Identification of BRCA1 interacting proteins

The breast cancer susceptibility gene, BRCA1, is implicated in multiple cellular processes including DNA repair, the transactivation of genes and maintenance of centrosomes. To further elucidate the function and regulation of BRCA1, I have characterized protein interactions with BRCA1 that were previously identified in a yeast two-hybrid study. Protein Phosphatase 1ß (PP1ß), a serine threonine phosphatase, and the Circadian Clock proteins Per1 and Per2, interact with protein encoded by exon11 of BRCA1 and may, therefore, be involved in the development of breast cancer. A tumour-associated missense mutation in BRCAI abrogates its interaction with both Per1 and Per2. Preliminary analysis of this interaction suggests that BRCA1 interacts with Per2 following treatment of cells with ã-irradiation. Additionally, analysis of the expression levels of 'Per1' and ' 2' in relation to other genes that may be involved in the development of breast cancer indicates that their expression correlates significantly with that of 'BARD1', suggesting that the ubiquitinating role of the 'BRCA1/BARD1' heterodimer may be an important mediator of Per1 and 2 functions. Further characterization of the interaction of BRCA1 with PP1 revealed that BRCA1 also coimmunoprecipitates with PP1Ã, ß or ã. Use of BRCA1-GST fusion proteins identified the region as well as the specific PP1 interacting domain in BRCA1 mediating their interaction. PP1 dephosphorylates BRCA1 in an 'in vitro' phosphatase assay, and colocalization studies indicate that BRCA1, along with PP1Ã or ß, interact at the centrosome during mitosis along with other proteins such as Aurora-A kinase and ã-tubulin. The balance of kinase and phosphatase activity at the centrosome can have important consequences on its function and regulation. Consequently, I analyzed expression levels of the three PP1 isoforms, along with additional genes encoding proteins with centrosomal functions to elucidate their relationship with each other and centrosome amplification in breast tumours. I found a strong correlation between the expression of 'PP1 'Ã and ß with that of ã-tubulin. BRCA1 may regulate the function of PP1 at the centrosome, and loss of this regulation could result in aneuploidy, a common phenotype of breast tumours lacking functional BRCA1., The breast cancer susceptibility gene 'BRCA1' is mutated in many cases of familial breast and ovarian cancer. In order to increase our understanding of the function of this gene, a yeast two-hybrid study was performed to identify proteins that interact with the BRCA1 protein. The two-hybrid screen was performed using a human mammary gland cDNA library and a portion of the BRCA1 protein as bait. The screen yielded a total of 366 colonies, which grew on adenine media. The colonies were subsequently tested for lacZ reporter gene activation, autonomous activation of the reporter genes by the activating domain (AD) protein and for specific interaction by the AD-protein with BRCA1. A total of 16 putative positive interactions were identified, representing 12 distinct interacting proteins. These proteins included Protein Phosphatase 1ß, the proteasome p27 subunit, 2 circadian clock proteins, importin Ã, and several other proteins. Future studies would include further analysis of the protein interactions to characterize the functional significance of the association of these proteins with BRCA1.