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/ Raf-1 is a binding partner of DSCR1. Raf-1 is a binding partner of DSCR1. Arch. Biochem. Biophys. (0003-9861) 2005 Cho YJ., Abe M., Kim SY., Sato Y. Cancer - Interactions investigated in the context of cancer A unique UniProtKB entry could not be associated with calcineurin since the exact gene product of the interactor cannot be ascertained, as it is a multi-subunit protein. The isoform that corresponds to the sequence in fig. 3 B is P53805-2. y-sato@idac.tohoku.ac.jp davet in vitro In vitro pull down pull down weight by comassie molecular weight estimation by coomasie staining weight by comassie Cho YJ., Abe M., Kim SY., Sato Y. Cancer - Interactions investigated in the context of cancer A unique UniProtKB entry could not be associated with calcineurin since the exact gene product of the interactor cannot be ascertained, as it is a multi-subunit protein. The isoform that corresponds to the sequence in fig. 3 B is P53805-2. y-sato@idac.tohoku.ac.jp Accepted 2007-DEC-8 by DAVET. Arch. Biochem. Biophys. (0003-9861) 2005 davet Raf-1 is a binding partner of DSCR1. Raf-1 is a binding partner of DSCR1. Arch. Biochem. Biophys. (0003-9861) 2005 Cho YJ., Abe M., Kim SY., Sato Y. Cancer - Interactions investigated in the context of cancer A unique UniProtKB entry could not be associated with calcineurin since the exact gene product of the interactor cannot be ascertained, as it is a multi-subunit protein. The isoform that corresponds to the sequence in fig. 3 B is P53805-2. y-sato@idac.tohoku.ac.jp davet in vitro In vitro pull down pull down western blot western blot Immuno blot Immuno blot Cho YJ., Abe M., Kim SY., Sato Y. Cancer - Interactions investigated in the context of cancer A unique UniProtKB entry could not be associated with calcineurin since the exact gene product of the interactor cannot be ascertained, as it is a multi-subunit protein. The isoform that corresponds to the sequence in fig. 3 B is P53805-2. y-sato@idac.tohoku.ac.jp Accepted 2007-DEC-8 by DAVET. Arch. Biochem. Biophys. (0003-9861) 2005 davet Raf-1 is a binding partner of DSCR1. Raf-1 is a binding partner of DSCR1. Arch. Biochem. Biophys. (0003-9861) 2005 Cho YJ., Abe M., Kim SY., Sato Y. Cancer - Interactions investigated in the context of cancer A unique UniProtKB entry could not be associated with calcineurin since the exact gene product of the interactor cannot be ascertained, as it is a multi-subunit protein. The isoform that corresponds to the sequence in fig. 3 B is P53805-2. y-sato@idac.tohoku.ac.jp davet in vitro In vitro pull down pull down ms participant Identification by mass spectrometry ms participant Cho YJ., Abe M., Kim SY., Sato Y. Cancer - Interactions investigated in the context of cancer A unique UniProtKB entry could not be associated with calcineurin since the exact gene product of the interactor cannot be ascertained, as it is a multi-subunit protein. The isoform that corresponds to the sequence in fig. 3 B is P53805-2. y-sato@idac.tohoku.ac.jp Accepted 2007-DEC-8 by DAVET. Arch. Biochem. Biophys. (0003-9861) 2005 davet Raf-1 is a binding partner of DSCR1. Raf-1 is a binding partner of DSCR1. Arch. Biochem. Biophys. (0003-9861) 2005 Cho YJ., Abe M., Kim SY., Sato Y. Cancer - Interactions investigated in the context of cancer A unique UniProtKB entry could not be associated with calcineurin since the exact gene product of the interactor cannot be ascertained, as it is a multi-subunit protein. The isoform that corresponds to the sequence in fig. 3 B is P53805-2. y-sato@idac.tohoku.ac.jp davet in vitro In vitro enzymatic study enzymatic study weight silver stain molecular weight estimation by silver staining weight silver stain Cho YJ., Abe M., Kim SY., Sato Y. Cancer - Interactions investigated in the context of cancer A unique UniProtKB entry could not be associated with calcineurin since the exact gene product of the interactor cannot be ascertained, as it is a multi-subunit protein. The isoform that corresponds to the sequence in fig. 3 B is P53805-2. GST-DSCR1.4 was incubated with mu-calpain from human erythrocytes. Reactions terminated by adding SDS sample buffer. Samples were analyzed by SDS-PAGE and silver staining. A unique UniProtKB entry could not be associated with calcineurin since the exact gene product of the interactor cannot be ascertained, as it is a multi-subunit protein. y-sato@idac.tohoku.ac.jp Accepted 2007-DEC-8 by DAVET. Arch. Biochem. Biophys. (0003-9861) 2005 davet Raf-1 is a binding partner of DSCR1. Raf-1 is a binding partner of DSCR1. Arch. Biochem. Biophys. (0003-9861) 2005 Cho YJ., Abe M., Kim SY., Sato Y. Cancer - Interactions investigated in the context of cancer A unique UniProtKB entry could not be associated with calcineurin since the exact gene product of the interactor cannot be ascertained, as it is a multi-subunit protein. The isoform that corresponds to the sequence in fig. 3 B is P53805-2. y-sato@idac.tohoku.ac.jp davet bovin-gm_7373 Bos taurus endothelial aorta cell line gm_7373 Bovine endothelial aorta cell line Bovine endothelial aorta established from a male bovine fetus at 24-week gestation; vascular endothelium transformed with benzo(a)pyrene in vitro. anti tag coip anti tag coimmunoprecipitation anti tag coip western blot western blot Immuno blot Immuno blot Cho YJ., Abe M., Kim SY., Sato Y. Cancer - Interactions investigated in the context of cancer A unique UniProtKB entry could not be associated with calcineurin since the exact gene product of the interactor cannot be ascertained, as it is a multi-subunit protein. The isoform that corresponds to the sequence in fig. 3 B is P53805-2. y-sato@idac.tohoku.ac.jp Accepted 2007-DEC-8 by DAVET. Arch. Biochem. Biophys. (0003-9861) 2005 davet p53805-2 Calcipressin-1 RCAN1 ADAPT78 CSP1 DSC1 DSCR1 Adapt78 Down syndrome critical region protein 1 Myocyte-enriched calcineurin-interacting protein 1 Regulator of calcineurin 1 CALP1-S protein protein human Homo sapiens Human MHFRNFNYSFSSLIACVANSDIFSESETRAKFESLFRTYDKDITFQYFKSFKRVRINFSNPFSAADARLQLHKTEFLGKEMKLYFAQTLHIGSSHLAPPNPDKQFLISPPASPPVGWKQVEDATPVINYDLLYAISKLGPGEKYELHAATDTTPSVVVHVCESDQEKEEEEEMERMRRPKPKIIQTRRPEYTPIHLS 980A1FD7AA54A238 raf1_human RAF proto-oncogene serine/threonine-protein kinase RAF1 RAF Proto-oncogene c-RAF Raf-1 protein protein human Homo sapiens Human MEHIQGAWKTISNGFGFKDAVFDGSSCISPTIVQQFGYQRRASDDGKLTDPSKTSNTIRVFLPNKQRTVVNVRNGMSLHDCLMKALKVRGLQPECCAVFRLLHEHKGKKARLDWNTDAASLIGEELQVDFLDHVPLTTHNFARKTFLKLAFCDICQKFLLNGFRCQTCGYKFHEHCSTKVPTMCVDWSNIRQLLLFPNSTIGDSGVPALPSLTMRRMRESVSRMPVSSQHRYSTPHAFTFNTSSPSSEGSLSQRQRSTSTPNVHMVSTTLPVDSRMIEDAIRSHSESASPSALSSSPNNLSPTGWSQPKTPVPAQRERAPVSGTQEKNKIRPRGQRDSSYYWEIEASEVMLSTRIGSGSFGTVYKGKWHGDVAVKILKVVDPTPEQFQAFRNEVAVLRKTRHVNILLFMGYMTKDNLAIVTQWCEGSSLYKHLHVQETKFQMFQLIDIARQTAQGMDYLHAKNIIHRDMKSNNIFLHEGLTVKIGDFGLATVKSRWSGSQQVEQPTGSVLWMAPEVIRMQDNNPFSFQSDVYSYGIVLYELMTGELPYSHINNRDQIIFMVGRGYASPDLSKLYKNCPKAMKRLVADCVKKVKEERPLFPQILSSIELLQHSLPKINRSASEPSLHRAAHTEDINACTLTTSPRLPVF DIP protein P04049 original sequence version: 117 DIP protein P04049 original sequence version: 106 DIP protein P04049 original sequence version: 114 DIP protein P04049 original sequence version: 141 DIP protein P04049 original sequence version: 142 DIP protein P04049 original sequence version: 147 DIP protein P04049-1 original sequence version: 136 DIP protein P04049 original sequence version: 136 DIP protein P04049 original sequence version: 153 DIP protein P04049 original sequence version: 150 DIP protein P04049 original sequence version: 164 DIP protein P04049 original sequence version: 172 DIP protein P04049-1 original sequence version: 172 DIP protein P04049 original sequence version: 184 DIP protein P04049 original sequence version: 188 DIP protein P04049 original sequence version: 195 EF821B5349711BC3 can1_human Calpain-1 catalytic subunit CAPN1 CANPL1 Calpain-1 large subunit Calcium-activated neutral proteinase 1 Calpain mu-type Micromolar-calpain Cell proliferation-inducing gene 30 protein PIG30 protein protein human Homo sapiens Human MSEEIITPVYCTGVSAQVQKQRARELGLGRHENAIKYLGQDYEQLRVRCLQSGTLFRDEAFPPVPQSLGYKDLGPNSSKTYGIKWKRPTELLSNPQFIVDGATRTDICQGALGDCWLLAAIASLTLNDTLLHRVVPHGQSFQNGYAGIFHFQLWQFGEWVDVVVDDLLPIKDGKLVFVHSAEGNEFWSALLEKAYAKVNGSYEALSGGSTSEGFEDFTGGVTEWYELRKAPSDLYQIILKALERGSLLGCSIDISSVLDMEAITFKKLVKGHAYSVTGAKQVNYRGQVVSLIRMRNPWGEVEWTGAWSDSSSEWNNVDPYERDQLRVKMEDGEFWMSFRDFMREFTRLEICNLTPDALKSRTIRKWNTTLYEGTWRRGSTAGGCRNYPATFWVNPQFKIRLDETDDPDDYGDRESGCSFVLALMQKHRRRERRFGRDMETIGFAVYEVPPELVGQPAVHLKRDFFLANASRARSEQFINLREVSTRFRLPPGEYVVVPSTFEPNKEGDFVLRFFSEKSAGTVELDDQIQANLPDEQVLSEEEIDENFKALFRQLAGEDMEISVKELRTILNRIISKHKDLRTKGFSLESCRSMVNLMDRDGNGKLGLVEFNILWNRIRNYLSIFRKFDLDKSGSMSAYEMRMAIESAGFKLNKKLYELIITRYSEPDLAVDFDNFVCCLVRLETMFRFFKTLDTDLDGVVTFDLFKWLQLTMFA 1CB6D7C56D063498 raf1_bovin RAF proto-oncogene serine/threonine-protein kinase RAF1 Proto-oncogene c-RAF Raf-1 protein protein bovin Bos taurus (Bovine) Bovine MEHIQGAWKTISNGFGFKDTVFDGTSCISPTIVQQFGYQRRASDDGKLTDPSKTSNTIRVFLPNKQRTVVNVRNGMSLHDCLMKALKVRGLQPECCAVFRLLHEHKGKKARLDWNTDAASLIGEELQVDFLDHVPLTTHNFARKTFLKLAFCDICQKFLLNGFRCQTCGYKFHEHCSTKVPTMCVDWSNIRQLLLFPNSTVGDGGVPALPSLTMRRMRESVSRIPPGSQHRYSTPHAFTFSASSPSSEGSLSQRQRSTSTPNVHMVSATLPVDSRMIEDAIRSHSESGSPSALSSSPNNLSPTGWSQPKTPAPAQRERAPGSSTQEKNKIRPRGQRDSSYYWEIEASEVMLSTRIGSGSFGTVYKGKWHGDVAVKILKVVDPTPEQFQAFRNEVAVLRKTRHVNILLFMGYMTKDNLAIVTQWCEGSSLYKHLHVQETKFQMFQLIDIARQTAQGMDYLHAKNIIHRDMKSNNIFLHEGLTVKIGDFGLATVKSRWSGSQQVEQPTGSILWMAPEVIRMQDNNPFSFQSDVYSYGIVLYELMTGELPYSHINNRDQIIFMVGRGYASPDLSKLYKNCPKAMKRLVADCVKKVKEERPLFPQILSSIELLQHSLPKINRSASEPSLHRAAHTEDINACTLTTSPRLPVF 283F6CEBFD9274E4 rcan1-raf1 3 6 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) 7 unspecified role unspecified role prey prey human-uvec Human Umbilical Vein Endothelial Cells (HUVEC) huvec umbilical_vein_endothelium Umbilical vein endothelial cell [Umbilical vein endothelium] physical association physical association The association between DSCR1.4 and Raf-1 was conWrmed by immunoblot analysis with antibody against Raf-1 following pull-down analysis 1 A rcan1-raf1-1 2 6 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) 7 unspecified role unspecified role prey prey bovin Bos taurus (Bovine) Bovine physical association physical association The interaction between DSCR1.4 and endogenous Raf-1 was again observed in pull-down experiments using the lysates prepared from GM7373 and MS-1 endothelial cells 1 B rcan1-raf1-2 2 7 unspecified role unspecified role prey prey mouse-ms_1 Mus musculus pancreatic islet endothelial cell line ms_1 MS-1 (MILE SVEN 1) murine pancreatic islet endothelial cell line 6 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) physical association physical association The interaction between DSCR1.4 and endogenous Raf-1 was again observed in pull-down experiments using the lysates prepared from GM7373 and MS-1 endothelial cells 1 B rcan1-raf1-3 5 9 unspecified role unspecified role prey prey 6 unspecified role unspecified role bait bait n-terminal gfp tag green fluorescent protein tag GFP green fluorescent protein GFP green fluorescent protein gfp tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range n-terminal sufficient to bind sufficient binding region sufficient to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain c-terminal sufficient to bind sufficient binding region sufficient to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain ecoli Escherichia coli (strain K12) physical association physical association 1C, 4 These experiments revealed association between Raf-1 and DSCR1.4. The first six amino acids of DSCR1.4 are necessary for binding of the N-terminal fragment as GST-DSCR1.4 aa 7-133 did not bind RAF-1. Thus it seems cleavage between Phe-6 and Asn-7 abolishes Raf-1 binding through the N-terminal of DSCR1.4. It also seems cleavage between Tyr-133 and Ala-134 releases C-terminal Raf-1 binding domain from the inhibition by aa 115-133. rcan1-raf1-4 1 7 unspecified role unspecified role prey prey human-uvec Human Umbilical Vein Endothelial Cells (HUVEC) huvec umbilical_vein_endothelium Umbilical vein endothelial cell [Umbilical vein endothelium] 6 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 n-terminal sufficient to bind sufficient binding region sufficient to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain c-terminal sufficient to bind sufficient binding region sufficient to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain ecoli Escherichia coli (strain K12) physical association physical association 2 the results suggest that Raf-1 can associate with two regions of DSCR1.4: one in the N-terminal end (amino acids 1-84) and the other in the C-terminus (amino acids 134-156), and this is interfered with by amino acid sequence 115-133 of DSCR1.4. The results suggest that Raf-1 can associate with two regions of DSCR1.4: one in the N-terminal end (amino acids 1-84) and the other in the C-terminus (amino acids 134-156), and this is interfered with by amino acid sequence 115-133 of DSCR1.4. capn1-rcan1 4 6 enzyme target enzyme target substrate substrate unspecified role unspecified role 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) 8 enzyme enzyme unspecified role unspecified role human-erythrocyte Homo sapiens erythrocyte Erythrocyte, Red blood cell; Corpuscle; RBC protein cleavage protein cleavage 3 Incubation with -calpain rapidly cleaved GST-DSCR1.4 fusion protein into multiple fragments of a molecular mass that ranged from 40 to 12 kDa (Fig. 3A, lane 2) within 5min. GST-DSCR1.4 was cleaved into multiple fragments of a molecular mass that ranged from 40 to 12 kDa. Two of the cleavage sites are between phe-6 and asn-7 and between tyr-133 and ala-134.