Sang Meixiang, Ando Kiyohiro, Okoshi Rintaro, Koida Nami, Li Yuanyuan, Zhu Yuyan, Shimozato Osamu, Geng Cuizhi, Shan Baoen, Nakagawara Akira, Ozaki Toshinori
Division of Biochemistry, Chiba Cancer Center Research Institute, Chiba, Japan.
Genes Cells. 2009 Jul;14(7):775-88. doi: 10.1111/j.1365-2443.2009.01309.x. Epub 2009 May 28.
Plk3, one of Polo-like kinase family members, is involved in the regulation of cell cycle progression and DNA damage response. In this study, we found that Plk3 inhibits pro-apoptotic activity of p73 through physical interaction and phosphorylation. During cisplatin (CDDP)-mediated apoptosis, Plk3 was transcriptionally induced, whereas its protein level was kept at basal level, suggesting that Plk3 might rapidly degrade in response to CDDP. Immunoprecipitation and in vitro pull-down experiments demonstrated that Plk3 interacts with p73. Luciferase reporter assays and RT-PCR experiments revealed that Plk3 inhibits p73-mediated transcriptional activity. Consistent with these results, pro-apoptotic activity of p73 was blocked by Plk3. Additionally, Plk3 decreased the stability of p73. Intriguingly, kinase-deficient Plk3 failed to inhibit p73 function, indicating that kinase activity of Plk3 is required for Plk3-mediated inhibition of p73. Indeed, in vitro kinase reaction showed that NH(2)-terminal portion of p73 is phosphorylated by Plk3. In accordance with these observations, knocking down of Plk3 increased the stability of p73 and promoted CDDP-mediated apoptosis in association with up-regulation of p73. Collectively, our present findings suggest that Plk3 plays an important role in the regulation of cell fate determination in response to DNA damage through the inhibition of p73.
Polo样激酶家族成员之一的Plk3参与细胞周期进程调控和DNA损伤反应。在本研究中，我们发现Plk3通过物理相互作用和磷酸化抑制p73的促凋亡活性。在顺铂（CDDP）介导的细胞凋亡过程中，Plk3被转录诱导，但其蛋白水平保持在基础水平，这表明Plk3可能在响应CDDP时迅速降解。免疫沉淀和体外下拉实验表明Plk3与p73相互作用。荧光素酶报告基因检测和RT-PCR实验显示Plk3抑制p73介导的转录活性。与这些结果一致，p73的促凋亡活性被Plk3阻断。此外，Plk3降低了p73的稳定性。有趣的是，激酶缺陷型Plk3未能抑制p73功能，表明Plk3的激酶活性是Plk3介导的对p73抑制作用所必需的。事实上，体外激酶反应表明p73的氨基末端部分被Plk3磷酸化。根据这些观察结果，敲低Plk3增加了p73的稳定性，并通过上调p73促进了CDDP介导的细胞凋亡。总的来说，我们目前的研究结果表明Plk3通过抑制p73在响应DNA损伤时的细胞命运决定调控中发挥重要作用。
