Mass spectrometric and mutational analyses reveal Lys-6-linked polyubiquitin chains catalyzed by BRCA1-BARD1 ubiquitin ligase.

Division of Breast and Endocrine Surgery, Institute of Medical Science, St. Marianna University School of Medicine, Kawasaki 216-8511, Japan.
The breast and ovarian cancer suppressor BRCA1 acquires significant ubiquitin ligase activity when bound to BARD1 as a RING heterodimer. Although the activity may well be important for the role of BRCA1 as a tumor suppressor, the biochemical consequence of the activity is not yet known. Here we report that BRCA1-BARD1 catalyzes Lys-6-linked polyubiquitin chain formation. K6R mutation of ubiquitin dramatically reduces the polyubiquitin products mediated by BRCA1-BARD1 in vitro. BRCA1-BARD1 preferentially utilizes ubiquitin with a single Lys residue at Lys-6 or Lys-29 to mediate autoubiquitination of BRCA1 in vivo. Furthermore, mass spectrometry analysis identified the Lys-6-linked branched ubiquitin fragment from the polyubiquitin chain produced by BRCA1-BARD1 using wild type ubiquitin. The BRCA1-BARD1-mediated Lys-6-linked polyubiquitin chains are deubiquitinated by 26 S proteasome in vitro, whereas autoubiquitinated CUL1 through Lys-48-linked polyubiquitin chains is degraded. Proteasome inhibitors do not alter the steady state level of the autoubiquitinated BRCA1 in vivo. Hence, the results indicate that BRCA1-BARD1 mediates novel polyubiquitin chains that may be distinctly edited by 26 S proteasome from conventional Lys-48-linked polyubiquitin chains.
Mesh Terms:
Amino Acid Sequence, BRCA1 Protein, Carrier Proteins, Cell Line, Humans, Lysine, Mass Spectrometry, Mutagenesis, Site-Directed, Peptide Hydrolases, Polyubiquitin, Proteasome Endopeptidase Complex, Recombinant Proteins, Tumor Suppressor Proteins, Ubiquitin-Protein Ligases
J. Biol. Chem. Feb. 06, 2004; 279(6);3916-24 [PUBMED:14638690]
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