MINT MINT, Dpt of Biology, University of Rome Tor Vergata MINT http://mint.bio.uniroma2.it/mint url>http://mint.bio.uniroma2.it/mint/ https://mint.bio.uniroma2.it MINT-[0-9]+ https://mint.bio.uniroma2.it/index.php/results-interactions/?id=${ac} The Molecular INTeraction database (MINT) is a relational database designed to store interactions between biological molecules. http://mint.bio.uniroma2.it Mutational analysis reveals a dual role of Mdm2 acidic domain in the regulation of p53 stability. Mutational analysis reveals a dual role of Mdm2 acidic domain in the regulation of p53 stability. FEBS Lett. (0014-5793) 2012 Dolezelova P., Cetkovska K., Vousden KH., Uldrijan S. suggested by lperfetto 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 Dolezelova P., Cetkovska K., Vousden KH., Uldrijan S. suggested by lperfetto FEBS Lett. (0014-5793) 2012 psa3_human Proteasome subunit alpha type-3 PSMA3 PSC8 Proteasome component C8 Macropain subunit C8 Multicatalytic endopeptidase complex subunit C8 HC8 protein protein human Homo sapiens Human MSSIGTGYDLSASTFSPDGRVFQVEYAMKAVENSSTAIGIRCKDGVVFGVEKLVLSKLYEEGSNKRLFNVDRHVGMAVAGLLADARSLADIAREEASNFRSNFGYNIPLKHLADRVAMYVHAYTLYSAVRPFGCSFMLGSYSVNDGAQLYMIDPSGVSYGYWGCAIGKARQAAKTEIEKLQMKEMTCRDIVKEVAKIIYIVHDEVKDKAFELELSWVGELTNGRHEIVPKDIREEAEKYAKESLKEEDESDDDNM DIP protein P25788 original sequence version: 173 A1854ED3C0650FB1 p53_human Cellular tumor antigen p53 TP53 P53 Tumor suppressor p53 Phosphoprotein p53 Antigen NY-CO-13 protein protein human Homo sapiens Human MEEPQSDPSVEPPLSQETFSDLWKLLPENNVLSPLPSQAMDDLMLSPDDIEQWFTEDPGPDEAPRMPEAAPPVAPAPAAPTPAAPAPAPSWPLSSSVPSQKTYQGSYGFRLGFLHSGTAKSVTCTYSPALNKMFCQLAKTCPVQLWVDSTPPPGTRVRAMAIYKQSQHMTEVVRRCPHHERCSDSDGLAPPQHLIRVEGNLRVEYLDDRNTFRHSVVVPYEPPEVGSDCTTIHYNYMCNSSCMGGMNRRPILTIITLEDSSGNLLGRNSFEVRVCACPGRDRRTEEENLRKKGEPHHELPPGSTKRALPNNTSSSPQPKKKPLDGEYFTLQIRGRERFEMFRELNEALELKDAQAGKEPGGSRAHSSHLKSKKGQSTSRHKKLMFKTEGPDSD DIP protein P04637 original sequence version: 121 DIP protein P04637 original sequence version: 115 DIP protein P04637-1 original sequence version: 224 DIP protein P04637 original sequence version: 117 DIP protein P04637-1 original sequence version: 226 DIP protein P04637 original sequence version: 226 DIP protein P04637 original sequence version: 212 DIP protein P04637 original sequence version: 220 DIP protein P04637-1 original sequence version: 220 DIP protein P04637-1 original sequence version: 145 DIP protein P04637 original sequence version: 146 DIP protein P04637 original sequence version: 145 DIP protein P04637 original sequence version: 166 DIP protein P04637-1 original sequence version: 166 DIP protein P04637 original sequence version: 169 DIP protein P04637 original sequence version: 177 DIP protein P04637 original sequence version: 175 DIP protein P04637-1 original sequence version: 175 DIP protein P04637-1 original sequence version: 172 DIP protein P04637-1 original sequence version: 169 DIP protein P04637 original sequence version: 170 DIP protein P04637 original sequence version: 172 DIP protein P04637-1 original sequence version: 194 DIP protein P04637 original sequence version: 196 DIP protein P04637 original sequence version: 185 DIP protein P04637-1 original sequence version: 191 DIP protein P04637 original sequence version: 191 DIP protein P04637 original sequence version: 180 DIP protein P04637-1 original sequence version: 197 DIP protein P04637 original sequence version: 183 DIP protein P04637-1 original sequence version: 183 DIP protein P04637-1 original sequence version: 185 DIP protein P04637 original sequence version: 199 DIP protein P04637 original sequence version: 201 DIP protein P04637 original sequence version: 189 DIP protein P04637 original sequence version: 235 DIP protein P04637 original sequence version: 230 DIP protein P04637 original sequence version: 234 DIP protein P04637-1 original sequence version: 234 DIP protein P04637-1 original sequence version: 235 DIP protein P04637 original sequence version: 239 DIP protein P04637-1 original sequence version: 212 DIP protein P04637-1 original sequence version: 213 DIP protein P04637 original sequence version: 213 DIP protein P04637 original sequence version: 206 DIP protein P04637-1 original sequence version: 188 DIP protein P04637-1 original sequence version: 228 DIP protein P04637 original sequence version: 208 DIP protein P04637 original sequence version: 240 DIP protein P04637-1 original sequence version: 240 DIP protein P04637 original sequence version: 204 DIP protein P04637-1 original sequence version: 204 DIP protein P04637-1 original sequence version: 209 DIP protein P04637-1 original sequence version: 203 DIP protein P04637 original sequence version: 221 DIP protein P04637-1 original sequence version: 152 DIP protein P04637 original sequence version: 217 DIP protein P04637-1 original sequence version: 140 DIP protein P04637 original sequence version: 215 DIP protein P04637 original sequence version: 214 DIP protein P04637-1 original sequence version: 182 DIP protein P04637 original sequence version: 182 DIP protein P04637 original sequence version: 188 DIP protein P04637 original sequence version: 192 DIP protein P04637-1 original sequence version: 192 DIP protein P04637 original sequence version: 218 DIP protein P04637-1 original sequence version: 218 AD5C149FD8106131 psb6_human Proteasome subunit beta type-6 PSMB6 LMPY Proteasome delta chain Macropain delta chain Multicatalytic endopeptidase complex delta chain Proteasome subunit Y Y protein protein human Homo sapiens Human MAATLLAARGAGPAPAWGPEAFTPDWESREVSTGTTIMAVQFDGGVVLGADSRTTTGSYIANRVTDKLTPIHDRIFCCRSGSAADTQAVADAVTYQLGFHSIELNEPPLVHTAASLFKEMCYRYREDLMAGIIIAGWDPQEGGQVYSVPMGGMMVRQSFAIGGSGSSYIYGYVDATYREGMTKEECLQFTANALALAMERDGSSGGVIRLAAIAESGVERQVLLGDQIPKFAVATLPPA DIP protein P28072 original sequence version: 163 7DF4081DC735930C psa6_human Proteasome subunit alpha type-6 PSMA6 PROS27 Proteasome iota chain Macropain iota chain Multicatalytic endopeptidase complex iota chain 27 kDa prosomal protein protein protein human Homo sapiens Human MSRGSSAGFDRHITIFSPEGRLYQVEYAFKAINQGGLTSVAVRGKDCAVIVTQKKVPDKLLDSSTVTHLFKITENIGCVMTGMTADSRSQVQRARYEAANWKYKYGYEIPVDMLCKRIADISQVYTQNAEMRPLGCCMILIGIDEEQGPQVYKCDPAGYYCGFKATAAGVKQTESTSFLEKKVKKKFDWTFEQTVETAITCLSTVLSIDFKPSEIEVGVVTVENPKFRILTEAEIDAHLVALAERD DIP protein P60900 original sequence version: 129 DIP protein P60900 original sequence version: 80 94D1FD3C0A7CC72A mdm2_human E3 ubiquitin-protein ligase Mdm2 MDM2 p53-binding protein Mdm2 Oncoprotein Mdm2 Double minute 2 protein RING-type E3 ubiquitin transferase Mdm2 protein protein human Homo sapiens Human MCNTNMSVPTDGAVTTSQIPASEQETLVRPKPLLLKLLKSVGAQKDTYTMKEVLFYLGQYIMTKRLYDEKQQHIVYCSNDLLGDLFGVPSFSVKEHRKIYTMIYRNLVVVNQQESSDSGTSVSENRCHLEGGSDQKDLVQELQEEKPSSSHLVSRPSTSSRRRAISETEENSDELSGERQRKRHKSDSISLSFDESLALCVIREICCERSSSSESTGTPSNPDLDAGVSEHSGDWLDQDSVSDQFSVEFEVESLDSEDYSLSEEGQELSDEDDEVYQVTVYQAGESDTDSFEEDPEISLADYWKCTSCNEMNPPLPSHCNRCWALRENWLPEDKGKDKGEISEKAKLENSTQAEEGFDVPDCKKTIVNDSRESCVEENDDKITQASQSQESEDYSQPSTSSSIIYSSQEDVKEFEREETQDKEESVESSLPLNAIEPCVICQGRPKNGCIVHGKTGHLMACFTCAKKLKKRNKPCPVCRQPIQMIVLTYFP DIP protein Q00987 original sequence version: 93 DIP protein Q00987-1 original sequence version: 192 DIP protein Q00987 original sequence version: 88 DIP protein Q00987 original sequence version: 180 DIP protein Q00987 original sequence version: 188 DIP protein Q00987 original sequence version: 189 DIP protein Q00987 original sequence version: 115 DIP protein Q00987-1 original sequence version: null DIP protein Q00987 original sequence version: 149 DIP protein Q00987 original sequence version: 146 DIP protein Q00987 original sequence version: 140 DIP protein Q00987 original sequence version: 183 DIP protein Q00987 original sequence version: 145 DIP protein Q00987 original sequence version: 182 DIP protein Q00987 original sequence version: 151 DIP protein Q00987 original sequence version: 156 DIP protein Q00987-1 original sequence version: 160 DIP protein Q00987 original sequence version: 160 DIP protein Q00987 original sequence version: 168 DIP protein Q00987 original sequence version: 186 DIP protein Q00987 original sequence version: 206 F37CE163876BC983 tyy1_human Transcriptional repressor protein YY1 Yin and yang 1 Delta transcription factor NF-E1 YY1 INO80S INO80 complex subunit S protein protein human Homo sapiens Human MASGDTLYIATDGSEMPAEIVELHEIEVETIPVETIETTVVGEEEEEDDDDEDGGGGDHGGGGGHGHAGHHHHHHHHHHHPPMIALQPLVTDDPTQVHHHQEVILVQTREEVVGGDDSDGLRAEDGFEDQILIPVPAPAGGDDDYIEQTLVTVAAAGKSGGGGSSSSGGGRVKKGGGKKSGKKSYLSGGAGAAGGGGADPGNKKWEQKQVQIKTLEGEFSVTMWSSDEKKDIDHETVVEEQIIGENSPPDYSEYMTGKKLPPGGIPGIDLSDPKQLAEFARMKPRKIKEDDAPRTIACPHKGCTKMFRDNSAMRKHLHTHGPRVHVCAECGKAFVESSKLKRHQLVHTGEKPFQCTFEGCGKRFSLDFNLRTHVRIHTGDRPYVCPFDGCNKKFAQSTNLKSHILTHAKAKNNQ DIP protein P25490 original sequence version: 153 058C05A0AD2D04E6 mdm2-tp53-16 1 MDM2 6 western blot western blot Immuno blot Immuno blot predetermined predetermined participant predetermined unspecified role unspecified role bait bait over-expressed over expressed level over-expressed living cell living cell p53 3 western blot western blot Immuno blot Immuno blot predetermined predetermined participant predetermined unspecified role unspecified role prey prey over-expressed over expressed level over-expressed living cell living cell flag tag flag tag flag tag FLAG-tagged FLAG DYKDDDDKV epitope tag DYKDDDDKV epitope tag FLAG FLAG-tagged undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined physical association physical association homomint mint f5a f5c Physical interaction between Mdm2 and p53 is mediated mainly by binding domains located at their respective N-termini. However, several studies showed that the central region of Mdm2 contains an additional site contributing to p53 binding and p53 degradation [39, 45- 47]. We hypothesized that a defect in Mdm2-p53 binding outside their N-terminal interaction domains could provide an explanation for the observed inactivation of our Mdm2 mutants. We therefore tested the ability of the AD mutants to bind p53 in the context of proteins containing deletions disrupting the N-terminal binding sites: Mdm2Δ58-89, lacking critical amino acids 58-89 of the p53-binding domain, and p53ΔI, lacking a conserved box I involved in Mdm2 binding. Surprisingly, neither the short deletion mutants nor the point mutants exhibited in our tests any significant decrease in their capability to interact with p53 outside the main binding sites (Fig.5), suggesting that p53 binding may not be the only function for the Mdm2 acidic domain in p53 ubiquitylation and degradation. tp53-mdm2-15 1 MDM2 6 western blot western blot Immuno blot Immuno blot predetermined predetermined participant predetermined unspecified role unspecified role prey prey over-expressed over expressed level over-expressed living cell living cell p53 3 western blot western blot Immuno blot Immuno blot predetermined predetermined participant predetermined unspecified role unspecified role bait bait over-expressed over expressed level over-expressed living cell living cell flag tag flag tag flag tag FLAG-tagged FLAG DYKDDDDKV epitope tag DYKDDDDKV epitope tag FLAG FLAG-tagged undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined physical association physical association homomint mint f5b f5c Physical interaction between Mdm2 and p53 is mediated mainly by binding domains located at their respective N-termini. However, several studies showed that the central region of Mdm2 contains an additional site contributing to p53 binding and p53 degradation [39, 45- 47]. We hypothesized that a defect in Mdm2-p53 binding outside their N-terminal interaction domains could provide an explanation for the observed inactivation of our Mdm2 mutants. We therefore tested the ability of the AD mutants to bind p53 in the context of proteins containing deletions disrupting the N-terminal binding sites: Mdm2Δ58-89, lacking critical amino acids 58-89 of the p53-binding domain, and p53ΔI, lacking a conserved box I involved in Mdm2 binding. Surprisingly, neither the short deletion mutants nor the point mutants exhibited in our tests any significant decrease in their capability to interact with p53 outside the main binding sites (Fig.5), suggesting that p53 binding may not be the only function for the Mdm2 acidic domain in p53 ubiquitylation and degradation. mdm2-psma6 1 S6a 5 western blot western blot Immuno blot Immuno blot predetermined predetermined participant predetermined unspecified role unspecified role prey prey over-expressed over expressed level over-expressed living cell living cell v5 tag v5 tag v5 tag GKPIPNPLLGLDST epitope tag GKPIPNPLLGLDST epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined MDM2 6 western blot western blot Immuno blot Immuno blot predetermined predetermined participant predetermined unspecified role unspecified role bait bait over-expressed over expressed level over-expressed living cell living cell physical association physical association f6a homomint mint we did not detect any decrease in the binding of the ΔAD9 mutant to 19S proteasome subunits S5a, S6a and S6b, or the 20S proteasome subunit C8α in immunoprecipitation assays (Fig. 6), indicating that the AD9 mutants retain a normal capacity to form complexes with the proteasome. mdm2-psmb6 1 MDM2 6 western blot western blot Immuno blot Immuno blot predetermined predetermined participant predetermined unspecified role unspecified role bait bait over-expressed over expressed level over-expressed living cell living cell S6b 4 western blot western blot Immuno blot Immuno blot predetermined predetermined participant predetermined unspecified role unspecified role prey prey over-expressed over expressed level over-expressed living cell living cell v5 tag v5 tag v5 tag GKPIPNPLLGLDST epitope tag GKPIPNPLLGLDST epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined physical association physical association f6a homomint mint we did not detect any decrease in the binding of the ΔAD9 mutant to 19S proteasome subunits S5a, S6a and S6b, or the 20S proteasome subunit C8α in immunoprecipitation assays (Fig. 6), indicating that the AD9 mutants retain a normal capacity to form complexes with the proteasome. mdm2-psma3 1 C8α 2 western blot western blot Immuno blot Immuno blot predetermined predetermined participant predetermined unspecified role unspecified role prey prey over-expressed over expressed level over-expressed living cell living cell myc tag myc tag myc tag EUKLISEED epitope tag EUKLISEED epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined MDM2 6 western blot western blot Immuno blot Immuno blot predetermined predetermined participant predetermined unspecified role unspecified role bait bait over-expressed over expressed level over-expressed living cell living cell physical association physical association f6b homomint mint we did not detect any decrease in the binding of the ΔAD9 mutant to 19S proteasome subunits S5a, S6a and S6b, or the 20S proteasome subunit C8α in immunoprecipitation assays (Fig. 6), indicating that the AD9 mutants retain a normal capacity to form complexes with the proteasome. yy1-mdm2 1 MDM2 6 western blot western blot Immuno blot Immuno blot predetermined predetermined participant predetermined unspecified role unspecified role prey prey over-expressed over expressed level over-expressed living cell living cell YY1 7 western blot western blot Immuno blot Immuno blot predetermined predetermined participant predetermined unspecified role unspecified role bait bait over-expressed over expressed level over-expressed living cell living cell flag tag flag tag flag tag FLAG-tagged FLAG DYKDDDDKV epitope tag DYKDDDDKV epitope tag FLAG FLAG-tagged undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined physical association physical association homomint f7 mint we therefore tested whether the acidic domain regions identified in this study might be essential for its binding. Results presented in Fig.7 suggest that neither AD5 nor AD9 is required for Mdm2-YY1 physical interaction. Mdm2 deletion mutant of the region involved in p300 binding was previously shown to be capable of mediating p53 ubiquitylation but not degradation [38]. In our study, we covered this region (amino acids 218-246) with four shorter deletions ΔAD1, ΔAD2, ΔAD3, and ΔAD4, none of which had a significant impact on Mdm2-mediated p53 degradation psma3-mdm2 1 MDM2 6 western blot western blot Immuno blot Immuno blot predetermined predetermined participant predetermined unspecified role unspecified role prey prey over-expressed over expressed level over-expressed living cell living cell C8α 2 western blot western blot Immuno blot Immuno blot predetermined predetermined participant predetermined unspecified role unspecified role bait bait over-expressed over expressed level over-expressed living cell living cell myc tag myc tag myc tag EUKLISEED epitope tag EUKLISEED epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined physical association physical association f6b homomint mint we did not detect any decrease in the binding of the ΔAD9 mutant to 19S proteasome subunits S5a, S6a and S6b, or the 20S proteasome subunit C8α in immunoprecipitation assays (Fig. 6), indicating that the AD9 mutants retain a normal capacity to form complexes with the proteasome.