Distinct DNA damage determines differential phosphorylation of Chk2
Distinct DNA damage determines differential phosphorylation of Chk2
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DOI：
10.4161/15384047.2014.972823
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发表时间：
2014-01-01
影响因子：
3.6
通讯作者：
Ouchi, Toru
中科院分区：
文献类型：
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作者：
Ouchi, Mutsuko;Ouchi, Toru
检查点激酶 2 (Chk2) 与 DNA 损伤信号传导有关。通过使用 BJ 人成纤维细胞、HCT116 结直肠癌细胞和 HeLa 宫颈癌细胞，我们进一步详细介绍了新抑癌素 (NCS) 或多柔比星 (Dox) 治疗下 Chk2 的磷酸化动力学。 NCS 处理后，Chk2 Thr68 在 3 分钟内发生磷酸化，随后 Ser19 和 Ser33/35 磷酸化。在ATM缺陷的成纤维细胞中，NCS不会诱导NBS1 Ser343和Chk2 Ser19和Ser33/35的磷酸化，但是Chk2 Thr68仍然被磷酸化，表明当用NCS处理时，ATM对于这些残基的磷酸化是必需的。通过使用重新表达磷酸突变体 Chk2 (T68A) 的 Chk2 缺陷型 HCT116 细胞，我们发现在 NCS 处理的细胞中，抑制 Thr68 磷酸化可增强 Ser19 磷酸化。有趣的是，与 NCS 相比，Dox 不会诱导 HeLa 和 HCT116 细胞中 Ser33/35 磷酸化。 Thr68 的磷酸化持续至 3 至 4 小时，Ser19 的磷酸化在 Dox 处理后 70 至 80 分钟发生。这些结果表明 Chk2 参与 DNA 损伤反应的早期阶段。这些残基的差异磷酸化动力学表明 DNA 损伤决定了 Chk2 的分子间和分子内相互作用，这可能调节磷酸化。
Checkpoint kinase 2 (Chk2) has been implicated in DNA damage signaling. By using BJ human fibroblasts, HCT116 colorectal cancer cells and HeLa cervical cancer cells, we further detailed phosphorylation kinetics of Chk2 under treatment with neocarcinostatin (NCS) or doxorubicin (Dox). After NCS treatment, phosphorylation of Chk2 Thr68 occurs in 3min, followed by phosphorylation of Ser19 and Ser33/35. In ATM deficient fibroblasts, NCS does not induce phosphorylation of NBS1 Ser343 and Chk2 Ser19 and Ser33/35, however Chk2 Thr68 is still phosphorylated, indicating that ATM is essential for phosphorylation of these residues when treated with NCS. By using Chk2-deficient HCT116 cells re-expressing phospho-mutant Chk2 (T68A), we found that inhibition of Thr68 phosphorylation enhances Ser19 phosphorylation in NCS treated cells. Interestingly, in contrast to NCS, Dox does not induce Ser33/35 phosphorylation in HeLa and HCT116 cells. Phosphorylation of Thr68 is sustained until 3 to 4hours, and phosphorylation of Ser19 occurs 70 to 80min after Dox treatment. These results demonstrate that Chk2s involved in the early stages of DNA damage response. Differential phosphorylation kinetics of these residues suggests that DNA damage determines intermolecular and intramolecular interaction of Chk2, which may regulate phosphorylation.