Li Liang, Feng Yi, Luo Rui
Department of Stomatology, Ankang City Central Hospital, No. 85, Jinzhou South Road, Hanbin District, Ankang 725000, China.
Protein Pept Lett. 2017;24(3):223-228. doi: 10.2174/0929866523666161213094427.
Checkpoint kinase 2 (Chk2) is a significant mediator of diverse responses to DNA damage. The present study was aimed to identify possible interactive proteins of Chk2 and try to clarify the underlying mechanism regarding Chk2 chromatin loading and its phosphorylation to DNA damage response in oral squamous cell carcinoma (OSCC). Differently tagged Chk2 and minichromosome maintenance (MCM) complex (MCM2, MCM3, MCM5, and MCM6) were overexpressed into SCC-4 cells. After 48 h of transfection cell fractionation was performed to localize proteins. In addition, immunoreactive species were detected by immunoprecipitation (IP) and immunoblot (IB) analysis, and protein-protein interaction between Chk2 and MCM complex was ensured by glutathione S-transferase (GST) pull-down assay. Expression of MCM2 and MCM6 was downregulated by small interfering RNA (siRNA), and the chromatin and non-chromatin fraction were analyzed. The expression of Chk2 phosphorylation (pT68-Chk2) was measured after administration of different dosages of siMCM2 (0.5 μg, 1 μg, and 2.5 μg) and camptothecin (CPT). Our results showed that Chk2 directly interacts with MCM2, MCM3, MCM5, and MCM6 in SCC-4 cells. Downregulation of MCM2 and MCM6 markedly reduced Chk2 chromatin fraction, and downregulation of MCM2 decreased the expression of pT68-Chk2 to DNA damage response in a dose manner. Our results suggest that the interaction between Chk2 and MCM complex is required for Chk2 chromatin loading and its phosphorylation to DNA damage response in SCC-4 cells.
检查点激酶2（Chk2）是多种DNA损伤反应的重要介导因子。本研究旨在鉴定Chk2可能的相互作用蛋白，并试图阐明口腔鳞状细胞癌（OSCC）中Chk2染色质加载及其对DNA损伤反应磷酸化的潜在机制。将不同标签的Chk2和微小染色体维持（MCM）复合物（MCM2、MCM3、MCM5和MCM6）过表达至SCC-4细胞中。转染48小时后进行细胞分级分离以定位蛋白。此外，通过免疫沉淀（IP）和免疫印迹（IB）分析检测免疫反应性物种，并通过谷胱甘肽S-转移酶（GST）下拉试验确定Chk2与MCM复合物之间的蛋白质-蛋白质相互作用。用小干扰RNA（siRNA）下调MCM2和MCM6的表达，并分析染色质和非染色质部分。在给予不同剂量的siMCM2（0.5μg、1μg和2.5μg）和喜树碱（CPT）后，测量Chk2磷酸化（pT68-Chk2）的表达。我们的结果表明，Chk2在SCC-4细胞中直接与MCM2、MCM3、MCM5和MCM6相互作用。MCM2和MCM6的下调显著降低了Chk2染色质部分，MCM2的下调以剂量方式降低了pT68-Chk2对DNA损伤反应的表达。我们的结果表明，Chk2与MCM复合物之间的相互作用是SCC-4细胞中Chk2染色质加载及其对DNA损伤反应磷酸化所必需的。
