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 AATF/Che-1 acts as a phosphorylation-dependent molecular modulator to repress p53-driven apoptosis. AATF/Che-1 acts as a phosphorylation-dependent molecular modulator to repress p53-driven apoptosis. EMBO J. (0261-4189) 2012 imex curation Höpker K., Hagmann H., Khurshid S., Chen S., Hasskamp P., Seeger-Nukpezah T., Schilberg K., Heukamp L., Lamkemeyer T., Sos ML., Thomas RK., Lowery D., Roels F., Fischer M., Liebau MC., Resch U., Kisner T., Röther F., Bartram MP., Müller RU., Fabretti F., Kurschat P., Schumacher B., Gaestel M., Medema RH., Yaffe MB., Schermer B., Reinhardt HC., Benzing T. Only protein-protein interactions in vitro In vitro protein kinase assay protein kinase assay experimental particp experimental participant identification experimental particp Höpker K., Hagmann H., Khurshid S., Chen S., Hasskamp P., Seeger-Nukpezah T., Schilberg K., Heukamp L., Lamkemeyer T., Sos ML., Thomas RK., Lowery D., Roels F., Fischer M., Liebau MC., Resch U., Kisner T., Röther F., Bartram MP., Müller RU., Fabretti F., Kurschat P., Schumacher B., Gaestel M., Medema RH., Yaffe MB., Schermer B., Reinhardt HC., Benzing T. EMBO J. (0261-4189) 2012 Only protein-protein interactions imex curation AATF/Che-1 acts as a phosphorylation-dependent molecular modulator to repress p53-driven apoptosis. AATF/Che-1 acts as a phosphorylation-dependent molecular modulator to repress p53-driven apoptosis. EMBO J. (0261-4189) 2012 imex curation Höpker K., Hagmann H., Khurshid S., Chen S., Hasskamp P., Seeger-Nukpezah T., Schilberg K., Heukamp L., Lamkemeyer T., Sos ML., Thomas RK., Lowery D., Roels F., Fischer M., Liebau MC., Resch U., Kisner T., Röther F., Bartram MP., Müller RU., Fabretti F., Kurschat P., Schumacher B., Gaestel M., Medema RH., Yaffe MB., Schermer B., Reinhardt HC., Benzing T. Only protein-protein interactions 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 anti tag western anti tag western blot anti tag western Höpker K., Hagmann H., Khurshid S., Chen S., Hasskamp P., Seeger-Nukpezah T., Schilberg K., Heukamp L., Lamkemeyer T., Sos ML., Thomas RK., Lowery D., Roels F., Fischer M., Liebau MC., Resch U., Kisner T., Röther F., Bartram MP., Müller RU., Fabretti F., Kurschat P., Schumacher B., Gaestel M., Medema RH., Yaffe MB., Schermer B., Reinhardt HC., Benzing T. EMBO J. (0261-4189) 2012 Only protein-protein interactions imex curation AATF/Che-1 acts as a phosphorylation-dependent molecular modulator to repress p53-driven apoptosis. AATF/Che-1 acts as a phosphorylation-dependent molecular modulator to repress p53-driven apoptosis. EMBO J. (0261-4189) 2012 imex curation Höpker K., Hagmann H., Khurshid S., Chen S., Hasskamp P., Seeger-Nukpezah T., Schilberg K., Heukamp L., Lamkemeyer T., Sos ML., Thomas RK., Lowery D., Roels F., Fischer M., Liebau MC., Resch U., Kisner T., Röther F., Bartram MP., Müller RU., Fabretti F., Kurschat P., Schumacher B., Gaestel M., Medema RH., Yaffe MB., Schermer B., Reinhardt HC., Benzing T. Only protein-protein interactions yeast Saccharomyces cerevisiae Baker's yeast 2 hybrid two hybrid 2H 2h Gal4 transcription regeneration two-hybrid yeast two hybrid 2-hybrid classical two hybrid classical two hybrid 2H two-hybrid 2-hybrid Gal4 transcription regeneration 2h yeast two hybrid Y2H Y-2H 2 hybrid experimental particp experimental participant identification experimental particp Höpker K., Hagmann H., Khurshid S., Chen S., Hasskamp P., Seeger-Nukpezah T., Schilberg K., Heukamp L., Lamkemeyer T., Sos ML., Thomas RK., Lowery D., Roels F., Fischer M., Liebau MC., Resch U., Kisner T., Röther F., Bartram MP., Müller RU., Fabretti F., Kurschat P., Schumacher B., Gaestel M., Medema RH., Yaffe MB., Schermer B., Reinhardt HC., Benzing T. EMBO J. (0261-4189) 2012 Only protein-protein interactions imex curation aatf_human Protein AATF Apoptosis-antagonizing transcription factor Rb-binding protein Che-1 AATF CHE1 DED HSPC277 protein protein human Homo sapiens Human MAGPQPLALQLEQLLNPRPSEADPEADPEEATAARVIDRFDEGEDGEGDFLVVGSIRKLASASLLDTDKRYCGKTTSRKAWNEDHWEQTLPGSSDEEISDEEGSGDEDSEGLGLEEYDEDDLGAAEEQECGDHRESKKSRSHSAKTPGFSVQSISDFEKFTKGMDDLGSSEEEEDEESGMEEGDDAEDSQGESEEDRAGDRNSEDDGVVMTFSSVKVSEEVEKGRAVKNQIALWDQLLEGRIKLQKALLTTNQLPQPDVFPLFKDKGGPEFSSALKNSHKALKALLRSLVGLQEELLFQYPDTRYLVDGTKPNAGSEEISSEDDELVEEKKQQRRRVPAKRKLEMEDYPSFMAKRFADFTVYRNRTLQKWHDKTKLASGKLGKGFGAFERSILTQIDHILMDKERLLRRTQTKRSVYRVLGKPEPAAQPVPESLPGEPEILPQAPANAHLKDLDEEIFDDDDFYHQLLRELIERKTSSLDPNDQVAMGRQWLAIQKLRSKIHKKVDRKASKGRKLRFHVLSKLLSFMAPIDHTTMNDDARTELYRSLFGQLHPPDEGHGD EC493EF3B4C3A199 q6zwq9_mouse Myl12a 2900073G15Rik Myl12b Gm49909 protein protein mouse Mus musculus Mouse MSSKRAKTKTTKKRPQRATSNVFAMFDQSQIQEFKEAFNMIDQNRDGFIDKEDLHDMLASMGKNPTDEYLDAMMNEAPGPINFTMFLTMFGEKLNGTDPEDVIRNAFACFDEEAIGTIQEDYLRELLTTMGDRFTDEEVDELYREAPIDKKGNFNYIEFTRILKHGAKDKDD mint homomint 50DB8144B916141A mapk2_human MAP kinase-activated protein kinase 2 MAPKAPK2 protein protein human Homo sapiens Human MLSNSQGQSPPVPFPAPAPPPQPPTPALPHPPAQPPPPPPQQFPQFHVKSGLQIKKNAIIDDYKVTSQVLGLGINGKVLQIFNKRTQEKFALKMLQDCPKARREVELHWRASQCPHIVRIVDVYENLYAGRKCLLIVMECLDGGELFSRIQDRGDQAFTEREASEIMKSIGEAIQYLHSINIAHRDVKPENLLYTSKRPNAILKLTDFGFAKETTSHNSLTTPCYTPYYVAPEVLGPEKYDKSCDMWSLGVIMYILLCGYPPFYSNHGLAISPGMKTRIRMGQYEFPNPEWSEVSEEVKMLIRNLLKTEPTQRMTITEFMNHPWIMQSTKVPQTPLHTSRVLKEDKERWEDVKEEMTSALATMRVDYEQIKIKKIEDASNPLLLKRRKKARALEAAALAH E4EFFF11CCF288DC myl12a-aatf 2 MRLC3 5 anti tag western anti tag western blot anti tag western experimental particp experimental participant identification experimental particp 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 AATF 4 anti tag western anti tag western blot anti tag western experimental particp experimental participant identification experimental particp 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 homomint mint To confirm this interaction in mammalian cells, we performed co-immunoprecipitation experiments in HEK293T cells co-expressing V5.AATF and FLAG.MRLC3 or FLAG.GFP, as a control. While AATF could readily be detected in the FLAG.MRLC3 precipitates, it was undetectable in the FLAG.GFP precipitations, thus validating the interaction between AATF and MRLC3 (Supplementary Figure 1B). In agreement with the results of the phospho-proteomic screen, treatment of V5.AATF and FLAG.MRLC3-expressing cells with the Ser/ Thr phosphatase inhibitor okadaic acid, abrogated the AATF:MRLC3 interaction (Figure 1C). We then went on to investigate whether the phosphorylation- sensitive interaction between AATF and MRLC3 is regulated by checkpoint kinases in response to genotoxic stress and performed co-immunoprecipitation experiments before and after DNA damage. As we had observed before, V5.AATF co-precipitated with FLAG.MRLC3 in mock-treated cells. In contrast, this interaction was abolished when cells were pre-treated with UV-C, indicating that genotoxic stress negatively regulates MRLC3:AATF complex formation (Figure 1D). Disruption of the MLRC3:AATF complex was also observed following treatment of cells with doxorubicin, indicating that the complex is sensitive to multiple types of genotoxic stress (Supplementary Figure 1C). sf1b sf1c f1c f1d aatf-myl12a 2 MRLC3 5 anti tag western anti tag western blot anti tag western experimental particp experimental participant identification experimental particp 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 AATF 4 anti tag western anti tag western blot anti tag western experimental particp experimental participant identification experimental particp 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 f1e homomint mint Identical co-precipitation behaviour was observed when the FLAG and V5 tags were swapped (Figure 1E). myl12a-aatf-1 3 MRLC3 5 predetermined predetermined participant predetermined experimental particp experimental participant identification experimental particp unspecified role unspecified role bait bait over-expressed over expressed level over-expressed living cell living cell fusion protein fusion protein fusion protein undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined AATF 4 nucleotide sequence nucleotide sequence identification sequence cloning sequence cloning nucleotide sequence experimental particp experimental participant identification experimental particp unspecified role unspecified role prey prey over-expressed over expressed level over-expressed cdna library cdna library fusion protein fusion protein fusion protein undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined physical association physical association nfa homomint mint aatf-mapkapk2 1 MK2 6 predetermined predetermined participant predetermined experimental particp experimental participant identification experimental particp enzyme enzyme neutral component neutral component purified purified AATF 4 weight autoradiogra molecular weight estimation by autoradiography weight autoradiogra experimental particp experimental participant identification experimental particp enzyme target enzyme target substrate substrate neutral component neutral component purified purified his tag his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined binding site binding region binding-associated region binding region certain certain sequence position certain certain certain sequence position certain phosphorylation phosphorylation reaction phosphorylation homomint sf3 mint We next used in vitro kinase assays to test whether MK2 directly phosphorylated AATF. As shown in Supplementary Figure 3, MK2 readily phosphorylated AATF, while no significant 32P incorporation could be detected in control reactions lacking MK2.