BTG3, a candidate tumor suppressor, promotes methylation of checkpoint kinase CHK1 B. Sharan Sharma ⁎ Taiwan International Graduate Program (TIGP)-Molecular Medicine, Institute of Biomedical Sciences (IBMS), Academia Sinica, 128 Academia Road, Nankang, Taipei 11529, Taiwan, ROC abstract article info Article history: Received 15 December 2016 Received in revised form 25 March 2017 Accepted 29 March 2017 Available online 30 March 2017 B-cell translocation gene 3 (BTG3), recognized as a member of an antiproliferative B-cell translocation gene/ Transducer of ErbB2 (BTG/Tob) gene family, is a downstream target of p53 and is induced upon genotoxic stress in a p53 and Checkpoint kinase 1 (CHK1), a vital checkpoint kinase which contributes significantly in cell survival and cell cycle checkpoints, dependent manner. Post-translational modifications of CHK1 (phosphorylation and ubiquitination) facilitated by interaction with BTG3 have been observed suggesting their possible role in tumor- igenesis, although the underlying mechanisms are unclear. Methylation, as one of the types of post-translational modifications, is a critical event during cell cycle checkpoint controls and DNA damage repair. Here, for the first time, it is reported that overexpression of BTG3 vividly enhances the methylation of CHK1. Expression of CHK1 was detected in a cancer cell line in this study. This work also reveals the significant role of the kinase domain of CHK1 for BTG3 facilitated methylation as BTG3 overexpression only promoted the methylation of wild type CHK1 but failed to promote the methylation of CHK1 mutants which were impaired for their kinase domain. This novel finding would therefore greatly enhance our understanding of the mechanisms underlying interac- tions between important cancer biomarkers. © 2017 Elsevier Inc. All rights reserved. Keywords: BTG3 overexpression CHK1 methylation Immunoblotting Immunoprecipitation 1. Introduction CHK1 is a ubiquitous serine/threonine kinase, essential to normal cellular functions. It phosphorylates numerous targets, including Cdc25, to allow the cell to progress through cellular division. It is activat- ed predominantly via ATR-mediated phosphorylation but also by ATM in response to DNA damage or inhibition of DNA replication (Abraham, 2001; Shiloh, 2003). Its prevalence in the cell division cycle has led to it being the target for numerous anticancer therapies. Many drugs are being developed that are CHK1 inhibitors that bind to the ATP binding site. CHK1 inhibition is meant to stop the cell in S phase and G2/M to allow DNA damaging chemotherapeutic treatments to work on tumor cells. CHK1 is an evolutionarily conserved protein kinase. In addition to ATR-mediated CHK1 phosphorylation, previous studies have shown that CHK1 is also subjected to other post-translational modification in- cluding mono- and poly-ubiquitination (Puc et al., 2005; Puc & Parsons, 2005; Zhang et al., 2005; Zhang et al., 2009; Cheng et al., 2013). Proteins, which generally are post-translationally methylated at arginine and/or lysine, are involved in a number of different cellular processes, including transcriptional regulation, RNA metabolism and DNA damage repair (Bedford & Clarke, 2009). Several proteins involved in DNA repair (MRE11, p53, DNA polymerase b) have been shown to be regulated by arginine/lysine methylation (Bedford & Clarke, 2009). BTG3 (B-cell translocation gene 3), a candidate tumor suppressor gene, is a member of the antiproliferative BTG/Transducer of ErbB2 gene family and is induced by genotoxic stress in a p53- and CHK1-de- pendent manner (Cheng et al., 2013). BTG4 overexpression suppresses colony formation in colorectal cancer cells and its expression is fre- quently down-regulated in primary gastric cancers (Dong et al., 2009). BTG3 expression also has a suppressive role in cancer progression as ev- idenced from previous studies (Yoneda et al., 2009; Lin et al., 2012). Cheng et al. (2013) reported that BTG3 interacts with CHK1 and regu- lates its phosphorylation and activation. However, BTG3 mediated methylation of CHK1 have not been previously examined. In this study, a possible role in the interaction between BTG3 and CHK1, impor- tant cancer biomarkers, was identified in a cancer cell line. CHK1 was found to interact with and be methylated by BTG3. In addition to other types of post-translational modifications, methylation has an im- portant role to play in maintaining the integrity of the genome. This Gene Reports 7 (2017) 119–122 Abbreviations: BTG3, B-cell translocation gene 3; CHK-1, Checkpoint kinase 1; SDS, sodium dodecyl sulfate; PAGE, PA-gel electrophoresis; DTT, dithiothreitol. ⁎ Gujarat State Biotechnology Mission (GSBTM), Udyog Bhavan, 11th Block, 9th Floor, Sector – 11, Gandhinagar 382 017, Gujarat, India. E-mail address: bssgenetix@gmail.com. http://dx.doi.org/10.1016/j.genrep.2017.03.005 2452-0144/© 2017 Elsevier Inc. All rights reserved. Contents lists available at ScienceDirect Gene Reports journal homepage: www.elsevier.com/locate/genrep