Department of Medical Oncology, Dana-Farber Cancer Institute and Department of Medicine, Harvard Medical School, Boston, MA, USA.
Mol Cell Biol. 2012 Oct;32(19):3963-77. doi: 10.1128/MCB.00436-12. Epub 2012 Jul 30.
When explanted into culture, normal human cells exhibit a finite number of cell divisions before entering a proliferative arrest termed replicative senescence. To identify genes essential for entry into replicative senescence, we performed an RNA interference (RNAi)-based loss-of-function screen and found that suppression of the Never in Mitosis Gene A (NIMA)-related protein kinase gene NEK4 disrupted timely entry into senescence. NEK4 suppression extended the number of population doublings required to reach replicative senescence in several human fibroblast strains and resulted in decreased transcription of the cyclin-dependent kinase inhibitor p21. NEK4-suppressed cells displayed impaired cell cycle arrest in response to double-stranded DNA damage, and mass spectrometric analysis of Nek4 immune complexes identified a complex containing DNA-dependent protein kinase catalytic subunit [DNA-PK(cs)], Ku70, and Ku80. NEK4 suppression causes defects in the recruitment of DNA-PK(cs) to DNA upon induction of double-stranded DNA damage, resulting in reduced p53 activation and H2AX phosphorylation. Together, these observations implicate Nek4 as a novel regulator of replicative senescence and the response to double-stranded DNA damage.
在被植入培养物后，正常人类细胞在进入增殖停滞（称为复制性衰老）之前，只会进行有限次数的细胞分裂。为了鉴定进入复制性衰老所必需的基因，我们进行了基于 RNA 干扰（RNAi）的功能丧失筛选，发现抑制丝氨酸/苏氨酸激酶基因 NIMA 相关蛋白激酶 4（NEK4）会破坏衰老的适时进入。抑制 NEK4 会延长达到复制性衰老所需的细胞倍增数，并导致细胞周期蛋白依赖性激酶抑制剂 p21 的转录减少。NEK4 抑制的细胞在响应双链 DNA 损伤时表现出细胞周期阻滞受损，并且 Nek4 免疫复合物的质谱分析鉴定出含有 DNA 依赖性蛋白激酶催化亚基 [DNA-PK(cs)]、Ku70 和 Ku80 的复合物。在诱导双链 DNA 损伤时，抑制 NEK4 会导致 DNA-PK(cs)向 DNA 的募集缺陷，从而导致 p53 激活和 H2AX 磷酸化减少。综上所述，这些观察结果表明 Nek4 是复制性衰老和双链 DNA 损伤反应的新型调节剂。
