Ubiquitin biology represents a major mode of cellular regulation. The final step of ubiquitination involves the recognition of a substrate by a ubiquiti ligase. Humans encode over 700 such ligases, significantly exceeding the number of kinases encoded by the genome. Among these ubiquitin ligase genes are many oncogenes and tumor suppressors. In many cases the substrates of these ligases have not been well defined, in part due to a lack of robust technologies for identifying these substrates. We have developed a method, which we call Ligase Trapping which traps the nascent ubiquitinated substrate on the ubiquitin ligase and allows for its co-purification and identification. Here, we will apply this technology to a group of ubiquitin ligase specificity subunits called F box proteins. Human's encode about 80 such proteins, and we will focus on nine that have either been shown to be mutated or mis-expressed in tumors, or have targets whose functions have been linked to tumorigenesis (such as cell cycle regulators and DNA damage response proteins). In addition, we will use this technology to characterize the binding surfaces of these substrate binders. Together, these data will allow the identification of potential drug targets, and, more importantly will allow the generation of inhibitors of the F box proteins themselves in such a way as to affect the turnover of the most relevant substrates.
Many ubiquitin ligases, including F box proteins associated with the SCF, constitute oncogenes and tumor suppressors. While some substrates important for tumorigenesis have been identified, others remain. We will employ a novel method that we have developed to identify ubiquitin ligase substrates to address identify substrates of the SCF and the regulation of their turnover.
