title: ING5 is phosphorylated by CDK2 and controls cell proliferation independently of p53.
abstract: Inhibitor of growth (ING) proteins have multiple functions in the control of cell proliferation, mainly by regulating processes associated with chromatin regulation and gene expression. ING5 has been described to regulate aspects of gene transcription and replication. Moreover deregulation of ING5 is observed in different tumors, potentially functioning as a tumor suppressor. Gene transcription in late G1 and in S phase and replication is regulated by cyclin-dependent kinase 2 (CDK2) in complex with cyclin E or cyclin A. CDK2 complexes phosphorylate and regulate several substrate proteins relevant for overcoming the restriction point and promoting S phase. We have identified ING5 as a novel CDK2 substrate. ING5 is phosphorylated at a single site, threonine 152, by cyclin E/CDK2 and cyclin A/CDK2 in vitro. This site is also phosphorylated in cells in a cell cycle dependent manner, consistent with it being a CDK2 substrate. Furthermore overexpression of cyclin E/CDK2 stimulates while the CDK2 inhibitor p27KIP1 represses phosphorylation at threonine 152. This site is located in a bipartite nuclear localization sequence but its phosphorylation was not sufficient to deregulate the subcellular localization of ING5. Although ING5 interacts with the tumor suppressor p53, we could not establish p53-dependent regulation of cell proliferation by ING5 and by phospho-site mutants. Instead we observed that the knockdown of ING5 resulted in a strong reduction of proliferation in different tumor cell lines, irrespective of the p53 status. This inhibition of proliferation was at least in part due to the induction of apoptosis. In summary we identified a phosphorylation site at threonine 152 of ING5 that is cell cycle regulated and we observed that ING5 is necessary for tumor cell proliferation, without any apparent dependency on the tumor suppressor p53.
authors: Institute of Biochemistry and Molecular Biology, Medical School, RWTH Aachen University, Pauwelsstrasse 30, 52057, Aachen, Germany.
authors: Ulrike
authors: U
authors: Linzen
authors: Institute of Biochemistry and Molecular Biology, Medical School, RWTH Aachen University, Pauwelsstrasse 30, 52057, Aachen, Germany.
authors: Richard
authors: R
authors: Lilischkis
authors: Institute of Biochemistry and Molecular Biology, Medical School, RWTH Aachen University, Pauwelsstrasse 30, 52057, Aachen, Germany.
authors: Ruwin
authors: R
authors: Pandithage
authors: Institute of Biochemistry and Molecular Biology, Medical School, RWTH Aachen University, Pauwelsstrasse 30, 52057, Aachen, Germany.
authors: Britta
authors: B
authors: Schilling
authors: Institute of Biochemistry and Molecular Biology, Medical School, RWTH Aachen University, Pauwelsstrasse 30, 52057, Aachen, Germany.
authors: Andrea
authors: A
authors: Ullius
authors: Institute of Biochemistry and Molecular Biology, Medical School, RWTH Aachen University, Pauwelsstrasse 30, 52057, Aachen, Germany.
authors: Juliane
authors: J
authors: Lüscher-Firzlaff
authors: Helmholtz Zentrum München, Institute of Molecular Immunology, Marchioninistrasse 25, 81377, München, Germany.
authors: Elisabeth
authors: E
authors: Kremmer
authors: Institute of Biochemistry and Molecular Biology, Medical School, RWTH Aachen University, Pauwelsstrasse 30, 52057, Aachen, Germany.
authors: Bernhard
authors: B
authors: Lüscher
authors: Institute of Biochemistry and Molecular Biology, Medical School, RWTH Aachen University, Pauwelsstrasse 30, 52057, Aachen, Germany.
authors: Jörg
authors: J
authors: Vervoorts
mesh_terms: D019943:Amino Acid Substitution; D001665:Binding Sites; D002460:Cell Line; D049109:Cell Proliferation / Q000502:physiology; D019925:Cyclin A / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D019927:Cyclin E / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D051357:Cyclin-Dependent Kinase 2 / Q000378:metabolism*; D055785:Gene Knockdown Techniques; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D013912:Threonine / Q000737:chemistry; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism*; D025521:Tumor Suppressor Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D019925:Cyclin A; D019927:Cyclin E; C508131:ING5 protein, human; D011994:Recombinant Proteins; C495901:TP53 protein, human; D014157:Transcription Factors; D016159:Tumor Suppressor Protein p53; D025521:Tumor Suppressor Proteins; D013912:Threonine; C495894:CDK2 protein, human; D051357:Cyclin-Dependent Kinase 2