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.

Figure 1

HeLa cells possess an NEM-resistant, 1,10-o-phenanthroline-sensitive, K63-specific DUB activity. (A) HeLa cell extracts (1% NP40) were treated with NEM (lanes 3 and 6) or left untreated (lanes 2 and 5), and then incubated with ¹²⁵I-K48Ub₄ (lanes 2–3) or ¹²⁵I-K63Ub₄ (lanes 5–6). Input tetramers are shown (lanes 1 and 4). Products were visualised by SDS–PAGE autoradiography. (B) Cell extracts were left untreated (lane 2) or incubated with the indicated phenanthroline compounds +/− NEM (lanes 3–7), then mixed with ¹²⁵I-K63Ub₂. Products were visualised as in (A).

Figure 2

Three JAMM/MPN+ domain-containing complexes fractionate with the K63-specific DUB activity. (A) Scheme for purifying NEM-resistant, K63-specific DUB activity; see Materials and methods for details. (B) Silver-stained gel showing proteins in Superose 6 column fractions that overlap with the peak of K63-specific, NEM-resistant DUB activity. (C) (Upper panel) Fractions from the Superose 6 column were tested for their ability to disassemble ¹²⁵I-K63Ub₄. (Lower panels) An aliquot of each fraction was immunoblotted with antibodies to Poh1, Jab1 and Bre/Brcc45.

Figure 3

Three JAMM-MPN+-containing protein complexes possess K63-specific, NEM-resistant DUB activity. (A) Untreated (lanes 2–3 and 7–8) or NEM-treated (lanes 4–5 and 9–10) PA700 was incubated with ¹²⁵I-K63Ub₂ (lanes 2–5) or unlabelled K48Ub₂ (lanes 7–10) for the indicated times. Products were visualised by autoradiography (lanes 1–5) or by immunoblotting with anti-Ub (lanes 6–10). (B) Anti-Flag immunoprecipitations were performed on lysates from HeLa cells that were untransduced (lanes 4–5), or stably expressing C-terminally Flag and HA-tagged WT (lanes 6–7) or mutant (H122Q) Brcc36 (lanes 8–9); or WT (lanes 10–11) or mutant (H138Q) Jab1 (lanes 12–13). Immunoprecipitates were incubated with ¹²⁵I-K63Ub₄ in the absence (even lanes) or presence (odd lanes) of Ubal. Input (in) ¹²⁵I-K63Ub₄ is shown (lane 1). (C) Brcc36 wild-type (WT) (lanes 2–5 and 13–16), H122Q mutant (Mut) complexes (lanes 7–8), or Isopeptidase T (isoT) (lanes 10–11) were left untreated (lanes 2–3, 7–8, 10 and 13–14) or incubated with NEM (lanes 4–5, 11 and 15–16), and then mixed with either ²⁵I-K63Ub₂ (lanes 2–8) or unlabelled K48Ub₂ (lanes 10–11 and 13–16) for the indicated times. Reaction products were visualised either by autoradiography (lanes 1–8) or by immunoblotting with anti-Ub (lanes 9–16).

Figure 4

PA700 and BRISC specifically cleave K63 linkages within a mixed polyUb polymer and act as ‘endo' Ub isopeptidases. PA700 and BRISC were left untreated (lanes 3–4 and 8–9, respectively) or reacted with NEM (lanes 5–6 and 10–11), then incubated with K48–K63–K48-linked Ub₄ for the indicated times. Markers (lane 1) consist of substrate partially digested by Isopeptidase T (isoT). Reactions were immunoblotted with anti-Ub. Note that input substrate (lanes 2 and 7) consists of a doublet representing linear (upper band) and cyclic (lower band) Ub₄ (see Supplementary Figure S3).

Figure 5

Purified PA700 and BRISC are sensitive to 1,10-o-phenanthroline and unprotonated Tris. (A) PA700 (upper panel) or BRISC (lower panel) were left untreated (lane 2) or incubated with the indicated compounds (lanes 3–13) and then mixed with ¹²⁵I-K63Ub₂. Reactions lacking Tris contained 10 mM Hepes, pH 8 (lanes 2–6, 8–9 and 11–12) in addition to the indicated compounds. Products were visualised by SDS–PAGE and autoradiography. Input (in) ¹²⁵I-K63Ub₂ is shown. (B) Same as (A) except Jab1 WT (upper panel) or Jab1 H138Q-containing (lower panel) CSN complexes were used. (C) Anti-HA immunoblot indicates levels of Jab1 WT and H138Q subunits in the CSN preparations used in (B).

Figure 6

CSN and BRISC interact, and Jab1 WT CSN co-precipitates more Brcc36 than Jab1 mutant CSN. (A) Jab1 WT–Flag–HA immunoprecipitates were passed over a Superdex 200 size exclusion column. Column fractions were immunoblotted with anti-HA (to visualise the tagged Jab1 subunit) and anti-Bre (a BRISC subunit). The input Flag eluate before size exclusion (in) and the running positions of size standards are shown. (B) Equivalent amounts of Jab1 WT (lanes 1 and 3) or H138Q mutant (lanes 2 and 5) CSNs were run on SDS–PAGE and immunoblotted with anti-Jab1 (left panel) or anti-Brcc36 (right panel). Lane 5 contains the anti-Flag immunoprecipitate from Abro1–Flag–HA stable HeLa cells to indicate the migration position of endogenous Brcc36 in non-CSN-associated BRISC.

Figure 7

Brcc36 accounts for all of the Ubal-resistant K63 DUB activity associated with the CSN. (A) Jab1 WT or H138Q mutant (MUT) complexes were purified from HeLa cells stably expressing either a Brcc36- or GFP-directed shRNA, as indicated. Complexes were then incubated with neddylated cullin-1 (Cul1) for the indicated times and reactions were immunoblotted with anti-Cul1. Input substrate (lane 1) and co-precipitated, endogenous Cul1 are indicated. Note that the substrate was a truncated form of Cul1. (B) CSN preparations were run on SDS–PAGE and immunoblotted with anti-Jab1 to show equivalent loading. (C) Jab1 WT or H138Q mutant (MUT) complexes were purified from HeLa cells stably expressing either a Brcc36- or GFP-directed shRNA, as indicated. Complexes were then treated with Ubal and incubated with ¹²⁵I-K63Ub₂ for the indicated times. Products were visualised by autoradiography. (D) CSN preparations were run on SDS–PAGE and immunoblotted with anti-Brcc36.