Depiction of BAP1 constructs used in this study. UCH (ubiquitin C-terminal hydrolase), NLS (nuclear localization signal).

BAP1-mediated suppression of NCI-H226 cell tumorigenicity in vivo requires deubiquitinating activity and nuclear localization. Athymic nu/nu female mice (5–6 weeks old) were injected subcutaneously with 1×10⁶ NCI-H226 cells carrying vector alone (VC), or vector encoding wild-type BAP1 (WT), active-site point mutant BAP1 (C91A), or mutant BAP1 that does not localize to the nucleus (NLS2-Ala). (A) Tumor growth rates represented as mm³/day ± SD. (B) The mean tumor volume was plotted as a function of time using the formula (Length x width² x ½). P-values shown are compared to WT BAP1 tumors. Other p-values are as follows: VC vs. C19A=0.1143; VC vs. NLS2-Ala=0.1206; C91A vs. NLS2-Ala=0.6844. n = 8. (C) Representative tumors excised from mice. Numbers at top represent mouse number.

BAP1 alters cell cycle distribution and induces cell death in NCI-H226 cells. NCI-H226 (BAP1-null) cells (1×10⁶) were infected with lentiviruses carrying vector alone (VC) or vector encoding WT, C91A or NLS2-Ala BAP1. An additional control well was not infected with any lentivirus. Cells were grown in selective medium (until non-infected cells were dead) then analyzed by three parameters: (A) cells were fixed and treated with propidium iodide to study alterations in DNA content and cell cycle distribution. Gates are as follows: M1=Sub-G₀/G₁; M2=G₀/G₁; M3=S-phase; M4=G₂/M. Graph represents relative % of total cell counts in respective gates (B) cells were treated with AnnexinV-FITC and propidium iodide and analyzed by flow cytometry at the indicated time points. Quadrants are as follows: lower left=viable cells; lower right=early apoptotic; upper right=late apoptotic/necrotic. Numbers in quadrants represent percent of total cells. (C) Cells were infected with viruses carrying empty vector (VC) or WT BAP1 at an MOI of 3 so that almost 100% cells were infected. Cells were not subjected to antibiotic selection. Growth and cell viability were assessed by trypan blue exclusion.

Deubiquitinating activity and nuclear localization are required for BAP1-mediated growth suppression of NCI-H226 cells in vitro. (A) Immunoblot analysis of lentivirus-expressed BAP1 in NCI-H226 cells compared to endogenous WT BAP1 in cultured cell lines: NCI-H226 cells (BAP1-null) expressing vector alone (lane 1) or WT BAP1 (lane 2), HeLa cells (lane 3) and 293FT cells (lane 4) (5 µg total cell lysate per lane). (B) NCI-H226 cells (1×10⁵) were seeded in triplicate in six-well plates. After 24 h cells were infected with lentiviruses carrying vector alone (VC), or vector encoding wild-type BAP1 (WT) or one of the following mutants: C91A, active-site point mutant; A95D, cancer-associated, catalytically inactive mutant; G178V, another cancer-associated, catalytically inactive mutant or BAP1 NLS2-Ala, mutant BAP1 that does not localize to the nucleus. An additional control well was not infected with any virus. Immunoblot analysis shows similar expression levels of WT and mutant BAP1 protein is attained 48 h after infection (10 µg cell lysate per lane). (C) The number of surviving colonies was quantified after incubation in selective medium (until all non-infected cells were dead), using crystal violet according to the ViraPower kit protocol. Results are represented graphically as a percentage of the vector alone (VC) mean (mean ± SD. n = 3). (D). BAP1-mediated growth suppression is independent of BRCA1. Human breast carcinoma cells HCC1937 and HCC1937-BRCA1 cells (HCC1937 cells expressing Myc epitope-tagged wild-type BRCA1) were seeded in triplicate in six-well plates (1×10⁵ per well). After 24 h cells were infected with lentiviruses carrying vector alone (VC) or vector encoding wild-type BAP1 (WT). An additional control well was not infected with any virus. Immunoblot analysis was performed 48 h after infection (10 µg cell lysate per lane). After incubation in selective medium (until all non-infected cells were dead), the number of surviving colonies was quantified using crystal violet according to the ViraPower kit protocol. Results are represented graphically as a percentage of the HCC1937-VC mean. Data are mean ± SD. n = 3.

Cancer-associated mutations in BAP1 result in protein lacking deubiquitinating activity. (A) Alignment of BAP1 N-terminal region from different species showing that the catalytic cysteine 91 (yellow box) and residues mutated in cancer cell lines alanine 95 (blue box) and glycine 178 (green box) are conserved. The alignment was performed using ClustalW alignment and MegAlign 7.1.0 software. NCBI accession numbers are as follows: Human BAP1, AAH01596; Mouse BAP1, NP_081364; Rat BAP1, NP_001100762; Xenopus BAP1, NP_001008206. (B) Deubiquitinating activity assay. Wild-type (WT) and mutant BAP1 proteins were expressed in E. coli by IPTG induction. Cells were lysed and deubiquitinating activity of crude extracts was measured as follows: ubiquitin C-terminal 7-amido-4-methylcoumarin (Ub-AMC; 80 nM) was added to crude extracts (10 µg) and hydrolysis of Ub-AMC was monitored by measuring the increase in fluorescence at 440 nm (λex= 340 nm). C91A (yellow line) is the active site point mutant, while A95D (blue line) and G178V (green line) are two mutants associated with lung cancers. (C) Specific activities were calculated from the slope and expressed as µmoles AMC/min/mg lysate. (mean ± standard deviation (SD), n=3). Immunoblot of E. coli cell lysates shows that similar levels of each BAP1 variant were expressed (5 µg of cell lysate per lane). (D) DUB activity of purified BAP1 is inhibited by the irreversible inhibitor ubiquitin vinyl sulfone (Ub-VS). BAP1 was purified using Ni-NTA chromatography. Enzyme activity was measured by adding 0.1 µg purified BAP1 to 80 nM Ub-AMC. Hydrolysis of Ub-AMC (red line) was measured as described in (B). Ub-VS was able to label WT BAP1 and inhibit the hydrolysis of Ub-AMC (black line).

NLS2 is required for nuclear localization of BAP1. (A) Alignment of C-terminal region of BAP1 orthologs showing conservation of predicted nuclear targeting signals. The alignment was performed using ClustalW alignment and MegAlign 7.1.0 software. NCBI accession numbers are as follows: Human BAP1, AAH01596; Mouse BAP1, NP_081364; Rat BAP1, NP_001100762; Xenopus BAP1, NP_001008206. (B) Both putative nuclear localization signals (NLSs) of BAP1 can localize GFP to the nucleus. Wild-type (WT) NLS sequences or mutant NLS sequences (where all basic residues of the NLS were mutated to alanine) were cloned into the dGFP vector. Each construct (2 µg) was transfected into HeLa cells (3×10⁵ cells per well of six-well plate). After 24 h, DNA was stained with 5 ng/µl bis-benzimide (Hoechst 33258) and the localization of GFP was assessed by direct fluorescent microscopy. The scale bar is 20 µm. (C) Full-length WT or mutant GFP-tagged BAP1 was expressed in HeLa cells and protein localization was analyzed by direct fluorescence microscopy, as described in (B). BAP1 mutants are as follows: NLS1-Ala, mutant in which all basic residues in predicted NLS1 have been converted to alanine; NLS2-Ala, mutant in which all basic residues in predicted NLS2 have been converted to alanine; (1–393), truncation at amino acid 393, discovered in non-small cell lung carcinoma cell line NCI-H1466; C91A, active-site point mutant; A95D and G178V, point mutants found in cancer cell lines. The scale bar is 10 µm. (D) BAP1 mutants that affect nuclear localization retain deubiquitinating activity. E. coli cell lysates (10 µg/µl) expressing WT BAP1, BAP1 (1–393), BAP1 NLS1-Ala or BAP1 NLS2-Ala were used in the Ub-AMC hydrolysis assay. Specific activities of WT and mutant BAP1 are represented as µmoles AMC/min/mg lysate. Immunoblot analysis of E. coli cell lysates expressing wild-type or mutant BAP1 shows similar levels of expression (5 µg of cell lysate per lane; mean ± SD, n =3.)