Linzen Ulrike, Lilischkis Richard, Pandithage Ruwin, Schilling Britta, Ullius Andrea, Lüscher-Firzlaff Juliane, Kremmer Elisabeth, Lüscher Bernhard, Vervoorts Jörg
Institute of Biochemistry and Molecular Biology, Medical School, RWTH Aachen University, Pauwelsstrasse 30, 52057, Aachen, Germany.
Helmholtz Zentrum München, Institute of Molecular Immunology, Marchioninistrasse 25, 81377, München, Germany.
PLoS One. 2015 Apr 10;10(4):e0123736. doi: 10.1371/journal.pone.0123736. eCollection 2015.
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.
生长抑制因子（ING）蛋白在细胞增殖调控中具有多种功能，主要通过调节与染色质调控和基因表达相关的过程来实现。ING5已被描述为可调节基因转录和复制的多个方面。此外，在不同肿瘤中观察到ING5失调，它可能起着肿瘤抑制因子的作用。细胞周期蛋白依赖性激酶2（CDK2）与细胞周期蛋白E或细胞周期蛋白A形成复合物，可调节G1晚期和S期的基因转录以及复制。CDK2复合物使几种与克服限制点和促进S期相关的底物蛋白磷酸化并进行调节。我们已将ING5鉴定为一种新型的CDK2底物。在体外，ING5在单个位点苏氨酸152处被细胞周期蛋白E/CDK2和细胞周期蛋白A/CDK2磷酸化。该位点在细胞中也以细胞周期依赖性方式被磷酸化，这与它作为CDK2底物一致。此外，细胞周期蛋白E/CDK2的过表达会刺激苏氨酸152处的磷酸化，而CDK2抑制剂p27KIP1则会抑制该位点的磷酸化。该位点位于一个双分型核定位序列中，但其磷酸化不足以改变ING5的亚细胞定位。尽管ING5与肿瘤抑制因子p53相互作用，但我们无法确定ING5及其磷酸化位点突变体对细胞增殖的p53依赖性调控。相反，我们观察到敲低ING5会导致不同肿瘤细胞系中的增殖显著降低，而与p53状态无关。这种增殖抑制至少部分是由于凋亡的诱导。总之，我们鉴定出ING5苏氨酸152处的一个磷酸化位点，该位点受细胞周期调控，并且我们观察到ING5是肿瘤细胞增殖所必需的，且对肿瘤抑制因子p53没有明显依赖性。
