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/ Force sensing by mechanical extension of the Src family kinase substrate p130Cas. Force sensing by mechanical extension of the Src family kinase substrate p130Cas. Cell (0092-8674) 2006 imex curation Sawada Y., Tamada M., Dubin-Thaler BJ., Cherniavskaya O., Sakai R., Tanaka S., Sheetz MP. Apoptosis - Interactions involving proteins with a function related to apoptosis NC-biotinylated CasSD was either stretched or not on latex membrane and then incubated with recombinant kinase and analyzed for phosphorylation. I wrote to the contact author who supplied me with the Invitrogen product numbers for the recombinant kinases. These were expressed in insect cells, however, the cell type is not specified. ys454-ind@umin.ac.jp,ys454@columbia.edu 21-Dec-2006: Contacted by JYOTI. Only protein-protein interactions 3 in vitro In vitro enzymatic study enzymatic study predetermined predetermined participant predetermined Sawada Y., Tamada M., Dubin-Thaler BJ., Cherniavskaya O., Sakai R., Tanaka S., Sheetz MP. Apoptosis - Interactions involving proteins with a function related to apoptosis NC-biotinylated CasSD was either stretched or not on latex membrane and then incubated with recombinant kinase and analyzed for phosphorylation. I wrote to the contact author who supplied me with the Invitrogen product numbers for the recombinant kinases. These were expressed in insect cells, however, the cell type is not specified. ys454-ind@umin.ac.jp,ys454@columbia.edu ys454-ind@umin.ac.jp Accepted 2006-DEC-13 by DAVET. 21-Dec-2006: Contacted by JYOTI. Cell (0092-8674) 2006 Only protein-protein interactions 3 imex curation src_human Proto-oncogene tyrosine-protein kinase Src pp60c-src SRC SRC1 Proto-oncogene c-Src protein protein human Homo sapiens Human MGSNKSKPKDASQRRRSLEPAENVHGAGGGAFPASQTPSKPASADGHRGPSAAFAPAAAEPKLFGGFNSSDTVTSPQRAGPLAGGVTTFVALYDYESRTETDLSFKKGERLQIVNNTEGDWWLAHSLSTGQTGYIPSNYVAPSDSIQAEEWYFGKITRRESERLLLNAENPRGTFLVRESETTKGAYCLSVSDFDNAKGLNVKHYKIRKLDSGGFYITSRTQFNSLQQLVAYYSKHADGLCHRLTTVCPTSKPQTQGLAKDAWEIPRESLRLEVKLGQGCFGEVWMGTWNGTTRVAIKTLKPGTMSPEAFLQEAQVMKKLRHEKLVQLYAVVSEEPIYIVTEYMSKGSLLDFLKGETGKYLRLPQLVDMAAQIASGMAYVERMNYVHRDLRAANILVGENLVCKVADFGLARLIEDNEYTARQGAKFPIKWTAPEAALYGRFTIKSDVWSFGILLTELTTKGRVPYPGMVNREVLDQVERGYRMPCPPECPESLHDLMCQCWRKEPEERPTFEYLQAFLEDYFTSTEPQYQPGENL DIP protein P12931 original sequence version: 188 DIP protein P12931 original sequence version: 189 DIP protein P12931 original sequence version: 190 DIP protein P12931 original sequence version: 191 DIP protein P12931 original sequence version: 115 DIP protein P12931 original sequence version: 116 DIP protein P12931 original sequence version: 164 DIP protein P12931 original sequence version: 142 DIP protein P12931 original sequence version: 143 DIP protein P12931 original sequence version: 150 DIP protein P12931 original sequence version: 160 DIP protein P12931 original sequence version: 184 DIP protein P12931 original sequence version: 155 DIP protein P12931-1 original sequence version: 155 DIP protein P12931 original sequence version: 202 C1908084683E5DE8 bcar1_rat Breast cancer anti-estrogen resistance protein 1 CRK-associated substrate p130cas Bcar1 Cas Crkas protein protein rat Rattus norvegicus (Rat) Rat MKYLVSVGAGPARRAGGLEDVSWGPRVSRRPQSYRAARHVNESLPRSAFRVPAAHGASVTPSAALGSGLPETQPEAVCRGTEKPRFAEGCKPAASRDKNVLAKALYDNVAESPDELSFRKGDIMTVLERDTQGLDGWWLCSLHGRQGIVPGNRLKILVGMYDKKPAAPGPGPPATPPQPQPSLPQGVHTPVPPASQYSPMLPTAYQPQPDNVYLVPTPSKTQQGLYQAPGPNPQFQSPPAKQTSTFSKQTPHHSFPSPATDLYQVPPGPGSPAQDIYQVPPSAGTGHDIYQVPPSLDTRSWEGTKPPAKVVVPTRVGQGYVYEASQAEQDEYDTPRHLLAPGSQDIYDVPPVRGLLPNQYGQEVYDTPPMAVKGPNGRDPLLDVYDVPPSVEKGLPPSNHHSVYDVPPSVSKDVPDGPLLREETYDVPPAFAKPKPFDPTRHPLILAAPPPDSPPAEDVYDVPPPAPDLYDVPPGLRRPGPGTLYDVPRERVLPPEVADGSVIDDGVYAVPPPAEREAPTDGKRLSASSTGSTRSSQSASSLEVVVPGREPLELEVAVETLARLQQGVSTTVAHLLDLVGSASGPGGWRSTSEPQEPPVQDLKAAVAAVHGAVHELLEFARSAVSSATHTSDRTLHAKLSRQLQKMEDVYQTLVVHGQVLDSGRGGPGFTLDDLDRLVACSRAVPEDAKQLASFLHGNASLLFRRTKAPGPGPEGSSSLHLNPTDKASSIQSRPLPSPPKFTSQDSPDGQYENSEGGWMEDYDYVHLQGKEEFEKTQKELLEKGNIVRQGKGQLELQQLKQFERLEQEVSRPIDHDLANWTPAQPLVPGRTGGLGPSDRQLLLFYLEQCEANLTTLTDAVDAFFTAVATNQPPKIFVAHSKFVILSAHKLVFIGDTLSRQAKAADVRSKVTHYSNLLCDLLRGIVATTKAAALQYPSPSAAQDMVDRVKELGHSTQQFRRVLGQLAAA E861641BFD68D377 abl1_human Tyrosine-protein kinase ABL1 Abelson tyrosine-protein kinase 1 ABL1 ABL JTK7 Abelson murine leukemia viral oncogene homolog 1 Proto-oncogene c-Abl p150 protein protein human Homo sapiens Human MLEICLKLVGCKSKKGLSSSSSCYLEEALQRPVASDFEPQGLSEAARWNSKENLLAGPSENDPNLFVALYDFVASGDNTLSITKGEKLRVLGYNHNGEWCEAQTKNGQGWVPSNYITPVNSLEKHSWYHGPVSRNAAEYLLSSGINGSFLVRESESSPGQRSISLRYEGRVYHYRINTASDGKLYVSSESRFNTLAELVHHHSTVADGLITTLHYPAPKRNKPTVYGVSPNYDKWEMERTDITMKHKLGGGQYGEVYEGVWKKYSLTVAVKTLKEDTMEVEEFLKEAAVMKEIKHPNLVQLLGVCTREPPFYIITEFMTYGNLLDYLRECNRQEVNAVVLLYMATQISSAMEYLEKKNFIHRDLAARNCLVGENHLVKVADFGLSRLMTGDTYTAHAGAKFPIKWTAPESLAYNKFSIKSDVWAFGVLLWEIATYGMSPYPGIDLSQVYELLEKDYRMERPEGCPEKVYELMRACWQWNPSDRPSFAEIHQAFETMFQESSISDEVEKELGKQGVRGAVSTLLQAPELPTKTRTSRRAAEHRDTTDVPEMPHSKGQGESDPLDHEPAVSPLLPRKERGPPEGGLNEDERLLPKDKKTNLFSALIKKKKKTAPTPPKRSSSFREMDGQPERRGAGEEEGRDISNGALAFTPLDTADPAKSPKPSNGAGVPNGALRESGGSGFRSPHLWKKSSTLTSSRLATGEEEGGGSSSKRFLRSCSASCVPHGAKDTEWRSVTLPRDLQSTGRQFDSSTFGGHKSEKPALPRKRAGENRSDQVTRGTVTPPPRLVKKNEEAADEVFKDIMESSPGSSPPNLTPKPLRRQVTVAPASGLPHKEEAGKGSALGTPAAAEPVTPTSKAGSGAPGGTSKGPAEESRVRRHKHSSESPGRDKGKLSRLKPAPPPPPAASAGKAGGKPSQSPSQEAAGEAVLGAKTKATSLVDAVNSDAAKPSQPGEGLKKPVLPATPKPQSAKPSGTPISPAPVPSTLPSASSALAGDQPSSTAFIPLISTRVSLRKTRQPPERIASGAITKGVVLDSTEALCLAISRNSEQMASHSAVLEAGKNLYTFCVSYVDSIQQMRNKFAFREAINKLENNLRELQICPATAGSGPAATQDFSKLLSSVKEISDIVQR DIP protein P00519 original sequence version: 114 DIP protein P00519 original sequence version: 137 DIP protein P00519 original sequence version: 161 DIP protein P00519 original sequence version: 220 DIP protein P00519 original sequence version: 219 85FE6C1C0E483EA2 fyn_human Tyrosine-protein kinase Fyn p59-Fyn Proto-oncogene c-Fyn Src-like kinase FYN Proto-oncogene Syn protein protein human Homo sapiens Human MGCVQCKDKEATKLTEERDGSLNQSSGYRYGTDPTPQHYPSFGVTSIPNYNNFHAAGGQGLTVFGGVNSSSHTGTLRTRGGTGVTLFVALYDYEARTEDDLSFHKGEKFQILNSSEGDWWEARSLTTGETGYIPSNYVAPVDSIQAEEWYFGKLGRKDAERQLLSFGNPRGTFLIRESETTKGAYSLSIRDWDDMKGDHVKHYKIRKLDNGGYYITTRAQFETLQQLVQHYSERAAGLCCRLVVPCHKGMPRLTDLSVKTKDVWEIPRESLQLIKRLGNGQFGEVWMGTWNGNTKVAIKTLKPGTMSPESFLEEAQIMKKLKHDKLVQLYAVVSEEPIYIVTEYMNKGSLLDFLKDGEGRALKLPNLVDMAAQVAAGMAYIERMNYIHRDLRSANILVGNGLICKIADFGLARLIEDNEYTARQGAKFPIKWTAPEAALYGRFTIKSDVWSFGILLTELVTKGRVPYPGMNNREVLEQVERGYRMPCPQDCPISLHELMIHCWKKDPEERPTFEYLQSFLEDYFTATEPQYQPGENL DIP protein P06241 original sequence version: 195 DIP protein P06241 original sequence version: 147 DIP protein P06241 original sequence version: 164 4A1E443A4B5A0977 scr-bcar1 1 3 enzyme target enzyme target substrate substrate unspecified role unspecified role region CasSD (substrate domain) sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain n-terminal Biotin AviTag (Avidity) GLNDIFEAQKIEWHE biotin tag biotin tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range c-terminal Biotin AviTag (Avidity) GLNDIFEAQKIEWHE sufficient to bind sufficient binding region sufficient to bind c-term range C-terminal range c-term range c-term range C-terminal range c-term range ecobb Escherichia coli (strain B / BL21) 2 enzyme enzyme unspecified role unspecified role c-terminus his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range enzymatic reaction enzymatic reaction Biochemical reaction Biochemical reaction 4 A, B, C, S2 B we examined the effect of extension on tyrosine phosphorylation of CasSD by recombinant active c-Src. While the level of phosphorylation was low without stretching (Figure 4A, lane 1), CasSD phosphorylation increased in proportion to the magnitude of latex membrane stretching (25%, 50%, 75%, 100%, and 150%) The level of phosphorylation of CasSD by c-Src increased as strectching of CasSD increased. The recombinant c-Src protein used was purchased from Invitrogen (product no. P3044; GenBank Accession Number: NP_005148.2)and was expressed in insect cells. It has an extra LEACQLGTDDYDIPTTHHHHHH at the C-terminus compared to the UniprotKb sequence. CasSD was phosphorylated at Tyr-460 and sites recognized by the pCas-165 and pCas-410. abl1-bcar1 1 4 enzyme enzyme unspecified role unspecified role c-terminal his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range 3 enzyme target enzyme target substrate substrate unspecified role unspecified role n-terminal Biotin AviTag (Avidity) GLNDIFEAQKIEWHE biotin tag biotin tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range c-terminal Biotin AviTag (Avidity) GLNDIFEAQKIEWHE biotin tag biotin tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range region CasSD (substrate domain) sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain ecobb Escherichia coli (strain B / BL21) enzymatic reaction enzymatic reaction Biochemical reaction Biochemical reaction 4 B Phosphorylation of CasSD by Abl1 was only observed when there was stretching of CasSD. The recombinant Abl1 used was purchased from Invitrogen (product no. P3049; GenBank Accession Number: NP_005148.2) and was expressed in insect cells. The sequence of the recombinant Abl1 protein used has the sequence LEACQLGTDDYDIPTTHHHHHH extra at the C-terminus as compared with the UniprotKb sequence. a known Cas kinase, Abl (Mayer et al., 1995) and another SFK, FynT, phosphorylated CasSD in an extension-dependent manner pCasSD was phosphorylated at Tyr-460. fyn-bcar1-1 1 5 enzyme enzyme unspecified role unspecified role c-terminal his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range 3 enzyme target enzyme target substrate substrate unspecified role unspecified role c-terminal Biotin AviTag (Avidity) GLNDIFEAQKIEWHE biotin tag biotin tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range region CasSD (substrate domain) sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain n-terminal Biotin AviTag (Avidity) GLNDIFEAQKIEWHE biotin tag biotin tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range ecobb Escherichia coli (strain B / BL21) enzymatic reaction enzymatic reaction Biochemical reaction Biochemical reaction 4 B Phosphorylation of CasSD by FynT was only observed when there was stretching of CasSD. The recombinant FynT used was purchased from Invitrogen (product no. P3042; GenBank Accession Number: NP_694592) and was expressed in insect cells. The sequence of the recombinant FYN protein used has the sequence LEACQLGTDDYDIPTTHHHHHH extra at the C-terminus as compared with the UniprotKb sequence. a known Cas kinase, Abl (Mayer et al., 1995) and another SFK, FynT, phosphorylated CasSD in an extension-dependent manner CasSD was phosphorylated on Tyr-460.