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.