title: Minichromosome Maintenance Complex is Required for Checkpoint Kinase 2 Chromatin Loading and its Phosphorylation to DNA Damage Response in SCC-4 Cells.
abstract: 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.
authors.forename: Liang
authors.initials: L
authors.lastname: Li
authors.forename: Yi
authors.initials: Y
authors.lastname: Feng
authors.affiliation: Department of Stomatology, Ankang City Central Hospital, No. 85, Jinzhou South Road, Hanbin District, Ankang 725000, China.
authors.forename: Rui
authors.initials: R
authors.lastname: Luo
mesh_terms: D002166:Camptothecin / Q000494:pharmacology; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism; D045744:Cell Line, Tumor; D064447:Checkpoint Kinase 2 / Q000235:genetics* / Q000378:metabolism; D002843:Chromatin / Q000737:chemistry* / Q000187:drug effects; D047369:Chromatin Immunoprecipitation; D004249:DNA Damage; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D064111:Minichromosome Maintenance Complex Component 2 / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D064126:Minichromosome Maintenance Complex Component 3 / Q000235:genetics / Q000378:metabolism; D064151:Minichromosome Maintenance Complex Component 6 / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D009061:Mouth Mucosa / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D010766:Phosphorylation / Q000187:drug effects; D010957:Plasmids / Q000737:chemistry / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D059004:Topoisomerase I Inhibitors / Q000494:pharmacology; D014162:Transfection
chemical_list: D018797:Cell Cycle Proteins; D002843:Chromatin; C066470:MCM3 protein, human; C489423:MCM5 protein, human; D034741:RNA, Small Interfering; D059004:Topoisomerase I Inhibitors; D064447:Checkpoint Kinase 2; C578634:CHEK2 protein, human; C084859:MCM2 protein, human; C103258:MCM6 protein, human; D064111:Minichromosome Maintenance Complex Component 2; D064126:Minichromosome Maintenance Complex Component 3; D064151:Minichromosome Maintenance Complex Component 6; D002166:Camptothecin
keywords: Checkpoint kinase 2; chromatin loading; minichromosome maintenance complex; oral squamous cell carcinoma; phosphorylation