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/ NK lytic-associated molecule, involved in NK cytotoxic function, is an E3 ligase NK lytic-associated molecule, involved in NK cytotoxic function, is an E3 ligase J Immunol. 2006 imex curation Fortier JM, Kornbluth J. kornblut@slu.edu 23-Dec-2009: Contacted by JYOTI. jyoti Only protein-protein interactions Curation by: Molecular Connections Pvt. Ltd human-293 Homo sapiens transformed primary embryonal kidney cells Human 293 Transformed human primary embryonal kidney cells. The Cell type named 293 is synonymous with HEK-293. anti tag coip anti tag coimmunoprecipitation anti tag coip anti tag western anti tag western blot anti tag western Fortier JM, Kornbluth J. kornblut@slu.edu Accepted 2009-DEC-10 by JYOTI. 23-Dec-2009: Contacted by JYOTI. J Immunol. 2006 jyoti Only protein-protein interactions imex curation Curation by: Molecular Connections Pvt. Ltd NK lytic-associated molecule, involved in NK cytotoxic function, is an E3 ligase NK lytic-associated molecule, involved in NK cytotoxic function, is an E3 ligase J Immunol. 2006 imex curation Fortier JM, Kornbluth J. kornblut@slu.edu 23-Dec-2009: Contacted by JYOTI. jyoti Only protein-protein interactions Curation by: Molecular Connections Pvt. Ltd human-293 Homo sapiens transformed primary embryonal kidney cells Human 293 Transformed human primary embryonal kidney cells. The Cell type named 293 is synonymous with HEK-293. confocal microscopy confocal microscopy immunostaining immunostaining Fortier JM, Kornbluth J. kornblut@slu.edu Accepted 2009-DEC-10 by JYOTI. 23-Dec-2009: Contacted by JYOTI. J Immunol. 2006 jyoti Only protein-protein interactions imex curation Curation by: Molecular Connections Pvt. Ltd NK lytic-associated molecule, involved in NK cytotoxic function, is an E3 ligase NK lytic-associated molecule, involved in NK cytotoxic function, is an E3 ligase J Immunol. 2006 imex curation Fortier JM, Kornbluth J. kornblut@slu.edu 23-Dec-2009: Contacted by JYOTI. jyoti Only protein-protein interactions Curation by: Molecular Connections Pvt. Ltd human-293 Homo sapiens transformed primary embryonal kidney cells Human 293 Transformed human primary embryonal kidney cells. The Cell type named 293 is synonymous with HEK-293. anti bait coip anti bait coimmunoprecipitation anti bait coip anti tag western anti tag western blot anti tag western Fortier JM, Kornbluth J. kornblut@slu.edu Accepted 2009-DEC-10 by JYOTI. 23-Dec-2009: Contacted by JYOTI. J Immunol. 2006 jyoti Only protein-protein interactions imex curation Curation by: Molecular Connections Pvt. Ltd NK lytic-associated molecule, involved in NK cytotoxic function, is an E3 ligase NK lytic-associated molecule, involved in NK cytotoxic function, is an E3 ligase J Immunol. 2006 imex curation Fortier JM, Kornbluth J. kornblut@slu.edu 23-Dec-2009: Contacted by JYOTI. jyoti Only protein-protein interactions Curation by: Molecular Connections Pvt. Ltd human-nk3_3 Human NK cell line NK3.3 nk3_3 Natural Killer cell line, NK3.3 anti bait coip anti bait coimmunoprecipitation anti bait coip western blot western blot Immuno blot Immuno blot Fortier JM, Kornbluth J. kornblut@slu.edu Accepted 2009-DEC-10 by JYOTI. 23-Dec-2009: Contacted by JYOTI. J Immunol. 2006 jyoti Only protein-protein interactions imex curation Curation by: Molecular Connections Pvt. Ltd NK lytic-associated molecule, involved in NK cytotoxic function, is an E3 ligase NK lytic-associated molecule, involved in NK cytotoxic function, is an E3 ligase J Immunol. 2006 imex curation Fortier JM, Kornbluth J. kornblut@slu.edu 23-Dec-2009: Contacted by JYOTI. jyoti Only protein-protein interactions Curation by: Molecular Connections Pvt. Ltd 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 Fortier JM, Kornbluth J. kornblut@slu.edu Accepted 2009-DEC-10 by JYOTI. 23-Dec-2009: Contacted by JYOTI. Human spleen cDNA J Immunol. 2006 jyoti Only protein-protein interactions imex curation Curation by: Molecular Connections Pvt. Ltd ub2l3_human Ubiquitin-conjugating enzyme E2 L3 Ubiquitin-protein ligase L3 Ubiquitin carrier protein L3 UbcH7 Ubiquitin-conjugating enzyme E2-F1 L-UBC UBE2L3 UBCE7 UBCH7 E2 ubiquitin-conjugating enzyme L3 protein protein human Homo sapiens Human MAASRRLMKELEEIRKCGMKNFRNIQVDEANLLTWQGLIVPDNPPYDKGAFRIEINFPAEYPFKPPKITFKTKIYHPNIDEKGQVCLPVISAENWKPATKTDQVIQSLIALVNDPQPEHPLRADLAEEYSKDRKKFCKNAEEFTKKYGEKRPVD F5A30243BE3C9985 rn19b_human E3 ubiquitin-protein ligase RNF19B RING finger protein 19B IBR domain-containing protein 3 Natural killer lytic-associated molecule RNF19B IBRDC3 NKLAM protein protein human Homo sapiens Human MGSEKDSESPRSTSLHAAAPDPKCRSGGRRRRLTLHSVFSASARGRRARAKPQAEPPPPAAQPPPAPAPAAAQGPPPEALPAEPAAEAEAEAAAAAAEPGFDDEEAAEGGGPGAEEVECPLCLVRLPPERAPRLLSCPHRSCRDCLRHYLRLEISESRVPISCPECSERLNPHDIRLLLADPPLMHKYEEFMLRRYLASDPDCRWCPAPDCGYAVIAYGCASCPKLTCEREGCQTEFCYHCKQIWHPNQTCDMARQQRAQTLRVRTKHTSGLSYGQESGPADDIKPCPRCSAYIIKMNDGSCNHMTCAVCGCEFCWLCMKEISDLHYLSPSGCTFWGKKPWSRKKKILWQLGTLIGAPVGISLIAGIAIPAMVIGIPVYVGRKIHSRYEGRKTSKHKRNLAITGGVTLSVIASPVIAAVSVGIGVPIMLAYVYGVVPISLCRGGGCGVSTANGKGVKIEFDEDDGPITVADAWRALKNPSIGESSIEGLTSVLSTSGSPTDGLSVMQGPYSETASFAALSGGTLSGGILSSGKGKYSRLEVQADVQKEIFPKDTASLGAISDNASTRAMAGSIISSYNPQDRECNNMEIQVDIEAKPSHYQLVSGSSTEDSLHVHAQMAENEEEGSGGGGSEEDPPCRHQSCEQKDCLASKPWDISLAQPESIRSDLESSDAQSDDVPDITSDECGSPRSHTAACPSTPRAQGAPSPSAHMNLSALAEGQTVLKPEGGEARV EB30FBB46393C30B ub2l6_human Ubiquitin/ISG15-conjugating enzyme E2 L6 UBE2L6 UBCH8 Ubiquitin-protein ligase L6 Ubiquitin carrier protein L6 UbcH8 Retinoic acid-induced gene B protein E2 ubiquitin-conjugating enzyme L6 protein protein human Homo sapiens Human MMASMRVVKELEDLQKKPPPYLRNLSSDDANVLVWHALLLPDQPPYHLKAFNLRISFPPEYPFKPPMIKFTTKIYHPNVDENGQICLPIISSENWKPCTKTCQVLEALNVLVNRPNIREPLRMDLADLLTQNPELFRKNAEEFTLRFGVDRPS DIP protein O14933 original sequence version: 136 D79768775344EFAE uckl1_human Uridine-cytidine kinase-like 1 F538 UCKL1 URKL1 protein protein human Homo sapiens Human MAAPPARADADPSPTSPPTARDTPGRQAEKSETACEDRSNAESLDRLLPPVGTGRSPRKRTTSQCKSEPPLLRTSKRTIYTAGRPPWYNEHGTQSKEAFAIGLGGGSASGKTTVARMIIEALDVPWVVLLSMDSFYKVLTEQQQEQAAHNNFNFDHPDAFDFDLIISTLKKLKQGKSVKVPIYDFTTHSRKKDWKTLYGANVIIFEGIMAFADKTLLELLDMKIFVDTDSDIRLVRRLRRDISERGRDIEGVIKQYNKFVKPSFDQYIQPTMRLADIVVPRGSGNTVAIDLIVQHVHSQLEERELSVRAALASAHQCHPLPRTLSVLKSTPQVRGMHTIIRDKETSRDEFIFYSKRLMRLLIEHALSFLPFQDCVVQTPQGQDYAGKCYAGKQITGVSILRAGETMEPALRAVCKDVRIGTILIQTNQLTGEPELHYLRLPKDISDDHVILMDCTVSTGAAAMMAVRVLLDHDVPEDKIFLLSLLMAEMGVHSVAYAFPRVRIITTAVDKRVNDLFRIIPGIGNFGDRYFGTDAVPDGSDEEEVAYTG 0CD0351A802FE199 rnf19b-ube2l3-2 1 6 anti tag western anti tag western blot anti tag western western blot western blot Immuno blot Immuno blot unspecified role unspecified role prey prey region flag tag flag tag FLAG-tagged FLAG DYKDDDDKV epitope tag DYKDDDDKV epitope tag FLAG FLAG-tagged n-term range n-terminal range n-term range n-term range n-terminal range n-term range 7 anti tag western anti tag western blot anti tag western western blot western blot Immuno blot Immuno blot unspecified role unspecified role bait bait n terminal myc tag myc tag EUKLISEED epitope tag EUKLISEED epitope tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range cd2 region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain physical association physical association fig 2 Coimmunoprecipitation of UbcH7 and UbcH8 with NKLAM CRDs. rnf19b-ube2l6-1 3 7 enzyme enzyme bait bait 8 monoclonal western monoclonal antibody western blot monoclonal western anti tag western anti tag western blot anti tag western unspecified role unspecified role prey prey n-terminal flag tag flag tag FLAG-tagged FLAG DYKDDDDKV epitope tag DYKDDDDKV epitope tag FLAG FLAG-tagged n-term range n-terminal range n-term range n-term range n-terminal range n-term range physical association physical association fig 1b UbcH7 and UbcH8 coimmunoprecipitated with NKLAM (Fig. 1b) rnf19b-ube2l6 1 8 anti tag western anti tag western blot anti tag western western blot western blot Immuno blot Immuno blot unspecified role unspecified role prey prey n terminal flag tag flag tag FLAG-tagged FLAG DYKDDDDKV epitope tag DYKDDDDKV epitope tag FLAG FLAG-tagged n-term range n-terminal range n-term range n-term range n-terminal range n-term range 7 anti tag western anti tag western blot anti tag western western blot western blot Immuno blot Immuno blot unspecified role unspecified role bait bait n-terminal myc tag myc tag EUKLISEED epitope tag EUKLISEED epitope tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range cd2 sufficient to bind sufficient binding region sufficient to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain cd1 region sufficient to bind sufficient binding region sufficient to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain physical association physical association 2 Coimmunoprecipitation of UbcH7 and UbcH8 with NKLAM CRDs The CD2 region of RNF19B bound more strongly to UBE2l6 than did the CD1. rnf19b-uckl1-2 5 7 unspecified role unspecified role bait bait n terminal sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain greater-than greater-than 9 unspecified role unspecified role prey prey physical association physical association Using the yeast two-hybrid system, we identified uridine kinase like-1 (URKL-1) protein as a substrate for NKLAM. rnf19b-uckl1 3 7 monoclonal western monoclonal antibody western blot monoclonal western anti tag western anti tag western blot anti tag western unspecified role unspecified role bait bait engineered engineered 9 anti tag western anti tag western blot anti tag western anti tag western anti tag western blot anti tag western unspecified role unspecified role prey prey engineered engineered n-terminal flag tag flag tag FLAG-tagged FLAG DYKDDDDKV epitope tag DYKDDDDKV epitope tag FLAG FLAG-tagged n-term range n-terminal range n-term range n-term range n-terminal range n-term range physical association physical association fig 4a Coimmunoprecipitation of NKLAM and URKL-1 rnf19b-uckl1-1 1 9 anti tag western anti tag western blot anti tag western western blot western blot Immuno blot Immuno blot unspecified role unspecified role prey prey engineered engineered n-terminal flag tag flag tag FLAG-tagged FLAG DYKDDDDKV epitope tag DYKDDDDKV epitope tag FLAG FLAG-tagged n-term range n-terminal range n-term range n-term range n-terminal range n-term range 7 anti tag western anti tag western blot anti tag western western blot western blot Immuno blot Immuno blot unspecified role unspecified role bait bait engineered engineered cd2 region sufficient to bind sufficient binding region sufficient to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain n-terminal myc tag myc tag EUKLISEED epitope tag EUKLISEED epitope tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range cd1 region sufficient to bind sufficient binding region sufficient to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain human Homo sapiens Human physical association physical association fig 4b The results show that URKL-1 interaction with NKLAM is greatest with the second CRD (Fig. 4b) Very weak binding of UCKL1 was also observed to the third cysteine-rich domain of RNF19B, binding of the CD2 was stronger than with the CD1. rnf19b-uckl1-3 2 7 monoclonal immunost monoclonal antibody immunostaining monoclonal immunost immunostaining immunostaining unspecified role unspecified role neutral component neutral component 9 anti tag immunost anti tag immunostaining anti tag immunost immunostaining immunostaining unspecified role unspecified role neutral component neutral component n terminal flag tag flag tag FLAG-tagged FLAG DYKDDDDKV epitope tag DYKDDDDKV epitope tag FLAG FLAG-tagged n-term range n-terminal range n-term range n-term range n-terminal range n-term range colocalization colocalization fig 7 Localization of NKLAM and URKL-1 in 293 cells. rnf19b-ube2l6-2 4 8 polyclonal western polyclonal antibody western blot polyclonal western western blot western blot Immuno blot Immuno blot unspecified role unspecified role prey prey 7 monoclonal western monoclonal antibody western blot monoclonal western western blot western blot Immuno blot Immuno blot unspecified role unspecified role bait bait physical association physical association fig 3 IFN-beta and IL-2 treated NK 3.3 cells rnf19b-ube2l3-1 3 7 enzyme enzyme bait bait 6 unspecified role unspecified role prey prey n-terminal flag tag flag tag FLAG-tagged FLAG DYKDDDDKV epitope tag DYKDDDDKV epitope tag FLAG FLAG-tagged n-term range n-terminal range n-term range n-term range n-terminal range n-term range physical association physical association fig 1b UbcH7 and UbcH8 coimmunoprecipitated with NKLAM (Fig. 1b)