Schoenfeld Alan R, Apgar Sarah, Dolios Georgia, Wang Rong, Aaronson Stuart A
Derald H. Ruttenberg Cancer Center, Mount Sinai School of Medicine, One Gustave L. Levy Place, Box 1130, New York, NY 10029, USA.
Mol Cell Biol. 2004 Sep;24(17):7444-55. doi: 10.1128/MCB.24.17.7444-7455.2004.
Individuals carrying a germ line mutation of the breast cancer susceptibility gene BRCA2 are predisposed to breast, ovarian, and other types of cancer. The BRCA2 protein has been proposed to function in the repair of DNA double-strand breaks. Using an immunopurification-mass spectrometry approach to identify novel proteins that associate with the BRCA2 gene product, we found that a deubiquitinating enzyme, USP11, formed specific complexes with BRCA2. Moreover, BRCA2 was constitutively ubiquitinated in vivo in the absence of detectable proteasomal degradation. Mitomycin C (MMC) led to decreased BRCA2 protein levels associated with increased ubiquitination, consistent with proteasome-dependent degradation. While BRCA2 could be deubiquitinated by USP11 in transient overexpression assays, a catalytically inactive USP11 mutant had no effect on BRCA2 ubiquitination or protein levels. Antagonism of USP11 function either through expression of this mutant or through RNA interference increased cellular sensitivity to MMC in a BRCA2-dependent manner. All of these results imply that BRCA2 expression levels are regulated by ubiquitination in the cellular response to MMC-induced DNA damage and that USP11 participates in DNA damage repair functions within the BRCA2 pathway independently of BRCA2 deubiquitination.
携带乳腺癌易感基因BRCA2种系突变的个体易患乳腺癌、卵巢癌和其他类型的癌症。有人提出BRCA2蛋白在DNA双链断裂修复中发挥作用。我们使用免疫纯化-质谱方法来鉴定与BRCA2基因产物相关的新蛋白，发现一种去泛素化酶USP11与BRCA2形成了特异性复合物。此外，在体内，BRCA2在没有可检测到的蛋白酶体降解的情况下持续被泛素化。丝裂霉素C（MMC）导致BRCA2蛋白水平降低，同时泛素化增加，这与蛋白酶体依赖性降解一致。虽然在瞬时过表达实验中BRCA2可以被USP11去泛素化，但催化失活的USP11突变体对BRCA2的泛素化或蛋白水平没有影响。通过表达这种突变体或通过RNA干扰来拮抗USP11功能，会以BRCA2依赖性方式增加细胞对MMC的敏感性。所有这些结果表明，在细胞对MMC诱导的DNA损伤的反应中，BRCA2的表达水平受泛素化调节，并且USP11独立于BRCA2去泛素化参与BRCA2途径中的DNA损伤修复功能。
