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/ CRIPak, a novel endogenous Pak1 inhibitor. CRIPak, a novel endogenous Pak1 inhibitor. Oncogene (0950-9232) 2006 imex curation Talukder AH., Meng Q., Kumar R. Apoptosis - Interactions involving proteins with a function related to apoptosis Origin of PAK1 suppplied by author rkumar@mdanderson.org 10-APR-2007: Contacted by JYOTI. Human mammary gland cDNA Only protein-protein interactions human-mcf7 Homo sapiens mammary carcinoma cells mcf7 mammary carcinoma cells anti tag coip anti tag coimmunoprecipitation anti tag coip western blot western blot Immuno blot Immuno blot Talukder AH., Meng Q., Kumar R. Apoptosis - Interactions involving proteins with a function related to apoptosis rkumar@mdanderson.org Accepted 2007-APR-8 by JYOTI. 10-APR-2007: Contacted by JYOTI. Oncogene (0950-9232) 2006 Only protein-protein interactions imex curation CRIPak, a novel endogenous Pak1 inhibitor. CRIPak, a novel endogenous Pak1 inhibitor. Oncogene (0950-9232) 2006 imex curation Talukder AH., Meng Q., Kumar R. Apoptosis - Interactions involving proteins with a function related to apoptosis Origin of PAK1 suppplied by author rkumar@mdanderson.org 10-APR-2007: Contacted by JYOTI. Human mammary gland cDNA Only protein-protein interactions human-mcf7 Homo sapiens mammary carcinoma cells mcf7 mammary carcinoma cells confocal microscopy confocal microscopy immunostaining immunostaining Talukder AH., Meng Q., Kumar R. Apoptosis - Interactions involving proteins with a function related to apoptosis rkumar@mdanderson.org Accepted 2007-APR-20 by JYOTI. 10-APR-2007: Contacted by JYOTI. Oncogene (0950-9232) 2006 Only protein-protein interactions imex curation CRIPak, a novel endogenous Pak1 inhibitor. CRIPak, a novel endogenous Pak1 inhibitor. Oncogene (0950-9232) 2006 imex curation Talukder AH., Meng Q., Kumar R. Apoptosis - Interactions involving proteins with a function related to apoptosis Origin of PAK1 suppplied by author rkumar@mdanderson.org 10-APR-2007: Contacted by JYOTI. Human mammary gland cDNA 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 Talukder AH., Meng Q., Kumar R. Apoptosis - Interactions involving proteins with a function related to apoptosis Origin of PAK1 suppplied by author rkumar@mdanderson.org Accepted 2007-APR-7 by JYOTI. 10-APR-2007: Contacted by JYOTI. Human mammary gland cDNA Oncogene (0950-9232) 2006 Only protein-protein interactions imex curation CRIPak, a novel endogenous Pak1 inhibitor. CRIPak, a novel endogenous Pak1 inhibitor. Oncogene (0950-9232) 2006 imex curation Talukder AH., Meng Q., Kumar R. Apoptosis - Interactions involving proteins with a function related to apoptosis Origin of PAK1 suppplied by author rkumar@mdanderson.org 10-APR-2007: Contacted by JYOTI. Human mammary gland cDNA Only protein-protein interactions chlae-cos_7 Cercopithecus aethiops simian cells transformed with SV40 Grivet cos_7 cercopithecus aethiops, by transformation with SV40 anti tag coip anti tag coimmunoprecipitation anti tag coip anti tag western anti tag western blot anti tag western Talukder AH., Meng Q., Kumar R. Apoptosis - Interactions involving proteins with a function related to apoptosis rkumar@mdanderson.org Accepted 2007-APR-8 by JYOTI. 10-APR-2007: Contacted by JYOTI. Oncogene (0950-9232) 2006 Only protein-protein interactions imex curation CRIPak, a novel endogenous Pak1 inhibitor. CRIPak, a novel endogenous Pak1 inhibitor. Oncogene (0950-9232) 2006 imex curation Talukder AH., Meng Q., Kumar R. Apoptosis - Interactions involving proteins with a function related to apoptosis Origin of PAK1 suppplied by author rkumar@mdanderson.org 10-APR-2007: Contacted by JYOTI. Human mammary gland cDNA Only protein-protein interactions in vitro In vitro pull down pull down weight autoradiogra molecular weight estimation by autoradiography weight autoradiogra Talukder AH., Meng Q., Kumar R. Apoptosis - Interactions involving proteins with a function related to apoptosis rkumar@mdanderson.org Accepted 2007-APR-7 by JYOTI. 10-APR-2007: Contacted by JYOTI. Oncogene (0950-9232) 2006 Only protein-protein interactions imex curation CRIPak, a novel endogenous Pak1 inhibitor. CRIPak, a novel endogenous Pak1 inhibitor. Oncogene (0950-9232) 2006 imex curation Talukder AH., Meng Q., Kumar R. Apoptosis - Interactions involving proteins with a function related to apoptosis Origin of PAK1 suppplied by author rkumar@mdanderson.org 10-APR-2007: Contacted by JYOTI. Human mammary gland cDNA 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 Talukder AH., Meng Q., Kumar R. Apoptosis - Interactions involving proteins with a function related to apoptosis Origin of PAK1 suppplied by author rkumar@mdanderson.org Accepted 2007-APR-7 by JYOTI. 10-APR-2007: Contacted by JYOTI. Oncogene (0950-9232) 2006 Only protein-protein interactions imex curation CRIPak, a novel endogenous Pak1 inhibitor. CRIPak, a novel endogenous Pak1 inhibitor. Oncogene (0950-9232) 2006 imex curation Talukder AH., Meng Q., Kumar R. Apoptosis - Interactions involving proteins with a function related to apoptosis Origin of PAK1 suppplied by author rkumar@mdanderson.org 10-APR-2007: Contacted by JYOTI. Human mammary gland cDNA Only protein-protein interactions in vitro In vitro protein kinase assay protein kinase assay predetermined predetermined participant predetermined Talukder AH., Meng Q., Kumar R. Apoptosis - Interactions involving proteins with a function related to apoptosis Additional information on E.coli expressed PAK1 taken from manufacturers website rkumar@mdanderson.org Accepted 2007-APR-8 by JYOTI. 10-APR-2007: Contacted by JYOTI. Oncogene (0950-9232) 2006 Only protein-protein interactions imex curation crpak_human Putative protein CRIPAK CRIPAK Cysteine-rich PAK1 inhibitor protein protein human Homo sapiens Human MHEPSLCANVECPPAHTCPCGVPACSCAHVECPPAHTCRCGVPACSHMPMWSARLLTRAHVECPPAHTRVHVECPPAHVPMWSAHLLTCADVECHLLTHVPMWSARLLTCPCGVPACSHVPMRSARLLTRAHAECPPAHTCPCGVPACSHVPMRSARLLTRADVECPPAHTCPCGVPACSHVPTWSARLITRAHVECSPAHTCRCGVPACSHVPMWSVRLLTRADAECPPAHTCRCGVPACSHVPMWSARLLTCRCGVPACSHVPMWSARLLTCRCGVPACSHVPMWSARLLTRAHVECPPAHTCRRGVPACSRAHMECPPAHTCHCGVPACSHTCRCGVPACSHVPMWSARLLTRAHVECPPAHTRAHVECPPAHTCPCGVPACSHTCPCGVPACSHKALAWWFCRFPVLPAESDAVTVHSTHGGFLIRFYVKDPFYISLHLEIT 92EDA1B7B8037FDF pak1_human Serine/threonine-protein kinase PAK 1 PAK1 p21-activated kinase 1 p65-PAK Alpha-PAK protein protein human Homo sapiens Human MSNNGLDIQDKPPAPPMRNTSTMIGAGSKDAGTLNHGSKPLPPNPEEKKKKDRFYRSILPGDKTNKKKEKERPEISLPSDFEHTIHVGFDAVTGEFTGMPEQWARLLQTSNITKSEQKKNPQAVLDVLEFYNSKKTSNSQKYMSFTDKSAEDYNSSNALNVKAVSETPAVPPVSEDEDDDDDDATPPPVIAPRPEHTKSVYTRSVIEPLPVTPTRDVATSPISPTENNTTPPDALTRNTEKQKKKPKMSDEEILEKLRSIVSVGDPKKKYTRFEKIGQGASGTVYTAMDVATGQEVAIKQMNLQQQPKKELIINEILVMRENKNPNIVNYLDSYLVGDELWVVMEYLAGGSLTDVVTETCMDEGQIAAVCRECLQALEFLHSNQVIHRDIKSDNILLGMDGSVKLTDFGFCAQITPEQSKRSTMVGTPYWMAPEVVTRKAYGPKVDIWSLGIMAIEMIEGEPPYLNENPLRALYLIATNGTPELQNPEKLSAIFRDFLNRCLEMDVEKRGSAKELLQHQFLKIAKPLSSLTPLIAAAKEATKNNH DIP protein Q13153 original sequence version: 161 DIP protein Q13153 original sequence version: 122 DIP protein Q13153 original sequence version: 134 DIP protein Q13153 original sequence version: 165 1A95CD5F2195CD7B esr1_human Estrogen receptor ESR1 ESR NR3A1 Estradiol receptor ER-alpha Nuclear receptor subfamily 3 group A member 1 protein protein human Homo sapiens Human MTMTLHTKASGMALLHQIQGNELEPLNRPQLKIPLERPLGEVYLDSSKPAVYNYPEGAAYEFNAAAAANAQVYGQTGLPYGPGSEAAAFGSNGLGGFPPLNSVSPSPLMLLHPPPQLSPFLQPHGQQVPYYLENEPSGYTVREAGPPAFYRPNSDNRRQGGRERLASTNDKGSMAMESAKETRYCAVCNDYASGYHYGVWSCEGCKAFFKRSIQGHNDYMCPATNQCTIDKNRRKSCQACRLRKCYEVGMMKGGIRKDRRGGRMLKHKRQRDDGEGRGEVGSAGDMRAANLWPSPLMIKRSKKNSLALSLTADQMVSALLDAEPPILYSEYDPTRPFSEASMMGLLTNLADRELVHMINWAKRVPGFVDLTLHDQVHLLECAWLEILMIGLVWRSMEHPGKLLFAPNLLLDRNQGKCVEGMVEIFDMLLATSSRFRMMNLQGEEFVCLKSIILLNSGVYTFLSSTLKSLEEKDHIHRVLDKITDTLIHLMAKAGLTLQQQHQRLAQLLLILSHIRHMSNKGMEHLYSMKCKNVVPLYDLLLEMLDAHRLHAPTSRGGASVEETDQSHLATAGSTSSHSLQKYYITGEAEGFPATV DIP protein P03372 original sequence version: 214 DIP protein P03372 original sequence version: 139 DIP protein P03372 original sequence version: 167 5455C57AB0CCCAA7 pak1-cripak-1 3 8 unspecified role unspecified role prey prey 9 unspecified role unspecified role bait bait n-terminal region 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 By performing a yeast two-hybrid screen of a mammary gland library, we identified cysteine-rich inhibitor of Pak1 (CRIPak) as a novel Pak1-interacting protein. pak1-cripak-2 6 8 unspecified role unspecified role prey prey 9 unspecified role unspecified role bait bait n-terminal region 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 GBD-PAK1 interacts with GAD-CRIPak in yeast cells as shown bytheir growth on media lacking histidine (Figure 2a). 2A pak1-cripak-3 5 9 unspecified role unspecified role bait bait n-terminal region sufficient to bind sufficient binding region sufficient to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain 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 unspecified role unspecified role prey prey region 35s radiolabel 35s radiolabel 35S s35 radiolabelled S35 s35 radiolabelled 35S S35 undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined in vitro In vitro physical association physical association To test whether CRIPak can bind directly to PAK1 in vitro, PAK1 was expressed as a GST fusion protein in bacteria and tested for interaction with in vitro-transcribed and -translated [35S] methionine-labeled CRIPak. 2B and 3A pak1-cripak-4 4 9 unspecified role unspecified role prey prey region myc tag myc tag EUKLISEED epitope tag EUKLISEED epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 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 physical association physical association As shown in Figure 2c, the association of CRIPak with PAK1 in COS7 cells was demonstrated by co-immunoprecipitation. 2C pak1-cripak-5 2 9 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 8 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 undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined colocalization colocalization To further examine the association of CRIPak with PAK1 in vivo, we localized transiently expressed PAK1 and CRIPak by confocal microscopy 2D pak1-1 7 9 putative self putative self putative self putative self 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 true Autophosphorylation of PAK1 4A pak1-cripak-6 7 9 self self 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 inhibitor inhibitor 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 4 Inhibition of Pak1 activity by CRIPak in vitro and in vivo cripak-esr1-1 1 10 unspecified role unspecified role prey prey region t7 tag t7 tag MASMTGGQQMG epitope tag MASMTGGQQMG epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 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 physical association physical association We found that CRIPak binds to ER in a ligand-sensitive manner, as these effects were blocked by anti-estrogen ICI-182780 (Figure 7f). 7f ICI-182780, anti-estrogen pak1-2 7 9 putative self putative self putative self putative self human-mcf7 Homo sapiens mammary carcinoma cells mcf7 mammary carcinoma cells phosphorylation phosphorylation reaction phosphorylation true Autophosphorylation of PAK1 8b Upper panel; Pak1 autophosphorylation;