Tumor Suppressor function of the deubiquitinating enzyme BAP1 and its substrate gamma-tubulin In regulation of cell cycle and genome stability

In normal cells, the cell cycle is controlled by a complex series of signalling pathways by which a cell grows, replicates its DNA and divides. The critical task of the cell cycle is to ensure that DNA is faithfully replicated and that identical chromosomal copies are distributed equally to two daughter cells during mitosis with the help of the mitotic spindle. Thus, mitotic spindle formation is vital for proper chromosome congression, alignment and segregation during cell division. Dysregulation of any of the components involved in this process may cause mis-segregation of the chromosomes and chromosomal instability, which are commonly observed in cancer. There are a number of surveillance mechanisms to ensure errors are corrected, and if not, the cells commit suicide (apoptosis). In cancer, this tightly regulated process malfunctions as a result of genetic mutations, resulting in uncontrolled cell proliferation. g-Tubulin is a member of the tubulin family that is required for interphase αβ-tubulin nucleation, spindle formation and centrosomal duplication. The cell cycle is a closely controlled process, involving multiple components with regulation on several levels. The findings from our studies suggest that at the G1/S-phase transition, when retinoblastoma protein (RB) releases E2 promoter binding factors (E2Fs), g-tubulin associates with and captures E2F to ensure a transient transcription of genes necessary for S-phase entry. In addition, we found that the g-tubulin deubiquitination enzyme, BRCA1 Associated Protein 1 (BAP1), was downregulated in metastatic adenocarcinoma breast cell lines compared to non-cancerous human breast epithelial cells. Reduced expression of BAP1 in breast cancer cell lines was associated with mitotic abnormalities. Rescue experiments involving expression of BAP1 reduced ubiquitination of g-tubulin and prevented mitotic defects. Furthermore, we found that the tumour suppressor function of BAP1 in neuroblastoma is mediated through the arrest of cells in S phase, and further, it promoted cell death. In conclusion, our studies define important functions of g-tubulin and BAP1 in the regulation of the cell cycle and proper segregation of the chromosomes

Deubiquitination of γ-tubulin by BAP1 prevents chromosome instability in breast cancer cells.

Microtubule nucleation requires the γ-tubulin ring complex, and during the M phase (mitosis) this complex accumulates at the centrosome to support mitotic spindle formation. The post-translational modification of γ-tubulin through ubiquitination is vital for regulating microtubule nucleation and centrosome duplication. Blocking the BRCA1/BARD1-dependent ubiquitination of γ-tubulin causes centrosome amplification. In the present study, we identified BRCA1 associated protein-1 (BAP1) as a deubiquitination enzyme for γ-tubulin. BAP1 was downregulated in metastatic adenocarcinoma breast cell lines compared to non-cancerous human breast epithelial cells. Furthermore, low expression of BAP1 was associated with reduced overall survival of breast cancer patients. Reduced expression of BAP1 in breast cancer cell lines was associated with mitotic abnormalities. Importantly, rescue experiments including expression of full length but not the catalytic mutant of BAP1 reduced ubiquitination of γ-tubulin and prevented mitotic defects. Our study uncovers a new mechanism for BAP1 involved in deubiquitination of γ-tubulin, which is required to prevent abnormal mitotic spindle formation and genome instability

Association of nuclear-localized nemo-like kinase with heat-shock protein 27 inhibits apoptosis in human breast cancer cells

Nemo-like kinase (NLK), a proline-directed serine/threonine kinase regulated by phosphorylation, can be localized in the cytosol or in the nucleus. Whether the localization of NLK can affect cell survival or cell apoptosis is yet to be disclosed. In the present study we found that NLK was mainly localized in the nuclei of breast cancer cells, in contrast to a cytosolic localization in non-cancerous breast epithelial cells. The nuclear localization of NLK was mediated through direct interaction with Heat shock protein 27 (HSP27) which further protected cancer cells from apoptosis. The present study provides evidence of a novel mechanism by which HSP27 recognizes NLK in the breast cancer cells and prevents NLK-mediated cell apoptosis

Nuclear localization of γ-tubulin affects E2F transcriptional activity and S-phase progression

We show that the centrosome- and microtubule-regulating protein γ-tubulin interacts with E2 promoter binding factors (E2Fs) to modulate E2F transcriptional activity and thereby control cell cycle progression. γ-Tubulin contains a C-terminal signal that results in its translocation to the nucleus during late G1 to early S phase. γ-Tubulin mutants showed that the C terminus interacts with the transcription factor E2F1 and that the E2F1–γ-tubulin complex is formed during the G1/S transition, when E2F1 is transcriptionally active. Furthermore, E2F transcriptional activity is altered by reduced expression of γ-tubulin or by complex formation between γ-tubulin and E2F1, E2F2, or E2F3, but not E2F6. In addition, the γ-tubulin C terminus encodes a DNA-binding domain that interacts with E2F-regulated promoters, resulting in γ-tubulin-mediated transient activation of E2Fs. Thus, we report a novel mechanism regulating the activity of E2Fs, which can help explain how these proteins affect cell cycle progression in mammalian cells.—Höög, G., Zarrizi, R., von Stedingk, K., Jonsson, K., Alvarado-Kristensson, M. Nuclear localization of γ-tubulin affects E2F transcriptional activity and S-phase progression

BAP1 induces cell death via interaction with 14-3-3 in neuroblastoma article

BRCA1-associated protein 1 (BAP1) is a nuclear deubiquitinating enzyme that is associated with multiprotein complexes that regulate key cellular pathways, including cell cycle, cellular differentiation, cell death, and the DNA damage response. In this study, we found that the reduced expression of BAP1 pro6motes the survival of neuroblastoma cells, and restoring the levels of BAP1 in these cells facilitated a delay in S and G2/M phase of the cell cycle, as well as cell apoptosis. The mechanism that BAP1 induces cell death is mediated via an interaction with 14-3-3 protein. The association between BAP1 and 14-3-3 protein releases the apoptotic inducer protein Bax from 14-3-3 and promotes cell death through the intrinsic apoptosis pathway. Xenograft studies confirmed that the expression of BAP1 reduces tumor growth and progression in vivo by lowering the levels of pro-survival factors such as Bcl-2, which in turn diminish the survival potential of the tumor cells. Patient data analyses confirmed the finding that the high-BAP1 mRNA expression correlates with a better clinical outcome. In summary, our study uncovers a new mechanism for BAP1 in the regulation of cell apoptosis in neuroblastoma cells

Release of NLK to the cytosol by down-regulation of HSP27 augments apoptosis induction.

<p>(A) MCF7 cells were mock-treated, or transfected with two different siRNAs targeting HSP27 (siRNA1 HSP27 or siRNA2 HSP27) or with a control oligonucleotide, twice for 24 hours, followed by a Western blot analysis, using HSP27 and Actin. (B) MCF7 cells were transfected with siRNAs targeting HSP27 or with a control oligonucleotide, twice for 24 hours. Nuclear (N) and cytosolic (C) fraction from total cell lysate was fractionated and followed by a Western blot analysis, using NLK, lamin B, or tubulin antibodies. (C) The cellular distribution of NLK in MCF7 cells transfected two times within 48 hours with siRNA oligos against HSP27. After fixation, cells were permeabilized with 0.25% Triton X-100 solution, after which they were blocked with 1% BSA, and subsequently probed with antibodies against NLK (1:100) and HSP27 (1:100). After washing the coverslips, fluorescent antibodies: Alexa fluor 488 goat anti Rabbit (1:1000) or Alexa fluor 568 Donkey anti Goat (1:1000) were applied and nuclei were stained with DAPI. (D) MCF7 cells were transfected with siRNAs targeting NLK or with a control oligonucleotide, twice for 24 hours. Nuclear (N) and cytosolic (C) fraction from total cell lysate was fractionated and followed by a Western blot analysis, using NLK, HSP27, tubulin, or lamin B antibodies. (E) Endogenous levels of NLK and HSP27 in total cell lysates from MCF7 cells, mock-treated, or transfected with siRNA against HSP27 or with a control oligonucleotide, twice for 24 hours. (F) MCF7 cells, transfected with siRNA, were evaluated 24 hours after secondary transfections, using the NucleoCounter cell viability assay. Data (mean ± s.e.m., p<0.05, n = 3) are presented as the amount of viable cells in comparison with mock-transfected cells. (G) MCF7 cells, transfected with siRNA, were fixed and stained with propidium iodide, 24 hours after secondary transfections. Cells were visualized by fluorescence microscopy, and scored for apoptotic nuclear morphology. Data (mean ± s.e.m., p<0.05, n = 3) show the amount of apoptotic cells in comparison with mock-transfected cells. (H) The cellular distribution of NLK in MCF10A transfected with HSP27 for 24 hours using immunofluorescence staining and confocal microscopy (upper panel). Lower panel shows the levels of HSP27 in transfected MCF10A cells.</p

Cytosolic localization of NLK induces cell death.

<p>(A) MCF7 and MCF10A cells were transfected for 24 hours with vectors encoding FLAG-tag fusions of full-length NLK (WT), or catalytically inactive NLK mutants (K155M and T286V). NLK localization (Green) was visualized by immunofluorescence staining and confocal microscopy. The nuclei of the cells were counter-stained with DAPI (Blue). (B) MCF7 cells were transfected with vectors encoding FLAG-tag fusions of full-length NLK (WT) or catalytically inactive NLK mutants (K155M and T286V), and counted 24, 48, or 72 h post transfection, using a Countess Automated Cell Counter. Data (mean ± s.e.m., p<0.05, n = 4) are expressed as number of viable cells at each time point. (C) MCF7 cells were mock-treated or transfected for 24 hours with WT-, K155M-, or T286V-NLK. The cells were fixed and stained with propidium iodide in order to count the cells displaying an apoptotic nuclear morphology. Data (mean ± s.e.m., p<0.05, n = 3) show the percentage of apoptotic cells in comparison with cells of mock-treated controls. (D) MCF7 cells were mock-treated or transfected for 24 hours with WT-, K155M-, or T286V-NLK. After 48, or 72 hours cells were subjected to DNA fragmentation assay. Data (mean ± s.e.m., p<0.05, n = 3) represent the percent of sub G0 population. Right panels shows the levels of Flag tagged WT-, K155M-, or T286V-NLK overexpressed in MCF10A and MCF7 cells.</p

TNF-α and etoposide promotes accumulation of NLK in the cytoplasm.

<p>(A) MCF7 breast cancer cells were incubated in the absence or presence of 50 ng TNF-α for 18 hours. The localization of NLK was examined with immunofluorescence followed by confocal microscopy. (B) MCF10A cells were transfected with mock, shRNA targeting NLK, or with a control ShRNA for 24 hours, followed by a Western blot analysis of the total cell lysate, using NLK and Actin (left panel) or subjected to DNA fragmentation assay (right panel, n = 2). Data represent the percent of sub G0 population. (C) MCF7 cells were transfected with shRNA targeting NLK, or with a control ShRNA for 24 hours and treated with etoposide (upper panel, 10 µM for 16 hours) or TNF-α (lower panel, 50 ng TNF-α for 18 hours), followed by DNA fragmentation assay (n = 4). Data represent the percent of sub G0 population. (D) MCF10A cells were grown on glass coverslips in 6-well plates in triplicates. On the following day the cells were treated with DMSO or 100mM of etoposide for 12 hours. After fixation, cells were permeabilized with a 0.25% Triton X-100 solution. Cells were further blocked with 1% BSA, and probed with antibodies against NLK. Nuclei were counter-stained with DAPI. (E) MCF10A, MCF7 and MDA-231 cells were lysed and immunoprecipitated with anti-NLK antibody. Immunoprecipitates were purified by washing two times with lysis buffer containing high salt (0.625M NaCl), three times with PBS containing high salt (0.625 M NaCl), and two times with PBS. The pellet were incubated with 0.5 µg LEF1 and 1 mM ATP in 40 µl of kinase buffer (10 mM MgCl2, 10 mM HEPES (pH 7.4), and 1 mM DTT) for 30 minutes at 30°C. Samples were resolved by SDS-PAGE followed by a Western blot analysis, using phospho-LEF1 and total-LEF1 antibodies.</p