DIP Database of Interacting Proteins 611 Young Drive East; Los Angeles CA 90095; USA http://dip.doe-mbi.ucla.edu/ dip@mbi.ucla.edu Functional characterization of human PFTK1 as a cyclin-dependent kinase. Functional characterization of human PFTK1 as a cyclin-dependent kinase. Proc. Natl. Acad. Sci. U.S.A. (0027-8424) 2007 imex curation Shu F., Lv S., Qin Y., Ma X., Wang X., Peng X., Luo Y., Xu BE., Sun X., Wu J. junwu@shanghaigenomics.com 29-OCT-2007: Contacted by JYOTI. 3 human cDNA libraries - (heLa, human T/B lymphocyte and fetal brain. Only protein-protein interactions in vitro In vitro protein kinase assay protein kinase assay predetermined predetermined participant predetermined Shu F., Lv S., Qin Y., Ma X., Wang X., Peng X., Luo Y., Xu BE., Sun X., Wu J. Additional data showing PFTK1 phosphorylated p53 could not be annotated as source of p53 not given in paper. junwu@shanghaigenomics.com Accepted 2007-JUL-12 by JYOTI. 29-OCT-2007: Contacted by JYOTI. 3 human cDNA libraries - (heLa, human T/B lymphocyte and fetal brain. Proc. Natl. Acad. Sci. U.S.A. (0027-8424) 2007 IMEx Functional characterization of human PFTK1 as a cyclin-dependent kinase. Functional characterization of human PFTK1 as a cyclin-dependent kinase. Proc. Natl. Acad. Sci. U.S.A. (0027-8424) 2007 imex curation Shu F., Lv S., Qin Y., Ma X., Wang X., Peng X., Luo Y., Xu BE., Sun X., Wu J. junwu@shanghaigenomics.com 29-OCT-2007: Contacted by JYOTI. 3 human cDNA libraries - (heLa, human T/B lymphocyte and fetal brain. 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 Shu F., Lv S., Qin Y., Ma X., Wang X., Peng X., Luo Y., Xu BE., Sun X., Wu J. junwu@shanghaigenomics.com Accepted 2007-JUL-12 by JYOTI. 3 human cDNA libraries - (heLa, human T/B lymphocyte and fetal brain. Proc. Natl. Acad. Sci. U.S.A. (0027-8424) 2007 IMEx Functional characterization of human PFTK1 as a cyclin-dependent kinase. Functional characterization of human PFTK1 as a cyclin-dependent kinase. Proc. Natl. Acad. Sci. U.S.A. (0027-8424) 2007 imex curation Shu F., Lv S., Qin Y., Ma X., Wang X., Peng X., Luo Y., Xu BE., Sun X., Wu J. junwu@shanghaigenomics.com 29-OCT-2007: Contacted by JYOTI. 3 human cDNA libraries - (heLa, human T/B lymphocyte and fetal brain. 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 predetermined predetermined participant predetermined Shu F., Lv S., Qin Y., Ma X., Wang X., Peng X., Luo Y., Xu BE., Sun X., Wu J. junwu@shanghaigenomics.com Accepted 2007-JUL-12 by JYOTI. 29-OCT-2007: Contacted by JYOTI. 3 human cDNA libraries - (heLa, human T/B lymphocyte and fetal brain. Proc. Natl. Acad. Sci. U.S.A. (0027-8424) 2007 IMEx Functional characterization of human PFTK1 as a cyclin-dependent kinase. Functional characterization of human PFTK1 as a cyclin-dependent kinase. Proc. Natl. Acad. Sci. U.S.A. (0027-8424) 2007 imex curation Shu F., Lv S., Qin Y., Ma X., Wang X., Peng X., Luo Y., Xu BE., Sun X., Wu J. junwu@shanghaigenomics.com 29-OCT-2007: Contacted by JYOTI. 3 human cDNA libraries - (heLa, human T/B lymphocyte and fetal brain. Only protein-protein interactions human-sh_sy5y Homo sapiens SH-SY5Y cell line sh_sy5y Human neuroblastoma cells anti bait coip anti bait coimmunoprecipitation anti bait coip western blot western blot Immuno blot Immuno blot Shu F., Lv S., Qin Y., Ma X., Wang X., Peng X., Luo Y., Xu BE., Sun X., Wu J. junwu@shanghaigenomics.com Accepted 2007-JUL-12 by JYOTI. 29-OCT-2007: Contacted by JYOTI. 3 human cDNA libraries - (heLa, human T/B lymphocyte and fetal brain. Proc. Natl. Acad. Sci. U.S.A. (0027-8424) 2007 IMEx Functional characterization of human PFTK1 as a cyclin-dependent kinase. Functional characterization of human PFTK1 as a cyclin-dependent kinase. Proc. Natl. Acad. Sci. U.S.A. (0027-8424) 2007 imex curation Shu F., Lv S., Qin Y., Ma X., Wang X., Peng X., Luo Y., Xu BE., Sun X., Wu J. junwu@shanghaigenomics.com 29-OCT-2007: Contacted by JYOTI. 3 human cDNA libraries - (heLa, human T/B lymphocyte and fetal brain. Only protein-protein interactions 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 anti tag western anti tag western blot anti tag western Shu F., Lv S., Qin Y., Ma X., Wang X., Peng X., Luo Y., Xu BE., Sun X., Wu J. junwu@shanghaigenomics.com Accepted 2007-JUL-12 by JYOTI. 29-OCT-2007: Contacted by JYOTI. 3 human cDNA libraries - (heLa, human T/B lymphocyte and fetal brain. Proc. Natl. Acad. Sci. U.S.A. (0027-8424) 2007 IMEx Functional characterization of human PFTK1 as a cyclin-dependent kinase. Functional characterization of human PFTK1 as a cyclin-dependent kinase. Proc. Natl. Acad. Sci. U.S.A. (0027-8424) 2007 imex curation Shu F., Lv S., Qin Y., Ma X., Wang X., Peng X., Luo Y., Xu BE., Sun X., Wu J. junwu@shanghaigenomics.com 29-OCT-2007: Contacted by JYOTI. 3 human cDNA libraries - (heLa, human T/B lymphocyte and fetal brain. Only protein-protein interactions 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 tag coip anti tag coimmunoprecipitation anti tag coip western blot western blot Immuno blot Immuno blot Shu F., Lv S., Qin Y., Ma X., Wang X., Peng X., Luo Y., Xu BE., Sun X., Wu J. junwu@shanghaigenomics.com Accepted 2007-JUL-12 by JYOTI. 29-OCT-2007: Contacted by JYOTI. 3 human cDNA libraries - (heLa, human T/B lymphocyte and fetal brain. Proc. Natl. Acad. Sci. U.S.A. (0027-8424) 2007 IMEx cdn2a_human Cyclin-dependent kinase inhibitor 2A CDKN2A CDKN2 Cyclin-dependent kinase 4 inhibitor A p16-INK4a Multiple tumor suppressor 1 MTS1 protein protein human Homo sapiens Human MEPAAGSSMEPSADWLATAAARGRVEEVRALLEAGALPNAPNSYGRRPIQVMMMGSARVAELLLLHGAEPNCADPATLTRPVHDAAREGFLDTLVVLHRAGARLDVRDAWGRLPVDLAEELGHRDVARYLRAAAGGTRGSNHARIDAAEGPSDIPD E59C0E6174B48255 ccnd3_human G1/S-specific cyclin-D3 CCND3 protein protein human Homo sapiens Human MELLCCEGTRHAPRAGPDPRLLGDQRVLQSLLRLEERYVPRASYFQCVQREIKPHMRKMLAYWMLEVCEEQRCEEEVFPLAMNYLDRYLSCVPTRKAQLQLLGAVCMLLASKLRETTPLTIEKLCIYTDHAVSPRQLRDWEVLVLGKLKWDLAAVIAHDFLAFILHRLSLPRDRQALVKKHAQTFLALCATDYTFAMYPPSMIATGSIGAAVQGLGACSMSGDELTELLAGITGTEVDCLRACQEQIEAALRESLREASQTSSSPAPKAPRGSSSQGPSQTSTPTDVTAIHL DIP protein P30281 original sequence version: 150 DIP protein P30281 original sequence version: 111 16E7B1604FEB0029 cdk14_human Cyclin-dependent kinase 14 CDK14 KIAA0834 PFTK1 Serine/threonine-protein kinase PFTAIRE-1 Cell division protein kinase 14 protein protein human Homo sapiens Human MCDLIEPQPAEKIGKMKKLRRTLSESFSRIALKKDDTTFDEICVTKMSTRNCQGMDSVIKPLDTIPEDKKVRVQRTQSTFDPFEKPANQVKRVHSENNACINFKTSSTGKESPKVRRHSSPSSPTSPKFGKADSYEKLEKLGEGSYATVYKGKSKVNGKLVALKVIRLQEEEGTPFTAIREASLLKGLKHANIVLLHDIIHTKETLTLVFEYVHTDLCQYMDKHPGGLHPDNVKLFLFQLLRGLSYIHQRYILHRDLKPQNLLISDTGELKLADFGLARAKSVPSHTYSNEVVTLWYRPPDVLLGSTEYSTCLDMWGVGCIFVEMIQGVAAFPGMKDIQDQLERIFLVLGTPNEDTWPGVHSLPHFKPERFTLYSSKNLRQAWNKLSYVNHAEDLASKLLQCSPKNRLSAQAALSHEYFSDLPPRLWELTDMSSIFTVPNVRLQPEAGESMRAFGKNNSYGKSLSNSKH 32CF9B9B7BD9CE0E ccnd1_human G1/S-specific cyclin-D1 CCND1 BCL1 PRAD1 PRAD1 oncogene BCL-1 oncogene B-cell lymphoma 1 protein protein protein human Homo sapiens Human MEHQLLCCEVETIRRAYPDANLLNDRVLRAMLKAEETCAPSVSYFKCVQKEVLPSMRKIVATWMLEVCEEQKCEEEVFPLAMNYLDRFLSLEPVKKSRLQLLGATCMFVASKMKETIPLTAEKLCIYTDNSIRPEELLQMELLLVNKLKWNLAAMTPHDFIEHFLSKMPEAEENKQIIRKHAQTFVALCATDVKFISNPPSMVAAGSVVAAVQGLNLRSPNNFLSYYRLTRFLSRVIKCDPDCLRACQEQIEALLESSLRQAQQNMDPKAAEEEEEEEEEVDLACTPTDVRDVDI DIP protein P24385 original sequence version: 163 DIP protein P24385 original sequence version: 165 DIP protein P24385 original sequence version: 160 DIP protein P24385 original sequence version: 121 DIP protein P24385 original sequence version: 122 DIP protein P24385 original sequence version: 143 DIP protein P24385 original sequence version: 127 3CC00C9905F58D3A cdk4_human Cyclin-dependent kinase 4 CDK4 Cell division protein kinase 4 PSK-J3 protein protein human Homo sapiens Human MATSRYEPVAEIGVGAYGTVYKARDPHSGHFVALKSVRVPNGGGGGGGLPISTVREVALLRRLEAFEHPNVVRLMDVCATSRTDREIKVTLVFEHVDQDLRTYLDKAPPPGLPAETIKDLMRQFLRGLDFLHANCIVHRDLKPENILVTSGGTVKLADFGLARIYSYQMALTPVVVTLWYRAPEVLLQSTYATPVDMWSVGCIFAEMFRRKPLFCGNSEADQLGKIFDLIGLPPEDDWPRDVSLPRGAFPPRGPRPVQSVVPEMEESGAQLLLEMLTFNPHKRISAFRALQHSYLHKDEGNPE DIP protein P11802 original sequence version: 177 DIP protein P11802 original sequence version: 135 DIP protein P11802 original sequence version: 136 DIP protein P11802 original sequence version: 200 DIP protein P11802 original sequence version: 86 0916A0C07403A33A cdk6_human Cyclin-dependent kinase 6 CDK6 Serine/threonine-protein kinase PLSTIRE Cell division protein kinase 6 CDKN6 protein protein human Homo sapiens Human MEKDGLCRADQQYECVAEIGEGAYGKVFKARDLKNGGRFVALKRVRVQTGEEGMPLSTIREVAVLRHLETFEHPNVVRLFDVCTVSRTDRETKLTLVFEHVDQDLTTYLDKVPEPGVPTETIKDMMFQLLRGLDFLHSHRVVHRDLKPQNILVTSSGQIKLADFGLARIYSFQMALTSVVVTLWYRAPEVLLQSSYATPVDLWSVGCIFAEMFRRKPLFRGSSDVDQLGKILDVIGLPGEEDWPRDVALPRQAFHSKSAQPIEKFVTDIDELGKDLLLKCLTFNPAKRISAYSALSHPYFQDLERCKENLDSHLPPSQNTSELNTA 571733EE6BE7FD4A cdn1a_human Cyclin-dependent kinase inhibitor 1 CDKN1A CAP20 CDKN1 CIP1 MDA6 PIC1 SDI1 WAF1 p21 CDK-interacting protein 1 Melanoma differentiation-associated protein 6 protein protein human Homo sapiens Human MSEPAGDVRQNPCGSKACRRLFGPVDSEQLSRDCDALMAGCIQEARERWNFDFVTETPLEGDFAWERVRGLGLPKLYLPTGPRRGRDELGGGRRPGTSPALLQGTAEEDHVDLSLSCTLVPRSGEQAEGSPGGPGDSQGRKRRQTSMTDFYHSKRRLIFSKRKP DIP protein P38936 original sequence version: 120 DIP protein P38936 original sequence version: 165 DIP protein P38936 original sequence version: 160 98D1E7C519ADFCA9 pftk1-cdkn1a-1 2 9 unspecified role unspecified role bait bait 13 unspecified role unspecified role prey prey physical association physical association Table 1 Yeast two-hybrid screening of PFTK1 protein Protein fragments only encoded but no detail given as to length pftk1-ccnd3-1 2 8 unspecified role unspecified role prey prey 9 unspecified role unspecified role bait bait physical association physical association Table 1 Yeast two-hybrid screening of PFTK1 protein Protein fragments only encoded but no detail given as to length pftk1-cdkn1a-2 3 9 unspecified role unspecified role bait bait 13 unspecified role unspecified role prey prey physical association physical association Fig. 1 Interactions between full-length proteins were confirmed by direct yeast two-hybrid analysis (Fig. 1). pftk1-ccnd3-2 3 8 unspecified role unspecified role prey prey 9 unspecified role unspecified role bait bait physical association physical association Fig. 1 Interactions between full-length proteins were confirmed by direct two-hybrid analysis (Fig. 1). pftk1-cdkn1a-3 5 13 unspecified role unspecified role bait bait 9 unspecified role unspecified role prey prey n-terminal flag tag flag tag FLAG-tagged FLAG DYKDDDDKV epitope tag DYKDDDDKV epitope tag FLAG FLAG-tagged n-term range n-terminal range n-term range n-term range n-terminal range n-term range physical association physical association Fig. 2A To examine the in vivo interaction and physiological relevance between PFTK1 and p21Cip1 identified in the yeast two-hybrid screening, we first conducted semiendogenous coimmunoprecipitation experiments. pftk1-cdkn1a-4 6 9 unspecified role unspecified role bait bait n-terminal flag tag flag tag FLAG-tagged FLAG DYKDDDDKV epitope tag DYKDDDDKV epitope tag FLAG FLAG-tagged n-term range n-terminal range n-term range n-term range n-terminal range n-term range region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain 13 unspecified role unspecified role prey prey region myc tag myc tag EUKLISEED epitope tag EUKLISEED epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined physical association physical association Fig. 2B To examine the in vivo interaction and physiological relevance between PFTK1 and p21Cip1 identified in the yeast two-hybrid screening, we first conducted semiendogenous coimmunoprecipitation experiments. pftk1-cdkn1a-5 6 13 unspecified role unspecified role bait bait n-terminal myc tag myc tag EUKLISEED epitope tag EUKLISEED epitope tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range 9 unspecified role unspecified role prey prey n-terminal flag tag flag tag FLAG-tagged FLAG DYKDDDDKV epitope tag DYKDDDDKV epitope tag FLAG FLAG-tagged n-term range n-terminal range n-term range n-term range n-terminal range n-term range 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 Fig. 2C As shown in Fig. 2C, p21 cCip1 was coimmunoprecipitated with full-length PFTK1 and its D1 derivative pftk1-cdkn1a-6 6 13 unspecified role unspecified role prey prey region sufficient to bind sufficient binding region sufficient to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain n-terminal myc tag myc tag EUKLISEED epitope tag EUKLISEED epitope tag deletion analysis deletion analysis n-term range n-terminal range n-term range n-term range n-terminal range n-term range 9 unspecified role unspecified role bait bait n-terminal flag tag flag tag FLAG-tagged FLAG DYKDDDDKV epitope tag DYKDDDDKV epitope tag FLAG FLAG-tagged n-term range n-terminal range n-term range n-term range n-terminal range n-term range physical association physical association Fig. 2D As shown in Fig. 2d, PFTK1 interacted with D, D2 and N fragments of p21Cip1 cdk4-cdkn2a-2 6 11 unspecified role unspecified role prey prey n-terminal ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range 7 unspecified role unspecified role bait bait n-terminal myc tag myc tag EUKLISEED epitope tag EUKLISEED epitope tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range physical association physical association Fig. 2E On the other hand, another CDK inhibitor p16Ink4a failed to interact with PFTK1 although it associated with its known binding partner CDK4 readily, pftk1-ccnd3-3 6 9 unspecified role unspecified role bait bait n-terminal flag tag flag tag FLAG-tagged FLAG DYKDDDDKV epitope tag DYKDDDDKV epitope tag FLAG FLAG-tagged n-term range n-terminal range n-term range n-term range n-terminal range n-term range 8 unspecified role unspecified role prey prey n-terminal ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range physical association physical association Fig. 2F The association between CCND3 and PFTK1 was relatively weak (Fig. 2f) Weak interaction pftk1-ccnd3-4 6 9 unspecified role unspecified role bait bait n-terminal flag tag flag tag FLAG-tagged FLAG DYKDDDDKV epitope tag DYKDDDDKV epitope tag FLAG FLAG-tagged n-term range n-terminal range n-term range n-term range n-terminal range n-term range 13 unspecified role unspecified role prey prey n-terminal myc tag myc tag EUKLISEED epitope tag EUKLISEED epitope tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range 8 unspecified role unspecified role prey prey n-terminal ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range association association Fig. 2F PFTK1 forms a ternary complex with p21Cip1 and CCND3 pftk1-ccnd3-5 4 8 unspecified role unspecified role prey prey 9 unspecified role unspecified role bait bait physical association physical association Fig. 3C Endogenous interaction between PFTK1 and CCND3 Results confirmed by immunodepletion cdk6-ccdn1-1 4 8 unspecified role unspecified role prey prey 10 unspecified role unspecified role prey prey 12 unspecified role unspecified role bait bait association association In contrast, CDK6 was coimmunoprecipitated with both CCND1 and CCND3 (Fig 3C) Fig 3C pftk1-1 1 9 putative self putative self putative self putative self O94921:p.Lys164Ala mutation mutation mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain K A Wed May 02 16:19:19 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'lys164ala' Tue Jun 30 18:40:57 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Lys164Ala' 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. phosphorylation phosphorylation reaction phosphorylation true An 50 kDa 32P-labelled band corresponding to PFTK1 was observed only when the wild-type protein was immunoprecipitated Fig. 4a Activity enhanced when cells transfected with CCND3 and inhibted when transfected with p21Cip1 prior to immunoprecipitation of kinase