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/ Isolation and identification of L-dopa decarboxylase as a protein that binds to and enhances transcriptional activity of the androgen receptor using the repressed transactivator yeast two-hybrid system. Isolation and identification of L-dopa decarboxylase as a protein that binds to and enhances transcriptional activity of the androgen receptor using the repressed transactivator yeast two-hybrid system. Biochem. J. (0264-6021) 2003 imex curation Wafa LA., Cheng H., Rao MA., Nelson CC., Cox M., Hirst M., Sadowski I., Rennie PS. Cancer - Interactions investigated in the context of cancer A reverse yeast two-hybrid screen specifically designed for transactivator bait proteins was used. The AR NTD is fused to a GAL4 DBD to form the bait. The prey is created by fusion of the cDNA library with a TUP1 repressor protein. The N-terminus of TUP1 is sufficient to cause transcriptional repression, so only the first 200 amino acid residues were fused to the cDNA library. When there is no interaction between the bait and TUP1-library fusion protein, the bait activator allows constitutive expression of the URA3 reporter gene. This permits growth in the absence of uracil, but causes sensitivity to 5-FOA, which is converted into a toxic 5-flourouracil product through the enzyme encoded by the URA3 gene. Thus transcriptional repression can be genetically monitored by using a counter-selectable URA3 reporter. Where there is an interaction between the AR NTD and a library clone, TUP1 will cause repression of URA3 expression, which can be detected by growth of yeast in the presence of 5- FOA. Therefore, viability of the MAV103 strain, or colony growth, on 5-FOA media represents a positive interaction between the AR NTD bait and prey. prennie@interchange.ubc.ca 01-April-2008: Contacted by JYOTI. A cDNA library from the LNCaP human prostate carcinoma cell line was used. orchard Only protein-protein interactions in vitro In vitro pull down pull down weight autoradiogra molecular weight estimation by autoradiography weight autoradiogra Wafa LA., Cheng H., Rao MA., Nelson CC., Cox M., Hirst M., Sadowski I., Rennie PS. Cancer - Interactions investigated in the context of cancer A reverse yeast two-hybrid screen specifically designed for transactivator bait proteins was used. The AR NTD is fused to a GAL4 DBD to form the bait. The prey is created by fusion of the cDNA library with a TUP1 repressor protein. The N-terminus of TUP1 is sufficient to cause transcriptional repression, so only the first 200 amino acid residues were fused to the cDNA library. When there is no interaction between the bait and TUP1-library fusion protein, the bait activator allows constitutive expression of the URA3 reporter gene. This permits growth in the absence of uracil, but causes sensitivity to 5-FOA, which is converted into a toxic 5-flourouracil product through the enzyme encoded by the URA3 gene. Thus transcriptional repression can be genetically monitored by using a counter-selectable URA3 reporter. Where there is an interaction between the AR NTD and a library clone, TUP1 will cause repression of URA3 expression, which can be detected by growth of yeast in the presence of 5- FOA. Therefore, viability of the MAV103 strain, or colony growth, on 5-FOA media represents a positive interaction between the AR NTD bait and prey. prennie@interchange.ubc.ca Accepted 2008-MAR-6 by ORCHARD. 01-April-2008: Contacted by JYOTI. A cDNA library from the LNCaP human prostate carcinoma cell line was used. Biochem. J. (0264-6021) 2003 orchard Only protein-protein interactions imex curation Isolation and identification of L-dopa decarboxylase as a protein that binds to and enhances transcriptional activity of the androgen receptor using the repressed transactivator yeast two-hybrid system. Isolation and identification of L-dopa decarboxylase as a protein that binds to and enhances transcriptional activity of the androgen receptor using the repressed transactivator yeast two-hybrid system. Biochem. J. (0264-6021) 2003 imex curation Wafa LA., Cheng H., Rao MA., Nelson CC., Cox M., Hirst M., Sadowski I., Rennie PS. Cancer - Interactions investigated in the context of cancer A reverse yeast two-hybrid screen specifically designed for transactivator bait proteins was used. The AR NTD is fused to a GAL4 DBD to form the bait. The prey is created by fusion of the cDNA library with a TUP1 repressor protein. The N-terminus of TUP1 is sufficient to cause transcriptional repression, so only the first 200 amino acid residues were fused to the cDNA library. When there is no interaction between the bait and TUP1-library fusion protein, the bait activator allows constitutive expression of the URA3 reporter gene. This permits growth in the absence of uracil, but causes sensitivity to 5-FOA, which is converted into a toxic 5-flourouracil product through the enzyme encoded by the URA3 gene. Thus transcriptional repression can be genetically monitored by using a counter-selectable URA3 reporter. Where there is an interaction between the AR NTD and a library clone, TUP1 will cause repression of URA3 expression, which can be detected by growth of yeast in the presence of 5- FOA. Therefore, viability of the MAV103 strain, or colony growth, on 5-FOA media represents a positive interaction between the AR NTD bait and prey. prennie@interchange.ubc.ca 01-April-2008: Contacted by JYOTI. A cDNA library from the LNCaP human prostate carcinoma cell line was used. orchard Only protein-protein interactions human-lncap Human caucasian prostate carcinoma lncap Human caucasian prostate adenocarcinoma anti bait coip anti bait coimmunoprecipitation anti bait coip western blot western blot Immuno blot Immuno blot Wafa LA., Cheng H., Rao MA., Nelson CC., Cox M., Hirst M., Sadowski I., Rennie PS. Cancer - Interactions investigated in the context of cancer A reverse yeast two-hybrid screen specifically designed for transactivator bait proteins was used. The AR NTD is fused to a GAL4 DBD to form the bait. The prey is created by fusion of the cDNA library with a TUP1 repressor protein. The N-terminus of TUP1 is sufficient to cause transcriptional repression, so only the first 200 amino acid residues were fused to the cDNA library. When there is no interaction between the bait and TUP1-library fusion protein, the bait activator allows constitutive expression of the URA3 reporter gene. This permits growth in the absence of uracil, but causes sensitivity to 5-FOA, which is converted into a toxic 5-flourouracil product through the enzyme encoded by the URA3 gene. Thus transcriptional repression can be genetically monitored by using a counter-selectable URA3 reporter. Where there is an interaction between the AR NTD and a library clone, TUP1 will cause repression of URA3 expression, which can be detected by growth of yeast in the presence of 5- FOA. Therefore, viability of the MAV103 strain, or colony growth, on 5-FOA media represents a positive interaction between the AR NTD bait and prey. prennie@interchange.ubc.ca Accepted 2008-MAR-6 by ORCHARD. 01-April-2008: Contacted by JYOTI. A cDNA library from the LNCaP human prostate carcinoma cell line was used. Biochem. J. (0264-6021) 2003 orchard Only protein-protein interactions imex curation Isolation and identification of L-dopa decarboxylase as a protein that binds to and enhances transcriptional activity of the androgen receptor using the repressed transactivator yeast two-hybrid system. Isolation and identification of L-dopa decarboxylase as a protein that binds to and enhances transcriptional activity of the androgen receptor using the repressed transactivator yeast two-hybrid system. Biochem. J. (0264-6021) 2003 imex curation Wafa LA., Cheng H., Rao MA., Nelson CC., Cox M., Hirst M., Sadowski I., Rennie PS. Cancer - Interactions investigated in the context of cancer A reverse yeast two-hybrid screen specifically designed for transactivator bait proteins was used. The AR NTD is fused to a GAL4 DBD to form the bait. The prey is created by fusion of the cDNA library with a TUP1 repressor protein. The N-terminus of TUP1 is sufficient to cause transcriptional repression, so only the first 200 amino acid residues were fused to the cDNA library. When there is no interaction between the bait and TUP1-library fusion protein, the bait activator allows constitutive expression of the URA3 reporter gene. This permits growth in the absence of uracil, but causes sensitivity to 5-FOA, which is converted into a toxic 5-flourouracil product through the enzyme encoded by the URA3 gene. Thus transcriptional repression can be genetically monitored by using a counter-selectable URA3 reporter. Where there is an interaction between the AR NTD and a library clone, TUP1 will cause repression of URA3 expression, which can be detected by growth of yeast in the presence of 5- FOA. Therefore, viability of the MAV103 strain, or colony growth, on 5-FOA media represents a positive interaction between the AR NTD bait and prey. prennie@interchange.ubc.ca 01-April-2008: Contacted by JYOTI. A cDNA library from the LNCaP human prostate carcinoma cell line was used. orchard Only protein-protein interactions yeasx Saccharomyces cerevisiae (Baker's yeast) Baker's yeast reverse two hybrid reverse two hybrid nucleotide sequence nucleotide sequence identification sequence cloning sequence cloning nucleotide sequence Wafa LA., Cheng H., Rao MA., Nelson CC., Cox M., Hirst M., Sadowski I., Rennie PS. Cancer - Interactions investigated in the context of cancer A reverse yeast two-hybrid screen specifically designed for transactivator bait proteins was used. The AR NTD is fused to a GAL4 DBD to form the bait. The prey is created by fusion of the cDNA library with a TUP1 repressor protein. The N-terminus of TUP1 is sufficient to cause transcriptional repression, so only the first 200 amino acid residues were fused to the cDNA library. When there is no interaction between the bait and TUP1-library fusion protein, the bait activator allows constitutive expression of the URA3 reporter gene. This permits growth in the absence of uracil, but causes sensitivity to 5-FOA, which is converted into a toxic 5-flourouracil product through the enzyme encoded by the URA3 gene. Thus transcriptional repression can be genetically monitored by using a counter-selectable URA3 reporter. Where there is an interaction between the AR NTD and a library clone, TUP1 will cause repression of URA3 expression, which can be detected by growth of yeast in the presence of 5- FOA. Therefore, viability of the MAV103 strain, or colony growth, on 5-FOA media represents a positive interaction between the AR NTD bait and prey. prennie@interchange.ubc.ca Accepted 2008-MAR-6 by ORCHARD. 01-April-2008: Contacted by JYOTI. A cDNA library from the LNCaP human prostate carcinoma cell line was used. Biochem. J. (0264-6021) 2003 orchard Only protein-protein interactions imex curation andr_human Androgen receptor Dihydrotestosterone receptor Nuclear receptor subfamily 3 group C member 4 AR DHTR NR3C4 protein protein human Homo sapiens Human MEVQLGLGRVYPRPPSKTYRGAFQNLFQSVREVIQNPGPRHPEAASAAPPGASLLLLQQQQQQQQQQQQQQQQQQQQQQQETSPRQQQQQQGEDGSPQAHRRGPTGYLVLDEEQQPSQPQSALECHPERGCVPEPGAAVAASKGLPQQLPAPPDEDDSAAPSTLSLLGPTFPGLSSCSADLKDILSEASTMQLLQQQQQEAVSEGSSSGRAREASGAPTSSKDNYLGGTSTISDNAKELCKAVSVSMGLGVEALEHLSPGEQLRGDCMYAPLLGVPPAVRPTPCAPLAECKGSLLDDSAGKSTEDTAEYSPFKGGYTKGLEGESLGCSGSAAAGSSGTLELPSTLSLYKSGALDEAAAYQSRDYYNFPLALAGPPPPPPPPHPHARIKLENPLDYGSAWAAAAAQCRYGDLASLHGAGAAGPGSGSPSAAASSSWHTLFTAEEGQLYGPCGGGGGGGGGGGGGGGGGGGGGGGEAGAVAPYGYTRPPQGLAGQESDFTAPDVWYPGGMVSRVPYPSPTCVKSEMGPWMDSYSGPYGDMRLETARDHVLPIDYYFPPQKTCLICGDEASGCHYGALTCGSCKVFFKRAAEGKQKYLCASRNDCTIDKFRRKNCPSCRLRKCYEAGMTLGARKLKKLGNLKLQEEGEASSTTSPTEETTQKLTVSHIEGYECQPIFLNVLEAIEPGVVCAGHDNNQPDSFAALLSSLNELGERQLVHVVKWAKALPGFRNLHVDDQMAVIQYSWMGLMVFAMGWRSFTNVNSRMLYFAPDLVFNEYRMHKSRMYSQCVRMRHLSQEFGWLQITPQEFLCMKALLLFSIIPVDGLKNQKFFDELRMNYIKELDRIIACKRKNPTSCSRRFYQLTKLLDSVQPIARELHQFTFDLLIKSHMVSVDFPEMMAEIISVQVPKILSGKVKPIYFHTQ DIP protein P10275 original sequence version: 220 DIP protein P10275-1 original sequence version: 213 DIP protein P10275 original sequence version: 216 DIP protein P10275 original sequence version: 143 DIP protein P10275 original sequence version: 174 DIP protein P10275 original sequence version: 169 DIP protein P10275 original sequence version: 188 DIP protein P10275-1 original sequence version: 188 DIP protein P10275 original sequence version: 196 DIP protein P10275 original sequence version: 217 DIP protein P10275-1 original sequence version: 217 DIP protein P10275-1 original sequence version: 207 A73432C55D39AE06 andr_rat Androgen receptor Ar Nr3c4 Dihydrotestosterone receptor Nuclear receptor subfamily 3 group C member 4 protein protein rat Rattus norvegicus (Rat) Rat MEVQLGLGRVYPRPPSKTYRGAFQNLFQSVREAIQNPGPRHPEAASIAPPGACLQQRQETSPRRRRRQQHPEDGSPQAHIRGTTGYLALEEEQQPSQQQSASEGHPESGCLPEPGAATAPGKGLPQQPPAPPDQDDSAAPSTLSLLGPTFPGLSSCSADIKDILSEAGTMQLLQQQQQQQQQQQQQQQQQQQQQQEVISEGSSSVRAREATGAPSSSKDSYLGGNSTISDSAKELCKAVSVSMGLGVEALEHLSPGEQLRGDCMYASLLGGPPAVRPTPCAPLAECKGLSLDEGPGKGTEETAEYSSFKGGYAKGLEGESLGCSGSSEAGSSGTLEIPSSLSLYKSGAVDEAAAYQNRDYYNFPLALSGPPHPPPPTHPHARIKLENPSDYGSAWAAAAAQCRYGDLASLHGGSVAGPSTGSPPATASSSWHTLFTAEEGQLYGPGGGGGSSSPSDAGPVAPYGYTRPPQGLASQEGDFSASEVWYPGGVVNRVPYPSPSCVKSEMGPWMENYSGPYGDMRLDSTRDHVLPIDYYFPPQKTCLICGDEASGCHYGALTCGSCKVFFKRAAEGKQKYLCASRNDCTIDKFRRKNCPSCRLRKCYEAGMTLGARKLKKLGNLKLQEEGENSSAGSPTEDPSQKMTVSHIEGYECQPIFLNVLEAIEPGVVCAGHDNNQPDSFAALLSSLNELGERQLVHVVKWAKALPGFRNLHVDDQMAVIQYSWMGLMVFAMGWRSFTNVNSRMLYFAPDLVFNEYRMHKSRMYSQCVRMRHLSQEFGWLQITPQEFLCMKALLLFSIIPVDGLKNQKFFDELRMNYIKELDRIIACKRKNPTSCSRRFYQLTKLLDSVQPIARELHQFTFDLLIKSHMVSVDFPEMMAEIISVQVPKILSGKVKPIYFHTQ 43F4064759FDCBED ddc_human Aromatic-L-amino-acid decarboxylase DDC AADC DOPA decarboxylase protein protein human Homo sapiens Human MNASEFRRRGKEMVDYMANYMEGIEGRQVYPDVEPGYLRPLIPAAAPQEPDTFEDIINDVEKIIMPGVTHWHSPYFFAYFPTASSYPAMLADMLCGAIGCIGFSWAASPACTELETVMMDWLGKMLELPKAFLNEKAGEGGGVIQGSASEATLVALLAARTKVIHRLQAASPELTQAAIMEKLVAYSSDQAHSSVERAGLIGGVKLKAIPSDGNFAMRASALQEALERDKAAGLIPFFMVATLGTTTCCSFDNLLEVGPICNKEDIWLHVDAAYAGSAFICPEFRHLLNGVEFADSFNFNPHKWLLVNFDCSAMWVKKRTDLTGAFRLDPTYLKHSHQDSGLITDYRHWQIPLGRRFRSLKMWFVFRMYGVKGLQAYIRKHVQLSHEFESLVRQDPRFEICVEVILGLVCFRLKGSNKVNEALLQRINSAKKIHLVPCHLRDKFVLRFAICSRTVESAHVQRAWEHIKELAADVLRAERE DIP protein P20711 original sequence version: 132 0A2CFF9A39BDE298 gak_human Cyclin-G-associated kinase GAK protein protein human Homo sapiens Human MSLLQSALDFLAGPGSLGGASGRDQSDFVGQTVELGELRLRVRRVLAEGGFAFVYEAQDVGSGREYALKRLLSNEEEKNRAIIQEVCFMKKLSGHPNIVQFCSAASIGKEESDTGQAEFLLLTELCKGQLVEFLKKMESRGPLSCDTVLKIFYQTCRAVQHMHRQKPPIIHRDLKVENLLLSNQGTIKLCDFGSATTISHYPDYSWSAQRRALVEEEITRNTTPMYRTPEIIDLYSNFPIGEKQDIWALGCILYLLCFRQHPFEDGAKLRIVNGKYSIPPHDTQYTVFHSLIRAMLQVNPEERLSIAEVVHQLQEIAAARNVNPKSPITELLEQNGGYGSATLSRGPPPPVGPAGSGYSGGLALAEYDQPYGGFLDILRGGTERLFTNLKDTSSKVIQSVANYAKGDLDISYITSRIAVMSFPAEGVESALKNNIEDVRLFLDSKHPGHYAVYNLSPRTYRPSRFHNRVSECGWAARRAPHLHTLYNICRNMHAWLRQDHKNVCVVHCMDGRAASAVAVCSFLCFCRLFSTAEAAVYMFSMKRCPPGIWPSHKRYIEYMCDMVAEEPITPHSKPILVRAVVMTPVPLFSKQRSGCRPFCEVYVGDERVASTSQEYDKMRDFKIEDGKAVIPLGVTVQGDVLIVIYHARSTLGGRLQAKMASMKMFQIQFHTGFVPRNATTVKFAKYDLDACDIQEKYPDLFQVNLEVEVEPRDRPSREAPPWENSSMRGLNPKILFSSREEQQDILSKFGKPELPRQPGSTAQYDAGAGSPEAEPTDSDSPPSSSADASRFLHTLDWQEEKEAETGAENASSKESESALMEDRDESEVSDEGGSPISSEGQEPRADPEPPGLAAGLVQQDLVFEVETPAVLPEPVPQEDGVDLLGLHSEVGAGPAVPPQACKAPSSNTDLLSCLLGPPEAASQGPPEDLLSEDPLLLASPAPPLSVQSTPRGGPPAAADPFGPLLPSSGNNSQPCSNPDLFGEFLNSDSVTVPPSFPSAHSAPPPSCSADFLHLGDLPGEPSKMTASSSNPDLLGGWAAWTETAASAVAPTPATEGPLFSPGGQPAPCGSQASWTKSQNPDPFADLGDLSSGLQGSPAGFPPGGFIPKTATTPKGSSSWQTSRPPAQGASWPPQAKPPPKACTQPRPNYASNFSVIGAREERGVRAPSFAQKPKVSENDFEDLLSNQGFSSRSDKKGPKTIAEMRKQDLAKDTDPLKLKLLDWIEGKERNIRALLSTLHTVLWDGESRWTPVGMADLVAPEQVKKHYRRAVLAVHPDKAAGQPYEQHAKMIFMELNDAWSEFENQGSRPLF 0ACE45DF57A5F981 ar-ddc 3 6 unspecified role unspecified role prey prey c-terminal sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain 4 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 physical association physical association PREY gene NM_000790 DDC (L-dopa decarboxylase) was detected multiple times as a novel AR-interacting protein, ar-gak 3 7 unspecified role unspecified role prey prey 4 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 physical association physical association Approx. 3x106 transformants were screened with all three AR NTD baits and the positive clones were sequenced and compared with known sequences available in GenBankTM. A total of 40 unique in-frame positive clones were detected from the LNCaP cDNA library, some of which were picked up multiple times. These included: a steroid receptor chaperone Hsp (only detected with the DBD-containing AR1−646 bait); the cyclin G-associated Ser/Thr kinase ar-ddc-2 1 6 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 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 in vitro In vitro 5 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 region sufficient to bind sufficient binding region sufficient to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain ecobb Escherichia coli (strain B / BL21) direct interaction direct interaction 3 fulllength DDC interacts with all domains of AR The interaction strength for DDC with the AR constructs varied in strength: AR (541-919)> AR(1-559) > AR (234-665) > AR (541-665). This indicated the strongest interaction occured between DDC and the AR LBD. ar-ddc-3 2 6 unspecified role unspecified role prey prey 4 unspecified role unspecified role bait bait physical association physical association 4 B DDC co-immunoprecipitated with AR in the presence and absence of R1881 (compare lanes 4 and 6 in Figure 4B), but the association seems to be stronger with the androgen treatment. Androgen (R1881) treatment enhanced this interacton.