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Mechanisms of Signal Transduction: BRCA1 Interaction with Human Papillomavirus Oncoproteins

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Mechanisms of Signal Transduction: BRCA1 Interaction with Human Papillomavirus Oncoproteins
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  Schlegel and Eliot M. RosenYongxian Ma, Pragati Katiyar, Richard Yiyu Zhang, Saijun Fan, Qinghui Meng,  Papillomavirus OncoproteinsBRCA1 Interaction with Human Mechanisms of Signal Transduction: doi: 10.1074/jbc.M505124200 srcinally published online June 27, 2005 2005, 280:33165-33177.J. Biol. Chem. 10.1074/jbc.M505124200Access the most updated version of this article at doi: .JBC Affinity SitesFind articles, minireviews, Reflections and Classics on similar topics on the  Alerts: When a correction for this article is posted• When this article is cited• to choose from all of JBC's e-mail alertsClick here   http://www.jbc.org/content/280/39/33165.full.html#ref-list-1This article cites 57 references, 26 of which can be accessed free at   b  y g u e  s  t   onM a r  c h 2 2  ,2  0 1 4 h  t   t   p :  /   /   w w w . j   b  c  . or  g /  D o wnl   o a  d  e  d f  r  om  b  y g u e  s  t   onM a r  c h 2 2  ,2  0 1 4 h  t   t   p :  /   /   w w w . j   b  c  . or  g /  D o wnl   o a  d  e  d f  r  om  BRCA1InteractionwithHumanPapillomavirusOncoproteins * Received for publication,May 10, 2005, and in revised form, June 22, 2005  Published, JBC Papers in Press,June 27, 2005, DOI 10.1074/jbc.M505124200 YiyuZhang ‡ ,SaijunFan ‡ ,QinghuiMeng ‡ ,YongxianMa ‡ ,PragatiKatiyar ‡ ,RichardSchlegel § ,andEliotM.Rosen ‡1 FromtheDepartmentsof  ‡ Oncologyand  § Pathology,LombardiComprehensiveCancerCenter,GeorgetownUniversity,Washington,D.C.20057-1469 Previously, we reported that BRCA1 strongly represses the tran-scriptional activity of estrogen receptor-  (ER-  ) in human breastand prostate cancer cells but only weakly inhibits ER-  in cervicalcancer cells. We now report that introduction of the human papil-lomavirus  E7  or  E6  oncogenesintohumanpapillomavirus-negativecellsrescuestheBRCA1repressionofER-  activityandthattheE7andE6oncoproteinsinteractdirectlywithBRCA1 invitro andasso-ciatewithBRCA1 invivo inculturedcells.ThisinteractioninvolvesatleasttwocontactpointsonBRCA1,onewithinanN-terminalsiteshown previously to interact with ER-  and the other in a C-termi-nal region of BRCA1 containing the first BRCA1 C-terminaldomain. Point mutations within the zinc finger domains of E7 andE6inactivatedthebindingtotheNterminusofBRCA1andreducedtheir ability to rescue BRCA1 inhibition of ER-  . E6 and E7 alsoantagonized the ability of BRCA1 to inhibit c-Myc E-box-mediatedtransactivation and human telomerase reverse transcriptase pro-moter activity, in a manner dependent upon the zinc fingerdomains. Finally, the ability of E6 and E7 to antagonize BRCA1 didnot involve proteolytic degradation of BRCA1. These findings sug-gestfunctionalinteractionsofBRCA1withE7andE6.Thepotentialsignificance of these findings is discussed. Mutations of the breast cancer susceptibility gene 1 (  BRCA1 ) 2 (chro-mosome 17q21) are linked to a high risk for breast and ovarian cancersinhereditaryearlyonsetbreastandbreast-ovariancancerfamilies(1,2).ThesemutationsalsoconferandincreasedriskforthesecancertypesinAshkenaziJewishwomenunselectedforafamilyhistoryofcancer(3).AlargestudyofcancerriskinBRCA1cancerfamiliesinEuropeandNorthAmericarevealedthatBRCA1mutationcarriersarealsoatsignificantly increasedriskforthedevelopmentofseveralothercancertypes,includ-ingpancreaticcancer,uterinecancer,cervicalcancer,andprostatecan-cer(inmenyoungerthanage65)(4).Forcervicalcancer,therelativeriskof BRCA1 mutation carriers compared with noncarriers was 3.72 (95%confidenceinterval  2.26–6.10,  p  0.001,two-sidedtest).Asubsetof patientswithsporadicinvasivecervicalcancershowshypermethylationof the BRCA1 promoter (5), as do patients with sporadic breast andovarian cancers (6, 7). BRCA1 promoter methylation may predict aworse prognosis in cervical cancer (8), although this point requires fur-ther study. An earlier and smaller study of cancer incidence in the rela-tives of BRCA1 and BRCA2 mutation carriers revealed about a 4-foldincreasedriskofcervicalcancerinBRCA2-associatedfamilies,althoughthe risk in BRCA1 families was not similarly elevated (9). Most interest-ingly,lossofheterozygosityatchromosome17q,asitethatcontainsthe  BRCA1  gene, appears to be a common event in cervical cancer (10).Previously, we found that the overexpression of BRCA1 inhibits theestrogen (E 2 )-induced transcriptional activity of the estrogen recep-tor-  (ER-  ) and inhibits E 2 -stimulated gene expression (11–13). Mostunexpectedly,theabilityofBRCA1torepressER-  activitywasfoundtobe cell type-specific. Thus, BRCA1 virtually abolished E 2 -stimulatedER-   activity in four of four human breast cancer (MCF-7, T47D,MDA-MB-231, and MDA-MB-453) and four of four human prostatecancer (DU-145, LNCaP, PC-3, and TsuPr-1 (now known to be derivedfrom a bladder cancer)) cell lines; however, BRCA1 caused only a mod-est or no reduction of ER-   activity in four of four human cervical celllines (HeLa, SiHa, CaSki, and C33A) (11). Moreover, BRCA1 overex-pression inhibited the E 2 -stimulated activity of activation function-2,theligand-inducibletranscriptionalactivationdomainofER-  inbreastand prostate cancer cells but not in cervical cancer cell lines (11, 14).Three of the four cervical cancer cell lines that we studied (HeLa, SiHa,and Caski) are known to contain integrated oncogenic human papillo-mavirus (HPV) genomes: HPV-16 for SiHa and CaSki and HPV-18 forHeLa(15,16).ThetwomajorHPVoncoproteins,E7andE6,functioninpartbyinactivatinghostcelltumorsuppressorproteins,retinoblastoma1 (RB1) (and other retinoblastoma family proteins) and p53 (17). Takentogether, these findings raised the possibility that HPV proteins may functionally inactivate BRCA1 in cervical cancer cells.ThepurposeofthisstudywastoevaluatethehypothesisthattheHPV oncoproteins E6 and E7 can interact with and inactivate the function of BRCA1. MATERIALSANDMETHODS CellLinesandCulture Humanbreastcancer(MCF-7andT47D),prostatecancer(DU-145),and cervical cancer (SiHa, Caski, and HeLa) cell lines and 293T humanembryonalkidneycellswereobtainedfromtheAmericanTypeCultureCollection (Manassas, VA) and cultured as described before (11–14).Briefly, the cells were grown in Dulbecco’s modified Eagle’s medium(DMEM) supplemented with 5 or 10% (v/v) fetal calf serum,  L -gluta-mine (5 m M ), nonessential amino acids (5 m M ), penicillin (100 units/ml), and streptomycin (100   g/ml). All cell culture reagents wereobtained from BioWhittaker, Walkersville, MD). ExpressionVectorsandReporters  Expression Vectors for GST Proteins —The GST-E7 (HPV16, wild-type, full-length) expression plasmid was a gift from Dr. Karl Munger *  This work was supported in part by United States Public Health Service Grants R01-CA80000,R01-CA82599,andR01-ES09169andbyResearchGrantsBASIC99–003255and BCTR0201295 from the Susan G. Komen Breast Cancer Foundation (to E. M. R.). The costs of publication of this article were defrayed in part by the payment of pagecharges.Thisarticlemustthereforebeherebymarked“ advertisement  ”inaccordancewith 18 U.S.C. Section 1734 solely to indicate this fact. 1  To whom correspondence should be addressed: Dept. of Oncology, Lombardi Com-prehensive Cancer Center, Georgetown University, Preclinical Sciences Bldg., Rm.GM12B, 3970 Reservoir Rd., NW, Box 571469, Washington D. C. 20057. Tel.: 202-687-7695; Fax: 202-787-7256; E-mail: emr36@georgetown.edu. 2  The abbreviations used are: BRCA1, breast cancer susceptibility gene 1; BRCA2, breastcancersusceptibilitygene2;BRCT,BRCA1C-terminaldomain;E 2 ,17  -estradiol;ER-  ,estrogen receptor-  ; ERE-TK-Luc, estrogen-responsive luciferase reporter plasmid;GST, glutathione-sulfotransferase; hTERT, human telomerase reverse transcriptase(catalytic subunit of telomerase); HPV, human papillomavirus; IP, immunoprecipita-tion;IVT, in vitro transcribedandtranslated;L  X  C  X  E,consensusretinoblastomafamilyprotein-bindingsite;RB1,retinoblastomasusceptibilitygene1;wt,wildtype;DMEM,Dulbecco’s modified Eagle’s medium; HPV, human papillomavirus; siRNAs, smallinterfering RNAs.  THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 280, NO. 39, pp. 33165–33177, September 30, 2005© 2005 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A. SEPTEMBER 30, 2005• VOLUME 280•NUMBER 39  JOURNAL OF BIOLOGICAL CHEMISTRY   33165   b  y g u e  s  t   onM a r  c h 2 2  ,2  0 1 4 h  t   t   p :  /   /   w w w . j   b  c  . or  g /  D o wnl   o a  d  e  d f  r  om  (Dept. of Pathology, Harvard Medical School, Boston). The GST-E6(HPV16, wild-type, full-length) plasmid was a gift from Dr. Peter M.Howley (Dept. of Pathology, Harvard Medical School) (18). The GST-E7-(2–38), -(16–98), and -(38–98) vectors and the GST-E6-(2–40),-(16–83), and -(80–151) vectors were generated by PCR cloning.Briefly,BamHIandEcoRIdigestionsitesweredesignedonthe5  and3  primers,respectively;andtheBamHIandEcoRIdoubledigestionprod-ucts were inserted into BamHI and EcoRI site of the pGEX2T vector(Amersham Biosciences). The GST-E7 C91G expression vector, whichencodes a full-length chimeric E7 protein with an inactivating pointmutation of the C-terminal zinc finger domain, was provided by Dr.Tony Kouzarides (Wellcome/CR UK Gurdon Institute, CambridgeUniversity, Cambridge, UK) (19). The GST-E6 C66,136G vector, whichencodes a full-length chimeric E6 protein with an inactivating pointmutation in both zinc finger domains, was generated by PCR cloning,using the pBS-E6 C66,136G plasmid (a gift from Dr. Denise A. Gallo-way, Fred Hutchinson Cancer Research Center, Seattle, WA (20)) as atemplate.TheBamHIandEcoRIdoubledigestionproductwasinsertedinto the BamHI and EcoRI site of pGEX2T vector. Expression vectorsfor GST-BRCA1 protein fragments corresponding to BRCA1 aminoacids 1–324, 260–553, 502–802, 758–1064, 1005–1313, and 1314–1863werekindlyprovidedbyDr.ToruOuchi(RuttenbergCancerCen-ter,MountSinaiSchoolofMedicine,NewYork).Theseconstructshavebeen described earlier (21).  Expression Vectors for in Vitro Translation and/or Expression within Mammalian Cells —The wild-type BRCA1 expression vector(wtBRCA1)wascreatedbycloningtheBRCA1cDNAintothepcDNA3 vector (Invitrogen) by using artificially engineered 5   HindIII and 3  NotI sites (22). cDNAs for BRCA1-(1–1313), -(1–771), -(1–302), -(34–300), -(67–300), -(101–300), and -(134–300) within the pcDNA3 orpCMV-Tag2 vector (Stratagene, La Jolla, CA) have been described ear-lier(12,23,24).TheBRCA1-(1–320)cDNAfragmentplasmidwasgen-eratedbyPCRcloning,andtheBamHIandXbaIdoubledigestionprod-uct was inserted into the BamHI and XbaI site of the pcDNA3 vector.TheBRCA1-(310–806),-(802–1314),-(1314–1863),and-(1532–1749)cDNA fragments were generated by PCR cloning, followed by BamHIandEcoRIdoubledigestionandinsertionintotheBamHIandEcoRIsiteof pcDNA3 vector. The pcDNA3 FLAG-E7 expression vector was gen-erously provided by Dr. Tony Kouzarides. The pcDNA3-E6 expression vector has been described earlier (25). The p3XFLAG-E6 andp3XFLAG-E6-(C66,136G) expression vectors were generated by PCRcloning of the FLAG-E6 cDNAs, followed by HindIII and XbaI doubledigestion and insertion into the HindIII and XbaI site of the p3XFLAG vector (Sigma). The FLAG-E7-(C91G) expression plasmid was createdinasimilarmanner.ThecorrectinsertionofthecDNAswasconfirmedby sequencing of each of the subcloned plasmids. The ER-  expression vector pCMV-ER-  was used to express ER-  in transient transfectionassays of estrogen receptor transcriptional activity (12).Expression vectors encoding the wt adenovirus E1A-(243R) proteinand various mutant or truncated E1A-(243R) proteins (including singlepoint mutations RG2 and 928m; the double point mutants RG2/928mandYH47/928m;andthedeletionmutants  15–35and  38–67)wereprovided by Dr. Richard G. Pestell (Lombardi Comprehensive CancerCenter, Georgetown University, Washington, D. C.) (26). Expression vectors encoding the wild-type SV40 large T oncoprotein (SV40T) anda mutant protein defective for RB family protein binding (mut-SV40T)(27) were also provided by Dr. Pestell.  Reporters —The estrogen-responsive reporter ERE-TK-Luc is com-posed of the vitellogenin A2 estrogen-responsive enhancer (ERE), con-trolling a minimal thymidine kinase promoter (TK81) and luciferase, inplasmid pGL2 (28). Assays of ER-  transcriptional activity utilizing theERE-TK-Luc reporter are described below. The E-box-Luc reportercontainsacanonicalc-MycE-boxupstreamofaminimalpromoterandluciferase (29). Its activity is stimulated by c-Myc and inhibited by thec-Myc/Max repressor Mad1 (29). The hTERT-Luc reporter consists of the core human telomerase reverse transcriptase (hTERT) promoterupstream of the luciferase gene, within the pGL3 plasmid (29).  AssaysofEstrogen-dependentTranscriptionalActivity  Subconfluent proliferating cells in 24-well dishes were incubatedovernight with 0.25   g of each indicated vector in serum-free DMEMcontaining Lipofectamine TM (Invitrogen). The total transfected DNAwas kept constant by addition of the appropriate control vectors. Thecells were washed, incubated in phenolphthalein-free DMEM contain-ing5%charcoal-strippedserum(obtainedfromtheTissueCultureCoreFacility of the Lombardi Comprehensive Cancer Center) (0.2 ml perwell)  17  -estradiol (E 2 , 1   M  or 10 n M , as indicated) for 24 h, andharvested for luciferase assays. To control for transfection efficiency,plasmid pRSV-  -gal was co-transfected to allow normalization of lucif-erase values to   -galactosidase activity in the same sample. Values aremeans  S.E. of four replicate wells and are representative of two ormore independent experiments. Immunoprecipitation To study the association of the endogenous BRCA1 and oncoproteinE7, proliferating SiHa cells at about 80% of confluence in 100-mm plas-tic dishes were harvested; and whole cell extracts were prepared asdescribedbefore(12,14,24),usingRIPAbuffer.EachIPwascarriedoutasdescribedbeforebyusing10  gofantibody(anti-BRCA1(I-20,SantaCruz Biotechnology, Santa Cruz, CA), anti-E7 (ED-17, Santa Cruz Bio-technology), or nonimmune rabbit or mouse IgG (negative control))and 500  g of total cell extract protein. The precipitated proteins werecollected using protein A/G Plus-agarose beads (Santa Cruz Biotech-nology) and eluted using boiling Laemmli sample buffer. The elutedproteins were then subjected to Western blotting to detect the BRCA1or E7 proteins, as described below.To study the interaction of BRCA1 with E6, subconfluent proliferat-ing 293T cells were transfected overnight with the p3XFLAG-E6 andwtBRCA1 expression vectors (see above) using Lipofectamine TM ,washed, post-incubated for 24 h to allow gene expression, harvested,and then immunoprecipitated using anti-BRCA1 (as above) or nonim-mune rabbit IgG (control). The precipitated proteins were collectedusing protein A/G Plus-agarose beads or anti-FLAG M2 affinity gel(Sigma),washed,eluted,andsubjectedtoWesternblottingtodetecttheBRCA1 protein or the FLAG epitope (see below). WesternBlotting Westernblottingwascarriedoutasdescribedbefore(23,24).TheIPs(seeabove)wereelectrophoresedona4–12%SDS-polyacrylamidegra-dientgel,transferredtonitrocellulosemembranes(Millipore),andblot-ted using primary antibodies directed against BRCA1 (C-20, rabbitpolyclonal, Santa Cruz Biotechnology, 1:200); E7 (TVG710Y, SantaCruz Biotechnology), or the FLAG epitope (M2, mouse monoclonalantibody, Sigma, 1:500 dilution). As a control, nonprecipitated lysates(50  gofcellprotein)wereblottedonthesamegels.Theblottedproteinbands were visualized using the enhanced chemiluminescence (ECL)detection system (Amersham Biosciences), with colored markers (Bio-Rad) as size standards.In a separate experiment, cells were transfected overnight with wild-type or mutant FLAG-E6 or FLAG-E7, post-incubated for 24 h to allow gene expression, and Western-blotted (50   g of total cell protein perlane) to detect BRCA1 (C-20 antibody), E6 protein (anti-HPV16/18 E6, BRCA1InteractionwithHPV  33166  JOURNAL OF BIOLOGICAL CHEMISTRY   VOLUME 280•NUMBER 39• SEPTEMBER 30, 2005   b  y g u e  s  t   onM a r  c h 2 2  ,2  0 1 4 h  t   t   p :  /   /   w w w . j   b  c  . or  g /  D o wnl   o a  d  e  d f  r  om  Abcam Ltd.), FLAG-E7 (M2, Sigma), p53 (polyclonal antibody 240,Santa Cruz Biotechnology) (which detects both wild-type and mutantp53),RB1(C-15antibody,SantaCruzBiotechnology),and  -actin(I-19antibody, Santa Cruz Biotechnology) (control for loading and transfer). GSTCaptureAssays GST bead assays were performed essentially as described earlier (12,14, 24).  In vitro  translated (IVT) proteins were prepared by   in vitro transcription and translation, using the T7 promoter of the pcDNA3 vector or the T3 promoter of the pCMV-Tag2B vector. The proteinswere labeled using [ 35 S]methionine (Amersham Biosciences) orTransend TM tRNA (Promega Corp., Madison, WI), and  in vitro  tran-scription-translation was carried out using the T N T-coupled rabbitreticulocyte lysate system (Promega), according to the manufacturer’sinstructions. The GST fusion proteins were generated from cDNAsclonedintothepGEX2Tvector,expressedin  Escherichiacoli ,andpuri-fied by affinity chromatography using glutathione-Sepharose (Amer-shamBiosciences).Thesourceorconstructionoftheexpressionvectorsfor various GST-BRCA1, GST-E7, and GST-E6 proteins is describedabove.LabeledIVTproteinswereincubatedwithGSTprotein(negativecontrol) or GST fusion proteins for 4 h at 4 °C, recovered using GSH-agarose beads, eluted in boiling sample buffer, and analyzed by SDS-PAGE autoradiography. In all GST capture assays, the IVT input lanesshow 10% of the protein quantity used in the GST capture assays. Allexperiments included a lane corresponding to capture by beads coatedwith GST alone, as a negative control. The GST fusion proteins were visualized by Western blotting, using anti-GST mouse monoclonalantibody 27-4577-01 (Amersham Biosciences, 1:5000). Additionaldetailsrelevanttospecificexperimentsareprovidedinthetextorfigurelegends. KnockdownofBRCA1UsingsiRNAs The BRCA1 and corresponding control (scrambled sequence) siR-NAs have been described earlier (29). All siRNAs were chemically synthesized by Dharmacon, Inc. The sequences used to synthesizethe siRNAs were as follows: BRCA1 siRNA, 5  -AATGCCAAAG-TAGCTAATGTA-3  ,andcontrolsiRNA,5  -CGATAGATACACA-GATTGAAT-3  .For siRNA treatments, subconfluent proliferating cells were trans-fected with 50 n M  of siRNA using the siPORT Amine transfection rea-gent(Ambion).Themaximalreductionofproteinlevelsrequireda72-hincubation with the siRNA. Prior studies established that under theseconditions, none of the siRNAs caused cytotoxicity, based on cell mor-phologyand3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbro-mide (MTT) assays. StatisticalMethods Whereappropriate,statisticalcomparisonsweremadeusingthetwo-tailed Student’s  t   test. FIGURE 1. RescueofBRCA1inhibitionofER-  transcriptionalactivitybytheHPVE7andE6andbyotherDNAtumorviraloncoproteins.  A ,domainstructureoftheDNAtumorvirus oncoproteins studied.  B,  rescue of wtBRCA1 repression of ER-  activity by  HPV-E7   and  HPV-E6  oncogenes.  C  , rescue of wtBRCA1 repression of ER-  activity by adenovirus  E1A oncogene. D, rescueofwtBRCA1repressionofER-  activitybytheSV40largeToncogene. E, wtBRCA1onlyminimallyrepressesER-  activityinhumancervicalcancercelllines.Formethodology, subconfluent proliferating cells in 24-well dishes were transfected overnight with the indicated expression vectors and an estrogen-responsive reporter plasmid(ERE-TK-Luc)(0.25  gDNAforeachplasmid)usingLipofectamine  TM ,asdescribedunder“MaterialsandMethods.”Thecellswerewashed,incubatedwithoutorwith17  -estradiol(E 2 ,10 n M  or 1  M ) for  t   24 h, and harvested for luciferase assays. Luciferase activity was expressed relative to the negative control ( 0 E  2 ) ( B–D ) or as a percentage of the  E 2  positivecontrol( E  ).Thevaluesareexpressedasmeans  S.E.offourreplicatewells.Eachexperimentwasperformedatleasttwicetoensurereproducibilityofthefindings.Abbreviationsandacronyms are as follows:  CDK2 , cyclin-dependent kinase 2;  CR1/2 , conserved region 1/2;  CtBP  , C-terminal binding protein;  C-TERM , C-terminal domain; C  XX  C, one-half of a zinccoordinationmotif; cycA ,cyclinA; ETQL ,PDZfamilyprotein-bindingmotif; hDLG ,homologofdiscslarge, Drosophila ; hScrib ,homologofScribble, Drosophila ; HR ,hostrangedomain; Hsc70 , heat shock cognate protein 70 kDa; L  X  C  X  E, consensus retinoblastoma family protein-binding motif;  MAGI-1 , membrane-associated guanylate kinase inverted-1;  MUPP-1 ,multiple PDZ domain protein;  mut-SV40T  , mutant SV40 large T oncogene with defective for RB-binding;  NLS,  nuclear localization signal;  N-TERM , N-terminal domain;  PCAF  , p300/CBP-associated factor;  PLDLS , CtBP-binding motif;  RNA pol II,  RNA polymerase II;  SV40T  , simian virus 40 large T oncogene;  TBP  , TATA box-binding protein;  TEF-1 , transcriptionalenhancer factor-1;  TFIIB , transcription factor IIB;  wt  , wild-type. BRCA1InteractionwithHPV  SEPTEMBER 30, 2005• VOLUME 280•NUMBER 39  JOURNAL OF BIOLOGICAL CHEMISTRY   33167   b  y g u e  s  t   onM a r  c h 2 2  ,2  0 1 4 h  t   t   p :  /   /   w w w . j   b  c  . or  g /  D o wnl   o a  d  e  d f  r  om  RESULTS  Rescue (Reversal) of the BRCA1-mediated Repression of ER-   Tran- scriptional Activity by HPV Oncoproteins E7 and E6  —We tested theability of several different DNA tumor virus-encoded transformingoncoproteins to rescue (reverse) the BRCA1-mediated repression of ER-   activity, including HPV oncoproteins E6 and E7, the adenovirusE1A oncoprotein, and the SV40 large T protein. Schematic diagramsshowing the domain structure of these proteins are provided in Fig. 1A.We utilized a standard transient transfection assay of ER-   transcrip-tional activity using an estrogen-responsive reporter, ERE-TK-Luc (12,14, 24). As illustrated in Fig. 1  B , in the absence of E7 or E6, exogenouswtBRCA1 reduced the estrogen (E 2 )-stimulated reporter activity to lessthan 1% of the control value (  p    0.001, two-tailed  t   test) in humanbreast (MCF-7 and T47D) and prostate (DU-145) cancer cells, whereastheemptypcDNA3vectorhadlittleornoeffectonthereporteractivity.In contrast, expression vectors encoding the E7 and E6 oncoproteinsfrom HPV-16 (an oncogenic HPV strain) rescued the wtBRCA1-medi-ated repression of ER-   activity in HPV-negative human breast andprostate cancer cell lines (Fig. 1  B ).Besides E7 and E6, several other DNA viral oncoproteins were alsoable to rescue the BRCA1 inhibition of ER-   transcriptional activity.Thus, in studies of DU-145 human prostate cancer and T47D breastcancer cells, the adenovirus E1A-(243R) and SV40 large T oncogenesalsorescuedtheBRCA1inhibitionofER-  activity(  p  0.001)(Fig.1, C  and  D , respectively). The E1A-(243R) transforming protein is the prod-uct of an alternatively spliced mRNA, the full-length form of whichencodes the E1A-(289R) protein (30). E1A-(243R) differs from E1A-(289R) in that E1A-(243R) is missing a conserved region (CR3) that ispresent in E1A-(289R). Studies of a small series of mutant E1A-(243R)genesrevealedthatexpressionvectorsencodingproteinswithanN-ter-minal mutation or deletion (RG2,  38–67, RG2/928m, and  15–35)failedtorescuetheBRCA1repression,whereasthosecontainingseveralother mutations (928m and YH47/928m) retained the ability to reversethe BRCA1-mediated repression of ER-  activity (  p  0.001) (Fig. 1 C  ).E1A-(928m) encodes a mutant protein with a defective retinoblastoma(RB) protein binding domain, suggesting that the RB binding domain isdispensable for the rescue of BRCA1-mediated repression. Expression vectors encoding wild-type SV40 large T and a mutant with a defectiveRB binding domain both rescued the BRCA1 repression (  p    0.001)(Fig. 1  D ), again suggesting that interaction with RB family proteins(RB1, p107, and p130) is not required for rescue.Forreference,Fig.1  E  showstheeffectofwtBRCA1onER-  signalingin three human cervical cancer cell lines, each of which contains anoncogenic HPV genome (  HPV-16   or  HPV-18 ) as follows: CaSki, SiHa,and HeLa. As compared with the HPV-negative breast and prostatecancer cell lines for which wtBRCA1 caused a reduction in estrogen-stimulated ER-   activity to close to the basal levels observed in theabsence of estrogen (2–3-log reduction), wtBRCA1 only had a modesteffectinthecervicalcancercells,reducingER-  activitybyonly0–40%(well under a 1-log reduction). Our previous studies indicate thatwtBRCA1 is well expressed in these cell lines (14).  DirectInteractionofBRCA1withtheHPV-E6andHPV-E7Oncopro-teins and Mapping of Interacting Sites —The findings described abovesuggest that several different DNA viral transforming oncoproteins arecapable of inactivating at least one function of BRCA1, i.e. its ability toinhibitER-  activity.BecauseE1AandSV40largeTcellshavenotbeenimplicatedinhumancarcinogenesis,wechosetofurtherinvestigatethemechanism(s) by which HPV-E6 and HPV-E7 can inactivate BRCA1function. GST-E7 and GST-E6 Proteins Capture C-terminally Truncated  BRCA1 Proteins —We used GST capture assays to determine whetherBRCA1caninteractphysicallywiththeE7orE6proteins.ThestructureoftheBRCA1proteinsusedintheseassaysisillustratedschematicallyinFig. 2A. Full-length GST-E7 and GST-E6 proteins were expressed in  E. coli , and the expression of these GST fusion proteins was confirmedby Western blotting, using an anti-GST antibody (Fig. 2  B ). Fig. 2 C  shows the expression of various  35 S-labeled IVT BRCA1 proteins, as visualizedbySDS-PAGEautoradiography.GSTcaptureassaysrevealedthat the full-length (1863 amino acids) BRCA1 protein was captured by beadscoatedwithGST-E7orGST-E6butnotbybeadscoatedwithGSTalone (negative control) (Fig. 2  D ). In addition to the full-length BRCA1,severalIVTC-terminallytruncatedBRCA1proteins(BRCA1-(1–1313),-(1–771), and -(1–302)) were also captured by GST-E7 and by GST-E6(Fig. 2  E  ). These findings suggest a physical interaction of BRCA1 withtheE7andE6proteins.Theyalsosuggestthepresenceofacontactpointwithin the N terminus of BRCA1. CaptureofDifferentBRCA1ProteinFragmentsbyFull-lengthGST-E7 and GST-E6  —To further delineate the binding site(s) for E7 and E6 onthe BRCA1 protein, we tested the ability of GST-E7 and GST-E6 tocapture different IVT BRCA1 fragments across the full length of theBRCA1 protein and within the N-terminal amino acids 1–302 (see Fig.3A). Studies of four protein fragments spanning the length of BRCA1revealedaninteractionbetweentheN-andC-terminalBRCA1proteins(amino acids 1–302 and 1314–1863) with both E7 (Fig. 3  B ) and E6 (Fig.3 C  ). In each case, GST alone failed to capture any of the IVT BRCA1proteins. Fig. 3,  D  and  E,  shows the ability of E7 and E6, respectively, tocapture each of four different IVT N-terminal BRCA1 fragments.Whereas BRCA1-(34–300) and -(67–300) were captured by bothGST-E7 and GST-E6, neither E7 nor E6 captured BRCA1-(101–300)and -(134–300). These findings suggest that the BRCA1 N-terminalRING domain (amino acids 20–64) is not required for the interactionwith E7 or E6, but the interaction does require amino acids 67–100.Finally,BRCA1-(1532–1749),whichcontainsthefirstBRCA1C-termi-nalrepeat(BRCT)domain,wassufficienttomediateaninteractionwithE7 and with E6 (Fig. 3  F  ); however, we cannot rule out the possibility of an additional contact point(s) for E7 or E6 within amino acids 1314–1863 of BRCA1. Capture of Full-length IVT E7 and E6 by Different GST-BRCA1 Pro-teins —To substantiate further the validity of the BRCA1-E7 andBRCA1-E6 interactions, we performed the reverse capture experiment,i.e. we tested the ability of a set of overlapping GST-BRCA1 proteins tocapture the full-length IVT E7 protein. The construction of these GST-BRCA1 expression vectors has been described earlier (21). Consistentwith our previous results, both N-terminal (amino acids 1–324) andC-terminal (amino acids 1314–1863) BRCA1 proteins captured full-lengthIVTE7(Fig.4  A ).TheabilityofdifferentGST-BRCA1proteinstocapture full-length IVT E6 is shown in Fig. 4  B . Here again the C-termi-nal fragment (amino acids 1314–1863) and the N-terminal fragment(amino acids 1–324) showed significant capture of IVT E6, whereasseveral other fragments showed a much smaller degree of capture. Theexpression of the different GST-BRCA1 proteins, visualized by anti-GSTWesternblotting,isshowninFig.4 C  .Insomecases,smallerbands,which may reflect degraded GST-BRCA1 proteins or cross-reactingspecies, were observed. The correct bands are indicated in the figure.This Western blot is typical of a number of repeat experiments. Takentogether, we can demonstrate using bi-directional GST capture assaysthat E7 and E6 each interact with N- and C-terminal domains withinBRCA1. BRCA1InteractionwithHPV  33168  JOURNAL OF BIOLOGICAL CHEMISTRY   VOLUME 280•NUMBER 39• SEPTEMBER 30, 2005   b  y g u e  s  t   onM a r  c h 2 2  ,2  0 1 4 h  t   t   p :  /   /   w w w . j   b  c  . or  g /  D o wnl   o a  d  e  d f  r  om
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