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/ CP110 cooperates with two calcium-binding proteins to regulate cytokinesis and genome stability. CP110 cooperates with two calcium-binding proteins to regulate cytokinesis and genome stability. Mol. Biol. Cell (1059-1524) 2006 Tsang WY., Spektor A., Luciano DJ., Indjeian VB., Chen Z., Salisbury JL., Sanchez I., Dynlacht BD. A unique UniProtKB entry could not be associated with human alpha-actinin, beta-spectrin, beta-tubulin or DnaJ/Hsp40 homolog since there are several different types of each protein present within the UniProtKB database and the authors have not been specific enough in order for the exact gene names of interactor to be ascertained. brian.dynlacht@med.nyu.edu 27-Aug-2008: Contacted by JYOTI. A human fetal brain cDNA library was used. in vitro In vitro pull down pull down autoradiography autoradiography Tsang WY., Spektor A., Luciano DJ., Indjeian VB., Chen Z., Salisbury JL., Sanchez I., Dynlacht BD. A unique UniProtKB entry could not be associated with human alpha-actinin, beta-spectrin, beta-tubulin or DnaJ/Hsp40 homolog since there are several different types of each protein present within the UniProtKB database and the authors have not been specific enough in order for the exact gene names of interactor to be ascertained. brian.dynlacht@med.nyu.edu Accepted 2008-SEP-6 by CDEROW. 27-Aug-2008: Contacted by JYOTI. A human fetal brain cDNA library was used. Mol. Biol. Cell (1059-1524) 2006 CP110 cooperates with two calcium-binding proteins to regulate cytokinesis and genome stability. CP110 cooperates with two calcium-binding proteins to regulate cytokinesis and genome stability. Mol. Biol. Cell (1059-1524) 2006 Tsang WY., Spektor A., Luciano DJ., Indjeian VB., Chen Z., Salisbury JL., Sanchez I., Dynlacht BD. A unique UniProtKB entry could not be associated with human alpha-actinin, beta-spectrin, beta-tubulin or DnaJ/Hsp40 homolog since there are several different types of each protein present within the UniProtKB database and the authors have not been specific enough in order for the exact gene names of interactor to be ascertained. brian.dynlacht@med.nyu.edu 27-Aug-2008: Contacted by JYOTI. A human fetal brain cDNA library was used. human-u2-os Human U2-OS osteosarcoma cell line u2-os U2-OS osteocaroma cell line Not in CAPRI and no primary literature reference fluorescence imaging fluorescence microscopy fluorescence imaging immunostaining immunostaining Tsang WY., Spektor A., Luciano DJ., Indjeian VB., Chen Z., Salisbury JL., Sanchez I., Dynlacht BD. A unique UniProtKB entry could not be associated with human alpha-actinin, beta-spectrin, beta-tubulin or DnaJ/Hsp40 homolog since there are several different types of each protein present within the UniProtKB database and the authors have not been specific enough in order for the exact gene names of interactor to be ascertained. brian.dynlacht@med.nyu.edu Accepted 2008-SEP-6 by CDEROW. 27-Aug-2008: Contacted by JYOTI. A human fetal brain cDNA library was used. Mol. Biol. Cell (1059-1524) 2006 CP110 cooperates with two calcium-binding proteins to regulate cytokinesis and genome stability. CP110 cooperates with two calcium-binding proteins to regulate cytokinesis and genome stability. Mol. Biol. Cell (1059-1524) 2006 Tsang WY., Spektor A., Luciano DJ., Indjeian VB., Chen Z., Salisbury JL., Sanchez I., Dynlacht BD. A unique UniProtKB entry could not be associated with human alpha-actinin, beta-spectrin, beta-tubulin or DnaJ/Hsp40 homolog since there are several different types of each protein present within the UniProtKB database and the authors have not been specific enough in order for the exact gene names of interactor to be ascertained. brian.dynlacht@med.nyu.edu 27-Aug-2008: Contacted by JYOTI. A human fetal brain cDNA library was used. 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 Tsang WY., Spektor A., Luciano DJ., Indjeian VB., Chen Z., Salisbury JL., Sanchez I., Dynlacht BD. A unique UniProtKB entry could not be associated with human alpha-actinin, beta-spectrin, beta-tubulin or DnaJ/Hsp40 homolog since there are several different types of each protein present within the UniProtKB database and the authors have not been specific enough in order for the exact gene names of interactor to be ascertained. brian.dynlacht@med.nyu.edu Accepted 2008-SEP-6 by CDEROW. A human fetal brain cDNA library was used. Mol. Biol. Cell (1059-1524) 2006 CP110 cooperates with two calcium-binding proteins to regulate cytokinesis and genome stability. CP110 cooperates with two calcium-binding proteins to regulate cytokinesis and genome stability. Mol. Biol. Cell (1059-1524) 2006 Tsang WY., Spektor A., Luciano DJ., Indjeian VB., Chen Z., Salisbury JL., Sanchez I., Dynlacht BD. A unique UniProtKB entry could not be associated with human alpha-actinin, beta-spectrin, beta-tubulin or DnaJ/Hsp40 homolog since there are several different types of each protein present within the UniProtKB database and the authors have not been specific enough in order for the exact gene names of interactor to be ascertained. brian.dynlacht@med.nyu.edu 27-Aug-2008: Contacted by JYOTI. A human fetal brain cDNA library was used. 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 Tsang WY., Spektor A., Luciano DJ., Indjeian VB., Chen Z., Salisbury JL., Sanchez I., Dynlacht BD. A unique UniProtKB entry could not be associated with human alpha-actinin, beta-spectrin, beta-tubulin or DnaJ/Hsp40 homolog since there are several different types of each protein present within the UniProtKB database and the authors have not been specific enough in order for the exact gene names of interactor to be ascertained. brian.dynlacht@med.nyu.edu Accepted 2008-SEP-6 by CDEROW. 27-Aug-2008: Contacted by JYOTI. A human fetal brain cDNA library was used. Mol. Biol. Cell (1059-1524) 2006 CP110 cooperates with two calcium-binding proteins to regulate cytokinesis and genome stability. CP110 cooperates with two calcium-binding proteins to regulate cytokinesis and genome stability. Mol. Biol. Cell (1059-1524) 2006 Tsang WY., Spektor A., Luciano DJ., Indjeian VB., Chen Z., Salisbury JL., Sanchez I., Dynlacht BD. A unique UniProtKB entry could not be associated with human alpha-actinin, beta-spectrin, beta-tubulin or DnaJ/Hsp40 homolog since there are several different types of each protein present within the UniProtKB database and the authors have not been specific enough in order for the exact gene names of interactor to be ascertained. brian.dynlacht@med.nyu.edu 27-Aug-2008: Contacted by JYOTI. A human fetal brain cDNA library was used. human-u2-os Human U2-OS osteosarcoma cell line u2-os U2-OS osteocaroma cell line Not in CAPRI and no primary literature reference anti tag coip anti tag coimmunoprecipitation anti tag coip western blot western blot Immuno blot Immuno blot Tsang WY., Spektor A., Luciano DJ., Indjeian VB., Chen Z., Salisbury JL., Sanchez I., Dynlacht BD. A unique UniProtKB entry could not be associated with human alpha-actinin, beta-spectrin, beta-tubulin or DnaJ/Hsp40 homolog since there are several different types of each protein present within the UniProtKB database and the authors have not been specific enough in order for the exact gene names of interactor to be ascertained. brian.dynlacht@med.nyu.edu Accepted 2008-SEP-6 by CDEROW. 27-Aug-2008: Contacted by JYOTI. A human fetal brain cDNA library was used. Mol. Biol. Cell (1059-1524) 2006 CP110 cooperates with two calcium-binding proteins to regulate cytokinesis and genome stability. CP110 cooperates with two calcium-binding proteins to regulate cytokinesis and genome stability. Mol. Biol. Cell (1059-1524) 2006 Tsang WY., Spektor A., Luciano DJ., Indjeian VB., Chen Z., Salisbury JL., Sanchez I., Dynlacht BD. A unique UniProtKB entry could not be associated with human alpha-actinin, beta-spectrin, beta-tubulin or DnaJ/Hsp40 homolog since there are several different types of each protein present within the UniProtKB database and the authors have not been specific enough in order for the exact gene names of interactor to be ascertained. brian.dynlacht@med.nyu.edu 27-Aug-2008: Contacted by JYOTI. A human fetal brain cDNA library was used. human-t98g Human caucasian glioblastoma line T98G t98g Human Caucasian glioblastoma T98G coip coimmunoprecipitation co-immunoprecipitation CoIp immunoprecipitation Co-IP co-immunoprecipitation CoIp immunoprecipitation Co-IP coip western blot western blot Immuno blot Immuno blot Tsang WY., Spektor A., Luciano DJ., Indjeian VB., Chen Z., Salisbury JL., Sanchez I., Dynlacht BD. To determine whether CP110 and CaM interact before mitosis, the cells were synchronized by serum deprivation and immunoprecipitated CP110 from extracts of cells synchronously traversing the cell cycle. A unique UniProtKB entry could not be associated with human alpha-actinin, beta-spectrin, beta-tubulin or DnaJ/Hsp40 homolog since there are several different types of each protein present within the UniProtKB database and the authors have not been specific enough in order for the exact gene names of interactor to be ascertained. brian.dynlacht@med.nyu.edu Accepted 2008-SEP-6 by CDEROW. 27-Aug-2008: Contacted by JYOTI. A human fetal brain cDNA library was used. Mol. Biol. Cell (1059-1524) 2006 CP110 cooperates with two calcium-binding proteins to regulate cytokinesis and genome stability. CP110 cooperates with two calcium-binding proteins to regulate cytokinesis and genome stability. Mol. Biol. Cell (1059-1524) 2006 Tsang WY., Spektor A., Luciano DJ., Indjeian VB., Chen Z., Salisbury JL., Sanchez I., Dynlacht BD. A unique UniProtKB entry could not be associated with human alpha-actinin, beta-spectrin, beta-tubulin or DnaJ/Hsp40 homolog since there are several different types of each protein present within the UniProtKB database and the authors have not been specific enough in order for the exact gene names of interactor to be ascertained. brian.dynlacht@med.nyu.edu 27-Aug-2008: Contacted by JYOTI. A human fetal brain cDNA library was used. 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. pull down pull down western blot western blot Immuno blot Immuno blot Tsang WY., Spektor A., Luciano DJ., Indjeian VB., Chen Z., Salisbury JL., Sanchez I., Dynlacht BD. A unique UniProtKB entry could not be associated with human alpha-actinin, beta-spectrin, beta-tubulin or DnaJ/Hsp40 homolog since there are several different types of each protein present within the UniProtKB database and the authors have not been specific enough in order for the exact gene names of interactor to be ascertained. brian.dynlacht@med.nyu.edu Accepted 2008-SEP-6 by CDEROW. 27-Aug-2008: Contacted by JYOTI. A human fetal brain cDNA library was used. Mol. Biol. Cell (1059-1524) 2006 myp0_human Myelin protein P0 Myelin protein zero Myelin peripheral protein MPZ protein protein human Homo sapiens Human MAPGAPSSSPSPILAVLLFSSLVLSPAQAIVVYTDREVHGAVGSRVTLHCSFWSSEWVSDDISFTWRYQPEGGRDAISIFHYAKGQPYIDEVGTFKERIQWVGDPRWKDGSIVIHNLDYSDNGTFTCDVKNPPDIVGKTSQVTLYVFEKVPTRYGVVLGAVIGGVLGVVLLLLLLFYVVRYCWLRRQAALQRRLSAMEKGKLHKPGKDASKRGRQTPVLYAMLDHSRSTKAVSEKKAKGLGESRKDKK A93F4744DACB0D5E 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 pnma1_human Paraneoplastic antigen Ma1 PNMA1 MA1 Neuron- and testis-specific protein 1 37 kDa neuronal protein protein protein human Homo sapiens Human MAMTLLEDWCRGMDVNSQRALLVWGIPVNCDEAEIEETLQAAMPQVSYRMLGRMFWREENAKAALLELTGAVDYAAIPREMPGKGGVWKVLFKPPTSDAEFLERLHLFLAREGWTVQDVARVLGFQNPTPTPGPEMPAEMLNYILDNVIQPLVESIWYKRLTLFSGRDIPGPGEETFDPWLEHTNEVLEEWQVSDVEKRRRLMESLRGPAADVIRILKSNNPAITTAECLKALEQVFGSVESSRDAQIKFLNTYQNPGEKLSAYVIRLEPLLQKVVEKGAIDKDNVNQARLEQVIAGANHSGAIRRQLWLTGAGEGPAPNLFQLLVQIREEEAKEEEEEAEATLLQLGLEGHF EB7F5B6AEDA25961 tfp11_human Tuftelin-interacting protein 11 TFIP11 HSPC006 STIP Septin and tuftelin-interacting protein 1 protein protein human Homo sapiens Human MSLSHLYRDGEGRIDDDDDERENFEITDWDLQNEFNPNRQRHWQTKEEATYGVWAERDSDDERPSFGGKRARDYSAPVNFISAGLKKGAAEEAELEDSDDEEKPVKQDDFPKDFGPRKLKTGGNFKPSQKGFAGGTKSFMDFGSWERHTKGIGQKLLQKMGYVPGRGLGKNAQGIINPIEAKQRKGKGAVGAYGSERTTQSMQDFPVVDSEEEAEEEFQKELSQWRKDPSGSKKKPKYSYKTVEELKAKGRISKKLTAPQKELSQVKVIDMTGREQKVYYSYSQISHKHNVPDDGLPLQSQQLPQSGKEAKAPGFALPELEHNLQLLIDLTEQEIIQNDRQLQYERDMVVNLFHELEKMTEVLDHEERVISNLSKVLEMVEECERRMQPDCSNPLTLDECARIFETLQDKYYEEYRMSDRVDLAVAIVYPLMKEYFKEWDPLKDCTYGTEIISKWKSLLENDQLLSHGGQDLSADAFHRLIWEVWMPFVRNIVTQWQPRNCDPMVDFLDSWVHIIPVWILDNILDQLIFPKLQKEVENWNPLTDTVPIHSWIHPWLPLMQARLEPLYSPIRSKLSSALQKWHPSDSSAKLILQPWKDVFTPGSWEAFMVKNIVPKLGMCLGELVINPHQQHMDAFYWVIDWEGMISVSSLVGLLEKHFFPKWLQVLCSWLSNSPNYEEITKWYLGWKSMFSDQVLAHPSVKDKFNEALDIMNRAVSSNVGAYMQPGARENIAYLTHTERRKDFQYEAMQERREAENMAQRGIGVAASSVPMNFKDLIETKAEEHNIVFMPVIGKRHEGKQLYTFGRIVIYIDRGVVFVQGEKTWVPTSLQSLIDMAK F4C44F8411517289 cetn2_human Centrin-2 CETN2 CALT CEN2 Caltractin isoform 1 protein protein human Homo sapiens Human MASNFKKANMASSSQRKRMSPKPELTEEQKQEIREAFDLFDADGTGTIDVKELKVAMRALGFEPKKEEIKKMISEIDKEGTGKMNFGDFLTVMTQKMSEKDTKEEILKAFKLFDDDETGKISFKNLKRVAKELGENLTDEELQEMIDEADRDGDGEVSEQEFLRIMKKTSLY 59CFD706AD7011B5 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 cep110-cam 3 9 unspecified role unspecified role bait bait 13 unspecified role unspecified role prey prey physical association physical association Figure 1A. To understand how CP110 functions at the centrosome and to identify potential upstream and downstream regulators, we performed a stringent yeast two-hybrid screen using full-length CP110 as bait. From ~380,000 transformants, 98 positive clones were identified, and their inserts were sequenced. Remarkably, 82 clones carried cDNAs that encoded CaM (Figure 1A). Several other well- or partially characterized proteins were also identified, but each was isolated once or in some cases a few times (Figure 1A). We focused on CaM because this protein has been previously implicated in centrosome function. cep110-pnma 3 9 unspecified role unspecified role bait bait 10 unspecified role unspecified role prey prey physical association physical association Figure 1A. To understand how CP110 functions at the centrosome and to identify potential upstream and downstream regulators, we performed a stringent yeast two-hybrid screen using full-length CP110 as bait. From ~380,000 transformants, 98 positive clones were identified, and their inserts were sequenced. Remarkably, 82 clones carried cDNAs that encoded CaM (Figure 1A). Several other well- or partially characterized proteins were also identified, but each was isolated once or in some cases a few times (Figure 1A). We focused on CaM because this protein has been previously implicated in centrosome function. cep110-tfip 3 11 unspecified role unspecified role prey prey 9 unspecified role unspecified role bait bait physical association physical association Figure 1A. To understand how CP110 functions at the centrosome and to identify potential upstream and downstream regulators, we performed a stringent yeast two-hybrid screen using full-length CP110 as bait. From ~380,000 transformants, 98 positive clones were identified, and their inserts were sequenced. Remarkably, 82 clones carried cDNAs that encoded CaM (Figure 1A). Several other well- or partially characterized proteins were also identified, but each was isolated once or in some cases a few times (Figure 1A). We focused on CaM because this protein has been previously implicated in centrosome function. cep110-cam-1 4 13 unspecified role unspecified role prey prey 9 unspecified role unspecified role bait bait physical association physical association Figure 1B, 1D, 1E. Immunoprecipitation of CP110 from 293T cell extract and subsequent immunoblot analysis suggested that CP110 and CaM association is robust and specific (Figure 1B). In addition, endogenous CP110 from 293T cell extracts binds specifically to a CaM agarose conjugate under low calcium (Figure 1D). Furthermore, we showed that CP110 and CaM interact in the presence or absence of calcium from extracts supplemented with either EGTA (-Ca2+) or calcium (+Ca2+), suggesting that CP110 may contain both Ca2+-dependent and Ca2+-independent CaM-binding domains (Figure 1E). Endogenous CP110 binds specifically to CaM in the presence or absence of Ca2+ suggesting that CP110 may contain both Ca2+-dependent and Ca2+-independent CaM-binding domains. cep110-cam-2 5 9 unspecified role unspecified role prey prey 13 unspecified role unspecified role bait bait region gfp tag green fluorescent protein tag GFP green fluorescent protein GFP green fluorescent protein gfp tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined physical association physical association Figure 1C. Furthermore, we generated a U2OS cell line stably expressing a GFP-CaM fusion protein to examine whether immunoprecipitates of the epitope-tagged protein contain endogenous CP110. We observed that endogenous CP110 specifically coimmunoprecipitates with recombinant GFP-CaM (Figure 1C). cep110-cam-4 2 9 unspecified role unspecified role neutral component neutral component 13 unspecified role unspecified role neutral component neutral component colocalization colocalization Figure 2A. Because CP110 is a centrosomal protein, we examined whether these proteins colocalize during mitosis. We found that CP110 and a portion of CaM colocalized, substantially overlapping during metaphase, anaphase, and telophase (Figure 2A). CP110 and a portion of CaM colocalized with substantial overlapping during the metaphase, anaphase and telophase stages of mitosis. cep110-cam-5 6 13 unspecified role unspecified role prey prey 9 unspecified role unspecified role bait bait physical association physical association Figure 2B. To determine whether CP110 and CaM interact before mitosis, we synchronized cells by serum deprivation and immunoprecipitated CP110 from extracts of cells synchronously traversing the cell cycle. CP110 and CaM were coimmunoprecipitated from extracts of cells in G, G, S, and G(Figure 2B). Interestingly, although CP110 levels vary during the cell cycle, the amount of immunoprecipitated CaM remains relatively unchanged (Figure 2B), suggesting that the levels of centrosomal CaM associated with CP110 may be constant throughout the cell cycle. CP110 and CaM associate at the centrosome throughout the cell cycle, although the interaction is most easily visualized at the cellular level during mitosis. cep110-cam-7 1 9 unspecified role unspecified role prey prey n-terminus sufficient to bind sufficient binding region sufficient to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain c-terminus sufficient to bind sufficient binding region sufficient to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain n- and c- termini required to bind necessary binding region required to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain certain certain sequence position certain certain certain sequence position certain region required to bind necessary binding region required to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain certain certain sequence position certain certain certain sequence position certain certain certain sequence position certain certain certain sequence position certain 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 13 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 ecoli Escherichia coli (strain K12) direct interaction direct interaction Figure 3A, 3B, 3C, 4A, 4B. These experiments demonstrated that CP110 contains independent amino-terminal (1�223) and carboxy-terminal CaM-binding domains (620 �991) that are Ca2-dependent (Figure 3, B and C, lanes 11, 12 and 20,21), consistent with database predictions. In contrast, the region spanning residues 200 �565 does not associate with CaM irrespective of calcium addition (Figure 3, B and C, lanes 14 and 15). We attempted to refine the identification of CaM-binding domains by deleting smaller regions of CP110. Interestingly, when the predicted CaM-binding domains were deleted singly or in combination, calcium-dependent binding to CaM was essentially abolished (Figure 4, A and B, compare lane 2 to lanes 5 and 8; compare lane 11 to lane 14). We detected a weak, residual interaction between CaM and full-length CP110 or carboxy-terminal fragments when calcium is absent (Figure 3, B and C, lanes 3, 18, and 21, and Figure 4, A and 4, lanes 3 and 18), and this interaction was observed even when the putative CaM-binding domains were deleted (Figure 4, A and B, lanes 9 and 21), suggesting that additional Ca2+-independent CaM binding sites, not revealed by the database, may be present at the carboxy terminus. This experiment showed that the cep110 protein contains independent n-terminal and c-terminal CaM-binding domains (3 CaM-binding domains were predicted by the Calmodulin Target Database at residues 64-82, 781-821 and 909-924) that are Ca2+-dependent and are required for the interaction. Additional Ca2+-independent CaM binding sites, not revealed by the database, may be present at the carboxy terminus as shown when weak residual interaction occurs between CaM and full-length CP110 or carboxy-terminal fragments when calcium is absent and even when the putative CaM-binding domains were all deleted together. To determine whether the interaction between CP110 and CaM is direct or is mediated by other proteins, we incubated in vitro-translated, radiolabeled CP110 with CaM agarose, and we observed robust binding (Figure 3A). No interaction was detected between CaM agarose and control proteins, strongly suggesting that the binding between CaM and CP110 is direct and specific (Figure 3A). cep110-centrin 4 9 unspecified role unspecified role prey prey 12 unspecified role unspecified role bait bait physical association physical association Figure 5C and 5E. We demonstrated that endogenous CP110 and centrin interact in cells by showing that both proteins coimmunoprecipated from 293T cell extracts with anti-centrin antibody but not with an irrelevant antibody (Figure 5C). Next, we asked whether centrin associated with CP110 in the very high-molecular-weight range fractions. Immunoprecipitation experiments indicated that CP110 indeed associated with centrin in a subset of the high-molecular-weight fractions in which the proteins coeluted (Figure 5E, fractions 16-18). Immunoprecipitation experiments indicated that CP110 associated with centrin in a subset of the high-molecular-weight fractions in which the proteins coeluted (fractions 16-18). cep110-centrin-1 7 12 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 ecoli Escherichia coli (strain K12) 9 unspecified role unspecified role prey prey 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. physical association physical association Figure 5D. Moreover, bacterially expressed GST-centrin incubated with 293T extracts associated with endogenous CP110, but not with a noncentrosomal protein, both in the absence (-Ca2+) and presence of calcium (+Ca2+; Figure 5D). cep110-centrin-2 4 12 unspecified role unspecified role bait bait 9 unspecified role unspecified role prey prey region flag tag flag tag FLAG-tagged FLAG DYKDDDDKV epitope tag DYKDDDDKV epitope tag FLAG FLAG-tagged western blot western blot Immuno blot Immuno blot undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined region sufficient to bind sufficient binding region sufficient to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain certain certain sequence position certain certain certain sequence position certain certain certain sequence position certain certain certain sequence position certain physical association physical association Figure 9C. Interestingly, both the wild type and CaM-binding mutant proteins interacted equally well with centrin (Figure 9C), rendering less likely the possibility that the mutant protein fails to interact with CaM owing to global misfolding. Both the full-length CEP110 and mutant CEP110 proteins (missing regions 67-82, 781-820 and 914-933) interacted with endogenous Centrin as detected using anti-centrin antibody. cep110-cam-8 4 13 unspecified role unspecified role prey prey 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. 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 region required to bind necessary binding region required to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain certain certain sequence position certain certain certain sequence position certain certain certain sequence position certain certain certain sequence position certain in vitro In vitro physical association physical association Figure 9D. Flag-1-991 123 exhibited significantly reduced affinity for CaM compared with the wild-type control in coimmunoprecipitation experiments (Figure 9D). Only the full-length CEP110 interacted with endogenous Calmodulin as detected using anti-FLAG antibody suggesting that the regions that were deleted were important for this interaction. The deletion mutant of CEP110 did display some residual binding. cep110-mpz 3 8 unspecified role unspecified role prey prey 9 unspecified role unspecified role bait bait physical association physical association Figure 1A. To understand how CP110 functions at the centrosome and to identify potential upstream and downstream regulators, we performed a stringent yeast two-hybrid screen using full-length CP110 as bait. From ~380,000 transformants, 98 positive clones were identified, and their inserts were sequenced. Remarkably, 82 clones carried cDNAs that encoded CaM (Figure 1A). Several other well- or partially characterized proteins were also identified, but each was isolated once or in some cases a few times (Figure 1A). We focused on CaM because this protein has been previously implicated in centrosome function.