BRCA2 Is Ubiquitinated In Vivo and Interacts with USP11, a Deubiquitinating Enzyme That Exhibits Prosurvival Function in the Cellular Response to DNA Damage

Abstract

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.Individuals who carry a germ line mutation in the BRCA2 gene are predisposed to breast, ovarian, and certain other cancers (42,49). Loss of function of the wild-type BRCA2 allele is observed in breast tumors that arise in these individuals (10), defining BRCA2 as a tumor suppressor gene. Additionally, certain biallelic BRCA2 mutations have been linked to the D1 and B subtypes of the cancer susceptibility disorder Fanconi anemia (19). The BRCA2 gene encodes a 3,418-amino-acid nuclear protein (42) with an approximate molecular mass of 460 kDa (24). The BRCA2 protein has been shown to bind to the mammalian homolog of the RecA recombinase, Rad51 (9,26,37,48). Hence, a BRCA2 function in the repair of DNA double-strand breaks through homologous recombination has been proposed (37). In support of this notion, mammalian cells lacking functional BRCA2 are sensitive to DNAdamaging agents (9, 32, 37, 52), show genomic instability (32,45,52), and are deficient in homology-directed DNA repair (28,44). Furthermore, BRCA2 has been shown to possess single-stranded DNA binding ability (51) and can influence Rad51 DNA binding and recombination activities (11). However, the precise role of BRCA2 in homologous recombination and/or DNA repair has not been elucidated.The study of interacting proteins is an important approach toward understanding the biological functions of proteins. Toward this end, we immunopurified a C-terminal region of BRCA2 and subjected copurifying proteins to mass spectrometry analysis. Here we report the identification of a novel BRCA2-interacting protein, USP11, and characterize its role in BRCA2-mediated DNA damage repair.

Effects on proliferation and melanogenesis by inhibition of mutant BRAF and expression of wild‐type INK4A in melanoma cells

“…Western blotting was performed as described. 26 The last lane (-) was control cell lysate from untransfected cells. (c) Endogenous BRAF inhibition by T1796A BRAF RNAi.…”

“…624Mel, 1363Mel and A375 melanoma cells were stably infected with luc-RNAi control (-) or T1796A BRAF RNAi retroviruses (þ) as described. 26 Western blots were probed with BRAF and tubulin antibodies. heterozygous (624Mel) or show LOH (A375) for the T1796A mutation.…”

“…For stable expression of RNAi and INK4A constructs, retroviruses were produced and used to infect melanoma cells, as described. 26 Mass culture was selected and maintained in medium containing 1 mg/ml puromycin (RNAi) or 750 mg/ml G418 (INK4A).…”

“…A 19 bp firefly luc sequence was used as RNAi control, as described. 26 Transient cotransfection experiments in 293T cells were done using Fugene (Roche, Nutley, NJ). Wild-type INK4A cDNA was subcloned into pBabe-neo retroviral vector.…”

Deubiquitinases in cancer: new functions and therapeutic options

“…the cellular response to mitomycin C-induced DNA damage within the BRCA2 pathway (Schoenfeld et al, 2004), and USP28 in the CHK2-p53-PUMA pathway (Zhang et al, 2006a). Finally, recent studies have identified USP47 as the enzyme responsible for the deubiquitylation of the base excision repair DNA polymerase (Polb), thus having an important role in DNA repair regulation and genome integrity maintenance (Parsons et al, 2011).…”