A new role of MEPE/OF45 as a co-factor of CHK1 for DNA damage response

描述（申请人提供）：DNA损伤是人类肿瘤发生的重要事件之一，也是人类癌症治疗的主要目的之一。我们最近的数据首次表明，基质细胞外磷酸糖蛋白/成骨细胞/骨细胞因子45 （MEPE/OF45）作为CHK1的辅助因子，存在于所有可分裂的人类细胞系中（在我们的实验室测试），影响细胞对DNA损伤的反应。然而，自从MEPE/OF45于2000年首次克隆以来，据报道其作为SIBLING（小整合素结合配体n-连接糖蛋白）成员的作用仅限于骨代谢。我们将在本研究中验证我们的假设，即MEPE/OF45通过CHK1通过两种机制影响细胞对DNA损伤的反应：1。

DESCRIPTION (provided by applicant): DNA damage is not only one of the essential events for human tumorigenesis but also one of the major therapeutic aims in human cancer treatment. Our recent data showed for the first time that Matrix extracellular phosphoglycoprotein/osteoblast/osteocyte factor 45 (MEPE/OF45), as a co-factor of CHK1, presents in all dividable human cell lines (tested in our laboratory) to affect cellular response to DNA damage. Since MEPE/OF45 was first cloned in 2000, however, its role as one member of SIBLING (Small Integrin-Binding Ligand N-linked Glycoprotein), has been reported to be limited to bone metabolism. We will test our hypothesis in this study that MEPE/OF45 affects cellular response to DNA damage through CHK1 by two mechanisms: 1. MEPE/OF45 interacts with CHK1 and stabilizes CHK1; 2. MEPE/OF45 undergoes degradation after phosphorylation by CHK1. In this way MEPE/OF45 maintains normal cellular response to DNA damage. Through these two mechanisms, MEPE/OF45 keeps the CHK1 level balanced following DNA damage and thus maintains normal cellular response to DNA damage. Our preliminary data strongly supports our hypothesis. Three specific aims will be performed: A. Determine whether MEPE/OF45 affecting cell response to DNA damage depends on the interaction of MEPE/OF45 with CHK1. B. Determine whether MEPE/OF45 affecting cell response to DNA damage is through protecting CHK1 from the ubiquitin-mediated degradation. C. Determine whether MEPE/OF45 affecting cell response to DNA damage is involved in the phosphorylation of MEPE/OF45 by CHK1. We will combine approaches of GST pull-down, site-directed mutagenesis, immunoprecipitation, kinase assay, ubiquitination, phosphorylation, Mass-Spectrum and crystal structure analysis to identify the interaction site(s) between MEPE/OF45 and CHK1, the ubiquitination site(s) as well as degron of CHK1 and the phosphorylation site(s) of MEPE/OF45 by CHK1. We will examine the effects of these key site(s) and key domain(s) on cellular DNA damage response by observing CHK1 half-life, MEPE/OF45 half-life, survival sensitivity of MEPE/OF45-/- or Chk1 conditional knock out cells transfected with the gene mutated at the key site(s) or key domain(s) to DNA damage. These described studies will elucidate the mechanism by which MEPE/OF45 affects cellular response to DNA damage. We believe that these results will add a functional link between matrix extracellular protein and DNA damage response. Therefore, our results not will only benefit cancer prevention and cancer treatment but will also open a new field for studying how matrix extracellular protein to maintain normal cell function.