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/ Identification of Mrj, a DnaJ/Hsp40 family protein, as a keratin 8/18 filament regulatory protein. Identification of Mrj, a DnaJ/Hsp40 family protein, as a keratin 8/18 filament regulatory protein. J. Biol. Chem. (0021-9258) 2000 imex curation Izawa I., Nishizawa M., Ohtakara K., Ohtsuka K., Inada H., Inagaki M. Source of K18 deduced from previous work by the gifting author minagaki@aichi-cc.pref.aichi.jp 29-OCT-2007: Contacted by JYOTI. A human liver cDNA library was used. orchard Only protein-protein interactions in vitro In vitro cosedimentation cosedimentation weight by comassie molecular weight estimation by coomasie staining weight by comassie Izawa I., Nishizawa M., Ohtakara K., Ohtsuka K., Inada H., Inagaki M. As the separation of K18 and recombinant Mrj protein produced as a GST fusion protein (GST-Mrj) on Coomassie Brilliant Blue-stained gel after SDS-polyacrylamide gel electrophoresis was not clear the samples were also detected with anti-GST antibody by immunoblotting to more clearly show the proportions of supernatant and precipitated GST-Mrj. Incubation in filament assembly buffer induced a rapid polymerization of K8 and K18. Source of K18 deduced from previous work by the gifting author minagaki@aichi-cc.pref.aichi.jp Accepted 2007-JUN-29 by ORCHARD. 29-OCT-2007: Contacted by JYOTI. A human liver cDNA library was used. J. Biol. Chem. (0021-9258) 2000 orchard Only protein-protein interactions imex curation Identification of Mrj, a DnaJ/Hsp40 family protein, as a keratin 8/18 filament regulatory protein. Identification of Mrj, a DnaJ/Hsp40 family protein, as a keratin 8/18 filament regulatory protein. J. Biol. Chem. (0021-9258) 2000 imex curation Izawa I., Nishizawa M., Ohtakara K., Ohtsuka K., Inada H., Inagaki M. Source of K18 deduced from previous work by the gifting author minagaki@aichi-cc.pref.aichi.jp 29-OCT-2007: Contacted by JYOTI. A human liver cDNA library was used. orchard Only protein-protein interactions 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 full dna sequence full identification by DNA sequencing full dna sequence Izawa I., Nishizawa M., Ohtakara K., Ohtsuka K., Inada H., Inagaki M. Source of K18 deduced from previous work by the gifting author minagaki@aichi-cc.pref.aichi.jp Accepted 2007-JUN-29 by ORCHARD. A human liver cDNA library was used. J. Biol. Chem. (0021-9258) 2000 orchard Only protein-protein interactions imex curation Identification of Mrj, a DnaJ/Hsp40 family protein, as a keratin 8/18 filament regulatory protein. Identification of Mrj, a DnaJ/Hsp40 family protein, as a keratin 8/18 filament regulatory protein. J. Biol. Chem. (0021-9258) 2000 imex curation Izawa I., Nishizawa M., Ohtakara K., Ohtsuka K., Inada H., Inagaki M. Source of K18 deduced from previous work by the gifting author minagaki@aichi-cc.pref.aichi.jp 29-OCT-2007: Contacted by JYOTI. A human liver cDNA library was used. orchard Only protein-protein interactions human-hela Homo sapiens epitheloid cervix carcinoma cells Human hela Human epitheloid cervix carcinoma HeLa cells derivation: cervix; epithelial; adenocarcinoma ATCC no:CCL-2 anti bait coip anti bait coimmunoprecipitation anti bait coip immunostaining immunostaining Izawa I., Nishizawa M., Ohtakara K., Ohtsuka K., Inada H., Inagaki M. Source of K18 deduced from previous work by the gifting author minagaki@aichi-cc.pref.aichi.jp Accepted 2007-JUN-29 by ORCHARD. 29-OCT-2007: Contacted by JYOTI. A human liver cDNA library was used. J. Biol. Chem. (0021-9258) 2000 orchard Only protein-protein interactions imex curation Identification of Mrj, a DnaJ/Hsp40 family protein, as a keratin 8/18 filament regulatory protein. Identification of Mrj, a DnaJ/Hsp40 family protein, as a keratin 8/18 filament regulatory protein. J. Biol. Chem. (0021-9258) 2000 imex curation Izawa I., Nishizawa M., Ohtakara K., Ohtsuka K., Inada H., Inagaki M. Source of K18 deduced from previous work by the gifting author minagaki@aichi-cc.pref.aichi.jp 29-OCT-2007: Contacted by JYOTI. A human liver cDNA library was used. orchard Only protein-protein interactions human-hela Homo sapiens epitheloid cervix carcinoma cells Human hela Human epitheloid cervix carcinoma HeLa cells derivation: cervix; epithelial; adenocarcinoma ATCC no:CCL-2 anti bait coip anti bait coimmunoprecipitation anti bait coip monoclonal immunost monoclonal antibody immunostaining monoclonal immunost Izawa I., Nishizawa M., Ohtakara K., Ohtsuka K., Inada H., Inagaki M. Source of K18 deduced from previous work by the gifting author minagaki@aichi-cc.pref.aichi.jp Accepted 2007-JUN-29 by ORCHARD. 29-OCT-2007: Contacted by JYOTI. A human liver cDNA library was used. J. Biol. Chem. (0021-9258) 2000 orchard Only protein-protein interactions imex curation Identification of Mrj, a DnaJ/Hsp40 family protein, as a keratin 8/18 filament regulatory protein. Identification of Mrj, a DnaJ/Hsp40 family protein, as a keratin 8/18 filament regulatory protein. J. Biol. Chem. (0021-9258) 2000 imex curation Izawa I., Nishizawa M., Ohtakara K., Ohtsuka K., Inada H., Inagaki M. Source of K18 deduced from previous work by the gifting author minagaki@aichi-cc.pref.aichi.jp 29-OCT-2007: Contacted by JYOTI. A human liver cDNA library was used. orchard Only protein-protein interactions 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 predetermined predetermined participant predetermined Izawa I., Nishizawa M., Ohtakara K., Ohtsuka K., Inada H., Inagaki M. Source of K18 deduced from previous work by the gifting author minagaki@aichi-cc.pref.aichi.jp Accepted 2007-JUN-29 by ORCHARD. 29-OCT-2007: Contacted by JYOTI. A human liver cDNA library was used. J. Biol. Chem. (0021-9258) 2000 orchard Only protein-protein interactions imex curation dnjb6_human DnaJ homolog subfamily B member 6 Heat shock protein J2 MSJ-1 HHDJ1 DNAJB6 HSJ2 MSJ1 MRJ protein protein human Homo sapiens Human MVDYYEVLGVQRHASPEDIKKAYRKLALKWHPDKNPENKEEAERKFKQVAEAYEVLSDAKKRDIYDKYGKEGLNGGGGGGSHFDSPFEFGFTFRNPDDVFREFFGGRDPFSFDFFEDPFEDFFGNRRGPRGSRSRGTGSFFSAFSGFPSFGSGFSSFDTGFTSFGSLGHGGLTSFSSTSFGGSGMGNFKSISTSTKMVNGRKITTKRIVENGQERVEVEEDGQLKSLTINGVADDDALAEERMRRGQNALPAQPAGLRPPKPPRPASLLRHAPHCLSEEEGEQDRPRAPGPWDPLASAAGLKEGGKRKKQKQREESKKKKSTKGNH ECF1628BF7A524F3 1433t_human 14-3-3 protein theta YWHAQ 14-3-3 protein tau 14-3-3 protein T-cell Protein HS1 protein protein human Homo sapiens Human MEKTELIQKAKLAEQAERYDDMATCMKAVTEQGAELSNEERNLLSVAYKNVVGGRRSAWRVISSIEQKTDTSDKKLQLIKDYREKVESELRSICTTVLELLDKYLIANATNPESKVFYLKMKGDYFRYLAEVACGDDRKQTIDNSQGAYQEAFDISKKEMQPTHPIRLGLALNFSVFYYEILNNPELACTLAKTAFDEAIAELDTLNEDSYKDSTLIMQLLRDNLTLWTSDSAGEECDAAEGAEN DIP protein P27348 original sequence version: 164 DIP protein P27348 original sequence version: 173 175534325E9E37C4 k2c8_human Keratin, type II cytoskeletal 8 Keratin-8 KRT8 CYK8 Cytokeratin-8 Type-II keratin Kb8 protein protein human Homo sapiens Human MSIRVTQKSYKVSTSGPRAFSSRSYTSGPGSRISSSSFSRVGSSNFRGGLGGGYGGASGMGGITAVTVNQSLLSPLVLEVDPNIQAVRTQEKEQIKTLNNKFASFIDKVRFLEQQNKMLETKWSLLQQQKTARSNMDNMFESYINNLRRQLETLGQEKLKLEAELGNMQGLVEDFKNKYEDEINKRTEMENEFVLIKKDVDEAYMNKVELESRLEGLTDEINFLRQLYEEEIRELQSQISDTSVVLSMDNSRSLDMDSIIAEVKAQYEDIANRSRAEAESMYQIKYEELQSLAGKHGDDLRRTKTEISEMNRNISRLQAEIEGLKGQRASLEAAIADAEQRGELAIKDANAKLSELEAALQRAKQDMARQLREYQELMNVKLALDIEIATYRKLLEGEESRLESGMQNMSIHTKTTSGYAGGLSSAYGGLTSPGLSYSLGSSFGSGAGSSSFSRTSSSRAVVVKKIETRDGKLVSESSDVLPK DIP protein P05787 original sequence version: 186 DIP protein P05787 original sequence version: 146 B0BC730B65929D37 hsp7c_human Heat shock cognate 71 kDa protein HSPA8 HSC70 HSP73 HSPA10 Heat shock 70 kDa protein 8 Lipopolysaccharide-associated protein 1 protein protein human Homo sapiens Human MSKGPAVGIDLGTTYSCVGVFQHGKVEIIANDQGNRTTPSYVAFTDTERLIGDAAKNQVAMNPTNTVFDAKRLIGRRFDDAVVQSDMKHWPFMVVNDAGRPKVQVEYKGETKSFYPEEVSSMVLTKMKEIAEAYLGKTVTNAVVTVPAYFNDSQRQATKDAGTIAGLNVLRIINEPTAAAIAYGLDKKVGAERNVLIFDLGGGTFDVSILTIEDGIFEVKSTAGDTHLGGEDFDNRMVNHFIAEFKRKHKKDISENKRAVRRLRTACERAKRTLSSSTQASIEIDSLYEGIDFYTSITRARFEELNADLFRGTLDPVEKALRDAKLDKSQIHDIVLVGGSTRIPKIQKLLQDFFNGKELNKSINPDEAVAYGAAVQAAILSGDKSENVQDLLLLDVTPLSLGIETAGGVMTVLIKRNTTIPTKQTQTFTTYSDNQPGVLIQVYEGERAMTKDNNLLGKFELTGIPPAPRGVPQIEVTFDIDANGILNVSAVDKSTGKENKITITNDKGRLSKEDIERMVQEAEKYKAEDEKQRDKVSSKNSLESYAFNMKATVEDEKLQGKINDEDKQKILDKCNEIINWLDKNQTAEKEEFEHQQKELEKVCNPIITKLYQSAGGMPGGMPGGFPGGGAPPSGGASSGPTIEEVD 9AA27B210730670C fiba_human Fibrinogen alpha chain FGA protein protein human Homo sapiens Human MFSMRIVCLVLSVVGTAWTADSGEGDFLAEGGGVRGPRVVERHQSACKDSDWPFCSDEDWNYKCPSGCRMKGLIDEVNQDFTNRINKLKNSLFEYQKNNKDSHSLTTNIMEILRGDFSSANNRDNTYNRVSEDLRSRIEVLKRKVIEKVQHIQLLQKNVRAQLVDMKRLEVDIDIKIRSCRGSCSRALAREVDLKDYEDQQKQLEQVIAKDLLPSRDRQHLPLIKMKPVPDLVPGNFKSQLQKVPPEWKALTDMPQMRMELERPGGNEITRGGSTSYGTGSETESPRNPSSAGSWNSGSSGPGSTGNRNPGSSGTGGTATWKPGSSGPGSTGSWNSGSSGTGSTGNQNPGSPRPGSTGTWNPGSSERGSAGHWTSESSVSGSTGQWHSESGSFRPDSPGSGNARPNNPDWGTFEEVSGNVSPGTRREYHTEKLVTSKGDKELRTGKEKVTSGSTTTTRRSCSKTVTKTVIGPDGHKEVTKEVVTSEDGSDCPEAMDLGTLSGIGTLDGFRHRHPDEAAFFDTASTGKTFPGFFSPMLGEFVSETESRGSESGIFTNTKESSSHHPGIAEFPSRGKSSSYSKQFTSSTSYNRGDSTFESKSYKMADEAGSEADHEGTHSTKRGHAKSRPVRDCDDVLQTHPSGTQSGIFNIKLPGSSKIFSVYCDQETSLGGWLLIQQRMDGSLNFNRTWQDYKRGFGSLNDEGEGEFWLGNDYLHLLTQRGSVLRVELEDWAGNEAYAEYHFRVGSEAEGYALQVSSYEGTAGDALIEGSVEEGAEYTSHNNMQFSTFDRDADQWEENCAEVYGGGWWYNNCQAANLNGIYYPGGSYDPRNNSPYEIENGVVWVSFRGADYSLRAVRMKIRPLVTQ DIP protein P02671 original sequence version: 118 DIP protein P02671-1 original sequence version: 160 DIP protein P02671 original sequence version: 157 EA73A81204D8AEC4 k1c18_human Keratin, type I cytoskeletal 18 KRT18 CYK18 Keratin-18 PIG46 Cytokeratin-18 Cell proliferation-inducing gene 46 protein protein protein human Homo sapiens Human MSFTTRSTFSTNYRSLGSVQAPSYGARPVSSAASVYAGAGGSGSRISVSRSTSFRGGMGSGGLATGIAGGLAGMGGIQNEKETMQSLNDRLASYLDRVRSLETENRRLESKIREHLEKKGPQVRDWSHYFKIIEDLRAQIFANTVDNARIVLQIDNARLAADDFRVKYETELAMRQSVENDIHGLRKVIDDTNITRLQLETEIEALKEELLFMKKNHEEEVKGLQAQIASSGLTVEVDAPKSQDLAKIMADIRAQYDELARKNREELDKYWSQQIEESTTVVTTQSAEVGAAETTLTELRRTVQSLEIDLDSMRNLKASLENSLREVEARYALQMEQLNGILLHLESELAQTRAEGQRQAQEYEALLNIKVKLEAEIATYRRLLEDGEDFNLGDALDSSNSMQTIQKTTTRRIVDGKVVSETNDTKVLRH DIP protein P05783 original sequence version: 186 DIP protein P05783 original sequence version: 175 1E5604C6BCC7A17A dnajb6-krt18 2 11 unspecified role unspecified role bait bait 6 unspecified role unspecified role prey prey c-terminal sufficient to bind sufficient binding region sufficient to bind full dna sequence full identification by DNA sequencing full dna sequence certain certain sequence position certain certain certain sequence position certain physical association physical association To investigate the function of K8/18, we screened a human liver cDNA library using the yeast two-hybrid technique and full-length K18. dnajb6-krt18-1 1 8 unspecified role unspecified role neutral component neutral component ecoli Escherichia coli (strain K12) 6 unspecified role unspecified role neutral component neutral component region gst tag glutathione s tranferase tag glutathione S-tranferase tag glutathione S-tranferase tag gst tag western blot western blot Immuno blot Immuno blot undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined ecoli Escherichia coli (strain K12) 11 unspecified role unspecified role neutral component neutral component ecoli Escherichia coli (strain K12) physical association physical association Because Mrj interacted with K18, but not with K8, in the two-hybrid analysis, the Mrj sedimenting with K8/18 was thought to be associated with the K8/18 polymer via direct binding to K18. In addition, Mrj did not sediment with vimentin. Figure 2A, 2B Recombinant GST (which served as a control) did not sediment in the presence of K8/K18 (Fig. 2, A and B, lanes 3). In the absence of K8/18, GST-Mrj did not sediment (Fig. 2, A and B, lanes 4); but in the presence of K8/18, a substantial portion of GST-Mrj was precipitated with K8/18 (lanes 5). dnajb6-hspc70 4 11 unspecified role unspecified role prey prey 6 unspecified role unspecified role bait bait human-hela Homo sapiens epitheloid cervix carcinoma cells Human hela Human epitheloid cervix carcinoma HeLa cells derivation: cervix; epithelial; adenocarcinoma ATCC no:CCL-2 9 unspecified role unspecified role prey prey human-hela Homo sapiens epitheloid cervix carcinoma cells Human hela Human epitheloid cervix carcinoma HeLa cells derivation: cervix; epithelial; adenocarcinoma ATCC no:CCL-2 association association Mrj is associated with Hsp/c70 in-vivo. Figure 3B Hsp/c70 was indeed detected in the Mrj immune complex. These results indicate that Mrj is associated with K18 and Hsp/c70 in vivo. dnajb6-krt18-3 5 6 unspecified role unspecified role bait bait 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 11 unspecified role unspecified role prey prey physical association physical association The interaction between dnajb6 and krt18 required the c-terminal of dnajb6. Figure 1B, 5B To further examine the function of Mrj, we determined which region of Mrj interacts with Hsp/c70 and K18 using the two-hybrid system. As shown in Fig. 5 (A and B), Mrj-F interacted with both K18 and Hsc70, but not with K8. Mrj-C (aa 99-242) strongly interacted with K18, but Mrj-N1 (aa 1-100) and Mrj-N2 (aa 1-146) did not bind to K18. In contrast, Mrj-N1 and Mrj-N2 bound to Hsc70, but Mrj-C did not interact with Hsc70. dnajb6-hspc70-1 5 9 unspecified role unspecified role prey prey 6 unspecified role unspecified role bait bait 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 physical association physical association The interaction between dnajb6 and hsp/c70 required the n-terminal of dnajb6. Figure 5B To further examine the function of Mrj, we determined which region of Mrj interacts with Hsp/c70 and K18 using the two-hybrid system. As shown in Fig. 5 (A and B), Mrj-F interacted with both K18 and Hsc70, but not with K8. Mrj-C (aa 99-242) strongly interacted with K18, but Mrj-N1 (aa 1-100) and Mrj-N2 (aa 1-146) did not bind to K18. In contrast, Mrj-N1 and Mrj-N2 bound to Hsc70, but Mrj-C did not interact with Hsc70. fga-krt18-1 2 10 unspecified role unspecified role prey prey 11 unspecified role unspecified role bait bait physical association physical association To investigate the function of K8/18, we screened a human liver cDNA library using the yeast two-hybrid technique and full-length K18. krt8-krt18-1 2 11 unspecified role unspecified role bait bait 8 unspecified role unspecified role prey prey physical association physical association To investigate the function of K8/18, we screened a human liver cDNA library using the yeast two-hybrid technique and full-length K18. ywhaq-krt18-1 2 7 unspecified role unspecified role prey prey 11 unspecified role unspecified role bait bait physical association physical association To investigate the function of K8/18, we screened a human liver cDNA library using the yeast two-hybrid technique and full-length K18. dnajb6-krt18-4 5 6 unspecified role unspecified role prey prey 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 11 unspecified role unspecified role bait bait region sufficient to bind sufficient binding region sufficient to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain 46 44 physical association physical association The interaction between dnajb6 and krt18 required the c-terminal of dnajb6. Figure 1D D, identification of the coil II region of K18 that is sufficient for binding to Mrj. krt8-krt18-2 1 11 unspecified role unspecified role neutral component neutral component ecoli Escherichia coli (strain K12) 8 unspecified role unspecified role neutral component neutral component ecoli Escherichia coli (strain K12) physical association physical association Krt18 interacted with K8 independantly of dnajb6. Figure 2A, 2B Incubation in filament assembly buffer induced a rapid polymerization of K8 and K18 (Fig. 2A, lanes 1). Recombinant GST (which served as a control) did not sediment in the presence of K8/K18 (Fig. 2, A and B, lanes 3). dnajb6-krt18-5 3 11 unspecified role unspecified role prey prey human-hela Homo sapiens epitheloid cervix carcinoma cells Human hela Human epitheloid cervix carcinoma HeLa cells derivation: cervix; epithelial; adenocarcinoma ATCC no:CCL-2 6 unspecified role unspecified role bait bait human-hela Homo sapiens epitheloid cervix carcinoma cells Human hela Human epitheloid cervix carcinoma HeLa cells derivation: cervix; epithelial; adenocarcinoma ATCC no:CCL-2 physical association physical association Using anti-Mrj polyclonal and anti-krt18 monoclonal antibodies, it was discovered that dnajb6 (Mrj) colocalized with Krt18 around the perinuclear region. Figure 4. and double-stained with anti-Mrj polyclonal antibody and anti-K18 monoclonal antibody. Mrj was present in both the cytoplasm and nucleus (Fig. 4a). In the cytoplasm, Mrj was dispersed mainly in the perinuclear area and showed some filamentous patterns. There was a significant overlap between filamentous Mrj and K18 (Fig. 4, a�d), where Mrj was more remarkably associated with K8/18 filament bundles around perinuclear areas than with finer filament structures in the cell periphery.