Identification of novel breast cancer-related genes using 2D-DIGE system and their functional analyses

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Analyses for regulation of protein stability of breast tumor suppressor BRCA1 is important to understand the mechanisms underlying breast carcinogenesis. The purposes of the project were as following: 1) Screen of substrates ubiquitinated by BRCA1 or of factors that regulates BRCA1. 2) Functional analyses of HERC2-mediated regulation of BRCA1. 3) Phenotypic analyses of HERC2 transgenic mice. Results: 1) Rabbit polyclonal antibodies to BRCA1 were generated to immunoprecipitate large amounts of BRCA1 complexes. However, I could not identify any novel factors that regulates or were regulated by BRCA1 using the screens. 2) The interaction between endogenous HERC2 and BRCA1 was confirmed by immunoprecipitation followed by immunoblotting. HERC2-BRCA1 interaction in cells maximized in the S phase of the cell cycle, when the BRCA1 expression minimized. Fragment analyses revealed that C-terminus of HERC2 that contains HECT domain interacts with N-terminal degron domain of BRCA1. Endogenous HERC2 mainly localized in the cytoplasm and was imported to the nuclear by the Crm1 inhibitor leptomycin B, suggesting that HERC2 was a cytoplasmic and nuclear shuttle protein. These data suggest that HERC2 is a possible E3 ligase for BRCA1 that targets BRCA1 for degradation in S phase. 3) We obtained 13 clones of transgenic mice and selected mice that expressed mRNA of HERC2 C-terminus in the breast tissue under MMTV induction. However, no significant phenotype was observed.