Proteomic Approach to Identify Phosphorylation-Dependent Targets of BRCT Domains

摘要

The importance of protein phosphorylation to breast cancer has been reaffirmed by recent findings that the BRCA1 C-terminal (BRCT) domains are novel phosphopeptide binding modules. Cancer-associated missense and deletion mutations have been found in the BRCT repeat regions of BRCA1, suggesting an essential role of BRCT domains in regulating BRCA1 activity. In addition, BRCT domains are found in many proteins that regulate DNA damage repair, cell cycle, and genome stability, implying a more global role of BRCT domains in genome stability surveillance. These results suggest that the BRCT domain acts as a sensor to protein phosphorylation in response to DNA damage, recruits phosphorylated cellular targets, and mediates signaling complex formation. However, the identities of the in vivo BRCT domain targets are largely unknown. We propose to use several approaches utilizing peptide libraries and peptide arrays to systematically identify phosphoproteins that can interact with BRCT domains. In addition to potential new regulators of genome stability, the approaches can identify phosphorylated sequences on proteins that are important for DNA damage responses and cell cycle. These phosphorylated sites can then used to generate phospho-specific antibodies for breast cancer research as well as diagnostic purposes.