K63-specific deubiquitination by two JAMM/MPN+ complexes: BRISC-associated Brcc36 and proteasomal Poh1

Authors

  • Eric M Cooper,

    Corresponding author
    1. Department of Biochemistry and Molecular Biology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD, USA
    • Corresponding authors. Department of Biochemistry and Molecular Biology, Johns Hopkins University, Bloomberg School of Public Health, 615 N. Wolfe St, Baltimore, MD 21205, USA. Tel.: +410 955 2926; Fax: +410 614 3734; E-mail: emcooper@jhsph.edu or E-mail: rcohen@jhsph.edu

    Search for more papers by this author
  • Colleen Cutcliffe,

    1. Department of Biochemistry and Molecular Biology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD, USA
    Search for more papers by this author
    • Present address: Elan Pharmaceuticals, South San Francisco, CA 94080, USA
  • Troels Z Kristiansen,

    1. McKusick-Nathans Institute of Genetic Medicine and Department of Biological Chemistry, Johns Hopkins University, Baltimore, MD, USA
    Search for more papers by this author
  • Akhilesh Pandey,

    1. McKusick-Nathans Institute of Genetic Medicine and Department of Biological Chemistry, Johns Hopkins University, Baltimore, MD, USA
    Search for more papers by this author
  • Cecile M Pickart,

    1. Department of Biochemistry and Molecular Biology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD, USA
    Search for more papers by this author
    • Deceased
  • Robert E Cohen

    Corresponding author
    1. Department of Biochemistry and Molecular Biology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD, USA
    • Corresponding authors. Department of Biochemistry and Molecular Biology, Johns Hopkins University, Bloomberg School of Public Health, 615 N. Wolfe St, Baltimore, MD 21205, USA. Tel.: +410 955 2926; Fax: +410 614 3734; E-mail: emcooper@jhsph.edu or E-mail: rcohen@jhsph.edu

    Search for more papers by this author

Abstract

An unusual deubiquitinating (DUB) activity exists in HeLa cell extracts that is highly specific for cleaving K63-linked but not K48-linked polyubiquitin chains. The activity is insensitive to both N-ethyl-maleimide and ubiquitin aldehyde, indicating that it lacks an active site cysteine residue, and gel filtration experiments show that it resides in a high molecular weight (∼600 kDa) complex. Using a biochemical approach, we found that the K63-specific DUB activity co-fractionated through seven chromatographic steps with three multisubunit complexes: the 19S (PA700) portion of the 26S proteasome, the COP9 signalosome (CSN) and a novel complex that includes the JAMM/MPN+ domain-containing protein Brcc36. When we analysed the individual complexes, we found that the activity was intrinsic to PA700 and the Brcc36 isopeptidase complex (BRISC), but that the CSN-associated activity was due entirely to an interaction with Brcc36. None of the complexes cleave K6, K11, K29, K48 or α-linked polyubiquitin, but they do cleave K63 linkages within mixed-linkage chains. Our results suggest that specificity for K63-linked polyubiquitin is a common property of the JAMM/MPN+ family of DUBs.

Ancillary