title: K63-specific deubiquitination by two JAMM/MPN+ complexes: BRISC-associated Brcc36 and proteasomal Poh1.
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 (approximately 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 alpha-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.
authors.affiliation: Department of Biochemistry and Molecular Biology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD 21205, USA. emcooper@jhsph.edu
authors.forename: Eric M
authors.initials: EM
authors.lastname: Cooper
authors.forename: Colleen
authors.initials: C
authors.lastname: Cutcliffe
authors.forename: Troels Z
authors.initials: TZ
authors.lastname: Kristiansen
authors.forename: Akhilesh
authors.initials: A
authors.lastname: Pandey
authors.forename: Cecile M
authors.initials: CM
authors.lastname: Pickart
authors.forename: Robert E
authors.initials: RE
authors.lastname: Cohen
mesh_terms: D000075686:COP9 Signalosome Complex; D002457:Cell Extracts; D000072017:Deubiquitinating Enzymes; D005033:Ethylmaleimide / Q000494:pharmacology; D006367:HeLa Cells; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism; D008239:Lysine / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism*; D046912:Multiprotein Complexes / Q000302:isolation & purification / Q000378:metabolism*; D010447:Peptide Hydrolases / Q000378:metabolism; D010618:Phenanthrolines / Q000494:pharmacology; D025821:Polyubiquitin / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D011485:Protein Binding / Q000187:drug effects; D013379:Substrate Specificity / Q000187:drug effects; D015534:Trans-Activators / Q000378:metabolism*; D054875:Ubiquitination / Q000187:drug effects
chemical_list: D002457:Cell Extracts; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D046912:Multiprotein Complexes; C085834:PA700 proteasome activator; C109285:PSMD14 protein, human; D010618:Phenanthrolines; D015534:Trans-Activators; D025821:Polyubiquitin; D010447:Peptide Hydrolases; C501488:COPS5 protein, human; C518017:BRCC3 protein, human; D000075686:COP9 Signalosome Complex; D000072017:Deubiquitinating Enzymes; D046988:Proteasome Endopeptidase Complex; D008239:Lysine; D005033:Ethylmaleimide; C025205:1,10-phenanthroline