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 http://mint.bio.uniroma2.it/mint anonymous-2014f-1 Ubiquitylation-dependent downregulation of Nck regulates its functional activity human Homo sapiens Human anti tag coip anti tag coimmunoprecipitation anti tag coip western blot western blot Immuno blot Immuno blot Mira.Barda-Saad@biu.ac.il 2014 imex curation Joseph N., Reicher B., David A., Matalon O., Barda-Saad M. FEBS Lett. (0014-5793) Only protein-protein interactions nck1_human Cytoplasmic protein NCK1 NCK1 NCK NCK adaptor protein 1 SH2/SH3 adaptor protein NCK-alpha protein protein human Homo sapiens Human MAEEVVVVAKFDYVAQQEQELDIKKNERLWLLDDSKSWWRVRNSMNKTGFVPSNYVERKNSARKASIVKNLKDTLGIGKVKRKPSVPDSASPADDSFVDPGERLYDLNMPAYVKFNYMAEREDELSLIKGTKVIVMEKCSDGWWRGSYNGQVGWFPSNYVTEEGDSPLGDHVGSLSEKLAAVVNNLNTGQVLHVVQALYPFSSSNDEELNFEKGDVMDVIEKPENDPEWWKCRKINGMVGLVPKNYVTVMQNNPLTSGLEPSPPQCDYIRPSLTGKFAGNPWYYGKVTRHQAEMALNERGHEGDFLIRDSESSPNDFSVSLKAQGKNKHFKVQLKETVYCIGQRKFSTMEELVEHYKKAPIFTSEQGEKLYLVKHLS cblb_human E3 ubiquitin-protein ligase CBL-B Signal transduction protein CBL-B SH3-binding protein CBL-B Casitas B-lineage lymphoma proto-oncogene b RING finger protein 56 CBLB RNF56 Nbla00127 RING-type E3 ubiquitin transferase CBL-B protein protein human Homo sapiens Human MANSMNGRNPGGRGGNPRKGRILGIIDAIQDAVGPPKQAAADRRTVEKTWKLMDKVVRLCQNPKLQLKNSPPYILDILPDTYQHLRLILSKYDDNQKLAQLSENEYFKIYIDSLMKKSKRAIRLFKEGKERMYEEQSQDRRNLTKLSLIFSHMLAEIKAIFPNGQFQGDNFRITKADAAEFWRKFFGDKTIVPWKVFRQCLHEVHQISSGLEAMALKSTIDLTCNDYISVFEFDIFTRLFQPWGSILRNWNFLAVTHPGYMAFLTYDEVKARLQKYSTKPGSYIFRLSCTRLGQWAIGYVTGDGNILQTIPHNKPLFQALIDGSREGFYLYPDGRSYNPDLTGLCEPTPHDHIKVTQEQYELYCEMGSTFQLCKICAENDKDVKIEPCGHLMCTSCLTAWQESDGQGCPFCRCEIKGTEPIIVDPFDPRDEGSRCCSIIDPFGMPMLDLDDDDDREESLMMNRLANVRKCTDRQNSPVTSPGSSPLAQRRKPQPDPLQIPHLSLPPVPPRLDLIQKGIVRSPCGSPTGSPKSSPCMVRKQDKPLPAPPPPLRDPPPPPPERPPPIPPDNRLSRHIHHVESVPSRDPPMPLEAWCPRDVFGTNQLVGCRLLGEGSPKPGITASSNVNGRHSRVGSDPVLMRKHRRHDLPLEGAKVFSNGHLGSEEYDVPPRLSPPPPVTTLLPSIKCTGPLANSLSEKTRDPVEEDDDEYKIPSSHPVSLNSQPSHCHNVKPPVRSCDNGHCMLNGTHGPSSEKKSNIPDLSIYLKGDVFDSASDPVPLPPARPPTRDNPKHGSSLNRTPSDYDLLIPPLGEDAFDALPPSLPPPPPPARHSLIEHSKPPGSSSRPSSGQDLFLLPSDPFVDLASGQVPLPPARRLPGENVKTNRTSQDYDQLPSCSDGSQAPARPPKPRPRRTAPEIHHRKPHGPEAALENVDAKIAKLMGEGYAFEEVKRALEIAQNNVEVARSILREFAFPPPVSPRLNL cblb-nck1 1566271 n/a Nck 1566272 western blot western blot Immuno blot Immuno blot unspecified role unspecified role bait bait over-expressed over expressed level over-expressed living cell living cell P16333:p.Trp38Lys mutation disrupting mutation disrupting interaction mutation disrupting certain certain sequence position certain certain certain sequence position certain false yfp tag yfp tag yellow fluorescent protein tag YFP yellow fluorescent protein yellow fluorescent protein YFP yfp tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false n/a Cbl-b 1566273 western blot western blot Immuno blot Immuno blot unspecified role unspecified role prey prey over-expressed over expressed level over-expressed living cell living cell association association false false false f6b f6c f6d 293T cells were co-transfected with YFP-tagged Nck constructs encoding Nck-wt, or each of the SH3 mutant molecules, together with HA-Ub and Cbl-b. As shown in Figure 6B, each of the Nck-SH3-mutants exhibited a substantial decrease in their 11 interaction with Cbl-b, and in Nck ubiquitylation. Moreover, a triple Nck-SH3 mutant (3SH3*) did not interact with Cbl-b at all, and was substantially less ubiquitylated (Fig. 6C). nck1-cblb-3 1566271 n/a Cbl-b 1566273 western blot western blot Immuno blot Immuno blot unspecified role unspecified role prey prey over-expressed over expressed level over-expressed living cell living cell n/a Nck 1566272 western blot western blot Immuno blot Immuno blot unspecified role unspecified role bait bait over-expressed over expressed level over-expressed living cell living cell yfp tag yfp tag yellow fluorescent protein tag YFP yellow fluorescent protein yellow fluorescent protein YFP yfp tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false P16333:p.Trp143Lys mutation disrupting mutation disrupting interaction mutation disrupting certain certain sequence position certain certain certain sequence position certain false association association false false false f6b f6c f6d 293T cells were co-transfected with YFP-tagged Nck constructs encoding Nck-wt, or each of the SH3 mutant molecules, together with HA-Ub and Cbl-b. As shown in Figure 6B, each of the Nck-SH3-mutants exhibited a substantial decrease in their 11 interaction with Cbl-b, and in Nck ubiquitylation. Moreover, a triple Nck-SH3 mutant (3SH3*) did not interact with Cbl-b at all, and was substantially less ubiquitylated (Fig. 6C). nck1-cblb-4 1566271 n/a Nck 1566272 western blot western blot Immuno blot Immuno blot unspecified role unspecified role bait bait over-expressed over expressed level over-expressed living cell living cell yfp tag yfp tag yellow fluorescent protein tag YFP yellow fluorescent protein yellow fluorescent protein YFP yfp tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false P16333:p.Trp229Lys mutation disrupting mutation disrupting interaction mutation disrupting certain certain sequence position certain certain certain sequence position certain false n/a Cbl-b 1566273 western blot western blot Immuno blot Immuno blot unspecified role unspecified role prey prey over-expressed over expressed level over-expressed living cell living cell association association false false false f6b f6c f6d 293T cells were co-transfected with YFP-tagged Nck constructs encoding Nck-wt, or each of the SH3 mutant molecules, together with HA-Ub and Cbl-b. As shown in Figure 6B, each of the Nck-SH3-mutants exhibited a substantial decrease in their 11 interaction with Cbl-b, and in Nck ubiquitylation. Moreover, a triple Nck-SH3 mutant (3SH3*) did not interact with Cbl-b at all, and was substantially less ubiquitylated (Fig. 6C). nck1-cblb-5 1566271 n/a Cbl-b 1566273 western blot western blot Immuno blot Immuno blot unspecified role unspecified role prey prey over-expressed over expressed level over-expressed living cell living cell n/a Nck 1566272 western blot western blot Immuno blot Immuno blot unspecified role unspecified role bait bait over-expressed over expressed level over-expressed living cell living cell P16333:p.[Trp38Lys;Trp143Lys;Trp229Lys] mutation disrupting mutation disrupting interaction mutation disrupting certain certain sequence position certain certain certain sequence position certain false certain certain sequence position certain certain certain sequence position certain false certain certain sequence position certain certain certain sequence position certain false yfp tag yfp tag yellow fluorescent protein tag YFP yellow fluorescent protein yellow fluorescent protein YFP yfp tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false association association false false false f6b f6c f6d 293T cells were co-transfected with YFP-tagged Nck constructs encoding Nck-wt, or each of the SH3 mutant molecules, together with HA-Ub and Cbl-b. As shown in Figure 6B, each of the Nck-SH3-mutants exhibited a substantial decrease in their interaction with Cbl-b, and in Nck ubiquitylation. Moreover, a triple Nck-SH3 mutant (3SH3*) did not interact with Cbl-b at all, and was substantially less ubiquitylated (Fig. 6C). To confirm this observation in T cells, Jurkat T-cell lines stably expressing the YFPNck constructs were prepared and analyzed (Fig. 6D). As in 293T cells, the molecular association of Cbl-b with each individual Nck-SH3-mutant was abolished. Furthermore, Nck ubiquitylation was correspondingly decreased. Thus, the three SH3 domains of Nck are all required for stable interaction with Cbl-b, and consequently, for Nck ubiquitylation.