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.
