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/ Identification of the E1A-regulated transcription factor p120 E4F as an interacting partner of the RASSF1A candidate tumor suppressor gene. Identification of the E1A-regulated transcription factor p120 E4F as an interacting partner of the RASSF1A candidate tumor suppressor gene. Cancer Res. (0008-5472) 2004 Fenton SL., Dallol A., Agathanggelou A., Hesson L., Ahmed-Choudhury J., Baksh S., Sardet C., Dammann R., Minna JD., Downward J., Maher ER., Latif F. Apoptosis - Interactions involving proteins with a function related to apoptosis Cancer - Interactions investigated in the context of cancer Note the UniProtKB entry Q66K89 for E4F1 has a higher degree of similarity to NP_004415 (the identifier given by the authors) than the other unreviewed entry of 784 aa. flatif@hgmp.mrc.ac.uk 23-MAY-2007: Contacted by JYOTI. Returned mail. A MATCHMAKER human adult brain cDNA library (BD-Clontech) was used. in vitro In vitro pull down pull down anti tag western anti tag western blot anti tag western Fenton SL., Dallol A., Agathanggelou A., Hesson L., Ahmed-Choudhury J., Baksh S., Sardet C., Dammann R., Minna JD., Downward J., Maher ER., Latif F. Apoptosis - Interactions involving proteins with a function related to apoptosis Note the UniProtKB entry Q66K89 for E4F1 has a higher degree of similarity to NP_004415 (the identifier given by the authors) than the other unreviewed entry of 784 aa. flatif@hgmp.mrc.ac.uk Accepted 2007-MAY-30 by JYOTI. 23-MAY-2007: Contacted by JYOTI. Returned mail. A MATCHMAKER human adult brain cDNA library (BD-Clontech) was used. Cancer Res. (0008-5472) 2004 Identification of the E1A-regulated transcription factor p120 E4F as an interacting partner of the RASSF1A candidate tumor suppressor gene. Identification of the E1A-regulated transcription factor p120 E4F as an interacting partner of the RASSF1A candidate tumor suppressor gene. Cancer Res. (0008-5472) 2004 Fenton SL., Dallol A., Agathanggelou A., Hesson L., Ahmed-Choudhury J., Baksh S., Sardet C., Dammann R., Minna JD., Downward J., Maher ER., Latif F. Apoptosis - Interactions involving proteins with a function related to apoptosis Cancer - Interactions investigated in the context of cancer Note the UniProtKB entry Q66K89 for E4F1 has a higher degree of similarity to NP_004415 (the identifier given by the authors) than the other unreviewed entry of 784 aa. flatif@hgmp.mrc.ac.uk 23-MAY-2007: Contacted by JYOTI. Returned mail. A MATCHMAKER human adult brain cDNA library (BD-Clontech) was used. yeasx Saccharomyces cerevisiae (Baker's yeast) 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 nucleotide sequence nucleotide sequence identification sequence cloning sequence cloning nucleotide sequence Fenton SL., Dallol A., Agathanggelou A., Hesson L., Ahmed-Choudhury J., Baksh S., Sardet C., Dammann R., Minna JD., Downward J., Maher ER., Latif F. Apoptosis - Interactions involving proteins with a function related to apoptosis Note the UniProtKB entry Q66K89 for E4F1 has a higher degree of similarity to NP_004415 (the identifier given by the authors) than the other unreviewed entry of 784 aa. Note the UniProtKB entry Q66K89 for E4F1 has a higher degree of similarity to NP_004415 (the identifier given by the authors) than the other unreviewed entry of 784 aa. flatif@hgmp.mrc.ac.uk Accepted 2007-MAY-30 by JYOTI. 23-MAY-2007: Contacted by JYOTI. Returned mail. A MATCHMAKER human adult brain cDNA library (BD-Clontech) was used. Cancer Res. (0008-5472) 2004 Identification of the E1A-regulated transcription factor p120 E4F as an interacting partner of the RASSF1A candidate tumor suppressor gene. Identification of the E1A-regulated transcription factor p120 E4F as an interacting partner of the RASSF1A candidate tumor suppressor gene. Cancer Res. (0008-5472) 2004 Fenton SL., Dallol A., Agathanggelou A., Hesson L., Ahmed-Choudhury J., Baksh S., Sardet C., Dammann R., Minna JD., Downward J., Maher ER., Latif F. Apoptosis - Interactions involving proteins with a function related to apoptosis Cancer - Interactions investigated in the context of cancer Note the UniProtKB entry Q66K89 for E4F1 has a higher degree of similarity to NP_004415 (the identifier given by the authors) than the other unreviewed entry of 784 aa. flatif@hgmp.mrc.ac.uk 23-MAY-2007: Contacted by JYOTI. Returned mail. A MATCHMAKER human adult brain cDNA library (BD-Clontech) was used. mouse-3t3 Mus musculus embryo cells 3t3 Mouse embryo fibroblast cells anti bait coip anti bait coimmunoprecipitation anti bait coip western blot western blot Immuno blot Immuno blot Fenton SL., Dallol A., Agathanggelou A., Hesson L., Ahmed-Choudhury J., Baksh S., Sardet C., Dammann R., Minna JD., Downward J., Maher ER., Latif F. Apoptosis - Interactions involving proteins with a function related to apoptosis Note the UniProtKB entry Q66K89 for E4F1 has a higher degree of similarity to NP_004415 (the identifier given by the authors) than the other unreviewed entry of 784 aa. flatif@hgmp.mrc.ac.uk Accepted 2007-MAY-30 by JYOTI. 23-MAY-2007: Contacted by JYOTI. Returned mail. A MATCHMAKER human adult brain cDNA library (BD-Clontech) was used. Cancer Res. (0008-5472) 2004 Identification of the E1A-regulated transcription factor p120 E4F as an interacting partner of the RASSF1A candidate tumor suppressor gene. Identification of the E1A-regulated transcription factor p120 E4F as an interacting partner of the RASSF1A candidate tumor suppressor gene. Cancer Res. (0008-5472) 2004 Fenton SL., Dallol A., Agathanggelou A., Hesson L., Ahmed-Choudhury J., Baksh S., Sardet C., Dammann R., Minna JD., Downward J., Maher ER., Latif F. Apoptosis - Interactions involving proteins with a function related to apoptosis Cancer - Interactions investigated in the context of cancer Note the UniProtKB entry Q66K89 for E4F1 has a higher degree of similarity to NP_004415 (the identifier given by the authors) than the other unreviewed entry of 784 aa. flatif@hgmp.mrc.ac.uk 23-MAY-2007: Contacted by JYOTI. Returned mail. A MATCHMAKER human adult brain cDNA library (BD-Clontech) was used. in vitro In vitro anti tag coip anti tag coimmunoprecipitation anti tag coip anti tag western anti tag western blot anti tag western Fenton SL., Dallol A., Agathanggelou A., Hesson L., Ahmed-Choudhury J., Baksh S., Sardet C., Dammann R., Minna JD., Downward J., Maher ER., Latif F. Apoptosis - Interactions involving proteins with a function related to apoptosis Note the UniProtKB entry Q66K89 for E4F1 has a higher degree of similarity to NP_004415 (the identifier given by the authors) than the other unreviewed entry of 784 aa. flatif@hgmp.mrc.ac.uk Accepted 2007-MAY-30 by JYOTI. 23-MAY-2007: Contacted by JYOTI. Returned mail. A MATCHMAKER human adult brain cDNA library (BD-Clontech) was used. Cancer Res. (0008-5472) 2004 Identification of the E1A-regulated transcription factor p120 E4F as an interacting partner of the RASSF1A candidate tumor suppressor gene. Identification of the E1A-regulated transcription factor p120 E4F as an interacting partner of the RASSF1A candidate tumor suppressor gene. Cancer Res. (0008-5472) 2004 Fenton SL., Dallol A., Agathanggelou A., Hesson L., Ahmed-Choudhury J., Baksh S., Sardet C., Dammann R., Minna JD., Downward J., Maher ER., Latif F. Apoptosis - Interactions involving proteins with a function related to apoptosis Cancer - Interactions investigated in the context of cancer Note the UniProtKB entry Q66K89 for E4F1 has a higher degree of similarity to NP_004415 (the identifier given by the authors) than the other unreviewed entry of 784 aa. flatif@hgmp.mrc.ac.uk 23-MAY-2007: Contacted by JYOTI. Returned mail. A MATCHMAKER human adult brain cDNA library (BD-Clontech) was used. human-a_549 Homo sapiens Caucasian lung carcinoma cell line a_549 Human Caucasian lung carcinoma anti bait coip anti bait coimmunoprecipitation anti bait coip western blot western blot Immuno blot Immuno blot Fenton SL., Dallol A., Agathanggelou A., Hesson L., Ahmed-Choudhury J., Baksh S., Sardet C., Dammann R., Minna JD., Downward J., Maher ER., Latif F. Apoptosis - Interactions involving proteins with a function related to apoptosis Note the UniProtKB entry Q66K89 for E4F1 has a higher degree of similarity to NP_004415 (the identifier given by the authors) than the other unreviewed entry of 784 aa. flatif@hgmp.mrc.ac.uk Accepted 2007-MAY-30 by JYOTI. 23-MAY-2007: Contacted by JYOTI. Returned mail. A MATCHMAKER human adult brain cDNA library (BD-Clontech) was used. Cancer Res. (0008-5472) 2004 Identification of the E1A-regulated transcription factor p120 E4F as an interacting partner of the RASSF1A candidate tumor suppressor gene. Identification of the E1A-regulated transcription factor p120 E4F as an interacting partner of the RASSF1A candidate tumor suppressor gene. Cancer Res. (0008-5472) 2004 Fenton SL., Dallol A., Agathanggelou A., Hesson L., Ahmed-Choudhury J., Baksh S., Sardet C., Dammann R., Minna JD., Downward J., Maher ER., Latif F. Apoptosis - Interactions involving proteins with a function related to apoptosis Cancer - Interactions investigated in the context of cancer Note the UniProtKB entry Q66K89 for E4F1 has a higher degree of similarity to NP_004415 (the identifier given by the authors) than the other unreviewed entry of 784 aa. flatif@hgmp.mrc.ac.uk 23-MAY-2007: Contacted by JYOTI. Returned mail. A MATCHMAKER human adult brain cDNA library (BD-Clontech) was used. human-nci_h1792 Homo sapiens non-small cell lung adenocarcinoma cell line NCI-H1792 nci_h1792 human non-small cell lung adenocarcinoma cell line ATCC number is CRL-5895. Tumour stage 4. Derived from pleural effusion. ATCC number is CRL-5895. anti bait coip anti bait coimmunoprecipitation anti bait coip western blot western blot Immuno blot Immuno blot Fenton SL., Dallol A., Agathanggelou A., Hesson L., Ahmed-Choudhury J., Baksh S., Sardet C., Dammann R., Minna JD., Downward J., Maher ER., Latif F. Apoptosis - Interactions involving proteins with a function related to apoptosis Note the UniProtKB entry Q66K89 for E4F1 has a higher degree of similarity to NP_004415 (the identifier given by the authors) than the other unreviewed entry of 784 aa. flatif@hgmp.mrc.ac.uk Accepted 2007-MAY-30 by JYOTI. 23-MAY-2007: Contacted by JYOTI. Returned mail. A MATCHMAKER human adult brain cDNA library (BD-Clontech) was used. Cancer Res. (0008-5472) 2004 stk3_human Serine/threonine-protein kinase 3 STE20-like kinase MST2 Mammalian STE20-like protein kinase 2 Serine/threonine-protein kinase Krs-1 STK3 MST2 KRS1 protein protein human Homo sapiens Human MEQPPAPKSKLKKLSEDSLTKQPEEVFDVLEKLGEGSYGSVFKAIHKESGQVVAIKQVPVESDLQEIIKEISIMQQCDSPYVVKYYGSYFKNTDLWIVMEYCGAGSVSDIIRLRNKTLIEDEIATILKSTLKGLEYLHFMRKIHRDIKAGNILLNTEGHAKLADFGVAGQLTDTMAKRNTVIGTPFWMAPEVIQEIGYNCVADIWSLGITSIEMAEGKPPYADIHPMRAIFMIPTNPPPTFRKPELWSDDFTDFVKKCLVKNPEQRATATQLLQHPFIKNAKPVSILRDLITEAMEIKAKRHEEQQRELEEEEENSDEDELDSHTMVKTSVESVGTMRATSTMSEGAQTMIEHNSTMLESDLGTMVINSEDEEEEDGTMKRNATSPQVQRPSFMDYFDKQDFKNKSHENCNQNMHEPFPMSKNVFPDNWKVPQDGDFDFLKNLSLEELQMRLKALDPMMEREIEELRQRYTAKRQPILDAMDAKKRRQQNF DIP protein Q13188 original sequence version: 150 DIP protein Q13188 original sequence version: 139 84F598ED3617292C stk4_human Serine/threonine-protein kinase 4 STK4 MST1 STE20-like kinase MST1 Mammalian STE20-like protein kinase 1 Serine/threonine-protein kinase Krs-2 KRS2 protein protein human Homo sapiens Human METVQLRNPPRRQLKKLDEDSLTKQPEEVFDVLEKLGEGSYGSVYKAIHKETGQIVAIKQVPVESDLQEIIKEISIMQQCDSPHVVKYYGSYFKNTDLWIVMEYCGAGSVSDIIRLRNKTLTEDEIATILQSTLKGLEYLHFMRKIHRDIKAGNILLNTEGHAKLADFGVAGQLTDTMAKRNTVIGTPFWMAPEVIQEIGYNCVADIWSLGITAIEMAEGKPPYADIHPMRAIFMIPTNPPPTFRKPELWSDNFTDFVKQCLVKSPEQRATATQLLQHPFVRSAKGVSILRDLINEAMDVKLKRQESQQREVDQDDEENSEEDEMDSGTMVRAVGDEMGTVRVASTMTDGANTMIEHDDTLPSQLGTMVINAEDEEEEGTMKRRDETMQPAKPSFLEYFEQKEKENQINSFGKSVPGPLKNSSDWKIPQDGDYEFLKSWTVEDLQKRLLALDPMMEQEIEEIRQKYQSKRQPILDAIEAKKRRQQNF 150758EBC5F77D5C q9ns23-2 Ras association domain-containing protein 1 RASSF1A RASSF1 RDA32 protein protein human Homo sapiens Human MSGEPELIELRELAPAGRAGKGRTRLERANALRIARGTACNPTRQLVPGRGHRFQPAGPATHTWCDLCGDFIWGVVRKGLQCAHCKFTCHYRCRALVCLDCCGPRDLGWEPAVERDTNVDEPVEWETPDLSQAEIEQKIKEYNAQINSNLFMSLNKDGSYTGFIKVQLKLVRPVSVPSSKKPPSLQDARRGPGRGTSVRRRTSFYLPKDAVKHLHVLSRTRAREVIEALLRKFLVVDDPRKFALFERAERHGQVYLRKLLDDEQPLRLRLLAGPSDKALSFVLKENDSGEVNWDAFSMPELHNFLRILQREEEEHLRQILQKYSYCRQKIQEALHACPLG Produced by alternative splicing of isoform D. 73FB7C7D87507D9F e4f1_human Transcription factor E4F1 E4F1 E4F Putative E3 ubiquitin-protein ligase E4F1 E4F transcription factor 1 Transcription factor E4F p120E4F p50E4F RING-type E3 ubiquitin transferase E4F1 protein protein human Homo sapiens Human MEGAMAVRVTAAHTAEAQAEAGREAGEGAVAAVAAALAPSGFLGLPAPFSEEDEDDVHRCGRCQAEFTALEDFVQHKIQKACQRAPPEALPATPATTALLGQEVVPAAPGPEEPITVAHIVVEAASLAADISHASDLVGGGHIKEVIVAAEAELGDGEMAEAPGSPRQQGLGLAGEGEQAQVKLLVNKDGRYVCALCHKTFKTGSILKAHMVTHSSRKDHECKLCGASFRTKGSLIRHHRRHTDERPYKCSKCGKSFRESGALTRHLKSLTPCTEKIRFSVSKDVVVSKEDARAGSGAGAAGLGTATSSVTGEPIETSPVIHLVTDAKGTVIHEVHVQMQELSLGMKALAPEPPVSQELPCSSEGSRENLLHQAMQNSGIVLERAAGEEGALEPAPAAGSSPQPLAVAAPQLPVLEVQPLETQVASEASAVPRTHPCPQCSETFPTAATLEAHKRGHTGPRPFACAQCGKAFPKAYLLKKHQEVHVRERRFRCGDCGKLYKTIAHVRGHRRVHSDERPYPCPKCGKRYKTKNAQQVHFRTHLEEKPHVCQFCSRGFREKGSLVRHVRHHTGEKPFKCYKCGRGFAEHGTLNRHLRTKGGCLLEVEELLVSEDSPAAATTVLTEDPHTVLVEFSSVVADTQEYIIEATADDAETSEATEIIEGTQTEVDSHIMKVVQQIVHQASAGHQIIVQNVTMDEETALGPEAAAADTITIATPESLTEQVAMTLASAISEGTVLAARAGTSGTEQATVTMVSSEDIEILEHAGELVIASPEGQLEVQTVIV 60F6E711F2748FD8 rassf1-e4f1 2 10 unspecified role unspecified role prey prey c-terminal sufficient to bind sufficient binding region sufficient to bind range range range range 9 unspecified role unspecified role bait bait n-terminal required to bind necessary binding region required to bind certain certain sequence position certain certain certain sequence position certain physical association physical association The bait RASSF1A-BD was then used to screen a pretransformed MATCHMAKER human adult brain cDNA library 103 p120E4F AS AN INTERACTING PARTNER OF RASSF1A (BD-Clontech). Approximately 6 106 independent transformations were screened, of which 103 clones were positive for -galactosidase expression. Nucleotide sequence analysis revealed that two separate clones (designated E4F)contained a 1-Kb insert with an open reading frame of 1113 bp encoding a protein fragment of 324 amino acids, which corresponds to the COOH-terminus half of the E1A regulated transcription factor p120E4F (NP 004415). PREY protein NP_004415 Isoform C of RASS1F as described by the authors lacks 119 aa at the N-terminus as compared to RASSF1A and did not interact with E4F1 (461-784). Note that the isoform C of RASS1F described in the UniProtKB entry Q9NS23 does not conform with the isoform described by the authors. The authors state E4F is 683 aa in length, whereas the protein they reference (NP_004415) is 784 aa, as is the corresponding UniProtKB entry. The predicted C-terminal zinc finger range for NP_004415 is aa 416-595. The range given above for E4FDelta is approximate only as precise mapping was not possible as the sequence of E4FDelta was not provided. rassf1-stk4 2 9 unspecified role unspecified role bait bait 8 unspecified role unspecified role prey prey physical association physical association We also identified a number of other interesting targets including the proapoptotic protein kinases MST1 and MST2 (10 clones) already known to interact with RASSF1A rassf1-stk3 2 9 unspecified role unspecified role bait bait 7 unspecified role unspecified role prey prey physical association physical association We also identified a number of other interesting targets including the proapoptotic protein kinases MST1 and MST2 (10 clones) already known to interact with RASSF1A rassf1-e4f1-1 4 10 unspecified role unspecified role prey prey c-terminal sufficient to bind sufficient binding region sufficient to bind range range range range region ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined in vitro In vitro 9 unspecified role unspecified role bait bait region myc tag myc tag EUKLISEED epitope tag EUKLISEED epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined in vitro In vitro direct interaction direct interaction PREY protein NP_004415 in vitro translated RASSF1A-myc and E4F-HA were tested in a coimmunoprecipitation before resolving on a 10% SDS-PAGE gel and Western blotting. In agreement with the data obtained in the yeast two-hybrid system an interaction was observed generating specific bands close to the predicated sizes of Mr 41,000 and Mr 60,000. 1 B e4f1-rassf1 4 9 unspecified role unspecified role prey prey region myc tag myc tag EUKLISEED epitope tag EUKLISEED epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined in vitro In vitro 10 unspecified role unspecified role bait bait c-terminal sufficient to bind sufficient binding region sufficient to bind range range range range region ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined in vitro In vitro direct interaction direct interaction BAIT protein NP_004415 in vitro translated RASSF1A-myc and E4F-HA were tested in a coimmunoprecipitation before resolving on a 10% SDS-PAGE gel and Western blotting. In agreement with the data obtained in the yeast two-hybrid system an interaction was observed generating specific bands close to the predicated sizes of Mr 41,000 and Mr 60,000. 1 B rassf1-e4f1-2 1 10 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 region required to bind necessary binding region required to bind range range range range in vitro In vitro 9 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 ecoli Escherichia coli (strain K12) direct interaction direct interaction PREY protein NP_004415 1 C, 4 A A GST fusion protein corresponding to full-length RASSF1A (1-340 amino acids) was created and incubated with in vitro translated E4F and p120E4F protein. E4F and wild-type p120E4F specifically bound to the GSTRASSF1A (1-340) fusion protein but not to the GST alone The range for E4F is approximate only. It is based on the end of the fourth C-terminal zinc finger (found using zinc finger consensus sequence CX2-4C....HX2-4H) and the next 26 aa (as the deletion analysis reveals the 26 aa after the end of this fourth zinc finger are required to bind.) rassf1-e4f1-3 3 9 unspecified role unspecified role bait bait 10 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 p120E4F-HA was efficiently coprecipitated in both NIH 3T3 and A549 cells with RASSF1A but not control IgG Q66K89; Q9NS23 transfection Q66K89; Q9NS23 over-expressed rassf1-e4f1-4 5 10 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 9 unspecified role unspecified role bait bait physical association physical association 2 A p120E4F-HA was efficiently coprecipitated in both NIH 3T3 and A549 cells with RASSF1A but not control IgG Q66K89; Q9NS23 transfection Q66K89; Q9NS23 over-expressed rassf1a-e4f1 6 10 unspecified role unspecified role prey prey 9 unspecified role unspecified role bait bait physical association physical association 2 B, C immunoprecipitation was performed using human breast carcinoma cell line MCF7 (RASSF1A negative) and non-small cell lung tumor cell line NCI-H1792 (RASSF1A positive). Cell lysates were prepared and subjected to immunoprecipitation with RASSF1A-specific polyclonal antibody. As shown in Fig. 2B endogenous levels of RASSF1A interact with p120E4F in vivo. This interaction is inhibited by RASSF1A-siRNA.