丝/苏氨酸蛋白磷酸酶4(Protein Serine/threonine Phosphatase 4, PP4)是蛋白磷酸酶PP2A家族的重要成员之一，在进化过程中具有一定的保守性。PP4催化亚基(PP4C)通过与不同的调节亚基结合形成磷酸酶复合体，参与调节多种细胞信号通路和中心体成熟，剪接体复合物组装以及同源染色体重组在内的重要功能。最近在酵母的研究中发现PP4C复合体的同源物在DNA损伤修复过程中起到重要的作用。但是对于这一过程的具体分子机制以及在其它物种中是否有保守性还不是很清楚。本文利用人类细胞和白色念珠菌两种模式生物对PP4C和其调节亚基在DNA损伤修复过程中的功能进行研究。白色念珠菌是一种多形态的致病真菌，形态之间的转化对其致病性与感染能力非常关键。而最近研究表明DNA损伤、细胞周期紊乱会诱导白色念珠菌极性生长。我们对PP4复合体在白色念珠菌中的同源物CaPph3与CaPsy2在DNA损伤检验点以及形态发生中的功能进行研究。发现Capph3Δ和Capsy2Δ不影响正常生长条件下细胞的形态、生长以及血清诱导菌丝的过程。Capph3Δ和Capsy2Δ对MMS，cisplatin以及UV等DNA损伤试剂处理具有很高的敏感性，表现出更加剧烈的假菌丝生长，细胞周期阻滞现象，侵入性增加。在DNA损伤后的恢复过程，Capph3Δ和Capsy2Δ不能恢复起始的酵母形态，不能重新进入细胞周期。我们发现CaPph3与CaPsy2不参与DNA损伤检验点上游组蛋白γH2AX的去磷酸化调控。CaPph3与CaPsy2参与了DNA损伤检验点下游激酶Rad53的去磷酸化调节，但是不参与HU导致DNA复制异常介导的Rad53的去磷酸化调节。这些结果为白色念珠菌DNA损伤检验点和极性生长之间的分子机制提供了新的线索。在人类细胞中，DNA检验点功能的缺陷或者紊乱导致的遗传不稳定性与细胞癌变密切相关。因此深入研究DNA损伤检验点有助于揭示细胞癌变的分子机制，对癌症的预防和治疗等方面都起到重要的意义。我们利用RNAi技术，以人肺腺癌细胞A549为实验材料，构建PP4C稳定低表达细胞株。广泛应用与临床的抗肿瘤药物cisplatin处理细胞会导致DNA双链断裂，细胞周期阻滞以及细胞凋亡。PP4C稳定抑制的细胞株对cisplatin处理非常敏感，并且这种敏感性有时间和浓度依赖性。PP4C在cisplatin处理后有向细胞核内聚集的趋势。PP4C参与了cisplatin诱导的γH2AX的去磷酸化调节。我们在不同的生物模型中研究蛋白磷酸酶PP4在DNA损伤检验点中的功能。对深入理解PP4C在进化过程中保守重要的功能有一定的意义。

Protein Serine/threonine Phosphatase 4 (PP4) is an important member of Serine/threonine Phosphatase 2A(PP2A) family. It is highly conserved during the process of evolution. Previously studies showed that PP4 complex participated in regulation of several cellular signaling pathways and played an important role in centrosome maturation, splicesome assembly. Recently studies have indicated that the PP2A phosphatase Pph3, the homologue of PP4C, plays an important role in DNA damage response pathway in S. cerevisiae . The ability of the pathogenic fungus Candida albicans to switch cellular morphologies is important for infection and virulence. Recent studies have revealed that C. albicans yeast cells can switch to filamentous growth under genotoxic stress in a manner dependent on the DNA replication/damage checkpoint. To date, the role of PP2A-related phosphatases in DNA damage response has not been studied in C. albicans. Here, we describe the identification and functional characterization of C. albicans PPH3 and PSY2. We found that deletion of PPH3 or PSY2 significantly increased the sensitivity to MMS, cisplatin and UV. Interestingly, after removing the DNA- damaging agents, both pph3Δ and psy2Δ mutants continued to grow in filamentous form and could not reenter the cell cycle, while wild-type cells switched to apparently normal yeast growth. We found that Pph3 and Psy2 are dispensable for the dephosphorylation of γH2AX during DNA damage recovery. In addition, Rad53 dephosphorylation was blocked during recovery from DNA damage in the mutants, suggesting a role for Pph3 and Psy2 in deactivating Rad53. Pph3 and Psy2 may not deactivate the Rad53 in DNA replication checkpoint. These results provide new insights into the molecular mechanisms underlying the deactivation of DNA damage checkpoint and filamentous growth in C. albicans response to genotoxic stresses. Cellular responses to DNA damage contribute one of the most important fields in cancer biology. Cisplatin is potent chemotherapeutic agents currently used in the treatment of many cancers.Stable knockdown of PP4C using shRNA approach in the lung cancer cells A549 also sensitized them to cisplatin treatment. Cisplatin form adducts with DNA that produce intrastrand and interstrand nucleotide crosslinks, caused arrest of the cell cuycle and triggered apoptosis of cells. Treatment with cisplatin led to PP4C became more concentrated in the nucleus.The γH2AX is hyperphosphorylated in stable knockdown of PP4C mutant during the recovery of DNA damage. The studies on PP4C in DNA damage checkpoint in different model organisms is useful for understand the function of PP4.