Yuan Liangping, Yu Wen-Mei, Qu Cheng-Kui
Department of Hematopoiesis, Jerome H. Holland Laboratory for the Biomedical Sciences, American Red Cross, Rockville, Maryland 20855, USA.
J Biol Chem. 2003 Oct 31;278(44):42812-20. doi: 10.1074/jbc.M305075200. Epub 2003 Aug 22.
DNA damage induced by radiation or DNA-damaging agents leads to apoptosis and cell cycle arrest. However, DNA damage-triggered signal transduction involved in these cellular responses is not well understood. We previously demonstrated an important role for SHP-2, a ubiquitously expressed SH2 domain-containing tyrosine phosphatase, in the DNA damage-induced apoptotic response. Here we report a potential role for SHP-2 in a DNA damage-activated cell cycle checkpoint. Cell cycle analysis and the mitotic index assay showed that following DNA damage induced by cisplatin or gamma-irradiation, the G2 (but not S) arrest response was diminished in SV40 large T antigen-immortalized embryonic fibroblast cells lacking functional SHP-2. Notably, reintroduction of wild-type SHP-2 into the mutant cells fully restored the DNA damage-induced G2 arrest response, suggesting a direct role of SHP-2 in the G2/M checkpoint. Further biochemical analysis revealed that SHP-2 constitutively associated with 14-3-3beta, and that Cdc25C cytoplasmic translocation induced by DNA damage was essentially blocked in SHP-2 mutant cells. Additionally, we showed that following DNA damage, activation of p38 kinase was significantly elevated, while Erk kinase activation was decreased in mutant cells, and treatment of SHP-2 mutant cells with SB203580, a selective inhibitor for p38 kinase, partially restored the DNA damage-induced G2 arrest response. These results together provide the first evidence that SHP-2 tyrosine phosphatase enhances the DNA damage G2/M checkpoint in SV40 large T antigen immortalized murine embryonic fibroblast cells.
辐射或DNA损伤剂诱导的DNA损伤会导致细胞凋亡和细胞周期停滞。然而，参与这些细胞反应的DNA损伤触发的信号转导尚未得到充分理解。我们之前证明了SHP-2（一种普遍表达的含SH2结构域的酪氨酸磷酸酶）在DNA损伤诱导的凋亡反应中起重要作用。在此，我们报告SHP-2在DNA损伤激活的细胞周期检查点中的潜在作用。细胞周期分析和有丝分裂指数测定表明，在用顺铂或γ射线诱导DNA损伤后，在缺乏功能性SHP-2的SV40大T抗原永生化胚胎成纤维细胞中，G2期（而非S期）停滞反应减弱。值得注意的是，将野生型SHP-2重新引入突变细胞中可完全恢复DNA损伤诱导的G2期停滞反应，表明SHP-2在G2/M检查点中起直接作用。进一步的生化分析表明，SHP-2与14-3-3β组成性结合，并且DNA损伤诱导的Cdc25C细胞质转位在SHP-2突变细胞中基本被阻断。此外，我们表明，在DNA损伤后，p38激酶的激活显著升高，而突变细胞中Erk激酶的激活降低，用p38激酶的选择性抑制剂SB203580处理SHP-2突变细胞可部分恢复DNA损伤诱导的G2期停滞反应。这些结果共同提供了首个证据，即SHP-2酪氨酸磷酸酶增强了SV40大T抗原永生化小鼠胚胎成纤维细胞中的DNA损伤G2/M检查点。
