Melixetian Marina, Klein Ditte Kjaersgaard, Sørensen Claus Storgaard, Helin Kristian
Department of Experimental Oncology, European Institute of Oncology, Via Adamello 16, 20135, Milan, Italy.
Nat Cell Biol. 2009 Oct;11(10):1247-53. doi: 10.1038/ncb1969. Epub 2009 Sep 6.
DNA damage-induced cell-cycle checkpoints have a critical role in maintaining genomic stability. A key target of the checkpoints is the CDC25A (cell division cycle 25 homologue A) phosphatase, which is essential for the activation of cyclin-dependent kinases and cell-cycle progression. To identify new genes involved in the G2/M checkpoint we performed a large-scale short hairpin RNA (shRNA) library screen. We show that NIMA (never in mitosis gene A)-related kinase 11 (NEK11) is required for DNA damage-induced G2/M arrest. Depletion of NEK11 prevents proteasome-dependent degradation of CDC25A, both in unperturbed and DNA-damaged cells. We show that NEK11 directly phosphorylates CDC25A on residues whose phosphorylation is required for beta-TrCP (beta-transducin repeat-containing protein)-mediated polyubiquitylation and degradation of CDC25A. Furthermore, we demonstrate that CHK1 (checkpoint kinase 1) directly activates NEK11 by phosphorylating it on Ser 273, indicating that CHK1 and NEK11 operate in a single pathway that controls proteolysis of CDC25A. Taken together, these results demonstrate that NEK11 is an important component of the pathway enforcing the G2/M checkpoint, suggesting that genetic mutations in NEK11 may contribute to the development of human cancer.
DNA损伤诱导的细胞周期检查点在维持基因组稳定性方面起着关键作用。这些检查点的一个关键靶点是细胞分裂周期25同源物A（CDC25A）磷酸酶，它对于细胞周期蛋白依赖性激酶的激活和细胞周期进程至关重要。为了鉴定参与G2/M检查点的新基因，我们进行了大规模短发夹RNA（shRNA）文库筛选。我们发现，NIMA（有丝分裂中从不出现基因A）相关激酶11（NEK11）是DNA损伤诱导的G2/M期阻滞所必需的。在未受干扰和DNA损伤的细胞中，NEK11的缺失均会阻止蛋白酶体依赖性的CDC25A降解。我们发现，NEK11直接磷酸化CDC25A上的残基，而这些残基的磷酸化是β-TrCP（含β-转导蛋白重复序列的蛋白）介导的CDC25A多聚泛素化和降解所必需的。此外，我们证明，CHK1（检查点激酶1）通过磷酸化NEK11的Ser 273位点直接激活NEK11，这表明CHK1和NEK11在控制CDC25A蛋白水解的单一途径中发挥作用。综上所述，这些结果表明NEK11是执行G2/M检查点途径的重要组成部分，提示NEK11中的基因突变可能与人类癌症的发生有关。
