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/ Activation of the tumour suppressor kinase LKB1 by the STE20-like pseudokinase STRAD. Activation of the tumour suppressor kinase LKB1 by the STE20-like pseudokinase STRAD. EMBO J. (0261-4189) 2003 imex curation Baas AF., Boudeau J., Sapkota GP., Smit L., Medema R., Morrice NA., Alessi DR., Clevers HC. Apoptosis - Interactions involving proteins with a function related to apoptosis Source of LKB1 determined from previous work by the authors and mapping of mutants. clevers@niob.knaw.nl 13-NOV-2006: Contacted by JYOTI. Foetal brain. Only protein-protein interactions in vitro In vitro protein kinase assay protein kinase assay western blot western blot Immuno blot Immuno blot Baas AF., Boudeau J., Sapkota GP., Smit L., Medema R., Morrice NA., Alessi DR., Clevers HC. Apoptosis - Interactions involving proteins with a function related to apoptosis Antibodies to phospho-sites Thr336 and Thr363 in LKB1 were used to quantitate the reaction Source of LKB1 determined from previous work by the authors and mapping of mutants. clevers@niob.knaw.nl Accepted 2006-NOV-3 by JYOTI. 13-NOV-2006: Contacted by JYOTI. Foetal brain. EMBO J. (0261-4189) 2003 Only protein-protein interactions imex curation Activation of the tumour suppressor kinase LKB1 by the STE20-like pseudokinase STRAD. Activation of the tumour suppressor kinase LKB1 by the STE20-like pseudokinase STRAD. EMBO J. (0261-4189) 2003 imex curation Baas AF., Boudeau J., Sapkota GP., Smit L., Medema R., Morrice NA., Alessi DR., Clevers HC. Apoptosis - Interactions involving proteins with a function related to apoptosis Source of LKB1 determined from previous work by the authors and mapping of mutants. clevers@niob.knaw.nl 13-NOV-2006: Contacted by JYOTI. Foetal brain. Only protein-protein interactions chlae-cos_7 Cercopithecus aethiops simian cells transformed with SV40 Grivet cos_7 cercopithecus aethiops, by transformation with SV40 fluorescence imaging fluorescence microscopy fluorescence imaging immunostaining immunostaining Baas AF., Boudeau J., Sapkota GP., Smit L., Medema R., Morrice NA., Alessi DR., Clevers HC. Apoptosis - Interactions involving proteins with a function related to apoptosis Source of LKB1 determined from previous work by the authors and mapping of mutants. clevers@niob.knaw.nl Accepted 2006-NOV-3 by JYOTI. 13-NOV-2006: Contacted by JYOTI. Foetal brain. EMBO J. (0261-4189) 2003 Only protein-protein interactions imex curation Activation of the tumour suppressor kinase LKB1 by the STE20-like pseudokinase STRAD. Activation of the tumour suppressor kinase LKB1 by the STE20-like pseudokinase STRAD. EMBO J. (0261-4189) 2003 imex curation Baas AF., Boudeau J., Sapkota GP., Smit L., Medema R., Morrice NA., Alessi DR., Clevers HC. Apoptosis - Interactions involving proteins with a function related to apoptosis Source of LKB1 determined from previous work by the authors and mapping of mutants. clevers@niob.knaw.nl 13-NOV-2006: Contacted by JYOTI. Foetal brain. 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 nucleotide sequence nucleotide sequence identification sequence cloning sequence cloning nucleotide sequence Baas AF., Boudeau J., Sapkota GP., Smit L., Medema R., Morrice NA., Alessi DR., Clevers HC. Apoptosis - Interactions involving proteins with a function related to apoptosis Source of LKB1 determined from previous work by the authors and mapping of mutants. clevers@niob.knaw.nl Accepted 2006-NOV-3 by JYOTI. 13-NOV-2006: Contacted by JYOTI. Foetal brain. EMBO J. (0261-4189) 2003 Only protein-protein interactions imex curation Activation of the tumour suppressor kinase LKB1 by the STE20-like pseudokinase STRAD. Activation of the tumour suppressor kinase LKB1 by the STE20-like pseudokinase STRAD. EMBO J. (0261-4189) 2003 imex curation Baas AF., Boudeau J., Sapkota GP., Smit L., Medema R., Morrice NA., Alessi DR., Clevers HC. Apoptosis - Interactions involving proteins with a function related to apoptosis Source of LKB1 determined from previous work by the authors and mapping of mutants. clevers@niob.knaw.nl 13-NOV-2006: Contacted by JYOTI. Foetal brain. Only protein-protein interactions human-293t Homo sapiens 293 cells transformed with SV40 large T antigen 293t 293 cells expressing SV40 large T antigen. The original designation of this cell line was 293tsA1609neo. It is not in Cabri,ATCC or HyperCLDB. anti tag coip anti tag coimmunoprecipitation anti tag coip western blot western blot Immuno blot Immuno blot Baas AF., Boudeau J., Sapkota GP., Smit L., Medema R., Morrice NA., Alessi DR., Clevers HC. Apoptosis - Interactions involving proteins with a function related to apoptosis Source of LKB1 determined from previous work by the authors and mapping of mutants. clevers@niob.knaw.nl Accepted 2006-NOV-3 by JYOTI. 13-NOV-2006: Contacted by JYOTI. Foetal brain. EMBO J. (0261-4189) 2003 Only protein-protein interactions imex curation Activation of the tumour suppressor kinase LKB1 by the STE20-like pseudokinase STRAD. Activation of the tumour suppressor kinase LKB1 by the STE20-like pseudokinase STRAD. EMBO J. (0261-4189) 2003 imex curation Baas AF., Boudeau J., Sapkota GP., Smit L., Medema R., Morrice NA., Alessi DR., Clevers HC. Apoptosis - Interactions involving proteins with a function related to apoptosis Source of LKB1 determined from previous work by the authors and mapping of mutants. clevers@niob.knaw.nl 13-NOV-2006: Contacted by JYOTI. Foetal brain. Only protein-protein interactions in vitro In vitro protein kinase assay protein kinase assay predetermined predetermined participant predetermined Baas AF., Boudeau J., Sapkota GP., Smit L., Medema R., Morrice NA., Alessi DR., Clevers HC. Apoptosis - Interactions involving proteins with a function related to apoptosis Source of LKB1 determined from previous work by the authors and mapping of mutants. clevers@niob.knaw.nl Accepted 2006-NOV-3 by JYOTI. 13-NOV-2006: Contacted by JYOTI. Foetal brain. EMBO J. (0261-4189) 2003 Only protein-protein interactions imex curation Activation of the tumour suppressor kinase LKB1 by the STE20-like pseudokinase STRAD. Activation of the tumour suppressor kinase LKB1 by the STE20-like pseudokinase STRAD. EMBO J. (0261-4189) 2003 imex curation Baas AF., Boudeau J., Sapkota GP., Smit L., Medema R., Morrice NA., Alessi DR., Clevers HC. Apoptosis - Interactions involving proteins with a function related to apoptosis Source of LKB1 determined from previous work by the authors and mapping of mutants. clevers@niob.knaw.nl 13-NOV-2006: Contacted by JYOTI. Foetal brain. Only protein-protein interactions human-293t Homo sapiens 293 cells transformed with SV40 large T antigen 293t 293 cells expressing SV40 large T antigen. The original designation of this cell line was 293tsA1609neo. It is not in Cabri,ATCC or HyperCLDB. anti bait coip anti bait coimmunoprecipitation anti bait coip western blot western blot Immuno blot Immuno blot Baas AF., Boudeau J., Sapkota GP., Smit L., Medema R., Morrice NA., Alessi DR., Clevers HC. Apoptosis - Interactions involving proteins with a function related to apoptosis Source of LKB1 determined from previous work by the authors and mapping of mutants. clevers@niob.knaw.nl Accepted 2006-NOV-3 by JYOTI. 13-NOV-2006: Contacted by JYOTI. Foetal brain. EMBO J. (0261-4189) 2003 Only protein-protein interactions imex curation Activation of the tumour suppressor kinase LKB1 by the STE20-like pseudokinase STRAD. Activation of the tumour suppressor kinase LKB1 by the STE20-like pseudokinase STRAD. EMBO J. (0261-4189) 2003 imex curation Baas AF., Boudeau J., Sapkota GP., Smit L., Medema R., Morrice NA., Alessi DR., Clevers HC. Apoptosis - Interactions involving proteins with a function related to apoptosis Source of LKB1 determined from previous work by the authors and mapping of mutants. clevers@niob.knaw.nl 13-NOV-2006: Contacted by JYOTI. Foetal brain. Only protein-protein interactions rat-rat-2 Rat R2 embryo cells rat-2 Rat embryo cell line anti bait coip anti bait coimmunoprecipitation anti bait coip western blot western blot Immuno blot Immuno blot Baas AF., Boudeau J., Sapkota GP., Smit L., Medema R., Morrice NA., Alessi DR., Clevers HC. Apoptosis - Interactions involving proteins with a function related to apoptosis Source of LKB1 determined from previous work by the authors and mapping of mutants. clevers@niob.knaw.nl Accepted 2006-NOV-3 by JYOTI. 13-NOV-2006: Contacted by JYOTI. Foetal brain. EMBO J. (0261-4189) 2003 Only protein-protein interactions imex curation stk11_human Serine/threonine-protein kinase STK11 STK11 LKB1 PJS Liver kinase B1 Renal carcinoma antigen NY-REN-19 protein protein human Homo sapiens Human MEVVDPQQLGMFTEGELMSVGMDTFIHRIDSTEVIYQPRRKRAKLIGKYLMGDLLGEGSYGKVKEVLDSETLCRRAVKILKKKKLRRIPNGEANVKKEIQLLRRLRHKNVIQLVDVLYNEEKQKMYMVMEYCVCGMQEMLDSVPEKRFPVCQAHGYFCQLIDGLEYLHSQGIVHKDIKPGNLLLTTGGTLKISDLGVAEALHPFAADDTCRTSQGSPAFQPPEIANGLDTFSGFKVDIWSAGVTLYNITTGLYPFEGDNIYKLFENIGKGSYAIPGDCGPPLSDLLKGMLEYEPAKRFSIRQIRQHSWFRKKHPPAEAPVPIPPSPDTKDRWRSMTVVPYLEDLHGADEDEDLFDIEDDIIYTQDFTVPGQVPEEEASHNGQRRGLPKAVCMNGTEAAQLSTKSRAEGRAPNPARKACSASSKIRRLSACKQQ DIP protein Q15831 original sequence version: 117 DIP protein Q15831 original sequence version: 154 DIP protein Q15831 original sequence version: 174 DIP protein Q15831 original sequence version: 168 6DF4C37AB7A89569 stk11_rat Serine/threonine-protein kinase STK11 Lkb1 Liver kinase B1 homolog Stk11 peptide peptide polypeptide oligopeptide oligopeptide polypeptide rat Rattus norvegicus (Rat) Rat MDVADPQPLGLFPEGELMSVGMDTFIHRIDSTEVIYQPRRKRAKLIGKYLMGDLLGEGSYGKVKEVLDSETLCRRAVKILKKKKLRRIPNGEANVKKEIQLLRRLRHRNVIQLVDVLYNEEKQKMYMVMEYCVCGMQEMLDSVPEKRFPVCQAHGYFRQLIDGLEYLHSQGIVHKDIKPGNLLLTTNGTLKISDLGVAEALHPFAVDDTCRTSQGSPAFQPPEIANGLDTFSGFKVDIWSAGVTLYNITTGLYPFEGDNIYKLFENIGRGDFTIPCDCAPPLSDLLRGMLEYEPAKRFSIRQIRQHSWFRKKHPLAEALVPIPPSPDTKDRWRSMTVVPYLEDLHGRAEEEEDEDLFDIEDGIIYTQDFTVPGQVLEEEVGQNGQSHSLPKAVCVNGTEPQLSSKVKPEGRPGAANPARKVCSSNKIRRLSACKQQ 3CB4B3D86583CF8A straa_rat STE20-related kinase adapter protein alpha Strada Strad Lyk5 STE20-related adapter protein protein protein rat Rattus norvegicus (Rat) Rat MSFLANEASSESIASFSKPEIMSSFLPEGGCYELLSVIGKGFEDLMTVNSRYKPTGEYVTVRRINLEACSNEMVTFLQGELHVSKLFSHPNIVPYRATFIADNELWAVTSFMAYGSAKDLIGTHFMDGMSELAIAYILQGVLKALDYIHHMGYVHRSVKASHILISTDGKVYLSGLRSNLSMISHGQRQRAVHDFPKYSIKVLPWLSPEVLQQNLQGYDAKSDIYSVGITACELANGHVPFKDMPATQMLLEKLNGTVPCLLDTSTIPAEELTMSPSRSIANPGLNDSLAAGSLRPANGDSPSHPYHRTFSPHFHNFVEQCLQRNPDARPNASTLLNHSFFKQIKRRASEALPELLRPVTPITSFEGSQSQDHSGILGLVTNLEDLEVDDWEF B92AB012E2A8AD34 straa_human STE20-related kinase adapter protein alpha STRADA Serologically defined breast cancer antigen NY-BR-96 STRAD LYK5 STE20-related adapter protein protein protein human Homo sapiens Human MSFLVSKPERIRRWVSEKFIVEGLRDLELFGEQPPGDTRRKTNDASSESIASFSKQEVMSSFLPEGGCYELLTVIGKGFEDLMTVNLARYKPTGEYVTVRRINLEACSNEMVTFLQGELHVSKLFNHPNIVPYRATFIADNELWVVTSFMAYGSAKDLICTHFMDGMNELAIAYILQGVLKALDYIHHMGYVHRSVKASHILISVDGKVYLSGLRSNLSMISHGQRQRVVHDFPKYSVKVLPWLSPEVLQQNLQGYDAKSDIYSVGITACELANGHVPFKDMPATQMLLEKLNGTVPCLLDTSTIPAEELTMSPSRSVANSGLSDSLTTSTPRPSNGDSPSHPYHRTFSPHFHHFVEQCLQRNPDARPSASTLLNHSFFKQIKRRASEALPELLRPVTPITNFEGSQSQDHSGIFGLVTNLEELEVDDWEF 9CC0A78D9CC6FC2C lkb1-strad-1 3 8 unspecified role unspecified role bait bait 11 unspecified role unspecified role prey prey physical association physical association We screened a fetal brain library using kinase-dead (KD) LKB1 (D176Y) as bait, and identified a single clone that interacted specifically with LKB1 and not with control baits. Kinase dead (Asp176Tyr) STK11 (Q15831) used as bait. lkb1-strad-2 6 8 unspecified role unspecified role bait bait 11 unspecified role unspecified role prey prey physical association physical association Endogenous LKB1 (55 kDa) interacts with STRAD (45�48 kDa) in Rat-2 and HEK-293T cells. 2A lkb1-strad-3 7 9 unspecified role unspecified role bait bait 10 unspecified role unspecified role prey prey physical association physical association Endogenous LKB1 (55 kDa) interacts with STRAD (45�48 kDa) in Rat-2 and HEK-293T cells. 2A lkb1-strad-4 4 8 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 region required to bind necessary binding region required to bind certain certain sequence position certain certain certain sequence position certain 11 unspecified role unspecified role prey prey physical association physical association A GST�LKB1 fusion protein, transfected into HEK-293T cells, associated with endogenous STRAD (Figure 2B). 2B & 2C stk11-strad-5 4 8 unspecified role unspecified role bait bait region required to bind necessary binding region required to bind mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain region myc tag myc tag EUKLISEED epitope tag EUKLISEED epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 11 unspecified role unspecified role prey prey region flag tag flag tag FLAG-tagged FLAG DYKDDDDKV epitope tag DYKDDDDKV epitope tag FLAG FLAG-tagged undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined physical association physical association Flag-tagged STRAD and myc-tagged LKB1 could be co-immunoprecipitated following co-transfection of HEK- 293T cells (Figure 3A). 3A lkb1-1 5 8 putative self putative self putative self putative self region myc tag myc tag EUKLISEED epitope tag EUKLISEED epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined human-293t Homo sapiens 293 cells transformed with SV40 large T antigen 293t 293 cells expressing SV40 large T antigen. The original designation of this cell line was 293tsA1609neo. It is not in Cabri,ATCC or HyperCLDB. phosphorylation phosphorylation reaction phosphorylation true Remarkably, when the isolated complexes were analysed in an in vitro kinase assay in the presence of [γ-32P]ATP, STRAD became phosphorylated and LKB1 autophosphorylation was strongly enhanced (Figure 3A and B). 3A & 3B Autophosphorylation of STK11 (Q15831). Subsequent mapping by mass spectrometry showed this to me at Thr-185 and Thr-402. lkb1-strad-7 5 11 enzyme target enzyme target substrate substrate unspecified role unspecified role region flag tag flag tag FLAG-tagged FLAG DYKDDDDKV epitope tag DYKDDDDKV epitope tag FLAG FLAG-tagged undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined human-293t Homo sapiens 293 cells transformed with SV40 large T antigen 293t 293 cells expressing SV40 large T antigen. The original designation of this cell line was 293tsA1609neo. It is not in Cabri,ATCC or HyperCLDB. 8 enzyme enzyme unspecified role unspecified role region required to bind necessary binding region required to bind mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain region myc tag myc tag EUKLISEED epitope tag EUKLISEED epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined human-293t Homo sapiens 293 cells transformed with SV40 large T antigen 293t 293 cells expressing SV40 large T antigen. The original designation of this cell line was 293tsA1609neo. It is not in Cabri,ATCC or HyperCLDB. phosphorylation phosphorylation reaction phosphorylation Remarkably, when the isolated complexes were analysed in an in vitro kinase assay in the presence of [γ-32P]ATP, STRAD became phosphorylated and LKB1 autophosphorylation was strongly enhanced (Figure 3A and B). 3A & 3B Phosphorylation of STARD (Q7RTN6). Subsequent mapping of phosphorylation sites by mass spectrometry (Fig. 6A) were shown to be Thr-329 and Thr-419. lkb1-strad-8 5 11 enzyme target enzyme target substrate substrate unspecified role unspecified role region flag tag flag tag FLAG-tagged FLAG DYKDDDDKV epitope tag DYKDDDDKV epitope tag FLAG FLAG-tagged undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined ecoli Escherichia coli (strain K12) 8 enzyme enzyme 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) phosphorylation phosphorylation reaction phosphorylation An in vitro kinase analysis of the purified proteins showed that LKB1-WT directly phosphorylated STRAD, whereas LKB1-KD did not (Figure 3D). 3D Phosphorylation of STRAD (Q7RTN6) lkb1-2 1 8 putative self putative self putative self putative self region myc tag myc tag EUKLISEED epitope tag EUKLISEED epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined human-293t Homo sapiens 293 cells transformed with SV40 large T antigen 293t 293 cells expressing SV40 large T antigen. The original designation of this cell line was 293tsA1609neo. It is not in Cabri,ATCC or HyperCLDB. phosphorylation phosphorylation reaction phosphorylation true Remarkably, when the isolated complexes were analysed in an in vitro kinase assay in the presence of [γ-32P]ATP, STRAD became phosphorylated and LKB1 autophosphorylation was strongly enhanced (Figure 3A and B). 4A STRAD (Q7RTN6) Equal amounts of the protein complexes were subjected to an in vitro kinase assay in the presence of [γ-32P]ATP. Immunoblots were performed with phospho-specific antibodies against the major in vitro autophosphorylation sites of LKB1, namely Thr336 and Thr363 (Thr366 in mouse Lkb1) (Sapkota et al., 2002). Upon transfection of STRAD, we found a strong increase in LKB1 autophosphorylation at these sites (Figure 4A). Autophosphorylation of STK11 (Q15381) stk11-strad-6 2 8 unspecified role unspecified role neutral component neutral component region myc tag myc tag EUKLISEED epitope tag EUKLISEED epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined region required to bind necessary binding region required to bind mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain 11 unspecified role unspecified role neutral component neutral component region flag tag flag tag FLAG-tagged FLAG DYKDDDDKV epitope tag DYKDDDDKV epitope tag FLAG FLAG-tagged undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined colocalization colocalization Flag-tagged STRAD and myc-tagged LKB1 could be co-immunoprecipitated following co-transfection of HEK- 293T cells (Figure 3A). 3A