The interaction between ATRIP and MCM complex is essential for ATRIP chromatin loading and its phosphorylation in mantle cell lymphoma cells.
The ATR-interacting protein (ATRIP) is responsible for the recognition of DNA damage-induced structure and regulation of cellular responses to DNA damage and replication stress. The purpose of our study was to identify the underlying mechanism with respect to chromatin loading and phosphorylation of ATRIP in mantle cell lymphoma (MCL).JeKo cells ... were used in our study. Differently tagged ATRIP (Myc-, hemaglutinin (HA) or Flag) and minichromosome maintenance (MCM) complex (MCM2, MCM3, MCM5, and MCM6) were transfected into 293T cells. After 48 h, ATRIP-interacting protein was identified by mass spectrometry (MS). Cell fractionation was done to localize proteins inside the cells. Immunoprecipitation (IP) and immunoblot (IB) analysis were used to identify immunoreactive species, and Glutathione S-transferase (GST) pull-down assays were performed to detect protein-protein interaction between ATRIP and MCM complex. After silencing the expression of MCM2 and MCM6 by short hairpin RNA (shRNA), chromatin fraction were analyzed. The expression of ATRIP phosphorylation (pS224-ATRIP) was determined after application of different doses of MCM2 shRNA (0.5 ?g, 1 ?g, and 2.5 ?g).ATRIP directly interacts with MCM2, MCM3, MCM6, and MCM7 in JeKo cells. Downregulation of MCM2 and MCM6 significantly reduced ATRIP chromatin fraction. Downregulation of MCM2 statistically decreased the expression of ATRIP phosphorylation. The expression levels of pS224-ATRIP were regulated by MCM2 shRNA in a dose-dependent manner.Our results suggest that interaction between ATRIP and MCM complex is required for ATRIP chromatin loading and ATRIP phosphorylation.
Mesh Terms:
Adaptor Proteins, Signal Transducing, Cell Cycle Proteins, Cell Line, Tumor, Chromatin, DNA Damage, DNA-Binding Proteins, Down-Regulation, Gene Expression Regulation, Neoplastic, Gene Silencing, Glutathione Transferase, Humans, Lymphoma, Mantle-Cell, Minichromosome Maintenance Complex Component 2, Minichromosome Maintenance Complex Component 3, Minichromosome Maintenance Complex Component 6, Phosphorylation, RNA, Small Interfering
Adaptor Proteins, Signal Transducing, Cell Cycle Proteins, Cell Line, Tumor, Chromatin, DNA Damage, DNA-Binding Proteins, Down-Regulation, Gene Expression Regulation, Neoplastic, Gene Silencing, Glutathione Transferase, Humans, Lymphoma, Mantle-Cell, Minichromosome Maintenance Complex Component 2, Minichromosome Maintenance Complex Component 3, Minichromosome Maintenance Complex Component 6, Phosphorylation, RNA, Small Interfering
Pharmazie
Date: Nov. 01, 2017
PubMed ID: 29442041
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