Regulation of BRCA1 by Phosphorylation

This concept award aimed to identify endogenous BRCAl phosphorylation sites using a new technology based on mass spectrometry as a prelude for the analysis of regulation of BRCAI by phosphorylation. We made great effort to isolate sufficient amount of the BRCAl protein from cycling HeLa cells so that we can analyze phosphorylation of the protein and identify the exact phosphorylation sites by mass spectrometry. BRCAl is so scarce that we can only purify 200 ng of the protein from 50 L HeLa cells. Despite of the power of mass spectrometry, this amount of protein (1 pmol) only allowed us to identify a single phosphorylation site by mass spectrometry each time. We managed to identify one in vivo phosphorylation site as Si 189. Si 189 does not confer to phosphorylation consensus of any Imown BRCAi kinases, suggesting that BRCAl is subject to regulation by a novel BRCAI kinase. Mutagenesis of this site and complementation of the HCC1937 cell lines are underway to assess the functional significance of this phosphorylation site.