Mass spectrometric and mutational analyses reveal Lys-6-linked polyubiquitin chains catalyzed by BRCA1-BARD1 ubiquitin ligase.
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.
Nishikawa
Hiroyuki
H
Division of Breast and Endocrine Surgery, Institute of Medical Science, St. Marianna University School of Medicine, Kawasaki 216-8511, Japan.
Ooka
Seido
S
Sato
Ko
K
Arima
Kei
K
Okamoto
Joji
J
Klevit
Rachel E
RE
Fukuda
Mamoru
M
Ohta
Tomohiko
T
D000595:Amino Acid Sequence; D019313:BRCA1 Protein / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D006801:Humans; D066298:In Vitro Techniques; D008239:Lysine / Q000737:chemistry; D013058:Mass Spectrometry; D016297:Mutagenesis, Site-Directed; D010447:Peptide Hydrolases / Q000378:metabolism; D025821:Polyubiquitin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D046988:Proteasome Endopeptidase Complex; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D025521:Tumor Suppressor Proteins; D044767:Ubiquitin-Protein Ligases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D019313:BRCA1 Protein; D002352:Carrier Proteins; D011994:Recombinant Proteins; D025521:Tumor Suppressor Proteins; D025821:Polyubiquitin; C102449:BARD1 protein, human; D044767:Ubiquitin-Protein Ligases; D010447:Peptide Hydrolases; D046988:Proteasome Endopeptidase Complex; C061553:ATP dependent 26S protease; D008239:Lysine