IntAct European Bioinformatics Institute IntAct intact http://www.ebi.ac.uk/ http://www.ebi.ac.uk/intact/query/${ac} EBI-[0-9]+|IA:[0-9]+ INTerAction database (IntAct) provides an open source database and toolkit for the storage, presentation and analysis of molecular interactions. http://www.ebi.ac.uk/intact European Bioinformatics Institute; Wellcome Trust Genome Campus; Hinxton, Cambridge; CB10 1SD; United Kingdom http://www.ebi.ac.uk/intact/ HMGA2 induces pituitary tumorigenesis by enhancing E2F1 activity. HMGA2 induces pituitary tumorigenesis by enhancing E2F1 activity. Cancer Cell (1535-6108) 2006 imex curation Fedele M., Visone R., De Martino I., Troncone G., Palmieri D., Battista S., Ciarmiello A., Pallante P., Arra C., Melillo RM., Helin K., Croce CM., Fusco A. Chromatin - Epigenetic interactions resulting in chromatin modulation Apoptosis - Interactions involving proteins with a function related to apoptosis Cancer - Interactions investigated in the context of cancer The Excel sheet filled in by the M. Fedele and correspondence with Fedele, which provides information about the interactors, can be found in P:\pro4\intact\local\data\curation-material\fedele-2006. afusco@napoli.com 24-JAN-2007: Contacted by JYOTI. Only protein-protein interactions 8 mouse-pituitary-adenoma Mus musculus pituitary adenoma pituitary_adenoma pituitary adenoma anti bait coip anti bait coimmunoprecipitation anti bait coip western blot western blot Immuno blot Immuno blot Fedele M., Visone R., De Martino I., Troncone G., Palmieri D., Battista S., Ciarmiello A., Pallante P., Arra C., Melillo RM., Helin K., Croce CM., Fusco A. Chromatin - Epigenetic interactions resulting in chromatin modulation The Excel sheet filled in by the M. Fedele and correspondence with Fedele, which provides information about the interactors, can be found in P:\pro4\intact\local\data\curation-material\fedele-2006. afusco@napoli.com Accepted 2006-OCT-12 by DAVET. Cancer Cell (1535-6108) 2006 Only protein-protein interactions 8 imex curation HMGA2 induces pituitary tumorigenesis by enhancing E2F1 activity. HMGA2 induces pituitary tumorigenesis by enhancing E2F1 activity. Cancer Cell (1535-6108) 2006 imex curation Fedele M., Visone R., De Martino I., Troncone G., Palmieri D., Battista S., Ciarmiello A., Pallante P., Arra C., Melillo RM., Helin K., Croce CM., Fusco A. Chromatin - Epigenetic interactions resulting in chromatin modulation Apoptosis - Interactions involving proteins with a function related to apoptosis Cancer - Interactions investigated in the context of cancer The Excel sheet filled in by the M. Fedele and correspondence with Fedele, which provides information about the interactors, can be found in P:\pro4\intact\local\data\curation-material\fedele-2006. afusco@napoli.com 24-JAN-2007: Contacted by JYOTI. Only protein-protein interactions 8 human-293t Homo sapiens 293 cells transformed with SV40 large T antigen 293t 293 cells expressing SV40 large T antigen. The original designation of this cell line was 293tsA1609neo. It is not in Cabri,ATCC or HyperCLDB. anti bait coip anti bait coimmunoprecipitation anti bait coip western blot western blot Immuno blot Immuno blot Fedele M., Visone R., De Martino I., Troncone G., Palmieri D., Battista S., Ciarmiello A., Pallante P., Arra C., Melillo RM., Helin K., Croce CM., Fusco A. Chromatin - Epigenetic interactions resulting in chromatin modulation The Excel sheet filled in by the M. Fedele and correspondence with Fedele, which provides information about the interactors, can be found in P:\pro4\intact\local\data\curation-material\fedele-2006. afusco@napoli.com Accepted 2006-OCT-12 by DAVET. 24-JAN-2007: Contacted by JYOTI. Cancer Cell (1535-6108) 2006 Only protein-protein interactions 8 imex curation HMGA2 induces pituitary tumorigenesis by enhancing E2F1 activity. HMGA2 induces pituitary tumorigenesis by enhancing E2F1 activity. Cancer Cell (1535-6108) 2006 imex curation Fedele M., Visone R., De Martino I., Troncone G., Palmieri D., Battista S., Ciarmiello A., Pallante P., Arra C., Melillo RM., Helin K., Croce CM., Fusco A. Chromatin - Epigenetic interactions resulting in chromatin modulation Apoptosis - Interactions involving proteins with a function related to apoptosis Cancer - Interactions investigated in the context of cancer The Excel sheet filled in by the M. Fedele and correspondence with Fedele, which provides information about the interactors, can be found in P:\pro4\intact\local\data\curation-material\fedele-2006. afusco@napoli.com 24-JAN-2007: Contacted by JYOTI. Only protein-protein interactions 8 in vitro In vitro anti bait coip anti bait coimmunoprecipitation anti bait coip western blot western blot Immuno blot Immuno blot Fedele M., Visone R., De Martino I., Troncone G., Palmieri D., Battista S., Ciarmiello A., Pallante P., Arra C., Melillo RM., Helin K., Croce CM., Fusco A. Chromatin - Epigenetic interactions resulting in chromatin modulation The Excel sheet filled in by the M. Fedele and correspondence with Fedele, which provides information about the interactors, can be found in P:\pro4\intact\local\data\curation-material\fedele-2006. afusco@napoli.com Accepted 2006-OCT-12 by DAVET. 24-JAN-2007: Contacted by JYOTI. Cancer Cell (1535-6108) 2006 Only protein-protein interactions 8 imex curation HMGA2 induces pituitary tumorigenesis by enhancing E2F1 activity. HMGA2 induces pituitary tumorigenesis by enhancing E2F1 activity. Cancer Cell (1535-6108) 2006 imex curation Fedele M., Visone R., De Martino I., Troncone G., Palmieri D., Battista S., Ciarmiello A., Pallante P., Arra C., Melillo RM., Helin K., Croce CM., Fusco A. Chromatin - Epigenetic interactions resulting in chromatin modulation Apoptosis - Interactions involving proteins with a function related to apoptosis Cancer - Interactions investigated in the context of cancer The Excel sheet filled in by the M. Fedele and correspondence with Fedele, which provides information about the interactors, can be found in P:\pro4\intact\local\data\curation-material\fedele-2006. afusco@napoli.com 24-JAN-2007: Contacted by JYOTI. Only protein-protein interactions 8 human-293t Homo sapiens 293 cells transformed with SV40 large T antigen 293t 293 cells expressing SV40 large T antigen. The original designation of this cell line was 293tsA1609neo. It is not in Cabri,ATCC or HyperCLDB. anti tag coip anti tag coimmunoprecipitation anti tag coip western blot western blot Immuno blot Immuno blot Fedele M., Visone R., De Martino I., Troncone G., Palmieri D., Battista S., Ciarmiello A., Pallante P., Arra C., Melillo RM., Helin K., Croce CM., Fusco A. Chromatin - Epigenetic interactions resulting in chromatin modulation The Excel sheet filled in by the M. Fedele and correspondence with Fedele, which provides information about the interactors, can be found in P:\pro4\intact\local\data\curation-material\fedele-2006. afusco@napoli.com Accepted 2006-OCT-12 by DAVET. 24-JAN-2007: Contacted by JYOTI. Cancer Cell (1535-6108) 2006 Only protein-protein interactions 8 imex curation HMGA2 induces pituitary tumorigenesis by enhancing E2F1 activity. HMGA2 induces pituitary tumorigenesis by enhancing E2F1 activity. Cancer Cell (1535-6108) 2006 imex curation Fedele M., Visone R., De Martino I., Troncone G., Palmieri D., Battista S., Ciarmiello A., Pallante P., Arra C., Melillo RM., Helin K., Croce CM., Fusco A. Chromatin - Epigenetic interactions resulting in chromatin modulation Apoptosis - Interactions involving proteins with a function related to apoptosis Cancer - Interactions investigated in the context of cancer The Excel sheet filled in by the M. Fedele and correspondence with Fedele, which provides information about the interactors, can be found in P:\pro4\intact\local\data\curation-material\fedele-2006. afusco@napoli.com 24-JAN-2007: Contacted by JYOTI. Only protein-protein interactions 8 human-293t Homo sapiens 293 cells transformed with SV40 large T antigen 293t 293 cells expressing SV40 large T antigen. The original designation of this cell line was 293tsA1609neo. It is not in Cabri,ATCC or HyperCLDB. anti bait coip anti bait coimmunoprecipitation anti bait coip anti tag western anti tag western blot anti tag western Fedele M., Visone R., De Martino I., Troncone G., Palmieri D., Battista S., Ciarmiello A., Pallante P., Arra C., Melillo RM., Helin K., Croce CM., Fusco A. Chromatin - Epigenetic interactions resulting in chromatin modulation The Excel sheet filled in by the M. Fedele and correspondence with Fedele, which provides information about the interactors, can be found in P:\pro4\intact\local\data\curation-material\fedele-2006. afusco@napoli.com Accepted 2006-OCT-12 by DAVET. 24-JAN-2007: Contacted by JYOTI. Cancer Cell (1535-6108) 2006 Only protein-protein interactions 8 imex curation HMGA2 induces pituitary tumorigenesis by enhancing E2F1 activity. HMGA2 induces pituitary tumorigenesis by enhancing E2F1 activity. Cancer Cell (1535-6108) 2006 imex curation Fedele M., Visone R., De Martino I., Troncone G., Palmieri D., Battista S., Ciarmiello A., Pallante P., Arra C., Melillo RM., Helin K., Croce CM., Fusco A. Chromatin - Epigenetic interactions resulting in chromatin modulation Apoptosis - Interactions involving proteins with a function related to apoptosis Cancer - Interactions investigated in the context of cancer The Excel sheet filled in by the M. Fedele and correspondence with Fedele, which provides information about the interactors, can be found in P:\pro4\intact\local\data\curation-material\fedele-2006. afusco@napoli.com 24-JAN-2007: Contacted by JYOTI. Only protein-protein interactions 8 in vitro In vitro pull down pull down anti tag western anti tag western blot anti tag western Fedele M., Visone R., De Martino I., Troncone G., Palmieri D., Battista S., Ciarmiello A., Pallante P., Arra C., Melillo RM., Helin K., Croce CM., Fusco A. Chromatin - Epigenetic interactions resulting in chromatin modulation The Excel sheet filled in by the M. Fedele and correspondence with Fedele, which provides information about the interactors, can be found in P:\pro4\intact\local\data\curation-material\fedele-2006. afusco@napoli.com Accepted 2006-OCT-12 by DAVET. 24-JAN-2007: Contacted by JYOTI. Cancer Cell (1535-6108) 2006 Only protein-protein interactions 8 imex curation HMGA2 induces pituitary tumorigenesis by enhancing E2F1 activity. HMGA2 induces pituitary tumorigenesis by enhancing E2F1 activity. Cancer Cell (1535-6108) 2006 imex curation Fedele M., Visone R., De Martino I., Troncone G., Palmieri D., Battista S., Ciarmiello A., Pallante P., Arra C., Melillo RM., Helin K., Croce CM., Fusco A. Chromatin - Epigenetic interactions resulting in chromatin modulation Apoptosis - Interactions involving proteins with a function related to apoptosis Cancer - Interactions investigated in the context of cancer The Excel sheet filled in by the M. Fedele and correspondence with Fedele, which provides information about the interactors, can be found in P:\pro4\intact\local\data\curation-material\fedele-2006. afusco@napoli.com 24-JAN-2007: Contacted by JYOTI. Only protein-protein interactions 8 human-293t Homo sapiens 293 cells transformed with SV40 large T antigen 293t 293 cells expressing SV40 large T antigen. The original designation of this cell line was 293tsA1609neo. It is not in Cabri,ATCC or HyperCLDB. far western blotting far western blotting Affinity blotting Affinity blotting antibody detection identification by antibody antibody detection Fedele M., Visone R., De Martino I., Troncone G., Palmieri D., Battista S., Ciarmiello A., Pallante P., Arra C., Melillo RM., Helin K., Croce CM., Fusco A. Chromatin - Epigenetic interactions resulting in chromatin modulation The Excel sheet filled in by the M. Fedele and correspondence with Fedele, which provides information about the interactors, can be found in P:\pro4\intact\local\data\curation-material\fedele-2006. afusco@napoli.com Accepted 2006-OCT-12 by DAVET. 24-JAN-2007: Contacted by JYOTI. Cancer Cell (1535-6108) 2006 Only protein-protein interactions 8 imex curation hmga2_mouse High mobility group protein HMGI-C High mobility group AT-hook protein 2 Hmga2 Hmgic protein protein mouse Mus musculus Mouse MSARGEGAGQPSTSAQGQPAAPVPQKRGRGRPRKQQQEPTCEPSPKRPRGRPKGSKNKSPSKAAQKKAETIGEKRPRGRPRKWPQQVVQKKPAQETEETSSQESAEED 40A3E38F62268C09 rb_mouse Retinoblastoma-associated protein Rb1 Rb-1 pRb pp105 p110-RB1 protein protein mouse Mus musculus Mouse MPPKAPRRAAAAEPPPPPPPPPREDDPAQDSGPEELPLARLEFEEIEEPEFIALCQKLKVPDHVRERAWLTWEKVSSVDGILEGYIQKKKELWGICIFIAAVDLDEMPFTFTELQKSIETSVYKFFDLLKEIDTSTKVDNAMSRLLKKYNVLCALYSKLERTCELIYLTQPSSALSTEINSMLVLKISWITFLLAKGEVLQMEDDLVISFQLMLCVVDYFIKFSPPALLREPYKTAAIPINGSPRTPRRGQNRSARIAKQLENDTRIIEVLCKEHECNIDEVKNVYFKNFIPFINSLGIVSSNGLPEVESLSKRYEEVYLKNKDLDARLFLDHDKTLQTDPIDSFETERTPRKNNPDEEANVVTPHTPVRTVMNTIQQLMVILNSASDQPSENLISYFNNCTVNPKENILKRVKDVGHIFKEKFANAVGQGCVDIGVQRYKLGVRLYYRVMESMLKSEEERLSIQNFSKLLNDNIFHMSLLACALEVVMATYSRSTLQHLDSGTDLSFPWILNVLNLKAFDFYKVIESFIKVEANLTREMIKHLERCEHRIMESLAWLSDSPLFDLIKQSKDGEGPDNLEPACPLSLPLQGNHTAADMYLSPLRSPKKRTSTTRVNSAANTETQAASAFHTQKPLKSTSLALFYKKVYRLAYLRLNTLCARLLSDHPELEHIIWTLFQHTLQNEYELMRDRHLDQIMMCSMYGICKVKNIDLKFKIIVTAYKDLPHAAQETFKRVLIREEEFDSIIVFYNSVFMQRLKTNILQYASTRPPTLSPIPHIPRSPYKFSSSPLRIPGGNIYISPLKSPYKISEGLPTPTKMTPRSRILVSIGESFGTSEKFQKINQMVCNSDRVLKRSAEGGNPPKPLKKLRFDIEGADEADGSKHLPAESKFQQKLAEMTSTRTRMQKQRMNESKDVSNKEEK DIP protein P13405 original sequence version: 115 DIP protein P13405 original sequence version: 114 DIP protein P13405 original sequence version: 110 AE81D35A07ADB493 rb_human Retinoblastoma-associated protein pRb pp110 p105-Rb RB1 p110-RB1 protein protein human Homo sapiens Human MPPKTPRKTAATAAAAAAEPPAPPPPPPPEEDPEQDSGPEDLPLVRLEFEETEEPDFTALCQKLKIPDHVRERAWLTWEKVSSVDGVLGGYIQKKKELWGICIFIAAVDLDEMSFTFTELQKNIEISVHKFFNLLKEIDTSTKVDNAMSRLLKKYDVLFALFSKLERTCELIYLTQPSSSISTEINSALVLKVSWITFLLAKGEVLQMEDDLVISFQLMLCVLDYFIKLSPPMLLKEPYKTAVIPINGSPRTPRRGQNRSARIAKQLENDTRIIEVLCKEHECNIDEVKNVYFKNFIPFMNSLGLVTSNGLPEVENLSKRYEEIYLKNKDLDARLFLDHDKTLQTDSIDSFETQRTPRKSNLDEEVNVIPPHTPVRTVMNTIQQLMMILNSASDQPSENLISYFNNCTVNPKESILKRVKDIGYIFKEKFAKAVGQGCVEIGSQRYKLGVRLYYRVMESMLKSEEERLSIQNFSKLLNDNIFHMSLLACALEVVMATYSRSTSQNLDSGTDLSFPWILNVLNLKAFDFYKVIESFIKAEGNLTREMIKHLERCEHRIMESLAWLSDSPLFDLIKQSKDREGPTDHLESACPLNLPLQNNHTAADMYLSPVRSPKKKGSTTRVNSTANAETQATSAFQTQKPLKSTSLSLFYKKVYRLAYLRLNTLCERLLSEHPELEHIIWTLFQHTLQNEYELMRDRHLDQIMMCSMYGICKVKNIDLKFKIIVTAYKDLPHAVQETFKRVLIKEEEYDSIIVFYNSVFMQRLKTNILQYASTRPPTLSPIPHIPRSPYKFPSSPLRIPGGNIYISPLKSPYKISEGLPTPTKMTPRSRILVSIGESFGTSEKFQKINQMVCNSDRVLKRSAEGSNPPKPLKKLRFDIEGSDEADGSKHLPGESKFQQKLAEMTSTRTRMQKQKMNDSMDTSNKEEK DIP protein P06400 original sequence version: 102 DIP protein P06400 original sequence version: 195 DIP protein P06400 original sequence version: 204 DIP protein P06400 original sequence version: 126 DIP protein P06400 original sequence version: 151 DIP protein P06400 original sequence version: 153 DIP protein P06400 original sequence version: 181 DIP protein P06400 original sequence version: 184 DIP protein P06400 original sequence version: 213 DIP protein P06400 original sequence version: 208 DIP protein P06400 original sequence version: 174 DIP protein P06400 original sequence version: 212 DIP protein P06400 original sequence version: 179 DIP protein P06400 original sequence version: 146 C8E746111E19CC32 e2f1_mouse Transcription factor E2F1 E2f1 protein protein mouse Mus musculus Mouse MAVAPAGGQHAPALEALLGAGALRLLDSSQIVIISTAPDVGAPQLPAAPPTGPRDSDVLLFATPQAPRPAPSAPRPALGRPPVKRRLDLETDHQYLAGSSGPFRGRGRHPGKGVKSPGEKSRYETSLNLTTKRFLELLSRSADGVVDLNWAAEVLKVQKRRIYDITNVLEGIQLIAKKSKNHIQWLGSHTMVGIGKRLEGLTQDLQQLQESEQQLDHLMHICTTQLQLLSEDSDTQRLAYVTCQDLRSIADPAEQMVIVIKAPPETQLQAVDSSETFQISLKSKQGPIDVFLCPEESADGISPGKTSCQETSSGEDRTADSGPAGPPPSPPSTSPALDPSQSLLGLEQEAVLPRMGHLRVPMEEDQLSPLVAADSLLEHVKEDFSGLLPGEFISLSPPHEALDYHFGLEEGEGIRDLFDCDFGDLTPLDF C5DF18AD3B4DFEFA hdac1_human Histone deacetylase 1 HDAC1 RPD3L1 protein protein human Homo sapiens Human MAQTQGTRRKVCYYYDGDVGNYYYGQGHPMKPHRIRMTHNLLLNYGLYRKMEIYRPHKANAEEMTKYHSDDYIKFLRSIRPDNMSEYSKQMQRFNVGEDCPVFDGLFEFCQLSTGGSVASAVKLNKQQTDIAVNWAGGLHHAKKSEASGFCYVNDIVLAILELLKYHQRVLYIDIDIHHGDGVEEAFYTTDRVMTVSFHKYGEYFPGTGDLRDIGAGKGKYYAVNYPLRDGIDDESYEAIFKPVMSKVMEMFQPSAVVLQCGSDSLSGDRLGCFNLTIKGHAKCVEFVKSFNLPMLMLGGGGYTIRNVARCWTYETAVALDTEIPNELPYNDYFEYFGPDFKLHISPSNMTNQNTNEYLEKIKQRLFENLRMLPHAPGVQMQAIPEDAIPEESGDEDEDDPDKRISICSSDKRIACEEEFSDSEEEGEGGRKNSSNFKKAKRVKTEDEKEKDPEEKKEVTEEEKTKEEKPEAKGVKEEVKLA DIP protein Q13547 original sequence version: 106 DIP protein Q13547 original sequence version: 173 DIP protein Q13547 original sequence version: 100 DIP protein Q13547 original sequence version: 166 DIP protein Q13547 original sequence version: 153 DIP protein Q13547 original sequence version: 164 DIP protein Q13547 original sequence version: 175 DIP protein Q13547 original sequence version: 191 DIP protein Q13547 original sequence version: 185 DIP protein Q13547 original sequence version: 165 DIP protein Q13547 original sequence version: 162 DIP protein Q13547 original sequence version: 189 DIP protein Q13547 original sequence version: 158 DIP protein Q13547 original sequence version: 134 DIP protein Q13547 original sequence version: 124 4D35B7C1ED7838D6 rb1-hmga2 1 9 unspecified role unspecified role bait bait 8 unspecified role unspecified role prey prey physical association physical association we coimmunoprecipitated protein lysates from two HMGA2 pituitary adenomas and from a pool of pituitary glands from control mice. Immunoprecipitation of the protein lysates with anti-pRB antibodies resulted in the coimmunoprecipitation of HMGA2 from pituitary adenomas 2 A rb1-hmga2-1 5 10 unspecified role unspecified role bait bait 8 unspecified role unspecified role prey prey region ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined physical association physical association 2 A HEK293 cells were transiently cotransfected with hemagglutinin (HA)-tagged-HMGA2 and pRB expression vectors. Protein lysates were immunoprecipitated with antipRB or anti-HA antibodies and immunoblotted with anti-HA or anti-pRB, respectively (Figure 2A, right panels). Coexpression of pRB and HMGA2 resulted in coimmunoprecipitation of the two proteins. rb1-hmga2-2 4 8 unspecified role unspecified role bait bait region ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 10 unspecified role unspecified role prey prey physical association physical association 2 A HEK293 cells were transiently cotransfected with hemagglutinin (HA)-tagged-HMGA2 and pRB expression vectors. Protein lysates were immunoprecipitated with antipRB or anti-HA antibodies and immunoblotted with anti-HA or anti-pRB, respectively (Figure 2A, right panels). Coexpression of pRB and HMGA2 resulted in coimmunoprecipitation of the two proteins. rb1-hmga2-3 7 8 unspecified role unspecified role prey prey 10 unspecified role unspecified role bait bait region pRB A/B (340-834) required to bind necessary binding region required to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain physical association physical association 2 B, C we carried out Far-Western analyses in which we probed HMGA2 on pRB immunoprecipitates from cell lysates overexpressing wild-type and different mutant forms of the pRB protein (Figures 2B and 2C). Interestingly, HMGA2 was found to bind to the pRB mutant expressing only the pocket region (lane 5) as efficiently as to the wild-type pRB (lane 1). It did not bind to the naturally occurring mutant pRBD21 that carries a deletion of exon 21 and encodes a portion of the pocket region rb1-hmga2-4 6 10 unspecified role unspecified role bait bait n-terminal sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain region exon 21 binding region binding-associated region binding region certain certain sequence position certain certain certain sequence position certain 8 unspecified role unspecified role prey prey region ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined physical association physical association 2 D These results are supported by in vitro binding experiments in which GST-pRB fusion proteins containing various pRB domains (Figure 2B) were incubated with a lysate from cells overexpressing HMGA2 (Figure 2D). pRB(379-928), which contains the entire wild-type pocket region of pRB plus the C-terminal region, was able to bind efficiently to HMGA2 (lane 1). In contrast, pRB(379-928)D21, which lacks the region corresponding to exon 21, only weakly associated with HMGA2 (lane 3). pRB(768-928) and pRB(834-928), which carry the C-terminal region plus a small region of the pocket and the C-terminal region only, respectively, did not bind to HMGA2 at all (lanes 5 and 6). Finally, pRB(1-379) and pRB(379-792), carrying the N-terminal region and the pocket region, respectively, were able to bind efficiently to HMGA2 (lanes 2 and 4). These results indicate that HMGA2 binds to pRB in two regions: the N terminus and the pocket region between amino acids 701 and 737. The construct of pRb lacking the region coded for by exon 21 only bound weakly to HMGA2. rb1-hmga2-5 6 8 unspecified role unspecified role prey prey region ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined n-terminal binding region binding-associated region binding region deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain 10 unspecified role unspecified role bait bait region gst tag glutathione s tranferase tag glutathione S-tranferase tag glutathione S-tranferase tag gst tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined physical association physical association 2 F We used cell lysates expressing HA-HMGA2 mutants carrying serial deletions at the N and C terminus regions (Figure 2E) in a pull-down assay with a recombinant GST-pRB protein (Figure 2F, lanes 1�6). The only mutant that lost the capacity to bind to pRB was A2(1-44), The HMGA2 mutant lacking only the second AT-hook [A1(Delta45-56] displayed reduced binding. The only mutant that did not bind pRb was A2(1-44), which contains the N-terminus, the first AT-hook, and the region preceding the second AT-hook. rb1-e2f1 2 12 unspecified role unspecified role prey prey 11 unspecified role unspecified role prey prey 10 unspecified role unspecified role bait bait association association 4 A we performed coimmunoprecipitation experiments with HEK293 cells transiently cotransfected with pRB and E2F1 expression plasmids in the presence of E1A or HMGA2 recombinant proteins (Figure 4A). As previously reported (Putzer et al., 1997), E1A prevented the binding between pRB and E2F1 (lane 2), whereas HMGA2 did not E1A prevented interaction between pRb and E2F1. HMGA2 does not prevent binding between pRb and E2F1, which indicates that competition for binding with pRb is not the mechanism via which HGMA2 activates E2F1. Both E1A and HMGA2 reduced the binding of pRB to HDAC1. This shows that, similarly to the E1A-pRB interaction, the HMGA2-pRB complex can affect HDAC1 recruitment. rb1-hdac1 3 12 unspecified role unspecified role prey prey region 35S-methionine labeled 35s radiolabel 35s radiolabel 35S s35 radiolabelled S35 s35 radiolabelled 35S S35 undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined in vitro In vitro 10 unspecified role unspecified role bait bait ecoli Escherichia coli (strain K12) physical association physical association HDAC1 was partially or completely displaced by the binding to pRB in the presence of 5 mg and 10 mg of HMGA2, respectively. This result demonstrates that HMGA2 directly interferes with the binding between HDAC1 and pRB. Moreover, to correlate the effect of HMGA2 on HDAC1 displacement from pRB with the HMGA2/pRB interaction, we also performed the experiment by using the A2(1-44) mutant, unable to bind pRB. As shown in the same Figure 4B, the binding between HDAC1 and pRB was not affected at all by the HMGA2 mutant, 4 B HMGA2 interferes with the binding between HDAC1 and pRb. but the A2(1-44) mutant does not.