Monoubiquitination of ASXLs controls the deubiquitinase activity of the tumor suppressor BAP1

Our plain-English summary of Monoubiquitination of ASXLs controls the deubiquitinase activity of the tumor suppressor BAP1, by S. Daou, H. Barbour, O. Ahmed, et al (Nature Communications, 2018).

The study

• Authors: S. Daou, H. Barbour, O. Ahmed, L. Masclef, C. Baril, N. Sen Nkwe, D. Tchelougou, M. Uriarte, E. Bonneil, D. Ceccarelli, N. Mashtalir, M. Tanji, J.Y. Masson, P. Thibault, F. Sicheri, H. Yang, M. Carbone, M. Therrien, E.B. Affar
• Journal: Nature Communications
• Published: 2018;9(1):4385
• Cited by: 56 articles, as counted by NIH iCite
• Read it: PubMed · Publisher · Free full text

Abstract

The tumor suppressor and deubiquitinase (DUB) BAP1 and its Drosophila ortholog Calypso assemble DUB complexes with the transcription regulators Additional sex combs-like (ASXL1, ASXL2, ASXL3) and Asx respectively. ASXLs and Asx use their DEUBiquitinase ADaptor (DEUBAD) domain to stimulate BAP1/Calypso DUB activity. Here we report that monoubiquitination of the DEUBAD is a general feature of ASXLs and Asx. BAP1 promotes DEUBAD monoubiquitination resulting in an increased stability of ASXL2, which in turn stimulates BAP1 DUB activity. ASXL2 monoubiquitination is directly catalyzed by UBE2E family of Ubiquitin-conjugating enzymes and regulates mammalian cell proliferation. Remarkably, Calypso also regulates Asx monoubiquitination and transgenic flies expressing monoubiquitination-defective Asx mutant exhibit developmental defects. Finally, the protein levels of ASXL2, BAP1 and UBE2E enzymes are highly correlated in mesothelioma tumors suggesting the importance of this signaling axis for tumor suppression. We propose that monoubiquitination orchestrates a molecular symbiosis relationship between ASXLs and BAP1.

In plain English

This laboratory study examined how the tumour suppressor and deubiquitinase BAP1 and its partner proteins ASXL1, ASXL2, ASXL3 (and the fly counterparts Calypso and Asx) regulate each other. Working in human cell lines (HEK293T, U-2 OS, IMR90 and others), in test-tube reactions with purified proteins, in Drosophila S2 cells and transgenic flies, and in 20 human malignant mesothelioma biopsies, the authors reported that BAP1 promoted the attachment of a single ubiquitin to the DEUBAD domain of ASXL2, at lysine 370, and that this site was shared across ASXLs and conserved in the fly. Ubiquitin-conjugating enzymes UBE2E1, UBE2E2 and UBE2E3 carried out this modification directly in vitro. Monoubiquitinated DEUBAD increased BAP1 activity towards ubiquitinated histone H2A, and cells expressing the non-modifiable ASXL2 K370R mutant proliferated less and progressed more slowly from G1 to M. Flies expressing Asx K325R showed partial haltere-to-wing transformation and crumpled wings. Among the 20 biopsies, the strongest association of staining levels was between UBE2E2 and ASXL2 (r = 0.55, p = 0.01), while UBE2E3 and ASXL2 gave r = 0.32, p = .17; the authors said these results suggest the expression of BAP1, ASXL2 and UBE2Es is correlated in mesothelioma tissues.

We wrote this plain-English summary from the full article with the help of AI, and a second AI model checked each sentence against the article. It is not the authors' wording; the abstract and the article are their own account.

What the study did and found

• Design: Laboratory experimental study combining cell-culture work, in vitro biochemistry with purified proteins, siRNA/CRISPR screens, Drosophila S2 cell and transgenic fly experiments, and immunohistochemistry of human mesothelioma biopsies
• Who was studied: Human cell lines (HEK293T, U-2 OS, IMR90, LF1), 3T3-L1 mouse preadipocytes, Drosophila S2 cells and transgenic flies, and 20 human malignant mesothelioma biopsies (10 with wild-type BAP1, 10 with mutated BAP1)
• Exposure: No environmental or occupational exposure was studied; the manipulations were genetic and biochemical (overexpression of BAP1, ASXL1/2/3, Asx and Calypso constructs, lysine-to-arginine mutants such as ASXL2 K370R and Asx K325R, siRNA/dsRNA depletion, CRISPR/Cas9 knockout of UBE2E3, and in vitro ubiquitination with 32 recombinant UBE2 enzymes)
• Main results:
  • ASXL2 was monoubiquitinated on the DEUBAD domain in a BAP1-dependent way, with lysine 370 identified as the key site
  • Of 32 recombinant E2 enzymes tested in vitro, only UBE2E1, UBE2E2 and UBE2E3 monoubiquitinated the ASXL2 DEUBAD domain
  • In in vitro deubiquitination assays on H2A nucleosomes, the monoubiquitinated DEUBAD/BAP1 complex showed stronger BAP1 activity than the K370R mutant complex, which retained residual activity
  • In U-2 OS cells, expression of the monoubiquitination-defective ASXL2 K370R reduced colony formation and delayed progression from G1 to M after nocodazole treatment
  • In Drosophila, expression of Asx K325R raised H2Aub levels, repressed Ubx in haltere discs and produced partial haltere-to-wing transformation and crumpled wings
  • In the 20 mesothelioma biopsies, logistic regression with BAP1 status as outcome gave UBE2E3 (p = 0.04) and ASXL2 (p = 0.04) as best predictors; across all 20 samples the strongest correlation was UBE2E2 with ASXL2 (r = 0.55, p = 0.01), then UBE2E2 with UBE2E3 (r = 0.49, p = .03) and UBE2E3 with ASXL2 (r = 0.32, p = .17)
  • Restricted to the 10 BAP1 wild-type mesothelioma samples, UBE2E2 and ASXL2 correlated most strongly (r = 0.90, p = 0.0004), followed by UBE2E2 and UBE2E3 (r = 0.50, p = 0.14) and UBE2E3 and ASXL2 (r = 0.48, p = 0.16)
• The authors' conclusion: The authors concluded that monoubiquitination of the DEUBAD domain is a conserved mechanism that stabilises ASXL2 and stimulates BAP1 deubiquitinase activity, and they suggested that deregulation of the UBE2E-ASXL2/BAP1 ubiquitin signalling axis might participate in mesothelioma development
• Limitations the authors state:
  • The authors stated they could not rule out that an as-yet unidentified E3 ligase is needed for efficient monoubiquitination inside cells
  • The authors stated it was unclear whether the proliferation effects were due solely to loss of H2A deubiquitination or to effects on other substrates
  • The authors stated the DUB and E2 siRNA screens were each performed once
  • For the fly work, the authors noted that further studies would be needed, for example on why GFP expression in the discs was patchy

Topics

Medical terms in this study, explained in our Lexicon: BAP1, Mesothelioma.

Related studies

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• Functional differences between wild-type and mutant-type BRCA1-associated protein 1 tumor suppressor against malignant mesothelioma cells — S. Hakiri, H. Osada, et al. (2015)
• Different prognostic roles of tumor suppressor gene BAP1 in cancer: A systematic review with meta-analysis — C. Luchini, N. Veronese, et al. (2016)
• BAP1: case report and insight into a novel tumor suppressor — K. Ghosh, B. Modi, et al. (2017)
• Loss of BAP1 function leads to EZH2-dependent transformation — L.M. LaFave, W. Béguelin, et al. (2015)
• BAP1 regulates IP3R3-mediated Ca2+ flux to mitochondria suppressing cell transformation — A. Bononi, C. Giorgi, et al. (2017)

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