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/ Cep97 and CP110 suppress a cilia assembly program. Cep97 and CP110 suppress a cilia assembly program. Cell (0092-8674) 2007 imex curation Spektor A., Tsang WY., Khoo D., Dynlacht BD. Origin of CP110 taken from PMID: 12361598 brian.dynlacht@med.nyu.edu 01-Feb-2008: Contacted by JYOTI. orchard Only protein-protein interactions 10 human-293 Homo sapiens transformed primary embryonal kidney cells Human 293 Transformed human primary embryonal kidney cells. The Cell type named 293 is synonymous with HEK-293. anti bait coip anti bait coimmunoprecipitation anti bait coip western blot western blot Immuno blot Immuno blot Spektor A., Tsang WY., Khoo D., Dynlacht BD. Origin of CP110 taken from PMID: 12361598 brian.dynlacht@med.nyu.edu Accepted 2008-FEB-7 by ORCHARD. 01-Feb-2008: Contacted by JYOTI. Cell (0092-8674) 2007 orchard Only protein-protein interactions 10 imex curation Cep97 and CP110 suppress a cilia assembly program. Cep97 and CP110 suppress a cilia assembly program. Cell (0092-8674) 2007 imex curation Spektor A., Tsang WY., Khoo D., Dynlacht BD. Origin of CP110 taken from PMID: 12361598 brian.dynlacht@med.nyu.edu 01-Feb-2008: Contacted by JYOTI. orchard Only protein-protein interactions 10 in vitro In vitro anti bait coip anti bait coimmunoprecipitation anti bait coip de novo protein sequence de novo protein sequencing by mass spectrometry de novo protein sequence Spektor A., Tsang WY., Khoo D., Dynlacht BD. Origin of CP110 taken from PMID: 12361598 brian.dynlacht@med.nyu.edu Accepted 2008-FEB-7 by ORCHARD. 01-Feb-2008: Contacted by JYOTI. Cell (0092-8674) 2007 orchard Only protein-protein interactions 10 imex curation Cep97 and CP110 suppress a cilia assembly program. Cep97 and CP110 suppress a cilia assembly program. Cell (0092-8674) 2007 imex curation Spektor A., Tsang WY., Khoo D., Dynlacht BD. Origin of CP110 taken from PMID: 12361598 brian.dynlacht@med.nyu.edu 01-Feb-2008: Contacted by JYOTI. orchard Only protein-protein interactions 10 human-293 Homo sapiens transformed primary embryonal kidney cells Human 293 Transformed human primary embryonal kidney cells. The Cell type named 293 is synonymous with HEK-293. anti bait coip anti bait coimmunoprecipitation anti bait coip western blot western blot Immuno blot Immuno blot Spektor A., Tsang WY., Khoo D., Dynlacht BD. Origin of CP110 taken from PMID: 12361598 brian.dynlacht@med.nyu.edu Accepted 2008-FEB-7 by ORCHARD. 01-Feb-2008: Contacted by JYOTI. Cell (0092-8674) 2007 orchard Only protein-protein interactions 10 imex curation Cep97 and CP110 suppress a cilia assembly program. Cep97 and CP110 suppress a cilia assembly program. Cell (0092-8674) 2007 imex curation Spektor A., Tsang WY., Khoo D., Dynlacht BD. Origin of CP110 taken from PMID: 12361598 brian.dynlacht@med.nyu.edu 01-Feb-2008: Contacted by JYOTI. orchard Only protein-protein interactions 10 human-293 Homo sapiens transformed primary embryonal kidney cells Human 293 Transformed human primary embryonal kidney cells. The Cell type named 293 is synonymous with HEK-293. anti tag coip anti tag coimmunoprecipitation anti tag coip western blot western blot Immuno blot Immuno blot Spektor A., Tsang WY., Khoo D., Dynlacht BD. Origin of CP110 taken from PMID: 12361598 brian.dynlacht@med.nyu.edu Accepted 2008-FEB-7 by ORCHARD. 01-Feb-2008: Contacted by JYOTI. Cell (0092-8674) 2007 orchard Only protein-protein interactions 10 imex curation Cep97 and CP110 suppress a cilia assembly program. Cep97 and CP110 suppress a cilia assembly program. Cell (0092-8674) 2007 imex curation Spektor A., Tsang WY., Khoo D., Dynlacht BD. Origin of CP110 taken from PMID: 12361598 brian.dynlacht@med.nyu.edu 01-Feb-2008: Contacted by JYOTI. orchard Only protein-protein interactions 10 human-293 Homo sapiens transformed primary embryonal kidney cells Human 293 Transformed human primary embryonal kidney cells. The Cell type named 293 is synonymous with HEK-293. anti tag coip anti tag coimmunoprecipitation anti tag coip anti tag western anti tag western blot anti tag western Spektor A., Tsang WY., Khoo D., Dynlacht BD. Origin of CP110 taken from PMID: 12361598 brian.dynlacht@med.nyu.edu Accepted 2008-FEB-7 by ORCHARD. 01-Feb-2008: Contacted by JYOTI. Cell (0092-8674) 2007 orchard Only protein-protein interactions 10 imex curation Cep97 and CP110 suppress a cilia assembly program. Cep97 and CP110 suppress a cilia assembly program. Cell (0092-8674) 2007 imex curation Spektor A., Tsang WY., Khoo D., Dynlacht BD. Origin of CP110 taken from PMID: 12361598 brian.dynlacht@med.nyu.edu 01-Feb-2008: Contacted by JYOTI. orchard Only protein-protein interactions 10 in vitro In vitro anti bait coip anti bait coimmunoprecipitation anti bait coip western blot western blot Immuno blot Immuno blot Spektor A., Tsang WY., Khoo D., Dynlacht BD. Origin of CP110 taken from PMID: 12361598 brian.dynlacht@med.nyu.edu Accepted 2008-FEB-7 by ORCHARD. 01-Feb-2008: Contacted by JYOTI. Cell (0092-8674) 2007 orchard Only protein-protein interactions 10 imex curation cep97_human Centrosomal protein of 97 kDa LRRIQ2 Leucine-rich repeat and IQ domain-containing protein 2 CEP97 protein protein human Homo sapiens Human MAVARVDAALPPGEGSVVNWSGQGLQKLGPNLPCEADIHTLILDKNQIIKLENLEKCKRLIQLSVANNRLVRMMGVAKLTLLRVLNLPHNSIGCVEGLKELVHLEWLNLAGNNLKAMEQINSCTALQHLDLSDNNISQIGDLSKLVSLKTLLLHGNIITSLRMAPAYLPRSLAILSLAENEIRDLNEISFLASLTELEQLSIMNNPCVMATPSIPGFDYRPYIVSWCLNLRVLDGYVISQKESLKAEWLYSQGKGRAYRPGQHIQLVQYLATVCPLTSTLGLQTAEDAKLEKILSKQRFHQRQLMNQSQNEELSPLVPVETRASLIPEHSSPVQDCQISQESEPVIQVNSWVGINSNDDQLFAVKNNFPASVHTTRYSRNDLHLEDIQTDEDKLNCSLLSSESTFMPVASGLSPLSPTVELRLQGINLGLEDDGVADESVKGLESQVLDKEEEQPLWAANENSVQMMRSEINTEVNEKAGLLPCPEPTIISAILKDDNHSLTFFPESTEQKQSDIKKPENTQPENKETISQATSEKLPMILTQRSVALGQDKVALQKLNDAATKLQACWRGFYARNYNPQAKDVRYEIRLRRMQEHIVCLTDEIRRLRKERDEERIKKFVQEEAFRFLWNQVRSLQVWQQTVDQRLSSWHTDVPPISSTLVPSKHPLFTQSQESSCDQNADWFIASDVAPQEKSLPEFPDSGFHSSLTEQVHSLQHSLDFEKSSTEGSESSIMGNSIDTVRYGKESDLGDVSEEHGEWNKESSNNEQDNSLLEQYLTSVQQLEDADERTNFDTETRDSKLHIACFPVQLDTLSDGASVDESHGISPPLQGEISQTQENSKLNAEVQGQQPECDSTFQLLHVGVTV 98B1C230DF586DF7 calm_human Calmodulin CALM1 CALM CAM CAM1 CALM2 CAMB CALM3 CAM3 CAMC CAMIII CAM2 CALML2 protein protein human Homo sapiens Human MADQLTEEQIAEFKEAFSLFDKDGDGTITTKELGTVMRSLGQNPTEAELQDMINEVDADGNGTIDFPEFLTMMARKMKDTDSEEEIREAFRVFDKDGNGYISAAELRHVMTNLGEKLTDEEVDEMIREADIDGDGQVNYEEFVQMMTAK DIP protein P62158 original sequence version: 97 DIP protein P62158 original sequence version: 144 DIP protein P62158 original sequence version: 146 DIP protein P62158 original sequence version: 73 DIP protein P62158 original sequence version: 102 DIP protein P62158 original sequence version: 98 DIP protein P62158 original sequence version: 96 DIP protein P62158 original sequence version: 111 DIP protein P62158 original sequence version: 131 DIP protein P62158 original sequence version: 136 DIP protein P62158 original sequence version: 145 DIP protein P62158 original sequence version: 154 DIP protein P62158 original sequence version: 148 DIP protein P62158 original sequence version: 116 DIP protein P62158 original sequence version: 120 DIP protein P62158 original sequence version: 130 6B4BC3FCDE10727B cp110_human Centriolar coiled-coil protein of 110 kDa CP110 CCP110 CEP110 KIAA0419 Centrosomal protein of 110 kDa protein protein human Homo sapiens Human MEEYEKFCEKSLARIQEASLSTESFLPAQSESISLIRFHGVAILSPLLNIEKRKEMQQEKQKALDVEARKQVNRKKALLTRVQEILDNVQVRKAPNASDFDQWEMETVYSNSEVRNLNVPATFPNSFPSHTEHSTAAKLDKIAGILPLDNEDQCKTDGIDLARDSEGFNSPKQCDSSNISHVENEAFPKTSSATPQETLISDGPFSVNEQQDLPLLAEVIPDPYVMSLQNLMKKSKEYIEREQSRRSLRGSINRIVNESHLDKEHDAVEVADCVKEKGQLTGKHCVSVIPDKPSLNKSNVLLQGASTQASSMSMPVLASFSKVDIPIRTGHPTVLESNSDFKVIPTFVTENNVIKSLTGSYAKLPSPEPSMSPKMHRRRSRTSSACHILINNPINACELSPKGKEQAMDLIIQDTDENTNVPEIMPKLPTDLAGVCSSKVYVGKNTSEVKEDVVLGKSNQVCQSSGNHLENKVTHGLVTVEGQLTSDERGAHIMNSTCAAMPKLHEPYASSQCIASPNFGTVSGLKPASMLEKNCSLQTELNKSYDVKNPSPLLMQNQNTRQQMDTPMVSCGNEQFLDNSFEKVKRRLDLDIDGLQKENCPYVITSGITEQERQHLPEKRYPKGSGFVNKNKMLGTSSKESEELLKSKMLAFEEMRKRLEEQHAQQLSLLIAEQEREQERLQKEIEEQEKMLKEKKAMTAEASELDINNAVELEWRKISDSSLLETMLSQADSLHTSNSNSSGFTNSAMQYSFVSANEAPFYLWGSSTSGLTKLSVTRPFGRAKTRWSQVFSLEIQAKFNKITAVAKGFLTRRLMQTDKLKQLRQTVKDTMEFIRSFQSEAPLKRGIVSAQDASLQERVLAQLRAALYGIHDIFFVMDAAERMSILHHDREVRKEKMLRQMDKMKSPRVALSAATQKSLDRKKYMKAAEMGMPNKKFLVKQNPSETRVLQPNQGQNAPVHRLLSRQGTPKTSVKGVVQNRQKPSQSRVPNRVPVSGVYAGKIQRKRPNVATI DIP protein O43303 original sequence version: 78 5459F655CFB9DFD0 cep97-cp110 4 7 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 human-293 Homo sapiens transformed primary embryonal kidney cells Human 293 Transformed human primary embryonal kidney cells. The Cell type named 293 is synonymous with HEK-293. 9 unspecified role unspecified role prey prey human-293 Homo sapiens transformed primary embryonal kidney cells Human 293 Transformed human primary embryonal kidney cells. The Cell type named 293 is synonymous with HEK-293. physical association physical association S1B, lane 3. We confirmed the interaction between Cep97 and CP110 by ectopically expressing Flag-tagged Cep97 and immunoprecipitating this protein using antibodies against CP110 (Figure S1B). Similarly, anti-Flag antibodies coprecipitated both Cep97 and CP110. In addition, Cep97 associates with CaM, an observation anticipated by the presence of an IQ domain. cep97-cam-cep110 4 8 unspecified role unspecified role prey prey human-293 Homo sapiens transformed primary embryonal kidney cells Human 293 Transformed human primary embryonal kidney cells. The Cell type named 293 is synonymous with HEK-293. 7 unspecified role unspecified role bait bait c-terminal region flag tag flag tag FLAG-tagged FLAG DYKDDDDKV epitope tag DYKDDDDKV epitope tag FLAG FLAG-tagged c-term range C-terminal range c-term range c-term range C-terminal range c-term range human-293 Homo sapiens transformed primary embryonal kidney cells Human 293 Transformed human primary embryonal kidney cells. The Cell type named 293 is synonymous with HEK-293. 9 unspecified role unspecified role prey prey human-293 Homo sapiens transformed primary embryonal kidney cells Human 293 Transformed human primary embryonal kidney cells. The Cell type named 293 is synonymous with HEK-293. association association S1B, lane 2. We confirmed the interaction between Cep97 and CP110 by ectopically expressing Flag-tagged Cep97 and immunoprecipitating this protein using antibodies against CP110 (Figure S1B). Similarly, anti-Flag antibodies coprecipitated both Cep97 and CP110. In addition, Cep97 associates with CaM, an observation anticipated by the presence of an IQ domain. Cep97 was shown to react with both CAM and Cep110. cep97-cp110-1 6 9 unspecified role unspecified role bait bait in vitro In vitro 7 unspecified role unspecified role prey prey region 35s radiolabel 35s radiolabel 35S s35 radiolabelled S35 s35 radiolabelled 35S S35 autoradiography autoradiography undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined in vitro In vitro direct interaction direct interaction 1C To determine whether Cep97 and CP110 directly interact, we in vitro translated both proteins and performed CP110 immunoprecipitations, which resulted in very efficient recovery of both proteins, suggesting that the proteins most likely interact directly (Figure 1C). Since both CP110 and Cep97 are CaM-binding proteins, we tested the possibility that the CP110-Cep97 interaction was mediated by CaM in the reticulocyte lysate by performing in vitro binding assays with CP110 mutants harboring small deletions that abrogate CaM association (Tsang et al., 2006). These studies suggested that CaM was not required to bridge interactions between Cep97 and CP110, and likewise, Cep97 mutants that do not bind CaM retain the ability to interact with CP110 (Figure 1C, and data not shown). cep97-cp110-2 1 7 unspecified role unspecified role bait bait human-293 Homo sapiens transformed primary embryonal kidney cells Human 293 Transformed human primary embryonal kidney cells. The Cell type named 293 is synonymous with HEK-293. 9 unspecified role unspecified role prey prey human-293 Homo sapiens transformed primary embryonal kidney cells Human 293 Transformed human primary embryonal kidney cells. The Cell type named 293 is synonymous with HEK-293. physical association physical association 1E To determine whether CP110 and Cep97 interact in vivo, we performed immunoprecipitations with affinity purified CP110 and Cep97 antibodies. Both CP110 and Cep97 were immunoprecipitated by CP110 antibodies, and reciprocal immunoprecipitations with Cep97 antibodies confirmed this physiological interaction(Figure 1E). CP110 has been shown to associate with both CaM and centrin, and we therefore tested the ability of Cep97 to interact with a similar cohort of proteins. Although we readily detected CP110 in centrin immunoprecipitates, as expected (Tsang et al., 2006), we did not observe Cep97. Moreover, Cep97 failed to immunoprecipitate centrin, although CP110 and CaM were detected (Figure 1E, and data not shown), suggesting that CP110 associates with discrete centrin and Cep97 complexes. CP110 was immunoprecipitated by Cep97. It was also detected in the centrin immunoprecipitate. cep97-cp110-3 1 7 unspecified role unspecified role prey prey human-293 Homo sapiens transformed primary embryonal kidney cells Human 293 Transformed human primary embryonal kidney cells. The Cell type named 293 is synonymous with HEK-293. 9 unspecified role unspecified role bait bait human-293 Homo sapiens transformed primary embryonal kidney cells Human 293 Transformed human primary embryonal kidney cells. The Cell type named 293 is synonymous with HEK-293. physical association physical association 1E To determine whether CP110 and Cep97 interact in vivo, we performed immunoprecipitations with affinity purified CP110 and Cep97 antibodies. Both CP110 and Cep97 were immunoprecipitated by CP110 antibodies, and reciprocal immunoprecipitations with Cep97 antibodies confirmed this physiological interaction(Figure 1E). cep97-cp110-4 5 7 unspecified role unspecified role prey prey c-terminal gfp tag green fluorescent protein tag GFP green fluorescent protein GFP green fluorescent protein gfp tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range human-293 Homo sapiens transformed primary embryonal kidney cells Human 293 Transformed human primary embryonal kidney cells. The Cell type named 293 is synonymous with HEK-293. 9 unspecified role unspecified role bait bait region flag tag flag tag FLAG-tagged FLAG DYKDDDDKV epitope tag DYKDDDDKV epitope tag FLAG FLAG-tagged anti tag western anti tag western blot anti tag western undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined n-terminal required to bind necessary binding region required to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain human-293 Homo sapiens transformed primary embryonal kidney cells Human 293 Transformed human primary embryonal kidney cells. The Cell type named 293 is synonymous with HEK-293. physical association physical association 6A, 6B We cotransfected human cells with Cep97-GFP and a series of Flag-tagged CP110 truncation mutants, and performed immunoprecipitations with anti-Flag antibodies. Cep97-GFP was readily detected on Western blots after immunoprecipitation of Flag-CP110, as expected (Figures 6A and 6B). We found that a fragment containing 223 amino-terminal residues was necessary and sufficient to bind Cep97 (Figure 6B). The n-terminal region (AA 1-223) of CP110 was required for interaction with Cep97. cep97-cp110-5 4 7 unspecified role unspecified role bait bait central region required to bind necessary binding region required to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain region flag tag flag tag FLAG-tagged FLAG DYKDDDDKV epitope tag DYKDDDDKV epitope tag FLAG FLAG-tagged anti tag western anti tag western blot anti tag western undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined human-293 Homo sapiens transformed primary embryonal kidney cells Human 293 Transformed human primary embryonal kidney cells. The Cell type named 293 is synonymous with HEK-293. 9 unspecified role unspecified role prey prey human-293 Homo sapiens transformed primary embryonal kidney cells Human 293 Transformed human primary embryonal kidney cells. The Cell type named 293 is synonymous with HEK-293. physical association physical association 6C, 6D Next, we performed the reciprocal experiment to determinethe regions of Cep97 involved in binding CP110. We constructed and transfected a set of Flag-tagged Cep97 amino- and carboxy-terminal truncation mutants and performed anti-Flag immunoprecipitation and Western blotting to detect endogenous CP110. We found that a region of Cep97 containing residues 300-750 was required for stable binding to CP110 (Figures 6C and 6D). In total, our data indicate that an amino-terminal region of CP110 directly interacts with residues in the middle and carboxy-terminal portion of Cep97. A central region containing AA 300-375 within the Cep97 protein was necessary for interaction with CP110. cep97-cam-cep110-1 3 7 unspecified role unspecified role prey prey c-terminal region flag tag flag tag FLAG-tagged FLAG DYKDDDDKV epitope tag DYKDDDDKV epitope tag FLAG FLAG-tagged c-term range C-terminal range c-term range c-term range C-terminal range c-term range human-293 Homo sapiens transformed primary embryonal kidney cells Human 293 Transformed human primary embryonal kidney cells. The Cell type named 293 is synonymous with HEK-293. 8 unspecified role unspecified role prey prey human-293 Homo sapiens transformed primary embryonal kidney cells Human 293 Transformed human primary embryonal kidney cells. The Cell type named 293 is synonymous with HEK-293. 9 unspecified role unspecified role bait bait human-293 Homo sapiens transformed primary embryonal kidney cells Human 293 Transformed human primary embryonal kidney cells. The Cell type named 293 is synonymous with HEK-293. association association S1B, lane 5. We confirmed the interaction between Cep97 and CP110 by ectopically expressing Flag-tagged Cep97 and immunoprecipitating this protein using antibodies against CP110 (Figure S1B). Similarly, anti-Flag antibodies coprecipitated both Cep97 and CP110. In addition, Cep97 associates with CaM, an observation anticipated by the presence of an IQ domain. Cep110 was shown to react with both CAM and Cep97. cep97-cep110-2 2 9 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 human-293 Homo sapiens transformed primary embryonal kidney cells Human 293 Transformed human primary embryonal kidney cells. The Cell type named 293 is synonymous with HEK-293. 7 unspecified role unspecified role prey prey human-293 Homo sapiens transformed primary embryonal kidney cells Human 293 Transformed human primary embryonal kidney cells. The Cell type named 293 is synonymous with HEK-293. physical association physical association Figure 1A. After immunoaffinity purification of Flag-CP110, we eluted the resulting CP110-associated proteins with a Flag peptide, fractionated the eluate by SDS-PAGE (Figure 1A), and excised gel fragments which we subjected to proteolytic digestion and mass spectrometric (MS) sequencing. MS sequencing identified at least five known centrosomal and/or spindle pole proteins, including calmodulin (CaM). cam-cep110-1 3 9 unspecified role unspecified role bait bait human-293 Homo sapiens transformed primary embryonal kidney cells Human 293 Transformed human primary embryonal kidney cells. The Cell type named 293 is synonymous with HEK-293. 8 unspecified role unspecified role prey prey human-293 Homo sapiens transformed primary embryonal kidney cells Human 293 Transformed human primary embryonal kidney cells. The Cell type named 293 is synonymous with HEK-293. physical association physical association S1B, lane 4. We confirmed the interaction between Cep97 and CP110 by ectopically expressing Flag-tagged Cep97 and immunoprecipitating this protein using antibodies against CP110 (Figure S1B). Similarly, anti-Flag antibodies coprecipitated both Cep97 and CP110. In addition, Cep97 associates with CaM, an observation anticipated by the presence of an IQ domain.