Identification and analysis of BRCA1 ubiquitination targets

The tumor suppressor gene BRCA1 is mutated in 50-90% of hereditary breast and ovarian cancers. This indicates that BRCA1 plays a major role in preventing tumor formation. Despite intense research, the molecular basis for BRCA1`s tumor suppressor function is still unknown. A number of genetic and biochemical findings strongly suggest that the tumor suppressor function of BRCA1 is executed by its ubiquitin-ligase activity. However, the important in vivo ubiquitination targets of BRAC1 remain to be identified and only identification of these targets will help us to understand the molecular pathways that are regulated by BRCA1. Therefore, identification of BRCA1 in vivo ubiquitination targets is currently one of the most important aspects in understanding the tumor suppressor function of BRCA1. This will be crucial for the design of new approaches to breast cancer prevention and therapy as well as to develop effective genetic tests for cancer susceptibility. We propose here a proteome-wide approach to identify BRCA1 ubiquitination targets in vivo. To this end we will use a tandem-affinity purification strategy that has been specifically developed in the host laboratory at the University of California, Irvine for proteome-wide purification of ubiquitinated proteins. We will combine this very effective purification strategy with a quantitative mass spectrometric approach that is based on in vivo incorporation of stable isotope labeled amino acids, to detect quantitative differences in ubiquitination profiles between BRCA1+ and BRCA1- cells. This proposal is focused on the identification of BRCA1 ubiquitination targets. However, development and demonstration of feasibility of the proposed strategy will have a broad impact on many biological and medical disciplines because the strategy can be readily applied to the identification of ubiquitination targets of any ubiquitin ligase.