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J. Biol. Chem., Vol. 279, Issue 6, 3916-3924, February 6, 2004

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Mass Spectrometric and Mutational Analyses Reveal Lys-6-linked Polyubiquitin Chains Catalyzed by BRCA1-BARD1 Ubiquitin Ligase^*

Hiroyuki Nishikawa, Seido Ooka, Ko Sato, Kei Arima, Joji Okamoto, Rachel E. Klevit¶, Mamoru Fukuda, and Tomohiko Ohta||

From the Divisions of Breast and Endocrine Surgery and Immunoregulation, Institute of Medical Science, St. Marianna University School of Medicine, Kawasaki 216-8511, Japan and the ^¶Department of Biochemistry, University of Washington, Seattle, Washington 98195

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.

Received for publication, August 4, 2003 , and in revised form, November 18, 2003.

^* This work was supported by grants from the Japan Society for the Promotion of Science and from the Japanese Ministry of Education, Culture, Sports, Science, and Technology. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^|| To whom correspondence should be addressed. Tel.: 81-44-977-8111; Fax: 81-44-976-5964; E-mail: to{at}marianna-u.ac.jp.

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