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/ VRK1 Phosphorylates Tip60/KAT5 and Is Required for H4K16 Acetylation in Response to DNA Damage. VRK1 Phosphorylates Tip60/KAT5 and Is Required for H4K16 Acetylation in Response to DNA Damage. Cancers 2020 imex curation García-González R., Morejón-García P., Campillo-Marcos I., Salzano M., Lazo PA. 2020-04-06: Submitted by PA Lazo, CSIC-Universidad de Salamanca, Spain. pedro.lazo@csic.es 05-Jan-2021: Contacted by IntAct-Help. Only protein-protein interactions imex curation in vitro In vitro protein kinase assay protein kinase assay western blot western blot Immuno blot Immuno blot García-González R., Morejón-García P., Campillo-Marcos I., Salzano M., Lazo PA. 2020-04-06: Submitted by PA Lazo, CSIC-Universidad de Salamanca, Spain. pedro.lazo@csic.es Accepted 2020-NOV-09 AT 08:36 GMT AT 08:36 GMT by PPORRAS Cancers 2020 Only protein-protein interactions imex curation imex curation VRK1 Phosphorylates Tip60/KAT5 and Is Required for H4K16 Acetylation in Response to DNA Damage. VRK1 Phosphorylates Tip60/KAT5 and Is Required for H4K16 Acetylation in Response to DNA Damage. Cancers 2020 imex curation García-González R., Morejón-García P., Campillo-Marcos I., Salzano M., Lazo PA. 2020-04-06: Submitted by PA Lazo, CSIC-Universidad de Salamanca, Spain. pedro.lazo@csic.es 05-Jan-2021: Contacted by IntAct-Help. Only protein-protein interactions imex curation 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 García-González R., Morejón-García P., Campillo-Marcos I., Salzano M., Lazo PA. 2020-04-06: Submitted by PA Lazo, CSIC-Universidad de Salamanca, Spain. pedro.lazo@csic.es Accepted 2020-NOV-09 AT 08:36 GMT AT 08:36 GMT by PPORRAS Cancers 2020 Only protein-protein interactions imex curation imex curation VRK1 Phosphorylates Tip60/KAT5 and Is Required for H4K16 Acetylation in Response to DNA Damage. VRK1 Phosphorylates Tip60/KAT5 and Is Required for H4K16 Acetylation in Response to DNA Damage. Cancers 2020 imex curation García-González R., Morejón-García P., Campillo-Marcos I., Salzano M., Lazo PA. 2020-04-06: Submitted by PA Lazo, CSIC-Universidad de Salamanca, Spain. pedro.lazo@csic.es 05-Jan-2021: Contacted by IntAct-Help. Only protein-protein interactions imex curation human-a_549 Homo sapiens Caucasian lung carcinoma cell line a_549 Human Caucasian lung carcinoma anti tag coip anti tag coimmunoprecipitation anti tag coip western blot western blot Immuno blot Immuno blot García-González R., Morejón-García P., Campillo-Marcos I., Salzano M., Lazo PA. 2020-04-06: Submitted by PA Lazo, CSIC-Universidad de Salamanca, Spain. pedro.lazo@csic.es Accepted 2020-NOV-09 AT 08:36 GMT AT 08:36 GMT by PPORRAS Cancers 2020 Only protein-protein interactions imex curation imex curation VRK1 Phosphorylates Tip60/KAT5 and Is Required for H4K16 Acetylation in Response to DNA Damage. VRK1 Phosphorylates Tip60/KAT5 and Is Required for H4K16 Acetylation in Response to DNA Damage. Cancers 2020 imex curation García-González R., Morejón-García P., Campillo-Marcos I., Salzano M., Lazo PA. 2020-04-06: Submitted by PA Lazo, CSIC-Universidad de Salamanca, Spain. pedro.lazo@csic.es 05-Jan-2021: Contacted by IntAct-Help. Only protein-protein interactions imex curation in vitro In vitro pull down pull down anti tag western anti tag western blot anti tag western García-González R., Morejón-García P., Campillo-Marcos I., Salzano M., Lazo PA. 2020-04-06: Submitted by PA Lazo, CSIC-Universidad de Salamanca, Spain. pedro.lazo@csic.es Accepted 2020-NOV-09 AT 08:36 GMT AT 08:36 GMT by PPORRAS Cancers 2020 Only protein-protein interactions imex curation imex curation VRK1 Phosphorylates Tip60/KAT5 and Is Required for H4K16 Acetylation in Response to DNA Damage. VRK1 Phosphorylates Tip60/KAT5 and Is Required for H4K16 Acetylation in Response to DNA Damage. Cancers 2020 imex curation García-González R., Morejón-García P., Campillo-Marcos I., Salzano M., Lazo PA. 2020-04-06: Submitted by PA Lazo, CSIC-Universidad de Salamanca, Spain. pedro.lazo@csic.es 05-Jan-2021: Contacted by IntAct-Help. Only protein-protein interactions imex curation human-a_549 Homo sapiens Caucasian lung carcinoma cell line a_549 Human Caucasian lung carcinoma anti tag coip anti tag coimmunoprecipitation anti tag coip anti tag western anti tag western blot anti tag western García-González R., Morejón-García P., Campillo-Marcos I., Salzano M., Lazo PA. 2020-04-06: Submitted by PA Lazo, CSIC-Universidad de Salamanca, Spain. pedro.lazo@csic.es Accepted 2020-NOV-09 AT 08:36 GMT AT 08:36 GMT by PPORRAS Cancers 2020 Only protein-protein interactions imex curation imex curation kat5_human Histone acetyltransferase KAT5 KAT5 TIP60 HTATIP Lysine acetyltransferase 5 cPLA(2)-interacting protein Histone acetyltransferase HTATIP 60 kDa Tat-interactive protein protein protein human Homo sapiens Human MAEVGEIIEGCRLPVLRRNQDNEDEWPLAEILSVKDISGRKLFYVHYIDFNKRLDEWVTHERLDLKKIQFPKKEAKTPTKNGLPGSRPGSPEREVPASAQASGKTLPIPVQITLRFNLPKEREAIPGGEPDQPLSSSSCLQPNHRSTKRKVEVVSPATPVPSETAPASVFPQNGAARRAVAAQPGRKRKSNCLGTDEDSQDSSDGIPSAPRMTGSLVSDRSHDDIVTRMKNIECIELGRHRLKPWYFSPYPQELTTLPVLYLCEFCLKYGRSLKCLQRHLTKCDLRHPPGNEIYRKGTISFFEIDGRKNKSYSQNLCLLAKCFLDHKTLYYDTDPFLFYVMTEYDCKGFHIVGYFSKEKESTEDYNVACILTLPPYQRRGYGKLLIEFSYELSKVEGKTGTPEKPLSDLGLLSYRSYWSQTILEILMGLKSESGERPQITINEISEITSIKKEDVISTLQYLNLINYYKGQYILTLSEDIVDGHERAMLKRLLRIDSKCLHFTPKDWSKRGKW DIP protein Q92993 original sequence version: 153 DIP protein Q92993 original sequence version: 155 DIP protein Q92993 original sequence version: 86 DIP protein Q92993 original sequence version: 149 DIP protein Q92993-1 original sequence version: 129 DIP protein Q92993 original sequence version: 144 DIP protein Q92993 original sequence version: 173 63724F5E10B957D5 vrk1_human Serine/threonine-protein kinase VRK1 Vaccinia-related kinase 1 VRK1 protein protein human Homo sapiens Human MPRVKAAQAGRQSSAKRHLAEQFAVGEIITDMAKKEWKVGLPIGQGGFGCIYLADMNSSESVGSDAPCVVKVEPSDNGPLFTELKFYQRAAKPEQIQKWIRTRKLKYLGVPKYWGSGLHDKNGKSYRFMIMDRFGSDLQKIYEANAKRFSRKTVLQLSLRILDILEYIHEHEYVHGDIKASNLLLNYKNPDQVYLVDYGLAYRYCPEGVHKEYKEDPKRCHDGTIEFTSIDAHNGVAPSRRGDLEILGYCMIQWLTGHLPWEDNLKDPKYVRDSKIRYRENIASLMDKCFPEKNKPGEIAKYMETVKLLDYTEKPLYENLRDILLQGLKAIGSKDDGKLDLSVVENGGLKAKTITKKRKKEIEESKEPGVEDTEWSNTQTEEAIQTRSRTRKRVQK 5640C624BF059949 kat5-vrk1-1 4 6 unspecified role unspecified role prey prey purified purified n-terminus his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range ecoli Escherichia coli (strain K12) 7 unspecified role unspecified role bait bait purified purified n-terminus gst tag glutathione s tranferase tag glutathione S-tranferase tag glutathione S-tranferase tag gst tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range ecoli Escherichia coli (strain K12) direct interaction direct interaction Fig. 3a [...] we performed an interaction assay from proteins expressed in bacteria and purified. In this experiment, a fixed amount of GST-VRK1 was incubated with increasing amountsof His-Tip60. The GST-VRK1 pulldown detected an increase in its interaction with His-Tip60 as the concentration of the latter was higher, and reaching a plateau as their concentrations became equimolar. vrk1-kat5-1 5 6 unspecified role unspecified role bait bait over-expressed over expressed level over-expressed c-terminus v5 tag v5 tag GKPIPNPLLGLDST epitope tag GKPIPNPLLGLDST epitope tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range 7 unspecified role unspecified role prey prey over-expressed over expressed level over-expressed n-terminus ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range physical association physical association Fig. 3b-II To confirm the interaction in vivo, A549 cells were transfected with Tip60-V5 and VRK1-HA tagged human proteins, and a reciprocal immunoprecipitation was performed. The Tip60-VRK1 complex was detected in both reciprocal precipitations indicating it is stable by itself. kat5-vrk1-2 5 6 unspecified role unspecified role prey prey over-expressed over expressed level over-expressed c-terminus v5 tag v5 tag GKPIPNPLLGLDST epitope tag GKPIPNPLLGLDST epitope tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range 7 unspecified role unspecified role bait bait over-expressed over expressed level over-expressed n-terminus ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range physical association physical association Fig. 3b-I To confirm the interaction in vivo, A549 cells were transfected with Tip60-V5 and VRK1-HA tagged human proteins, and a reciprocal immunoprecipitation was performed. The Tip60-VRK1 complex was detected in both reciprocal precipitations indicating it is stable by itself. kat5-vrk1-3 2 6 unspecified role unspecified role bait bait physiological level physiological level endogenous level endogenous endogenous endogenous level 7 unspecified role unspecified role prey prey physiological level physiological level endogenous level endogenous endogenous endogenous level physical association physical association Fig. 3c, 3d [...] the endogenous Tip60 was immunoprecipitated and the endogenous VRK1 detected in the immunoprecipitate. vrk1-kat5-2 1 6 enzyme target enzyme target substrate substrate neutral component neutral component purified purified region opser O-phospho-L-serine (2S)-2-amino-3-(phosphonooxy)propanoic acid 2-amino-3-hydroxypropanoic acid 3-phosphate 2-azanyl-3-(phosphonooxy)propanoic acid ACT_SITE Phosphoserine intermediate MOD_RES Phosphoserine O-phospho-L-serine O-phosphonoserine O-phosphorylated L-serine O3-phosphoserine serine phosphate ester OPSer Phospho Phospho Seryl Phosphorylation phosphorylations undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined observed-ptm observed-ptm observed ptm observed-ptm n-terminus his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range ecoli Escherichia coli (strain K12) 7 enzyme enzyme neutral component neutral component purified purified n-terminus gst tag glutathione s tranferase tag glutathione S-tranferase tag glutathione S-tranferase tag gst tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range Q99986:p.Lys179Glu mutation disrupting rate mutation disrupting interaction rate mutation disrupting rate certain certain sequence position certain certain certain sequence position certain K E ecoli Escherichia coli (strain K12) phosphorylation phosphorylation reaction phosphorylation Fig. 3e Tip60 was phosphorylated by kinase-active VRK1, but not by kinase-dead VRK1 (K179E). kat5-vrk1-4 3 6 unspecified role unspecified role bait bait over-expressed over expressed level over-expressed c-terminus v5 tag v5 tag GKPIPNPLLGLDST epitope tag GKPIPNPLLGLDST epitope tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range 7 unspecified role unspecified role prey prey physiological level physiological level endogenous level endogenous endogenous endogenous level physical association physical association Fig. 4a The treatment with doxorubicin did not alter the intracellular complex formed between the endogenous VRK1 and the transfected Tip60 determined by immunoprecipitation (Figure 4A).