalovirusHAhemagglutininGSTglutathione S-transferaseMCSmultiple cloning sitesPAKp21-activated kinaseCKIcyclin-dependent kinase inhibitor and human herpesvirus-8 (HHV-8) also known as Kaposi's sarcoma herpesvirus, which are largely disseminated in the population. Infections by the γ-herpesviruses have been implicated in the development of lymphoproliferative diseases and in some cases, tumorigenesis (1Dourmishev L.A. Dourmishev A.L. Palmeri D. Schwartz R.A. Lukac D.M. Microbiol. Mol. Biol. Rev. 2003; 67: 175-212Crossref PubMed Scopus (293) Google Scholar, 2Verma S.C. Robertson E.S. FEMS Microbiol. Lett. 2003; 222: 155-163Crossref PubMed Scopus (91) Google Scholar). One of the characteristic biological features of the γ-herpesviruses is their ability to establish a life-long latent infection in lymphocytes, during which only a few viral genes are expressed. One hypothesis is that these viral genes play a major role in the establishment and maintenance of viral latency. Understanding the molecular mechanisms whereby latency-associated proteins alter the regulation of the cellular pathways of the infected host cell, as well as evade immune surveillance will provide essential information for devising therapeutic strategies against these viruses. γ-Herpesviruses employ similar strategies to subvert the infected host cell, encoding for viral proteins, which often interfere with cellular processes in the host cell, in particular targeting the transcriptional machinery and signaling pathways. In EBV, the EBNA-1, EBNA-2, EBN-3A, -B, -C, and EBNA-LP latency-associated proteins are involved in the altered transcriptional regulation of the host's genes (3Zimber-Strobl U. Strobl L.J. Semin. Cancer Biol. 2001; 11: 423-434Crossref PubMed Scopus (98) Google Scholar, 4Pokrovskaja K. Mattsson K. Kashuba E. Klein G. Szekely L. J. Gen. Virol. 2001; 82: 345-358Crossref PubMed Scopus (35) Google Scholar), whereas the LMP-1 and LMP-2A proteins are involved in the altered regulation of endogenous cellular signaling pathways. In the case of LMP-1, it functions as a constitutively active CD40 receptor. This viral protein binds and activates TRAF-2 and -3 proteins leading to NF-κB activation and consequently cell proliferation (5Klein E. Teramoto N. Gogolak P. Nagy N. Bjorkholm M. Immunol. Lett. 1999; 68: 147-154Crossref PubMed Scopus (19) Google Scholar, 6Young L.S. Dawson C.W. Eliopoulos A.G. Mol. Pathol. 2000; 53: 238-247Crossref PubMed Scopus (87) Google Scholar). It has been demonstrated that LMP-2A associates with the cellular tyrosine kinases Fyn, Lyn, and Syk, modulating their interactions with the B-cell receptor (7Fruehling S. Swart R. Dolwick K.M. Kremmer E. Longnecker R. J. Virol. 1998; 72: 7796-7806Crossref PubMed Google Scholar, 8Longnecker R. Merchant M. Brown M.E. Fruehling S. Bickford J.O. Ikeda M. Harty R.N. Exp. Cell Res. 2000; 257: 332-340Crossref PubMed Scopus (26) Google Scholar), thereby acting in the same manner as a constitutively active B-cell receptor. In B-cell receptor-negative B-cells, LMP-2A activates signals that are usually generated by the B-cell receptor to inhibit apoptosis of infected cells (9Caldwell R.G. Wilson J.B. Anderson S.J. Longnecker R. Immunity. 1998; 9: 405-411Abstract Full Text Full Text PDF PubMed Scopus (478) Google Scholar). LMP-2A has been shown to activate the phosphatidylinositol 3-kinase/Akt (also called PKB) signaling cascade, which has a major role in cell proliferation and survival (10Scholle F. Bendt K.M. Raab-Traub N. J. Virol. 2000; 74: 10681-10689Crossref PubMed Scopus (231) Google Scholar, 11Swart R. Ruf I.K. Sample J. Longnecker R. J. Virol. 2000; 74: 10838-10845Crossref PubMed Scopus (132) Google Scholar). During latency, HHV-8 expresses viral proteins, such as the D-type cyclin homologue (v-cyclin) (12Godden-Kent D. Talbot S.J. Boshoff C. Chang Y. Moore P. Weiss R.A. Mittnacht S. J. Virol. 1997; 71: 4193-4198Crossref PubMed Google Scholar, 13Li M. Lee H. Yoon D.W. Albrecht J.C. Fleckenstein B. Neipel F. Jung J.U. J. Virol. 1997; 71: 1984-1991Crossref PubMed Google Scholar), the transcription regulators LANA-1 and LANA-2 (vIRF-3) (14Rivas C. Thlick A.E. Parravicini C. Moore P.S. Chang Y. J. Virol. 2001; 75: 429-438Crossref PubMed Scopus (254) Google Scholar, 15Esteban M. Garcia M.A. Domingo-Gil E. Arroyo J. Nombela C. Rivas C. J. Gen. Virol. 2003; 84: 1463-1470Crossref PubMed Scopus (66) Google Scholar), kaposin A (16Muralidhar S. Veytsmann G. Chandran B. Ablashi D. Doniger J. Rosenthal L.J. J. Clin. Virol. 2000; 16: 203-213Crossref PubMed Scopus (54) Google Scholar), and vFLIP (17Field N. Low W. Daniels M. Howell S. Daviet L. Boshoff C. Collins M. J. Cell Sci. 2003; 116: 3721-3728Crossref PubMed Scopus (201) Google Scholar) to manipulate cellular signaling pathways. Another HHV-8 latent protein, LAMP, has been shown to inhibit B-cell receptor-dependent signal transduction and to associate with the cellular proteins TRAF-1, -2, and -3, suggesting that it might also mimic the function of the CD40 receptor (18Choi J.K. Lee B.S. Shim S.N. Li M. Jung J.U. J. Virol. 2000; 74: 436-446Crossref PubMed Scopus (108) Google Scholar, 19Sharp T.V. Wang H.W. Koumi A. Hollyman D. Endo Y. Ye H. Du M.Q. Boshoff C. J. Virol. 2002; 76: 802-816Crossref PubMed Scopus (168) Google Scholar).It is evident that the biological functions of some of these γ-herpesviruses latent proteins are to promote lymphocyte proliferation and survival to preserve and propagate the viral genomes during latent infection. Mammalian cell proliferation requires successful transition through cell cycle phases (G1, S, G2, and M) (20Sherr C.J. Roberts J.M. Genes Dev. 2004; 18: 2699-2711Crossref PubMed Scopus (899) Google Scholar, 21Sherr C.J. Roberts J.M. Genes Dev. 1999; 13: 1501-1512Crossref PubMed Scopus (5096) Google Scholar). The expression of D-type cyclins (cyclins D1, D2, and D3) is predominantly regulated by extracellular mitogenic (or inhibitory) signals and are rate-limiting for transition through G1. Once cells have reached late G1, the E-type cyclins (E1 and E2) accumulate and allow S phase initiation, followed by induction of cyclin A in S phase. The D-type cyclins preferentially associated with and activate CDK-4 and -6, whereas cyclin E/A with CDK2. The primary physiological substrate for the CDKs is the retinoblastoma (pRb) family of pocket proteins (pRb, p107, and p130). When hypophosphorylated, the pocket proteins bind to and inactivate the E2F family of transcription factors that are required for the transcription of genes that are necessary for entry into S-phase. The activities of the CDKs are negatively regulated by two families of CDK inhibitors, namely the Cip/Kip family (comprising p21Cip1, p27Kip1, and p57Kip2) and the INK4 family. The Cip/Kip family inhibits a broad range of cyclin-CDK complexes, whereas the INK4 family members specifically inhibit cyclin D-CDK4/6 (21Sherr C.J. Roberts J.M. Genes Dev. 1999; 13: 1501-1512Crossref PubMed Scopus (5096) Google Scholar). The HHV-8 v-cyclin D has been shown to be refractory to the inhibitory effects of the Cip/Kip family of CKIs, and has been shown to increase the rates of proliferation of the infected cells, thereby propagating the viral genome (22Child E.S. Mann D.J. Oncogene. 2001; 20: 3311-3322Crossref PubMed Scopus (33) Google Scholar). Another mechanism whereby the viral genome is propagated during latency is via inhibition of apoptosis.The Bcl-2 family of proteins is central to regulating the commitment of cells to apoptosis (programmed cell death) (23Harada H. Grant S. Rev. Clin. Exp. Hematol. 2003; 7: 117-138PubMed Google Scholar, 24Lutz R.J. Biochem. Soc. Trans. 2000; 28: 51-56Crossref PubMed Google Scholar). The Bcl-2 family of proteins contains both anti- and pro-apoptotic members and can be divided into three distinct classes: the anti-apoptotic sub-group (e.g. Bcl-2 and Bcl-XL); the pro-apoptotic, multi-Bcl-2 homology (BH) domain sub-group (e.g. Bak and Bax); and the pro-apoptotic, BH3 domain-only sub-group (e.g. Bad, Bik, Bid, Bim, PUMA, and Bmf). The pro-apoptotic BH3-only member of the Bcl-2 family, Bim, plays a crucial role in regulating apoptosis in lymphocytes during negative selection. Bim, like other BH3-only Bcl-2 proteins, mediates apoptosis through inducing mitochondrial cytochrome c release, which in turn activates caspase-9, caspase-3, and eventually the downstream cell death machinery (23Harada H. Grant S. Rev. Clin. Exp. Hematol. 2003; 7: 117-138PubMed Google Scholar, 25Dijkers P.F. Medema R.H. Lammers J.W. Koenderman L. Coffer P.J. Curr. Biol. 2000; 10: 1201-1204Abstract Full Text Full Text PDF PubMed Scopus (828) Google Scholar). Bim is believed to promote apoptosis by neutralizing pro-survival Bcl-2 proteins in a mechanism that requires members of the multi-BH-domain pro-apoptotic subgroup.The murine γ-herpesvirus 68 (MHV-68) is closely related to the human HHV-8 and EBV. Because γ-herpesviruses often share similar mechanisms to manipulate the host cellular signaling cascades, MHV-68 is often used as a rodent model of γ-herpesviral infection. M2 is a unique latency-associated protein of MHV-68. It has no known cellular or viral homologues and does not contain any previously defined conserved domains. This protein is expressed during both the establishment and long term maintenance of latency, indicating that it is likely to play a key role in viral persistence (26Flano E. Kim I.J. Moore J. Woodland D.L. Blackman M.A. J. Immunol. 2003; 170: 3828-3834Crossref PubMed Scopus (71) Google Scholar, 27Flano E. Kim I.J. Woodland D.L. Blackman M.A. J. Exp. Med. 2002; 196: 1363-1372Crossref PubMed Scopus (141) Google Scholar). To study the functional role of this viral protein we sought to identify cellular targets of M2. Yeast-two-hybrid screening of a cDNA library derived from MHV-68 latently infected B lymphocytes demonstrated M2 interaction with both the Vav1 and Vav2 guanine nucleotide exchange factors. In immature B-cell line WEHI-231, the interaction between M2 and Vav promotes cell proliferation and survival.MATERIALS AND METHODSConstruction of a Splenic B Cell cDNA Library from Latently Infected BALB/c MiceSplenic B220+ cells from three MHV-68 latently infected mice were purified at 15, 18, and 21 days post infection by the μMACS Streptavidin kit (Miltenyl Biotec, Surrey, UK). The percentage of B220+ cells over total cell number determined by fluorescence-activated cell sorting analysis was of 95%. mRNA was extracted by the mRNA Fast Track 2.0 mRNA isolation system (Invitrogen), and the cDNA was synthesized by the TimeSaver cDNA Synthesis kit (Amersham Biosciences). cDNAs were ligated to the oligonucleotide adaptors 5′-pCCG GTG CTT CCG G-3′ and 5′-pCGA GGC CAC GAA GGC C-3′, which allowed the insertion into the XhoI site of λACT2 vector (Clontech, Basingstoke, UK). Clones containing the cDNA molecules were released from λACT2 by cre-lox recombination in BNN132 Escherichia coli cells. A cDNA library containing 2 × 105 independent clones with an average insert size of 0.9 kb was obtained.PlasmidsYeast Two-hybrid System Plasmids—The following genes were PCR-amplified from the MHV-68 genome and cloned into the MCS of pGBT9 (Clontech, Palo Alto, CA) in-frame with Gal4BD: M2, M2 del1 encoding for amino acids 1–126 of M2, M2 del2 encoding for amino acids 131–199 of M2, M2 del3 encoding for amino acids 96–199 of M2, M2 del4 encoding for amino acids 44–129 of M2, M2 del5 encoding for amino acids 21–199 of M2, M2 del6 encoding for amino acids 41–199 of M2, M2 del7 encoding for amino acids 80–199 of M2, M2 del8 encoding for amino acids 1–179 of M2, M2 del9 encoding for amino acids 1–160 of M2, M2 del10 encoding for amino acids 1–138 of M2, M2 del11 encoding for amino acids 1–98 of M2, M2 del12 encoding for amino acids 1–98 and 126–199 of M2, M2ΔP1 encoding for amino acids 8–199 of M2 with the substitution of proline residues 167 and 170 for alanine residues and M2ΔP2 encoding for amino acids 8–199 of M2 with the substitution of proline residues 165, 167, 170, and 174 for alanine residues. vav2 gene was PCR-amplified from Mus musculus vav2 cDNA, using the primers 5′: CGG AAT TCA GCA GTG GCG GCA A and 3′: TCT AGA ATT CAG CAG AAA AGA CAG GCA and cloned into the EcoRI site of pACT2 MCS in-frame with Gal4AD, this plasmid was designated pACT2-Vav2. Restriction sites are highlighted in bold, and nucleotide modifications are underlined. vav2SH gene encoding for amino acids 536–869 of Vav2 was PCR-amplified from M. musculus vav2 cDNA using the primers 5′: GAG AAT TCT CAG GGG TAC CTT and 3′: CAC GAA TTC TGT CCA GGT CT and cloned into the EcoRI site of pACT2 MCS in-frame with Gal4AD, and this plasmid was designated pACT2-Vav2SH. vav3SH gene encoding for amino acids 531–860 of Vav3 was PCR amplified from M. musculus vav3 cDNA using the primers 5′: TAT CCC GGG TAC TTA TGT TTT and 3′:GTC CTC GAG ATT TTA GTG CA and cloned into the SmaI/XhoI sites of pACT2 MCS in-frame with Gal4AD, and was designated pACT2-Vav3SH.Plasmids for Protein Expression in Bacterial Cells—The M2 gene was PCR-amplified from the MHV-68 genome using the primers 5′: GCG TTA GCG AAT TCA CTG TT and 3′: CAG GAT CCA GTC TGA TGA GG, corresponding to bases 4010–4641 of MHV-68 genome, and cloned into the BamHI/EcoRI sites of pGEX-4T1 (Amersham Biosciences), and designated pGEX-M2. Plasmids for expression of proteins in eukaryotic cells: pCMV-FLAG-Vav1 was a kind gift of Dr. Martin Turner, Brabaham Institute, Brabaham, UK (Doody, et al. (48Doody G.M. Billadeau D.D. Clayton E. Hutchings A. Berland R. McAdam S. Leibson P.J. Turner M. EMBO J. 2000; 19: 6173-6184Crossref PubMed Scopus (67) Google Scholar)). M2 gene was PCR-amplified from MHV-68 genome using the primers 5′: ACG GTA CCT TCA CTG TTA CTC C and 3′: TTT CGA ATT CAG GTA ATG GC, corresponding to coordinates 4013–4622 of MHV-68 genome, and cloned into the EcoRI/KpnI sites of pCMV-Myc (Clontech) MCS in-frame with the Myc tag, and this plasmid was named pCMV-Myc-M2. M2ΔP1 and M2ΔP2 genes were PCR-amplified from pGBT9-M2ΔP1 and pGBT9-M2ΔP2, respectively, using the primers 5′: TCG ACG GTA CCT TAC TCC TC and 3′: TGT ATC TCG AGA ATT CAT GGC, representing coordinates 4017–4623 of MHV-68 genome, and cloned into XhoI/KpnI site of pCMV-Myc (Clontech) MCS in-frame with the Myc tag, these plasmids were designated pCMV-Myc-M2ΔP1 and pCMV-Myc-M2ΔP2. M2, M2ΔP1, and M2ΔP2 genes were subcloned from pCMV-Myc-M2, pCMV-Myc-M2ΔP1, and pCMV-Myc-M2ΔP2, respectively, into pCMV-HA (Clontech) MCS by digestion with the same restriction enzymes used for cloning into pCMV-Myc, and these plasmids were designated pCMV-HA-M2, pCMV-HA-M2ΔP1, and pCMV-HA-M2ΔP2, respectively. vav2 gene was subcloned from pACT2-Vav2 into pCMV-HA and pCMV-Myc MCS, in-frame with the HA and Myc tags, into the EcoRI sites, and these plasmids were designated by pCMV-HA-Vav2 and pCMV-Myc-Vav2, respectively.Retroviral Plasmids—M2 gene was PCR-amplified from the MHV-68 genomic DNA, M2ΔP1 gene was PCR-amplified from pGBT9-M2ΔP1 and M2ΔP2 gene was PCR-amplified from pGBT9-M2ΔP2, using the primers 5′: AAA GAA TTC ACA ATG GCA TCA ATG CAG AAG CTG ATC TCA GAG GAG GAC CTG GCC CCA ACA CCC CCA CAA GGA AGG AT and 3′: AAA CTC GAG TTA CTC CTC GCC CCA CTC CAC; the respective genes were cloned into the EcoRI/XhoI sites of pMSCV-Zeo MCS (28Stevenson P.G. Efstathiou S. Doherty P.C. Lehner P.J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8455-8460Crossref PubMed Scopus (192) Google Scholar), these plasmids were designated pMSCV-Zeo-M2, pMSCV-Zeo-M2ΔP1, and pMSCV-Zeo-M2ΔP2.Yeast Two-hybrid SystemAll experiments were performed in the yeast reporter strain Y190. The yeast vector pGBT9 is a Gal4 DNA-binding domain (Gal4BD) encoding vector that allows fusion of desired protein to the Gal4BD. The yeast vector pACT2 is a Gal4 activation domain (Gal4AD) encoding vector that allows fusion of desired protein to the Gal4AD. The yeast two-hybrid library screen and protocols were as recommended (Clontech).Cell Culture, Transfections, and Cell LinesWEHI-231 and WEHI-bclXL (described in Ref. 29Lam E.W. Glassford J. van der Sman J. Banerji L. Pizzey A.R. Shaun N. Thomas B. Klaus G.G. Thomas N.S. Eur. J. Immunol. 1999; 29: 3380-3389Crossref PubMed Scopus (23) Google Scholar were cultured in RPMI 1640 medium (Invitrogen) supplemented with 10% fetal calf serum, 2 mm glutamine, 100 units/ml penicillin/streptomycin and 50 μm β-mercaptoethanol, and were stimulated with 10 μg/ml of monoclonal anti-IgM (clone b.7.6 purchased from Cappel/ICN, Costa Mesa, CA). 293T cells were cultured in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% fetal calf serum, 2 mm glutamine, and 100 units/ml penicillin/streptomycin. For each transfection, 8 × 105 293T cells were seeded in 60-mm plates and incubated at 37 °C, 5% CO2 for 16 h. Cells were transfected with plasmid DNA using the FuGENE Reagent (Roche Applied Science) according to the manufacturer's instructions. The WEHI-231-M2, WEHI-231-M2ΔP1, and WEHI-231-M2ΔP2 cell lines were obtained by viral infection with high titer viral supernatants derived from 293T cells co-transfected with pMSCV-Zeo-M2, pMSCV-Zeo-M2ΔP1, or pMSCV-Zeo-M2ΔP2 retroviral vectors (28Stevenson P.G. Efstathiou S. Doherty P.C. Lehner P.J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8455-8460Crossref PubMed Scopus (192) Google Scholar) and the packaging vector pEQPAM3 (30Persons D.A. Mehaffey M.G. Kaleko M. Nienhuis A.W. Vanin E.F. Blood Cells Mol. Dis. 1998; 24: 167-182Crossref PubMed Scopus (72) Google Scholar) selected in zeocin (Invitrogen, UK) for 2 weeks. WEHI-bcl-XL-M2 cells were obtained by infection with retrovirus encoding for M2, as described above for WEHI-231-M2 cells. For growth curves, cells were seeded in triplicated flasks at about 0.4 × 106 cell/ml and cultured in growth medium in the presence of absence of 10 μg/ml of anti-IgM.Western Blot Analysis and AntibodiesWestern blot extracts were prepared by lysing cells with two times packed cell volume of Nonidet P-40 lysis buffer (1% Nonidet P-40, 150 mm NaCl, 20 mm Tris, pH 7.4, 10 mm NaF, 1 mm sodium orthovanadate, and Complete Mini protease inhibitors (Roche Applied Science)) on ice for 10 min. Protein yield was quantified by Dc protein assay (Bio-Rad). Cell lysates were size-fractionated by SDS-PAGE, transferred to Protran nitrocellulose membranes (Schleicher and Schuell, London, UK), and recognized by specific antibodies. The antibodies against pVav (Tyr-174), Vav1 (C-14), Vav2 (C-19), Bcl-XL (S-18), Bim (H-191), pRB (C-15), p107 (C-18), p130 (C-20), cyclin D2 (M-20), cyclin E (M-20), cyclin A (C-19), E2F1 (C-20), CDK2 (M2), CDK4 (C-22), CDK6 (C-21), and p27Kip1 (C-19) were purchased from Santa Cruz Biotechnology (Santa Crux, CA). The anti-phospho pRB (phosphorylation at the Ser-807/811 site is indicative of CDK4/6 activity) was purchased from Cell Signaling Technology (Hitchin, UK). Antibodies against phospho-44/42 MAPK (Thr-202/Tyr-204), total mitogen-activated protein kinase, phospho-AKT (Ser-473), and total AKT were purchased from Cell Signaling Technologies. The anti-GST-M2 serum was made by injecting GST-M2 protein into rabbits (AbCam) and exclusion of anti-GST-specific antibodies with glutathione Sepharose-GST beads. The antibody anti-B220-biotin was produced at the Gulbenkian Institute for Science, Oeiras, Portugal. The antibody anti-GST (27-4577-01) was purchased from Amersham Biosciences. The antibody against Rac clone 23A8 was purchased from Upstate. The antibodies were detected using horseradish peroxidase-linked anti-rabbit IgG-horseradish peroxidase, and anti-mouse IgG-horseradish peroxidase purchased from DAKO (Ely, UK), and visualized by the enhanced chemiluminescent (ECL) detection system (Amersham Biosciences).Immunoprecipitation and ImmunoblottingFor each immunoprecipitation, Cells were washed in phosphate-buffered saline and lysed with 1 ml Nonidet P-40 lysis buffer (1% Nonidet P-40, 150 mm NaCl, 20 mm Tris, pH 7.4, 10 mm sodium molybdate, 1 mm sodium orthovanadate, 5 mm iodoacetamide, 1 mm sodium fluoride, and Complete Mini protease inhibitors (Roche Applied Science)) for 15 min on ice. 293T cell lysates (500 μg) were pre-cleared for 1 h with protein G-Sepharose, then incubated with the antibody of interest for 1 h, and then with protein G-Sepharose for 1 h. Beads were washed four times with 1 ml of lysis buffer and resuspended in 50 μl of 2× SDS-PAGE loading buffer. Immunoprecipitates were then Western blotted as before.Cell Cycle AnalysisCell cycle analyses were performed by propidium iodide staining as described previously (29Lam E.W. Glassford J. van der Sman J. Banerji L. Pizzey A.R. Shaun N. Thomas B. Klaus G.G. Thomas N.S. Eur. J. Immunol. 1999; 29: 3380-3389Crossref PubMed Scopus (23) Google Scholar). Briefly, cells were washed with phosphate-buffered saline and fixed in 90% ethanol, 10% phosphate-buffered saline. Following fixation, cells were washed again and then incubated with 500 μg/ml DNase-free RNase A (Sigma) and 20 μg/ml propidium iodide (Sigma) for 30 min at 37 °C prior to analysis using a FACScan flow cytometer (BD Biosciences).GST-PAK Pull-down AssaysWEHI-231, WEHI-231-M2, WEHI-231-M2ΔP1, or WEHI-231-M2ΔP2 cells (5 × 106) were lysed in 750 μl of Nonidet P-40 lysis buffer (1% Nonidet P-40, 150 mm NaCl, 20 mm Tris-HCl, pH 7.4, 10 mm NaMoO4, 1 mm NaVO4, 1 mm NaF, 5 mm iodoacetamide, 1 tablet of Complete mini protease inhibitors (Roche Applied Science), per 10 ml of buffer). Cell lysates were incubated with 30 μl of glutathione-agarose beads and 5 μg of GST-PAK protein, at 4 °C for 1 h with rotation. Pull down assays were analyzed by Western blotting as described previously. The levels of pulled-down Rac were measured by band densitometry using the program, Image J (National Institutes of Health).RESULTSM2 Interacts with Vav1 and Vav2 Guanine Nucleotide Exchange Factors in the Yeast Two-hybrid Screens—M2 is a unique viral protein with no known mammalian or viral domain homology. To determine its function and cellular targets, we performed a yeast two-hybrid screen using M2 as bait. Full-length M2 protein was fused in-frame with the DNA binding domain of the GAL4 (GAL4-BD) yeast transcription factor and used as a bait to screen a cDNA library derived from MHV-68 latently infected mouse splenic B-lymphocytes fused with the activation domain of GAL4 (GAL4-AD) (TABLE ONE). A total of 8.7 × 107 transformants was analyzed in two independent rounds of screening. Of the seven positive clones selected for sequence analysis, four were found to contain the C-terminal region of Vav1 (amino acids 532–845), encoding for a fragment of the zinc finger (ZF) region and the entire SH3-SH2-SH3 domains, following comparison with the nucleotide data base at the National Library of Medicine using the BLAST algorithm (TABLE ONE). The other three clones did not match any of the published cDNAs, and their identities remained undefined. The Vav family of guanine nucleotide exchange factors is composed of three highly homologous proteins, namely Vav1, -2, and -3 (31Turner M. Adv. Exp. Med. Biol. 2002; 512: 29-34Crossref PubMed Scopus (4) Google Scholar). Additional interaction experiments using the yeast two-hybrid system showed that M2 is also able to bind to the C-terminal region of Vav2 (amino acids 536–868) encoding for the SH3-SH2-SH3 domains but not to Vav3.TABLE ONEScreening of an MHV-68 latently infected B-cell cDNA library with the latency-associated M2 protein Yeast cells (Y190) sequentially transformed with M2 in-frame with Gal4BD in pGBT9 (pGBT9-M2) and a spleen B-cell cDNA library in pACT2 (pACT2-cDNA/B220+) were selected on –Trp, –Leu, –His medium containing 50 mm 3-AT. Colonies growing in the selection medium were further selected by the expression of β-galactosidase by the filter lift assay as described by the manufacturer (Clontech). Clones were sequenced by a 377 DNA sequencer (ABI Prism).B-cell cDNA libraryGal4AD cloneFirst screenpACT2-unknown cDNApACT2-unknown cDNApACT2-Vav1-SH (a.a. 532-845)aa.a., amino acids.pACT2-Vav1-SH (a.a. 532-845)pACT2-Vav1-SH (a.a. 532-845)Second screenpACT2-unknown cDNApACT2-Vav1-SH (a.a. 532-845)a a.a., amino acids. Open table in a new tab The PXXP Motifs Located at the C Terminus of the Viral Protein Are Important for M2-Vav Interaction—After confirming interaction of Vav1 and -2 with M2, we examined the amino acid sequence of M2 required for binding to Vav. We observed that M2 protein contains 9 PXXP motifs, where P represents a proline residue and X any amino acid. These loosely defined motifs have been proposed to have a role in binding to signaling molecules through their SH3 domains (32Alexandropoulos K. Cheng G. Baltimore D. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3110-3114Crossref PubMed Scopus (250) Google Scholar). Given that the C terminus region of Vav proteins contain two SH3 domains, it is possible these moieties are mediating the M2 binding. To determine if these PXXP motifs were important for binding to Vav, we constructed several M2 deletion mutants lacking specific PXXP motifs. These mutants were cloned in-frame with the binding domain of GAL4 and used in a yeast two-hybrid experiment to study for their interactions with Vav1 and Vav2 SH3-SH2-SH3 regions. These results showed that amino acids 160–179 of M2 are essential for Vav1 and Vav2 SH3-SH2-SH3 binding (Fig. 1). This region of M2 contains three PXXP motifs, at amino acid residues 162–165, 167–170, and 174–176. To further investigate the importance of these motifs in Vav binding, we generated two M2 mutants in which the proline residues at positions 167 and 170 (M2ΔP1 mutant) and 165, 167, 170, and 174 (M2ΔP2 mutant) were replaced by alanine residues. As shown in Fig. 1, the M2ΔP1 and M2ΔP2 mutations partially or totally abrogated the binding of M2 to Vav1 and Vav2, respectively. These results demonstrated that the three PXXP motifs located at the C terminus of M2 are essential for binding to the SH3-SH2-SH3 region of both Vav1 and Vav2 proteins.M2 Interacts with Vav1 and Vav2 Proteins in Vivo—We next studied the in vivo interaction between M2 and Vav proteins in mammalian cells by co-immunoprecipitation (Fig. 2, A and B). To this end, 293T cells were transiently transfected with Vav mammalian expression vectors either alone or in combination with M2, M2ΔP1, or M2ΔP2. 48 h after transfection, cell lysates were immunoprecipitated with either an anti-M2 serum or with the pre-immune serum. The immunoprecipitates were then resolved by SDS-PAGE and immunoblotted with anti-Vav antibodies. As shown in" @default.
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• W1974967315 date "2005-11-01" @default.
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• W1974967315 title "Murine γ-Herpesvirus 68 Latency Protein M2 Binds to Vav Signaling Proteins and Inhibits B-cell Receptor-induced Cell Cycle Arrest and Apoptosis in WEHI-231 B Cells" @default.
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• W1974967315 doi "https://doi.org/10.1074/jbc.m507478200" @default.
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