Differential regulation of JAMM domain deubiquitinating enzyme activity within the RAP80 complex

Abstract

BRCC36 is a JAMM (JAB1/MPN/Mov34 metalloenzyme) domain, lysine 63-ubiquitin (K63-Ub)-specific deubiquitinating enzyme (DUB) and a member of two protein complexes: the DNA damage-responsive BRCA1-RAP80 complex, and the cytoplasmic BRCC36 isopeptidase complex (BRISC). The presence of several identical constituents in both complexes suggests common regulatory mechanisms and potential competition between K63-Ub-related signaling in cytoplasmic and nuclear compartments. Surprisingly, we discover that BRCC36 DUB activity requires different interactions within the context of each complex. Abraxas and BRCC45 were essential for BRCC36 DUB activity within the RAP80 complex, whereas KIAA0157/Abro was the only interaction required for DUB activity within the BRISC. Poh1 also required protein interactions for activity, suggesting a common regulatory mechanism for JAMM domain DUBs. Finally, BRISC deficiency enhanced formation of the BRCA1-RAP80 complex in vivo, increasing BRCA1 levels at DNA double strand breaks. These findings reveal that JAMM domain DUB activity and K63-Ub levels are regulated by multiple mechanisms within the cell.

FIGURE 1.

BRCC36 requires interactions within the RAP80 complex for DUB activity. A, Coomassie-stained gel representing purified proteins used for in vitro DUB assays. Recombinant Abraxas, BRCC36, BRCC45, MERIT40, and RAP80 (all of which are FLAG-HA-tagged) were purified from baculovirus-infected Sf9 cells by FLAG-IP and FLAG peptide elution and are estimated to be ∼95% pure. Cp, complex. A full-length gel is included in supplemental Fig. S1. B, BRCC36 Lys⁶³-specific DUB activity requires interactions within the RAP80 complex. FLAG-HA-tagged BRCC36 or the entire RAP80 complex was purified from baculovirus-infected Sf9 cells 48 h postinfection. FLAG-purified complexes were incubated with either hexa-K48-Ub or hexa-K63-Ub for 1 h, and the products were detected by IB with the P4D1 antibody to ubiquitin. The figure is representative of three independent experiments. C, an interaction-deficient BRCC36 mutant (ΔN13) lacks DUB activity. FLAG-HA-tagged BRCC36-ΔN13, and RAP80 complexes containing either BRCC36 (BRCC36 complex) or BRCC36 mutants (ΔN13 complex and QSQ complex) were incubated with hexa-K63-Ub for 1 h, and the products were detected by IB with the P4D1 antibody to detect ubiquitin. The figure is representative of three independent experiments. See supplemental Fig. S1 for a Coomassie-stained gel representing purified proteins. D, the RAP80 complex does not require ubiquitin targeting for BRCC36 DUB activity. FLAG-HA-tagged BRCC36, RAP80 complex, RAP80ΔE81 complex, or QSQ complex were purified from baculovirus-infected Sf9 cells 48 h postinfection. FLAG-purified complexes were incubated with hexa-K63-Ub for 1 h, and the products were detected by IB with the P4D1 antibody to ubiquitin. Increasing amounts of the RAP80, RAP80ΔE81, and QSQ complexes were employed as denoted by the arrow. The figure is representative of three independent experiments.

FIGURE 2.

Abraxas and BRCC45 interact with BRCC36 through MPN- and UEV domains respectively. A, schematic diagram of the domains and mutations used for Abraxas (top) and BRCC45 (bottom) interaction studies with BRCC36. Abraxas contains an MPN⁻ domain, a coiled coil domain, and a phosphoserine motif. BRCC45 contains two UEV domains, labeled UEV1 and UEV2 for ease of communication. B, interaction profile of BRCC45 UEV1 mutants. FLAG-tagged plasmids encoding wild type BRCC45 (Wt), BRCC45 S97A, and BRCC45 UEV1 WNP were transiently transfected into 293T cells, and FLAG-IP was performed 48 h later. RAP80, Abraxas, MERIT40, and BRCC36 were detected as BRCC45-associated proteins for both wild type BRCC45 and mutant BRCC45 S97A. BRCC45 UEV1 WNP demonstrated reduced interaction with RAP80, Abraxas, and BRCC36. C, interaction profile of BRCC45 UEV2 mutants. FLAG-tagged plasmids encoding WT BRCC45, BRCC45 S341A, and BRCC45 ΔUEV2 were transiently transfected into 293T cells, and IP was performed 48 h later. RAP80, Abraxas, MERIT40, and BRCC36 were detected as BRCC45-associated proteins for both WT BRCC45 and mutant BRCC45 S341A. BRCC45 ΔUEV2 demonstrated reduced interaction with RAP80, Abraxas, and MERIT40 while maintaining interaction with BRCC36. D, interaction profile of an Abraxas MPN domain mutant. FLAG-HA-tagged plasmids encoding wild type Abraxas and Abraxas W99E were transiently transfected into 293T cells, and IP was performed 48 h later. The Abraxas W99E mutant demonstrated loss of interaction with RAP80, MERIT40, and BRCC36.

FIGURE 3.

Abraxas and BRCC45 are essential for BRCC36 DUB activity within the RAP80 complex. A, assessment of the contribution of each core RAP80 complex constituent to BRCC36 DUB activity. FLAG-HA-tagged RAP80 complexes lacking a single component of the core RAP80 complex (Abraxas, BRCC45, MERIT40, or RAP80) were FLAG-purified from baculovirus-infected Sf9 cells 48 h postinfection. Complexes were incubated with increasing concentrations of hexa-K63-Ub for 1 h, as indicated, and the products were detected by IB with the P4D1 antibody. The appearance of diubiquitin was quantified using NIH Image J software, and DUB activity was calculated as a function of input hexa-K63-Ub. The figure is an average of two independent experiments. Enzyme kinetics were determined using nonlinear regression (Michaelis-Menten enzyme kinetics). The representative graph is displayed alongside the calculated V_max and K_m values. B, full BRCC36 DUB activity requires protein-protein interactions and WT BRCC45 UEV domains. RAP80 complexes, of which all components are FLAG-HA-tagged, containing wild type Abraxas/BRCC45 or mutants were purified from baculovirus-infected Sf9 cells 48 h postinfection. FLAG-purified complexes were incubated with hexa-K63-Ub for 1 h, and the products were detected by IB with the P4D1 antibody to ubiquitin. The appearance of diubiquitin was quantified using NIH Image J software, and DUB activity was calculated as a function of input hexa-K63-Ub. The figure is an average of two independent experiments. Enzyme kinetics were determined using nonlinear regression (Michaelis-Menten enzyme kinetics). The representative graph is displayed alongside the calculated V_max and K_m values. C, KIAA0157, but not Abraxas, is sufficient to impart K63-Ub DUB activity on BRCC36 in vitro. FLAG-purified complexes (right, Coomassie-stained gel representing FLAG-purified complexes) were incubated with hexa-K63-Ub for 1 h, and the products were detected by IB with the P4D1 antibody (left). The figure is representative of three independent experiments.

FIGURE 4.

Protein-protein interactions with MPN⁻ domain proteins are a common mechanism of regulating JAMM/JAB DUB activity. A, IB of ectopic Poh1 complexes after FLAG-IP from 293T cells. FLAG-HA-tagged plasmids encoding Poh1 were co-transfected with control (Ct) or Rpn8 siRNA, and IP was performed 48 h later (top). Depletion of Rpn8 from Poh1 complexes abrogates Poh1 DUB activity (bottom). Equal amounts of FLAG peptide-eluted Poh1 complexes were incubated with hexa-K63-Ub for 1 h, and the products were detected by IB with the P4D1 antibody to ubiquitin. B, IB of ectopic BRCC36 complexes after FLAG-IP from HeLa S3 cell nuclear extracts. Cells were treated with control or Abraxas siRNA, and IP was performed on nuclear extracts 48 h later (top). Abraxas deficiency abrogates BRCC36 DUB activity from nuclear extracts (bottom). Equal amounts FLAG peptide-eluted BRCC36 protein were incubated with hexa-K63-Ub for 1 h, and the products were detected by IB with the P4D1 antibody. C, IB of ectopic BRCC36 complexes after FLAG-IP from HeLa S3 cell nuclear extracts. Cells were treated with control or BRCC45 siRNA, and IP was performed on nuclear extracts 48 h later (top). BRCC45 deficiency abrogates BRCC36 DUB activity from nuclear extracts (bottom). Equal amounts of FLAG peptide-eluted BRCC36 protein were incubated with hexa-K63-Ub for 1 h, and the products were detected by IB with the P4D1 antibody.

FIGURE 5.

RAP80 is an in vivo target of BRCC36 Lys⁶³-specific DUB activity. A, FLAG-IP of ectopic BRCC36 complexes was performed and probed with anti-BRCC36 and anti-RAP80 antibodies as indicated. The anti-BRCC36 antibody (J86) recognizes the C-terminal 20 amino acids of BRCC36. C-terminal epitope tags strongly reduce J86 recognition on Western blot, accounting for the weak signal of BRCC36 species with C-terminal FLAG-HA tags (C-B36 and C-QSQ) compared with N-terminal FLAG-HA-tagged BRCC36 (N-B36 and N-QSQ). B, RAP80 is ubiquitinated by both WT and K48R mutated ubiquitin. 293T cells expressing FLAG-HA-RAP80 (RAP80) were mock-transfected or transfected with either WT ubiquitin or ubiquitin K48R mutant containing an N-terminal 6-histidine sequence. Ubiquitinated proteins were purified over a Ni²⁺-agarose column and eluted with imidazole-containing buffer. IB was performed with an anti-HA antibody. C, Ubc13 knockdown strongly decreases the polyubiquitinated form of RAP80 associated with BRCC36 QSQ. FLAG-IP of ectopic BRCC36 QSQ complex was performed following control (Ct) or Ubc13 siRNA-mediated knockdown. IB was performed on FLAG-purified BRCC36 complexes as indicated. D, FLAG and HA tandem immunoaffinity purification was performed for ectopic BRCC36 QSQ from HeLa S3 nuclear extracts. The RAP80-Ub2 Coomassie-stained band was excised and trypsin-digested prior to mass spectrometry analysis. M, molecular weight marker; Mock, mock-transfected cell line. E, peptide sequences obtained by mass spectrometry of tryptic digests from the RAP80-Ub2 band. K#, RAP80 lysine residues that are conjugated to ubiquitin. A peptide sequence indicating the presence of Lys⁶³-linked polyubiquitin was also detected by mass spectrometry from tryptic digests of the RAP80-Ub2 band. F, IB of ectopically expressed FLAG-HA BRCC36 or FLAG-HA BRCC36-QSQ after FLAG-IP. Increased RAP80 polyubiquination, specifically Lys⁶³-linked ubiquitin, is observed in association with BRCC36-QSQ compared with BRCC36.

FIGURE 6.

Relative levels of KIAA0157 influence the abundance and localization of the BRCA1-RAP80 complex. A, differential influence of BRISC and RAP80 complexes on K63-Ub levels at DSBs. IF was performed for γH2AX and K63-Ub following control, KIAA0157, Abraxas, BRCC45, MERIT40, and RAP80 siRNA treatment in laser-microirradiated U2OS cells. Knockdown of Abraxas, BRCC45, MERIT40, and RAP80 increased K63-Ub intensity at DSBs. Bars, 10 μm. B, quantification of K63-Ub stripe intensity displayed graphically from A. At least 100 cells were counted in triplicate for the analysis. Error bars, S.D. p values were calculated by Student's t test. C, IB of H2AX-Ub following siRNA knockdown of either Abraxas or KIAA0157. Compared with control knockdown cells (Ct), H2AX-Ub levels are increased following Abraxas knockdown and decreased following KIAA0157 knockdown. IB is representative of three independent experiments. D, IB of ectopic Abraxas following KIAA0157 siRNA-mediated depletion. FLAG-HA-tagged Abraxas was FLAG-immunoprecipitated and blotted as indicated. Knockdown of KIAA0157 increased interaction between Abraxas and BRCA1, RAP80, MERIT40, and BRCC36. E, KIAA0157 depletion increases BRCA1 localization to DSBs. IF was performed for 53BP1 and BRCA1 following control and KIAA0157 siRNA treatment in laser-microirradiated U2OS cells. Knockdown of KIAA0157 increased BRCA1 intensity compared with control. Bars, 10 μm. F, quantification of BRCA1 stripe intensity displayed graphically from E. At least 100 cells were counted in triplicate for the analysis. Error bars, S.D. p values were calculated by Student's t test.

FIGURE 7.

Model for Lys⁶³-specific DUB regulation in different cellular compartments. The BRISC and BRCA1-RAP80 complexes are differentially regulated by protein-protein interactions in the cytoplasm and nucleus, respectively. Formation of each complex is limited in part by the abundance of the other complex. In the nucleus, the ubiquitin binding of RAP80 targets the RAP80 complex to DSBs. We theorize that the same mechanism exists in the cytoplasm, where an unknown protein targets DUB activity within the BRISC to act on yet to be identified K63-Ub targets.