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/ Amplitude control of protein kinase C by RINCK, a novel E3 ubiquitin ligase. Amplitude control of protein kinase C by RINCK, a novel E3 ubiquitin ligase. J. Biol. Chem. (0021-9258) 2007 imex curation Chen D., Gould C., Garza R., Gao T., Hampton RY., Newton AC. Apoptosis - Interactions involving proteins with a function related to apoptosis Cancer - Interactions investigated in the context of cancer The authors provided the species of origin of all recombinant proteins. anewton@ucsd.edu 22-Jan-2008: Contacted by JYOTI. A mouse embryonic cDNA library was used. DAVET 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 Chen D., Gould C., Garza R., Gao T., Hampton RY., Newton AC. Apoptosis - Interactions involving proteins with a function related to apoptosis The authors provided the species of origin of all recombinant proteins. anewton@ucsd.edu Accepted 2007-DEC-22 by DAVET. A mouse embryonic cDNA library was used. J. Biol. Chem. (0021-9258) 2007 DAVET Only protein-protein interactions imex curation Amplitude control of protein kinase C by RINCK, a novel E3 ubiquitin ligase. Amplitude control of protein kinase C by RINCK, a novel E3 ubiquitin ligase. J. Biol. Chem. (0021-9258) 2007 imex curation Chen D., Gould C., Garza R., Gao T., Hampton RY., Newton AC. Apoptosis - Interactions involving proteins with a function related to apoptosis Cancer - Interactions investigated in the context of cancer The authors provided the species of origin of all recombinant proteins. anewton@ucsd.edu 22-Jan-2008: Contacted by JYOTI. A mouse embryonic cDNA library was used. DAVET Only protein-protein interactions human-tsa201 Homo sapiens subclone of human embryonic kidney 293 cell line that expresses simian virus 40 T antigen tsa201 Human embryonic kidney cell line subclone of 293 cell line that expresses simian virus 40 T antigen pull down pull down anti tag immunost anti tag immunostaining anti tag immunost Chen D., Gould C., Garza R., Gao T., Hampton RY., Newton AC. Apoptosis - Interactions involving proteins with a function related to apoptosis The authors provided the species of origin of all recombinant proteins. anewton@ucsd.edu Accepted 2007-DEC-22 by DAVET. 22-Jan-2008: Contacted by JYOTI. J. Biol. Chem. (0021-9258) 2007 DAVET Only protein-protein interactions imex curation Amplitude control of protein kinase C by RINCK, a novel E3 ubiquitin ligase. Amplitude control of protein kinase C by RINCK, a novel E3 ubiquitin ligase. J. Biol. Chem. (0021-9258) 2007 imex curation Chen D., Gould C., Garza R., Gao T., Hampton RY., Newton AC. Apoptosis - Interactions involving proteins with a function related to apoptosis Cancer - Interactions investigated in the context of cancer The authors provided the species of origin of all recombinant proteins. anewton@ucsd.edu 22-Jan-2008: Contacted by JYOTI. A mouse embryonic cDNA library was used. DAVET 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 western blot western blot Immuno blot Immuno blot Chen D., Gould C., Garza R., Gao T., Hampton RY., Newton AC. Apoptosis - Interactions involving proteins with a function related to apoptosis The authors provided the species of origin of all recombinant proteins. anewton@ucsd.edu Accepted 2007-DEC-22 by DAVET. 22-Jan-2008: Contacted by JYOTI. J. Biol. Chem. (0021-9258) 2007 DAVET Only protein-protein interactions imex curation Amplitude control of protein kinase C by RINCK, a novel E3 ubiquitin ligase. Amplitude control of protein kinase C by RINCK, a novel E3 ubiquitin ligase. J. Biol. Chem. (0021-9258) 2007 imex curation Chen D., Gould C., Garza R., Gao T., Hampton RY., Newton AC. Apoptosis - Interactions involving proteins with a function related to apoptosis Cancer - Interactions investigated in the context of cancer The authors provided the species of origin of all recombinant proteins. anewton@ucsd.edu 22-Jan-2008: Contacted by JYOTI. A mouse embryonic cDNA library was used. DAVET Only protein-protein interactions human-tsa201 Homo sapiens subclone of human embryonic kidney 293 cell line that expresses simian virus 40 T antigen tsa201 Human embryonic kidney cell line subclone of 293 cell line that expresses simian virus 40 T antigen anti bait coip anti bait coimmunoprecipitation anti bait coip anti tag immunost anti tag immunostaining anti tag immunost Chen D., Gould C., Garza R., Gao T., Hampton RY., Newton AC. Apoptosis - Interactions involving proteins with a function related to apoptosis The authors provided the species of origin of all recombinant proteins. anewton@ucsd.edu Accepted 2007-DEC-22 by DAVET. 22-Jan-2008: Contacted by JYOTI. J. Biol. Chem. (0021-9258) 2007 DAVET Only protein-protein interactions imex curation Amplitude control of protein kinase C by RINCK, a novel E3 ubiquitin ligase. Amplitude control of protein kinase C by RINCK, a novel E3 ubiquitin ligase. J. Biol. Chem. (0021-9258) 2007 imex curation Chen D., Gould C., Garza R., Gao T., Hampton RY., Newton AC. Apoptosis - Interactions involving proteins with a function related to apoptosis Cancer - Interactions investigated in the context of cancer The authors provided the species of origin of all recombinant proteins. anewton@ucsd.edu 22-Jan-2008: Contacted by JYOTI. A mouse embryonic cDNA library was used. DAVET Only protein-protein interactions human-tsa201 Homo sapiens subclone of human embryonic kidney 293 cell line that expresses simian virus 40 T antigen tsa201 Human embryonic kidney cell line subclone of 293 cell line that expresses simian virus 40 T antigen anti bait coip anti bait coimmunoprecipitation anti bait coip western blot western blot Immuno blot Immuno blot Chen D., Gould C., Garza R., Gao T., Hampton RY., Newton AC. Apoptosis - Interactions involving proteins with a function related to apoptosis The authors provided the species of origin of all recombinant proteins. anewton@ucsd.edu Accepted 2007-DEC-22 by DAVET. 22-Jan-2008: Contacted by JYOTI. J. Biol. Chem. (0021-9258) 2007 DAVET Only protein-protein interactions imex curation Amplitude control of protein kinase C by RINCK, a novel E3 ubiquitin ligase. Amplitude control of protein kinase C by RINCK, a novel E3 ubiquitin ligase. J. Biol. Chem. (0021-9258) 2007 imex curation Chen D., Gould C., Garza R., Gao T., Hampton RY., Newton AC. Apoptosis - Interactions involving proteins with a function related to apoptosis Cancer - Interactions investigated in the context of cancer The authors provided the species of origin of all recombinant proteins. anewton@ucsd.edu 22-Jan-2008: Contacted by JYOTI. A mouse embryonic cDNA library was used. DAVET Only protein-protein interactions human-tsa201 Homo sapiens subclone of human embryonic kidney 293 cell line that expresses simian virus 40 T antigen tsa201 Human embryonic kidney cell line subclone of 293 cell line that expresses simian virus 40 T antigen anti tag coip anti tag coimmunoprecipitation anti tag coip western blot western blot Immuno blot Immuno blot Chen D., Gould C., Garza R., Gao T., Hampton RY., Newton AC. Apoptosis - Interactions involving proteins with a function related to apoptosis The authors provided the species of origin of all recombinant proteins. anewton@ucsd.edu Accepted 2007-DEC-22 by DAVET. 22-Jan-2008: Contacted by JYOTI. J. Biol. Chem. (0021-9258) 2007 DAVET Only protein-protein interactions imex curation Amplitude control of protein kinase C by RINCK, a novel E3 ubiquitin ligase. Amplitude control of protein kinase C by RINCK, a novel E3 ubiquitin ligase. J. Biol. Chem. (0021-9258) 2007 imex curation Chen D., Gould C., Garza R., Gao T., Hampton RY., Newton AC. Apoptosis - Interactions involving proteins with a function related to apoptosis Cancer - Interactions investigated in the context of cancer The authors provided the species of origin of all recombinant proteins. anewton@ucsd.edu 22-Jan-2008: Contacted by JYOTI. A mouse embryonic cDNA library was used. DAVET Only protein-protein interactions in vitro In vitro pull down pull down western blot western blot Immuno blot Immuno blot Chen D., Gould C., Garza R., Gao T., Hampton RY., Newton AC. Apoptosis - Interactions involving proteins with a function related to apoptosis The authors provided the species of origin of all recombinant proteins. anewton@ucsd.edu Accepted 2007-DEC-22 by DAVET. 22-Jan-2008: Contacted by JYOTI. J. Biol. Chem. (0021-9258) 2007 DAVET Only protein-protein interactions imex curation p68403-2 Protein kinase C beta type Prkcb Pkcb Prkcb1 protein protein rat Rattus norvegicus (Rat) Rat MADPAAGPPPSEGEESTVRFARKGALRQKNVHEVKNHKFTARFFKQPTFCSHCTDFIWGFGKQGFQCQVCCFVVHKRCHEFVTFSCPGADKGPASDDPRSKHKFKIHTYSSPTFCDHCGSLLYGLIHQGMKCDTCMMNVHKRCVMNVPSLCGTDHTERRGRIYIQAHIDREVLIVVVRDAKNLVPMDPNGLSDPYVKLKLIPDPKSESKQKTKTIKCSLNPEWNETFRFQLKESDKDRRLSVEIWDWDLTSRNDFMGSLSFGISELQKAGVDGWFKLLSQEEGEYFNVPVPPEGSEGNEELRQKFERAKIGQGTKAPEEKTANTISKFDNNGNRDRMKLTDFNFLMVLGKGSFGKVMLSERKGTDELYAVKILKKDVVIQDDDVECTMVEKRVLALPGKPPFLTQLHSCFQTMDRLYFVMEYVNGGDLMYHIQQVGRFKEPHAVFYAAEIAIGLFFLQSKGIIYRDLKLDNVMLDSEGHIKIADFGMCKENIWDGVTTKTFCGTPDYIAPEIIAYQPYGKSVDWWAFGVLLYEMLAGQAPFEGEDEDELFQSIMEHNVAYPKSMSKEAVAICKGLMTKHPGKRLGCGPEGERDIKEHAFFRYIDWEKLERKEIQPPYKPKACGRNAENFDRFFTRHPPVLTPPDQEVIRNIDQSEFEGFSFVNSEFLKPEVKS Contains a phosphothreonine at position 641. Contains a phosphoserine at position 654. Contains a phosphoserine at position 660. Contains a phosphoserine at position 664. A65573AAF6E224C7 ubiq_yeast Ubiquitin UBI1 RPL40A YIL148W UBI2 RPL40B YKR094C UBI3 RPS31 YLR167W L9470.14 UBI4 SCD2 YLL039C protein protein yeasx Saccharomyces cerevisiae (Baker's yeast) Baker's yeast MQIFVKTLTGKTITLEVESSDTIDNVKSKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKESTLHLVLRLRGG obsolete protein 9AC936269D38403E kpcz_rat Protein kinase C zeta type Prkcz Pkcz nPKC-zeta protein protein rat Rattus norvegicus (Rat) Rat MPSRTDPKMDRSGGRVRLKAHYGGDILITSVDPTTTFQDLCEEVRDMCGLHQQHPLTLKWVDSEGDPCTVSSQMELEEAFRLACQGRDEVLIIHVFPSIPEQPGMPCPGEDKSIYRRGARRWRKLYRANGHLFQAKRFNRRAYCGQCSERIWGLARQGYRCINCKLLVHKRCHVLVPLTCRRHMDSVMPSQEPPVDDKNDGVDLPSEETDGIAYISSSRKHDNIKDDSEDLKPVIDGVDGIKISQGLGLQDFDLIRVIGRGSYAKVLLVRLKKNDQIYAMKVVKKELVHDDEDIDWVQTEKHVFEQASSNPFLVGLHSCFQTTSRLFLVIEYVNGGDLMFHMQRQRKLPEEHARFYAAEICIALNFLHERGIIYRDLKLDNVLLDADGHIKLTDYGMCKEGLGPGDTTSTFCGTPNYIAPEILRGEEYGFSVDWWALGVLMFEMMAGRSPFDIITDNPDMNTEDYLFQVILEKPIRIPRFLSVKASHVLKGFLNKDPKERLGCRPQTGFSDIKSHAFFRSIDWDLLEKKQTLPPFQPQITDDYGLDNFDTQFTSEPVQLTPDDEDVIKRIDQSEFEGFEYINPLLLSAEESV DIP protein P09217 original sequence version: 107 5A3021171C2FD7C7 tri41_human E3 ubiquitin-protein ligase TRIM41 TRIM41 RINCK Tripartite motif-containing protein 41 RING finger-interacting protein with C kinase RING-type E3 ubiquitin transferase TRIM41 protein protein human Homo sapiens Human MAAVAMTPNPVQTLQEEAVCAICLDYFTDPVSIGCGHNFCRVCVTQLWGGEDEEDRDELDREEEEEDGEEEEVEAVGAGAGWDTPMRDEDYEGDMEEEVEEEEEGVFWTSGMSRSSWDNMDYVWEEEDEEEDLDYYLGDMEEEDLRGEDEEDEEEVLEEVEEEDLDPVTPLPPPPAPRRCFTCPQCRKSFPRRSFRPNLQLANMVQVIRQMHPTPGRGSRVTDQGICPKHQEALKLFCEVDEEAICVVCRESRSHKQHSVVPLEEVVQEYKAKLQGHVEPLRKHLEAVQKMKAKEERRVTELKSQMKSELAAVASEFGRLTRFLAEEQAGLERRLREMHEAQLGRAGAAASRLAEQAAQLSRLLAEAQERSQQGGLRLLQDIKETFNRCEEVQLQPPEVWSPDPCQPHSHDFLTDAIVRKMSRMFCQAARVDLTLDPDTAHPALMLSPDRRGVRLAERRQEVADHPKRFSADCCVLGAQGFRSGRHYWEVEVGGRRGWAVGAARESTHHKEKVGPGGSSVGSGDASSSRHHHRRRRLHLPQQPLLQREVWCVGTNGKRYQAQSSTEQTLLSPSEKPRRFGVYLDYEAGRLGFYNAETLAHVHTFSAAFLGERVFPFFRVLSKGTRIKLCP 8E704CBEAB928BF5 kpca_human Protein kinase C alpha type PRKCA PKCA PRKACA protein protein human Homo sapiens Human MADVFPGNDSTASQDVANRFARKGALRQKNVHEVKDHKFIARFFKQPTFCSHCTDFIWGFGKQGFQCQVCCFVVHKRCHEFVTFSCPGADKGPDTDDPRSKHKFKIHTYGSPTFCDHCGSLLYGLIHQGMKCDTCDMNVHKQCVINVPSLCGMDHTEKRGRIYLKAEVADEKLHVTVRDAKNLIPMDPNGLSDPYVKLKLIPDPKNESKQKTKTIRSTLNPQWNESFTFKLKPSDKDRRLSVEIWDWDRTTRNDFMGSLSFGVSELMKMPASGWYKLLNQEEGEYYNVPIPEGDEEGNMELRQKFEKAKLGPAGNKVISPSEDRKQPSNNLDRVKLTDFNFLMVLGKGSFGKVMLADRKGTEELYAIKILKKDVVIQDDDVECTMVEKRVLALLDKPPFLTQLHSCFQTVDRLYFVMEYVNGGDLMYHIQQVGKFKEPQAVFYAAEISIGLFFLHKRGIIYRDLKLDNVMLDSEGHIKIADFGMCKEHMMDGVTTRTFCGTPDYIAPEIIAYQPYGKSVDWWAYGVLLYEMLAGQPPFDGEDEDELFQSIMEHNVSYPKSLSKEAVSVCKGLMTKHPAKRLGCGPEGERDVREHAFFRRIDWEKLENREIQPPFKPKVCGKGAENFDKFFTRGQPVLTPPDQLVIANIDQSDFEGFSYVNPQFVHPILQSAV 9EB157789A062349 kpca_rat Protein kinase C alpha type Prkca Pkca protein protein rat Rattus norvegicus (Rat) Rat MADVYPANDSTASQDVANRFARKGALRQKNVHEVKDHKFIARFFKQPTFCSHCTDFIWGFGKQGFQCQVCCFVVHKRCHEFVTFSCPGADKGPDTDDPRSKHKFKIHTYGSPTFCDHCGSLLYGLIHQGMKCDTCDMNVHKQCVINVPSLCGMDHTEKRGRIYLKAEVTDEKLHVTVRDAKNLIPMDPNGLSDPYVKLKLIPDPKNESKQKTKTIRSTLNPQWNESFTFKLKPSDKDRRLSVEIWDWDRTTRNDFMGSLSFGVSELMKMPASGWYKLLNQEEGEYYNVPIPEGDEEGNVELRQKFEKAKLGPAGNKVISPSEDRKQPSNNLDRVKLTDFNFLMVLGKGSFGKVMLADRKGTEELYAIKILKKDVVIQDDDVECTMVEKRVLALLDKPPFLTQLHSCFQTVDRLYFVMEYVNGGDLMYHIQQVGKFKEPQAVFYAAEISIGLFFLHKRGIIYRDLKLDNVMLDSEGHIKIADFGMCKEHMMDGVTTRTFCGTPDYIAPEIIAYQPYGKSVDWWAYGVLLYEMLAGQPPFDGEDEDELFQSIMEHNVSYPKSLSKEAVSICKGLMTKHPAKRLGCGPEGERDVREHAFFRRIDWEKLENREIQPPFKPKVCGKGAENFDKFFTRGQPVLTPPDQLVIANIDQSDFEGFSYVNPQFVHPILQSAV 94889E7339C17719 q8wv44-2 E3 ubiquitin-protein ligase TRIM41 TRIM41b TRIM41beta TRIM41 RINCK RING finger-interacting protein with C kinase Tripartite motif-containing protein 41 RINCK1 RING-type E3 ubiquitin transferase TRIM41 protein protein human Homo sapiens Human MAAVAMTPNPVQTLQEEAVCAICLDYFTDPVSIGCGHNFCRVCVTQLWGGEDEEDRDELDREEEEEDGEEEEVEAVGAGAGWDTPMRDEDYEGDMEEEVEEEEEGVFWTSGMSRSSWDNMDYVWEEEDEEEDLDYYLGDMEEEDLRGEDEEDEEEVLEEVEEEDLDPVTPLPPPPAPRRCFTCPQCRKSFPRRSFRPNLQLANMVQVIRQMHPTPGRGSRVTDQGICPKHQEALKLFCEVDEEAICVVCRESRSHKQHSVVPLEEVVQEYKAKLQGHVEPLRKHLEAVQKMKAKEERRVTELKSQMKSELAAVASEFGRLTRFLAEEQAGLERRLREMHEAQLGRAGAAASRLAEQAAQLSRLLAEAQERSQQGGLRLLQDIKETFNRCEEVQLQPPEVWSPDPCQPHSHDFLTDAIVRKMSRMFCQAARVDLTLDPDTAHPALMLSPDRRGVRLAERRQEVADHPKRFSADCCVLGAQGFRSGRHYWEEPKEPSWPPAQPSLTYYVCPTDRPEFSFT 88078577B9A3C98E kpcd_mouse Protein kinase C delta type nPKC-delta Tyrosine-protein kinase PRKCD Prkcd Pkcd protein protein mouse Mus musculus Mouse MAPFLRISFNSYELGSLQVEDEASQPFCAVKMKEALSTERGKTLVQKKPTMYPEWKTTFDAHIYEGRVIQIVLMRAAEDPVSEVTVGVSVLAERCKKNNGKAEFWLDLQPQAKVLMCVQYFLEDGDCKQSMRSEEEAKFPTMNRRGAIKQAKIHYIKNHEFIATFFGQPTFCSVCKEFVWGLNKQGYKCRQCNAAIHKKCIDKIIGRCTGTATNSRDTIFQKERFNIDMPHRFKVYNYMSPTFCDHCGSLLWGLVKQGLKCEDCGMNVHHKCREKVANLCGINQKLLAEALNQVTQRSSRKLDTTESVGIYQGFEKKPEVSGSDILDNNGTYGKIWEGSTRCTLENFTFQKVLGKGSFGKVLLAELKGKDKYFAIKCLKKDVVLIDDDVECTMVEKRVLALAWESPFLTHLICTFQTKDHLFFVMEFLNGGDLMFHIQDKGRFELYRATFYAAEIICGLQFLHSKGIIYRDLKLDNVMLDRDGHIKIADFGMCKENIFGEGRASTFCGTPDYIAPEILQGLKYSFSVDWWSFGVLLYEMLIGQSPFHGDDEDELFESIRVDTPHYPRWITKESKDIMEKLFERDPDKRLGVTGNIRIHPFFKTINWSLLEKRKVEPPFKPKVKSPSDYSNFDPEFLNEKPQLSFSDKNLIDSMDQEAFHGFSFVNPKFEQFLDI DIP protein P28867 original sequence version: 135 6E9F753348F03D59 prkcb1-rinck1 1 11 unspecified role unspecified role prey prey 8 unspecified role unspecified role bait bait n-terminal sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain physical association physical association we used a yeast two-hybrid approach to screen a mouse embryonic cDNA library with a sequence encoding the first 111 amino acids of rat PKC; as bait. This sequence encodes the N-terminal tail, the pseudosubstrate sequence, and C1A domain of PKC;. Five positive clones were obtained from approximately 100,000 screened. One of these encoded a 72 amino acid sequence which database analysis revealed was part of a novel gene predicted to encode a RING finger domain-containing protein. We named the putative protein RINCK for RING finger protein that interacts with C Kinase trim41-prkca 6 13 unspecified role unspecified role prey prey 14 unspecified role unspecified role bait bait 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 2 A Figure 2A reveals that recombinant PKC was present in immunoprecipitates of FLAGRINCK1 Q8WV44-2; P05696 transfection trim41-prkca-1 5 14 unspecified role unspecified role bait bait 12 unspecified role unspecified role prey prey physical association physical association 2 B RINCK was immunoprecipitated from tsA201 cells and probed for bound PKC ;. A discrete band co-migrating with PKC ; was visible in Western blots of immunoprecipitated RINCK (Figure 2B, lane 3), but not control beads incubated with pre-immune serum (Figure 2B, lane 2). Thus, RINCK forms a complex with PKC ; in cells. trim41-prkca-2 7 13 unspecified role unspecified role prey prey 11 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 ecobb Escherichia coli (strain B / BL21) direct interaction direct interaction 2 C PKC bound to GST-RINCK1 (lane 2) but not to GST alone (lane 1), revealing that PKC binds to RINCK directly. trim41-p68403 3 8 unspecified role unspecified role prey prey 14 unspecified role unspecified role bait bait 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 3 A TsA201 cells were transfected with the cDNAs encoding a PKC from each class: PKC βII (another conventional isozyme), PKC (novel), or PKC (atypical). The Western blot in Figure 3 shows that all three isoforms were present in immunoprecipitates of FLAG-RINCK1 Q8WV44-2 transfection trim41-prkcd 3 15 unspecified role unspecified role prey prey 14 unspecified role unspecified role bait bait 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 3 C TsA201 cells were transfected with the cDNAs encoding a PKC from each class: PKC βII (another conventional isozyme), PKC (novel), or PKC (atypical). The Western blot in Figure 3 shows that all three isoforms were present in immunoprecipitates of FLAG-RINCK1 Q8WV44-2 transfection trim41-pkc_zeta_rat 3 14 unspecified role unspecified role bait bait 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 10 unspecified role unspecified role prey prey physical association physical association 3 B TsA201 cells were transfected with the cDNAs encoding a PKC from each class: PKC βII (another conventional isozyme), PKC (novel), or PKC (atypical). The Western blot in Figure 3 shows that all three isoforms were present in immunoprecipitates of FLAG-RINCK1 Q8WV44-2 transfection trim41-p68403-1 6 14 unspecified role unspecified role bait bait 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 8 unspecified role unspecified role prey prey physical association physical association 3 D Q8WV44-2; P68403-2 transfection p68403-trim41 2 14 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 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 n-terminal sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain physical association physical association 4 All GST-tagged constructs were expressed in tsA201 cells and effectively precipitated by glutathione-Sepharose beads (Figure 4, upper panel) as judged by staining with anti-GST antibodies. RINCK1 associated with all the PKC constructs that contained the C1A domain ubi1-p68403 4 8 unspecified role unspecified role bait bait 9 unspecified role unspecified role prey prey region ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined physical association physical association 6 Increased levels of HAtagged ubiquitin were detected on PKC II in the presence of exogenous RINCK RINCK1 - Overexpression of RINCK1 enhanced the ubiquitination of PKC betaII. The C20A mutation abolished this enhancement. P61864; P68403-2 transfection