Cho Y-C, Park J E, Park B C, Kim J-H, Jeong D G, Park S G, Cho S
College of Pharmacy, Chung-Ang University, Seoul 156-756, Republic of Korea.
Medical Proteomics Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon 305-806, Republic of Korea.
Cell Death Differ. 2015 Oct;22(10):1605-17. doi: 10.1038/cdd.2015.2. Epub 2015 Jan 30.
Cdc25C (cell division cycle 25C) phosphatase triggers entry into mitosis in the cell cycle by dephosphorylating cyclin B-Cdk1. Cdc25C exhibits basal phosphatase activity during interphase and then becomes activated at the G2/M transition after hyperphosphorylation on multiple sites and dissociation from 14-3-3. Although the role of Cdc25C in mitosis has been extensively studied, its function in interphase remains elusive. Here, we show that during interphase Cdc25C suppresses apoptosis signal-regulating kinase 1 (ASK1), a member of mitogen-activated protein (MAP) kinase kinase kinase family that mediates apoptosis. Cdc25C phosphatase dephosphorylates phospho-Thr-838 in the activation loop of ASK1 in vitro and in interphase cells. In addition, knockdown of Cdc25C increases the activity of ASK1 and ASK1 downstream targets in interphase cells, and overexpression of Cdc25C inhibits ASK1-mediated apoptosis, suggesting that Cdc25C binds to and negatively regulates ASK1. Furthermore, we showed that ASK1 kinase activity correlated with Cdc25C activation during mitotic arrest and enhanced ASK1 activity in the presence of activated Cdc25C resulted from the weak association between ASK1 and Cdc25C. In cells synchronized in mitosis following nocodazole treatment, phosphorylation of Thr-838 in the activation loop of ASK1 increased. Compared with hypophosphorylated Cdc25C, which exhibited basal phosphatase activity in interphase, hyperphosphorylated Cdc25C exhibited enhanced phosphatase activity during mitotic arrest, but had significantly reduced affinity to ASK1, suggesting that enhanced ASK1 activity in mitosis was due to reduced binding of hyperphosphorylated Cdc25C to ASK1. These findings suggest that Cdc25C negatively regulates proapoptotic ASK1 in a cell cycle-dependent manner and may play a role in G2/M checkpoint-mediated apoptosis.
细胞分裂周期25C（Cdc25C）磷酸酶通过使细胞周期蛋白B-Cdk1去磷酸化来触发细胞进入有丝分裂。Cdc25C在间期表现出基础磷酸酶活性，然后在多个位点发生超磷酸化并与14-3-3解离后，在G2/M期转换时被激活。尽管Cdc25C在有丝分裂中的作用已得到广泛研究，但其在间期的功能仍不清楚。在此，我们表明在间期Cdc25C抑制凋亡信号调节激酶1（ASK1），ASK1是丝裂原活化蛋白（MAP）激酶激酶激酶家族的成员，介导细胞凋亡。Cdc25C磷酸酶在体外和间期细胞中使ASK1激活环中的磷酸化苏氨酸-838去磷酸化。此外，敲低Cdc25C会增加间期细胞中ASK1及其下游靶点的活性，而过表达Cdc25C会抑制ASK1介导的细胞凋亡，这表明Cdc25C与ASK1结合并对其进行负调控。此外，我们发现ASK1激酶活性与有丝分裂停滞期间Cdc25C的激活相关，并且在存在激活的Cdc25C时ASK1活性增强是由于ASK1与Cdc25C之间的弱结合。在用诺考达唑处理后同步于有丝分裂的细胞中，ASK1激活环中苏氨酸-838的磷酸化增加。与在间期表现出基础磷酸酶活性的低磷酸化Cdc25C相比，超磷酸化的Cdc25C在有丝分裂停滞期间表现出增强的磷酸酶活性，但与ASK1的亲和力显著降低，这表明有丝分裂中ASK1活性增强是由于超磷酸化的Cdc25C与ASK1的结合减少。这些发现表明Cdc25C以细胞周期依赖性方式对促凋亡的ASK1进行负调控，并可能在G2/M检查点介导的细胞凋亡中发挥作用。
