Phosphorylation and Subcellular Localization of p27Kip1 Regulated by Hydrogen Peroxide Modulation in Cancer Cells

The Cyclin-dependent kinase inhibitor 1B (p27Kip1) is a key protein in the decision between proliferation and cell cycle exit. Quiescent cells show nuclear p27Kip1, but this protein is exported to the cytoplasm in response to proliferating signals. We recently reported that catalase treatment increases the levels of p27Kip1 in vitro and in vivo in a murine model. In order to characterize and broaden these findings, we evaluated the regulation of p27Kip1 by hydrogen peroxide (H2O2) in human melanoma cells and melanocytes. We observed a high percentage of p27Kip1 positive nuclei in melanoma cells overexpressing or treated with exogenous catalase, while non-treated controls showed a cytoplasmic localization of p27Kip1. Then we studied the levels of p27Kip1 phosphorylated (p27p) at serine 10 (S10) and at threonine 198 (T198) because phosphorylation at these sites enables nuclear exportation of this protein, leading to accumulation and stabilization of p27pT198 in the cytoplasm. We demonstrated by western blot a decrease in p27pS10 and p27pT198 levels in response to H2O2 removal in melanoma cells, associated with nuclear p27Kip1. Melanocytes also exhibited nuclear p27Kip1 and lower levels of p27pS10 and p27pT198 than melanoma cells, which showed cytoplasmic p27Kip1. We also showed that the addition of H2O2 (0.1 µM) to melanoma cells arrested in G1 by serum starvation induces proliferation and increases the levels of p27pS10 and p27pT198 leading to cytoplasmic localization of p27Kip1. Nuclear localization and post-translational modifications of p27Kip1 were also demonstrated by catalase treatment of colorectal carcinoma and neuroblastoma cells, extending our findings to these other human cancer types. In conclusion, we showed in the present work that H2O2 scavenging prevents nuclear exportation of p27Kip1, allowing cell cycle arrest, suggesting that cancer cells take advantage of their intrinsic pro-oxidant state to favor cytoplasmic localization of p27Kip1.

细胞周期蛋白依赖性激酶抑制剂1B（Cyclin-dependent kinase inhibitor 1B，p27Kip1）是调控细胞增殖与细胞周期退出决策过程的关键蛋白。静息细胞中p27Kip1定位于细胞核，但在增殖信号刺激下，该蛋白会被转运至细胞质。本团队近期研究报道，过氧化氢酶处理可在体外及小鼠模型体内提升p27Kip1的表达水平。为进一步表征并拓展上述研究发现，我们针对人黑色素瘤细胞与黑素细胞，探究了过氧化氢（hydrogen peroxide，H₂O₂）对p27Kip1的调控作用。我们观察到，在过表达外源性过氧化氢酶或经外源性过氧化氢酶处理的黑色素瘤细胞中，p27Kip1阳性细胞核占比极高；而未处理的对照组细胞中，p27Kip1则呈细胞质定位。随后，我们研究了丝氨酸10（serine 10，S10）与苏氨酸198（threonine 198，T198）位点磷酸化的p27Kip1（p27p）的表达水平——因该蛋白的上述位点磷酸化可介导其核输出，进而导致细胞质中磷酸化p27pT198的积累与稳定。我们通过蛋白质印迹法（western blot）实验证实，黑色素瘤细胞在去除H₂O₂后，p27pS10与p27pT198的表达水平均出现下调，且该现象与p27Kip1的细胞核定位相关。黑素细胞同样呈现p27Kip1的细胞核定位，且其p27pS10与p27pT198的表达水平低于以p27Kip1呈细胞质定位的黑色素瘤细胞。我们还证实，对经血清饥饿诱导G1期阻滞的黑色素瘤细胞添加0.1 µM的H₂O₂，可诱导细胞增殖，并提升p27pS10与p27pT198的表达水平，进而使p27Kip1发生细胞质定位。通过对结直肠癌与神经母细胞瘤细胞进行过氧化氢酶处理，我们同样证实了p27Kip1的细胞核定位与翻译后修饰，将上述研究发现拓展至了其他两类人类癌症细胞类型。综上，本研究表明，清除H₂O₂可阻止p27Kip1的核输出，从而介导细胞周期阻滞；这提示癌细胞可利用其固有的促氧化状态，促使p27Kip1定位于细胞质。