INSERM U1016, 75014 Paris, France.
J Biol Chem. 2012 Sep 28;287(40):33812-25. doi: 10.1074/jbc.M112.367839. Epub 2012 Jul 30.
Neutrophils are deprived of proliferative capacity and have a tightly controlled lifespan to avoid their persistence at the site of injury. We have recently described that the proliferating cell nuclear antigen (PCNA), a nuclear factor involved in DNA replication and repair of proliferating cells, is a key regulator of neutrophil survival. In neutrophils, PCNA was localized exclusively in the cytoplasm due to its nuclear-to-cytoplasmic relocalization during granulocytic differentiation. We showed here that leptomycin B, an inhibitor of the chromosome region maintenance 1 (CRM1) exportin, inhibited PCNA relocalization during granulocytic differentiation of HL-60 and NB4 promyelocytic cell lines and of human CD34(+) primary cells. Using enhanced green fluorescent protein fusion constructs, we have demonstrated that PCNA relocalization involved a nuclear export signal (NES) located from Ile-11 to Ile-23 in the PCNA sequence. However, this NES, located at the inner face of the PCNA trimer, was not functional in wild-type PCNA, but instead, was fully active and leptomycin B-sensitive in the monomeric PCNAY114A mutant. To test whether a defect in PCNA cytoplasmic relocalization would affect its antiapoptotic activity in mature neutrophils, a chimeric PCNA fused with the SV40 nuclear localization sequence (NLS) was generated to preclude its cytoplasmic localization. As expected, neutrophil-differentiated PLB985 cells expressing ectopic SV40NLS-PCNA had an increased nuclear PCNA as compared with cells expressing wild-type PCNA. Accordingly, the nuclear PCNA mutant did not show any antiapoptotic activity as compared with wild-type PCNA. Nuclear-to-cytoplasmic relocalization that occurred during myeloid differentiation is essential for PCNA antiapoptotic activity in mature neutrophils and is dependent on the newly identified monomerization-dependent PCNA NES.
中性粒细胞的增殖能力受到剥夺，其寿命受到严格控制，以避免其在损伤部位持续存在。我们最近描述了增殖细胞核抗原（PCNA），一种参与增殖细胞的 DNA 复制和修复的核因子，是中性粒细胞存活的关键调节因子。在中性粒细胞中，由于 PCNA 在粒细胞分化过程中的核质重新分布，其仅定位于细胞质中。我们在这里表明，莱普霉素 B（一种染色体区域维持 1（CRM1）输出蛋白的抑制剂）抑制了 HL-60 和 NB4 早幼粒细胞系以及人 CD34+原代细胞的粒细胞分化过程中的 PCNA 重新分布。使用增强型绿色荧光蛋白融合构建体，我们已经证明 PCNA 重新分布涉及 PCNA 序列中从 Ile-11 到 Ile-23 的核输出信号（NES）。然而，这个位于 PCNA 三聚体内部的 NES 在野生型 PCNA 中没有功能，但在单体 PCNAY114A 突变体中是完全活跃且对莱普霉素 B 敏感的。为了测试 PCNA 细胞质重新分布的缺陷是否会影响其在成熟中性粒细胞中的抗凋亡活性，生成了与 SV40 核定位序列（NLS）融合的嵌合 PCNA，以排除其细胞质定位。正如预期的那样，与表达野生型 PCNA 的细胞相比，表达异位 SV40NLS-PCNA 的 PLB985 细胞分化的中性粒细胞具有更多的核 PCNA。因此，与野生型 PCNA 相比，核 PCNA 突变体没有显示出任何抗凋亡活性。在骨髓分化过程中发生的核质重新分布对于成熟中性粒细胞中 PCNA 的抗凋亡活性是必需的，并且依赖于新鉴定的单体依赖性 PCNA NES。
