Stegmeier F, Rape M, Draviam VM, Nalepa G, Sowa ME, Ang XL et al.. Anaphase initiation is regulated by antagonistic ubiquitination and deubiquitination activities. Nature 446: 876-881

Howard Hughes Medical Institute, Department of Genetics, Harvard Partners Center for Genetics and Genomics,Boston, Massachusetts 02115, USA.
Nature (Impact Factor: 41.46). 04/2007; 446(7138):876-881. DOI: 10.1038/nature05694


The spindle checkpoint prevents chromosome mis-segregation by delaying sister chromatid separation until all chromosomes have achieved bipolar attachment to the mitotic spindle. Its operation is essential for accurate chromosome segregation, whereas its dysregulation can contribute to birth defects and tumorigenesis. The target of the spindle checkpoint is the anaphase-promoting complex (APC), a ubiquitin ligase that promotes sister chromatid separation and progression to anaphase. Using a short hairpin RNA screen targeting components of the ubiquitin-proteasome pathway in human cells, we identified the deubiquitinating enzyme USP44 (ubiquitin-specific protease 44) as a critical regulator of the spindle checkpoint. USP44 is not required for the initial recognition of unattached kinetochores and the subsequent recruitment of checkpoint components. Instead, it prevents the premature activation of the APC by stabilizing the APC-inhibitory Mad2–Cdc20 complex. USP44 deubiquitinates the APC coactivator Cdc20 both in vitro and in vivo, and thereby directly counteracts the APC-driven disassembly of Mad2–Cdc20 complexes (discussed in an accompanying paper). Our findings suggest that a dynamic balance of ubiquitination by the APC and deubiquitination by USP44 contributes to the generation of the switch-like transition controlling anaphase entry, analogous to the way that phosphorylation and dephosphorylation of Cdk1 by Wee1 and Cdc25 controls entry into mitosis.

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Available from: Viji M Draviam
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    • "APC/C activator Cdc20 to maintain the SAC (Stegmeier et al. 2007). Although it seems plausible that reversal of APC/C ubiquitination is a conserved mechanism, there is no known USP44 homolog in non-mammals. "
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    ABSTRACT: Ubiquitination and deubiquitination of proteins are reciprocal events involved in many cellular processes, including the cell cycle. During mitosis, the metaphase to anaphase transition is regulated by the ubiquitin ligase activity of the anaphase-promoting complex/cyclosome (APC/C). While the E3 ubiquitin ligase function of the APC/C has been well characterized, it is not clear if deubiquitinating proteases (DUBs) play a role in reversing APC/C substrate ubiquitination. Here we performed a genetic screen to determine what DUB, if any, antagonizes the function of the APC/C in the fission yeast Schizosaccharomyces pombe. We found that deletion of ubp8, encoding the SAGA complex associated DUB, suppressed temperature sensitive phenotypes of APC/C mutants cut9-665, lid1-6, cut4-533, and slp1-362. Our analysis revealed that Ubp8 antagonizes APC/C function in a mechanism independent of the spindle assembly checkpoint and proteasome activity. Notably, suppression of APC/C mutants was linked to loss of Ubp8 catalytic activity and required histone H2B ubiquitination. On the basis of these data, we conclude that Ubp8 antagonizes APC/C function indirectly by modulating H2B ubiquitination status.
    Full-text · Article · Jun 2014 · G3-Genes Genomes Genetics
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    • "As the proteasome inhibitor, MG132 also promotes a SAC-dependent mitotic arrest, the authors of this article proposed that APC/C-dependent proteolysis was required to inactivate the SAC, and that APC/C inhibition activated the SAC. Interestingly, some laboratories have proposed that the APC/C-mediated ubiquitylation, but not degradation, of Cdc20 releases the APC/C from SAC inhibition (26, 27), though we and others suggest that APC/C-mediated ubiquitylation and degradation of Cdc20 sustains the SAC (28, 29). As pro-TAME activates the SAC in an APC/C-dependent manner, without chromosome misalignment at metaphase, it could be reasoned that the reduction in APC/C ligase activity in TIF1γ knockdown cells similarly activates the SAC through the stabilization of multiple APC/C substrates. "
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    ABSTRACT: The anaphase-promoting complex/cyclosome (APC/C) is an ubiquitin ligase that functions during mitosis. Here we identify the transcriptional regulator, transcriptional intermediary factor 1γ, TIF1γ, as an APC/C-interacting protein that regulates APC/C function. TIF1γ is not a substrate for APC/C-dependent ubiquitylation but instead, associates specifically with the APC/C holoenzyme and Cdc20 to affect APC/C activity and progression through mitosis. RNA interference studies indicate that TIF1γ knockdown results in a specific reduction in APC/C ubiquitin ligase activity, the stabilization of APC/C substrates, and an increase in the time taken for cells to progress through mitosis from nuclear envelope breakdown to anaphase. TIF1γ knockdown cells are also characterized by the inappropriate presence of cyclin A at metaphase, and an increase in the number of cells that fail to undergo metaphase-to-anaphase transition. Expression of a small interfering RNA-resistant TIF1γ species relieves the mitotic phenotype imposed by TIF1γ knockdown and allows for mitotic progression. Binding studies indicate that TIF1γ is also a component of the APC/C-mitotic checkpoint complex (MCC), but is not required for MCC dissociation from the APC/C once the spindle assembly checkpoint (SAC) is satisfied. TIF1γ inactivation also results in chromosome misalignment at metaphase and SAC activation; inactivation of the SAC relieves the mitotic block imposed by TIF1γ knockdown. Together these data define novel functions for TIF1γ during mitosis and suggest that a reduction in APC/C ubiquitin ligase activity promotes SAC activation.Oncogene advance online publication, 19 November 2012; doi:10.1038/onc.2012.501.
    Full-text · Article · Nov 2012 · Oncogene
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    • "Moreover, in a screen for interactors of USP44, histones H2A and H2B, and the subunit of the RNA polymerase II – POLR2G were significantly enriched (Sowa et al., 2009). Notably, in differentiated cells USP44 was identified as a critical regulator of the mitotic spindle checkpoint, through deubiquitylation of the Cdc20 protein (Stegmeier et al., 2007). It is plausible that Cdc20 is a high affinity USP44 target, rendering it a substrate for that DUB even in differentiated cells that express rather little USP44. "
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    ABSTRACT: Embryonic stem cells (ESCs) maintain high genomic plasticity, which is essential for their capacity to enter diverse differentiation pathways. Posttranscriptional modifications of chromatin histones play a pivotal role in maintaining this plasticity. We now report that one such modification, monoubiquitylation of histone H2B on lysine 120 (H2Bub1), catalyzed by the E3 ligase RNF20, increases during ESC differentiation and is required for efficient execution of this process. This increase is particularly important for the transcriptional induction of relatively long genes during ESC differentiation. Furthermore, we identify the deubiquitinase USP44 as a negative regulator of H2B ubiquitylation, whose downregulation during ESC differentiation contributes to the increase in H2Bub1. Our findings suggest that optimal ESC differentiation requires dynamic changes in H2B ubiquitylation patterns, which must occur in a timely and well-coordinated manner.
    Full-text · Article · Jun 2012 · Molecular cell
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