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/ http://www.ebi.ac.uk/ ahmed-2010-1 SIK2 Is a Centrosome Kinase Required for Bipolar Mitotic Spindle Formation that Provides a Potential Target for Therapy in Ovarian Cancer. in vitro In vitro protein kinase assay protein kinase assay sequence tag sequence tag identification sequence tag Ahmed AA., Lu Z., Jennings NB., Etemadmoghadam D., Capalbo L., Jacamo RO., Barbosa-Morais N., Le XF., null null., Vivas-Mejia P., Lopez-Berestein G., Grandjean G., Bartholomeusz G., Liao W., Andreeff M., Bowtell D., Glover DM., Sood AK., Bast RC. Cancer - Interactions investigated in the context of cancer A kinase assay was performed in the presence of gamma-32P-ATP followed by autoradiography. The bands representing GST-C-Nap1-ct were cut and subjected to LC-MS analysis for protein identification and to detect sites of phosphorylation. Source of SIK2 taken from manufacturers website. Source of NAP1 inferred from sequence length and residue mapping. Assays with gamma-tubulin could not be captured as this could not be ascribed to a single gene product. ahmed.ahmed@obs-gyn.ox.ac.uk Cancer Cell (1535-6108) 2010 Only protein-protein interactions imex curation ahmed-2010-2 SIK2 Is a Centrosome Kinase Required for Bipolar Mitotic Spindle Formation that Provides a Potential Target for Therapy in Ovarian Cancer. 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 Ahmed AA., Lu Z., Jennings NB., Etemadmoghadam D., Capalbo L., Jacamo RO., Barbosa-Morais N., Le XF., null null., Vivas-Mejia P., Lopez-Berestein G., Grandjean G., Bartholomeusz G., Liao W., Andreeff M., Bowtell D., Glover DM., Sood AK., Bast RC. Cancer - Interactions investigated in the context of cancer A kinase assay was performed in the presence of gamma-32P-ATP followed by autoradiography. The bands representing GST-C-Nap1-ct were cut and subjected to LC-MS analysis for protein identification and to detect sites of phosphorylation. Source of SIK2 taken from manufacturers website. Source of NAP1 inferred from sequence length and residue mapping. Assays with gamma-tubulin could not be captured as this could not be ascribed to a single gene product. ahmed.ahmed@obs-gyn.ox.ac.uk Cancer Cell (1535-6108) 2010 Only protein-protein interactions imex curation ahmed-2010-3 SIK2 Is a Centrosome Kinase Required for Bipolar Mitotic Spindle Formation that Provides a Potential Target for Therapy in Ovarian Cancer. 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 Ahmed AA., Lu Z., Jennings NB., Etemadmoghadam D., Capalbo L., Jacamo RO., Barbosa-Morais N., Le XF., null null., Vivas-Mejia P., Lopez-Berestein G., Grandjean G., Bartholomeusz G., Liao W., Andreeff M., Bowtell D., Glover DM., Sood AK., Bast RC. Cancer - Interactions investigated in the context of cancer A kinase assay was performed in the presence of gamma-32P-ATP followed by autoradiography. The bands representing GST-C-Nap1-ct were cut and subjected to LC-MS analysis for protein identification and to detect sites of phosphorylation. Source of SIK2 taken from manufacturers website. Source of NAP1 inferred from sequence length and residue mapping. Assays with gamma-tubulin could not be captured as this could not be ascribed to a single gene product. ahmed.ahmed@obs-gyn.ox.ac.uk Cancer Cell (1535-6108) 2010 Only protein-protein interactions imex curation ahmed-2010-4 SIK2 Is a Centrosome Kinase Required for Bipolar Mitotic Spindle Formation that Provides a Potential Target for Therapy in Ovarian Cancer. human-skov_3 Homo sapiens ovarian adenocarcinoma cell line skov_3 Human ovarian adenocarcinoma cell line Derived from the ascitic fluid from a 64 year old caucasian female with an ovarian tumour. Forms moderately well-differentiated adenocarcinoma consistent with ovarian primary cells. confocal microscopy confocal microscopy immunostaining immunostaining Ahmed AA., Lu Z., Jennings NB., Etemadmoghadam D., Capalbo L., Jacamo RO., Barbosa-Morais N., Le XF., null null., Vivas-Mejia P., Lopez-Berestein G., Grandjean G., Bartholomeusz G., Liao W., Andreeff M., Bowtell D., Glover DM., Sood AK., Bast RC. Cancer - Interactions investigated in the context of cancer A kinase assay was performed in the presence of gamma-32P-ATP followed by autoradiography. The bands representing GST-C-Nap1-ct were cut and subjected to LC-MS analysis for protein identification and to detect sites of phosphorylation. Source of SIK2 taken from manufacturers website. Source of NAP1 inferred from sequence length and residue mapping. Assays with gamma-tubulin could not be captured as this could not be ascribed to a single gene product. ahmed.ahmed@obs-gyn.ox.ac.uk Cancer Cell (1535-6108) 2010 Only protein-protein interactions imex curation ahmed-2010-5 SIK2 Is a Centrosome Kinase Required for Bipolar Mitotic Spindle Formation that Provides a Potential Target for Therapy in Ovarian Cancer. human-hela Homo sapiens epitheloid cervix carcinoma cells Human hela Human epitheloid cervix carcinoma HeLa cells derivation: cervix; epithelial; adenocarcinoma ATCC no:CCL-2 confocal microscopy confocal microscopy tag fluorescence tag visualisation by fluorescence tag fluorescence Ahmed AA., Lu Z., Jennings NB., Etemadmoghadam D., Capalbo L., Jacamo RO., Barbosa-Morais N., Le XF., null null., Vivas-Mejia P., Lopez-Berestein G., Grandjean G., Bartholomeusz G., Liao W., Andreeff M., Bowtell D., Glover DM., Sood AK., Bast RC. Cancer - Interactions investigated in the context of cancer A kinase assay was performed in the presence of gamma-32P-ATP followed by autoradiography. The bands representing GST-C-Nap1-ct were cut and subjected to LC-MS analysis for protein identification and to detect sites of phosphorylation. Source of SIK2 taken from manufacturers website. Source of NAP1 inferred from sequence length and residue mapping. Assays with gamma-tubulin could not be captured as this could not be ascribed to a single gene product. ahmed.ahmed@obs-gyn.ox.ac.uk Cancer Cell (1535-6108) 2010 Only protein-protein interactions imex curation ahmed-2010-6 SIK2 Is a Centrosome Kinase Required for Bipolar Mitotic Spindle Formation that Provides a Potential Target for Therapy in Ovarian Cancer. human-skov_3 Homo sapiens ovarian adenocarcinoma cell line skov_3 Human ovarian adenocarcinoma cell line Derived from the ascitic fluid from a 64 year old caucasian female with an ovarian tumour. Forms moderately well-differentiated adenocarcinoma consistent with ovarian primary cells. confocal microscopy confocal microscopy tag fluorescence tag visualisation by fluorescence tag fluorescence Ahmed AA., Lu Z., Jennings NB., Etemadmoghadam D., Capalbo L., Jacamo RO., Barbosa-Morais N., Le XF., null null., Vivas-Mejia P., Lopez-Berestein G., Grandjean G., Bartholomeusz G., Liao W., Andreeff M., Bowtell D., Glover DM., Sood AK., Bast RC. Cancer - Interactions investigated in the context of cancer A kinase assay was performed in the presence of gamma-32P-ATP followed by autoradiography. The bands representing GST-C-Nap1-ct were cut and subjected to LC-MS analysis for protein identification and to detect sites of phosphorylation. Source of SIK2 taken from manufacturers website. Source of NAP1 inferred from sequence length and residue mapping. Assays with gamma-tubulin could not be captured as this could not be ascribed to a single gene product. ahmed.ahmed@obs-gyn.ox.ac.uk Cancer Cell (1535-6108) 2010 Only protein-protein interactions imex curation cp250_human Centrosome-associated protein CEP250 Centrosomal protein 2 Centrosomal Nek2-associated protein 1 CEP250 CEP2 CNAP1 250 kDa centrosomal protein protein protein human Homo sapiens Human METRSPGLNNMKPQSLQLVLEEQVLALQQQMAENQAASWRKLKNSQEAQQRQATLVRKLQAKVLQYRSWCQELEKRLEATGGPIPQRWENVEEPNLDELLVRLEEEQQRCESLAEVNTQLRLHMEKADVVNKALREDVEKLTVDWSRARDELMRKESQWQMEQEFFKGYLKGEHGRLLSLWREVVTFRRHFLEMKSATDRDLMELKAEHVRLSGSLLTCCLRLTVGAQSREPNGSGRMDGREPAQLLLLLAKTQELEKEAHERSQELIQLKSQGDLEKAELQDRVTELSALLTQSQKQNEDYEKMIKALRETVEILETNHTELMEHEASLSRNAQEEKLSLQQVIKDITQVMVEEGDNIAQGSGHENSLELDSSIFSQFDYQDADKALTLVRSVLTRRRQAVQDLRQQLAGCQEAVNLLQQQHDQWEEEGKALRQRLQKLTGERDTLAGQTVDLQGEVDSLSKERELLQKAREELRQQLEVLEQEAWRLRRVNVELQLQGDSAQGQKEEQQEELHLAVRERERLQEMLMGLEAKQSESLSELITLREALESSHLEGELLRQEQTEVTAALARAEQSIAELSSSENTLKTEVADLRAAAVKLSALNEALALDKVGLNQQLLQLEEENQSVCSRMEAAEQARNALQVDLAEAEKRREALWEKNTHLEAQLQKAEEAGAELQADLRDIQEEKEEIQKKLSESRHQQEAATTQLEQLHQEAKRQEEVLARAVQEKEALVREKAALEVRLQAVERDRQDLAEQLQGLSSAKELLESSLFEAQQQNSVIEVTKGQLEVQIQTVTQAKEVIQGEVRCLKLELDTERSQAEQERDAAARQLAQAEQEGKTALEQQKAAHEKEVNQLREKWEKERSWHQQELAKALESLEREKMELEMRLKEQQTEMEAIQAQREEERTQAESALCQMQLETEKERVSLLETLLQTQKELADASQQLERLRQDMKVQKLKEQETTGILQTQLQEAQRELKEAARQHRDDLAALQEESSSLLQDKMDLQKQVEDLKSQLVAQDDSQRLVEQEVQEKLRETQEYNRIQKELEREKASLTLSLMEKEQRLLVLQEADSIRQQELSALRQDMQEAQGEQKELSAQMELLRQEVKEKEADFLAQEAQLLEELEASHITEQQLRASLWAQEAKAAQLQLRLRSTESQLEALAAEQQPGNQAQAQAQLASLYSALQQALGSVCESRPELSGGGDSAPSVWGLEPDQNGARSLFKRGPLLTALSAEAVASALHKLHQDLWKTQQTRDVLRDQVQKLEERLTDTEAEKSQVHTELQDLQRQLSQNQEEKSKWEGKQNSLESELMELHETMASLQSRLRRAELQRMEAQGERELLQAAKENLTAQVEHLQAAVVEARAQASAAGILEEDLRTARSALKLKNEEVESERERAQALQEQGELKVAQGKALQENLALLTQTLAEREEEVETLRGQIQELEKQREMQKAALELLSLDLKKRNQEVDLQQEQIQELEKCRSVLEHLPMAVQEREQKLTVQREQIRELEKDRETQRNVLEHQLLELEKKDQMIESQRGQVQDLKKQLVTLECLALELEENHHKMECQQKLIKELEGQRETQRVALTHLTLDLEERSQELQAQSSQIHDLESHSTVLARELQERDQEVKSQREQIEELQRQKEHLTQDLERRDQELMLQKERIQVLEDQRTRQTKILEEDLEQIKLSLRERGRELTTQRQLMQERAEEGKGPSKAQRGSLEHMKLILRDKEKEVECQQEHIHELQELKDQLEQQLQGLHRKVGETSLLLSQREQEIVVLQQQLQEAREQGELKEQSLQSQLDEAQRALAQRDQELEALQQEQQQAQGQEERVKEKADALQGALEQAHMTLKERHGELQDHKEQARRLEEELAVEGRRVQALEEVLGDLRAESREQEKALLALQQQCAEQAQEHEVETRALQDSWLQAQAVLKERDQELEALRAESQSSRHQEEAARARAEALQEALGKAHAALQGKEQHLLEQAELSRSLEASTATLQASLDACQAHSRQLEEALRIQEGEIQDQDLRYQEDVQQLQQALAQRDEELRHQQEREQLLEKSLAQRVQENMIQEKQNLGQEREEEEIRGLHQSVRELQLTLAQKEQEILELRETQQRNNLEALPHSHKTSPMEEQSLKLDSLEPRLQRELERLQAALRQTEAREIEWREKAQDLALSLAQTKASVSSLQEVAMFLQASVLERDSEQQRLQDELELTRRALEKERLHSPGATSTAELGSRGEQGVQLGEVSGVEAEPSPDGMEKQSWRQRLEHLQQAVARLEIDRSRLQRHNVQLRSTLEQVERERRKLKREAMRAAQAGSLEISKATASSPTQQDGRGQKNSDAKCVAELQKEVVLLQAQLTLERKQKQDYITRSAQTSRELAGLHHSLSHSLLAVAQAPEATVLEAETRRLDESLTQSLTSPGPVLLHPSPSTTQAASR sik2_human Serine/threonine-protein kinase SIK2 SIK2 Qin-induced kinase SNF1LK2 KIAA0781 QIK Serine/threonine-protein kinase SNF1-like kinase 2 Salt-inducible kinase 2 protein protein human Homo sapiens Human MVMADGPRHLQRGPVRVGFYDIEGTLGKGNFAVVKLGRHRITKTEVAIKIIDKSQLDAVNLEKIYREVQIMKMLDHPHIIKLYQVMETKSMLYLVTEYAKNGEIFDYLANHGRLNESEARRKFWQILSAVDYCHGRKIVHRDLKAENLLLDNNMNIKIADFGFGNFFKSGELLATWCGSPPYAAPEVFEGQQYEGPQLDIWSMGVVLYVLVCGALPFDGPTLPILRQRVLEGRFRIPYFMSEDCEHLIRRMLVLDPSKRLTIAQIKEHKWMLIEVPVQRPVLYPQEQENEPSIGEFNEQVLRLMHSLGIDQQKTIESLQNKSYNHFAAIYFLLVERLKSHRSSFPVEQRLDGRQRRPSTIAEQTVAKAQTVGLPVTMHSPNMRLLRSALLPQASNVEAFSFPASGCQAEAAFMEEECVDTPKVNGCLLDPVPPVLVRKGCQSLPSNMMETSIDEGLETEGEAEEDPAHAFEAFQSTRSGQRRHTLSEVTNQLVVMPGAGKIFSMNDSPSLDSVDSEYDMGSVQRDLNFLEDNPSLKDIMLANQPSPRMTSPFISLRPTNPAMQALSSQKREVHNRSPVSFREGRRASDTSLTQGIVAFRQHLQNLARTKGILELNKVQLLYEQIGPEADPNLAPAAPQLQDLASSCPQEEVSQQQESVSTLPASVHPQLSPRQSLETQYLQHRLQKPSLLSKAQNTCQLYCKEPPRSLEQQLQEHRLQQKRLFLQKQSQLQAYFNQMQIAESSYPQPSQQLPLPRQETPPPSQQAPPFSLTQPLSPVLEPSSEQMQYSPFLSQYQEMQLQPLPSTSGPRAAPPLPTQLQQQQPPPPPPPPPPRQPGAAPAPLQFSYQTCELPSAASPAPDYPTPCQYPVDGAQQSDLTGPDCPRSPGLQEAPSSYDPLALSELPGLFDCEMLDAVDPQHNGYVLVN cetn3_human Centrin-3 CETN3 CEN3 protein protein human Homo sapiens Human MSLALRSELVVDKTKRKKRRELSEEQKQEIKDAFELFDTDKDEAIDYHELKVAMRALGFDVKKADVLKILKDYDREATGKITFEDFNEVVTDWILERDPHEEILKAFKLFDDDDSGKISLRNLRRVARELGENMSDEELRAMIEEFDKDGDGEINQEEFIAIMTGDI sik2-c_nap1 976985 n/a 976991 enzyme target enzyme target substrate substrate neutral component neutral component n terminus 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 false c terminal sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false ecoli Escherichia coli (strain K12) n/a 976992 enzyme enzyme neutral component neutral component n terminus 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 false spofr-sf_9 Spodoptera frugiperda insect cells Fall armyworm sf_9 insect cells Derived from pupal ovarian tissue of the fall armyworm Cloned from the parent cell line, IPLB-SF-21-AE (SF-21) derived from immature ovaries of fall armyworm Spodoptera frugiperda pupae cells are susceptible to Baculovirus infection phosphorylation phosphorylation reaction phosphorylation false false false Fig 4D, 4E. Phosphorylation of enzyme targets; the predominant serine phosphorylation site of C-Nap1 is S2392 at the predicted consensus phosphorylation sequence and to a lesser extent S2394 (LHHSLSHSLL). In addition, a second site (S2234) at the fifth potential coiled coil region of C-Nap1 was confirmed. sik2-c_nap1-1 976986 n/a 976991 unspecified role unspecified role bait bait n terminus protein a tag protein a tag protein A protein A undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false c terminal sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false n/a 976992 unspecified role unspecified role prey prey physical association physical association false false false Fig 4F. sik2-c_nap1-2 976987 n/a 976992 unspecified role unspecified role prey prey n/a 976991 unspecified role unspecified role bait bait region myc tag myc tag EUKLISEED epitope tag EUKLISEED epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false ecoli Escherichia coli (strain K12) physical association physical association false false false Fig 4G. Co-expression of the kinase mutant SIK2 did not result in phosphorylation of the coexpressed C-Nap1. sik2-c_nap-1 976988 n/a 976992 unspecified role unspecified role neutral component neutral component n/a 976991 unspecified role unspecified role neutral component neutral component ecoli Escherichia coli (strain K12) colocalization colocalization false false false Fig 4I. sik2-centrin 976989 n/a 976993 unspecified role unspecified role neutral component neutral component 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 false n/a 976992 unspecified role unspecified role neutral component neutral component colocalization colocalization false false false Fig S3D. sik2-c_nap 976990 n/a 976992 unspecified role unspecified role neutral component neutral component n/a 976991 unspecified role unspecified role neutral component neutral component 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 false ecoli Escherichia coli (strain K12) colocalization colocalization false false false Fig S4I.