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Anaphase initiation is regulated by antagonistic ubiquitination and deubiquitination activities

Abstract

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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Figure 1: Validation of candidate genes from Taxol screen.
Figure 2: USP44 activity is cell-cycle regulated and is required for proper spindle checkpoint function and anaphase timing.
Figure 3: USP44 regulates APC Cdc20 downstream of Mad2 recruitment to kinetochores.
Figure 4: USP44 inhibits APCCdc20 activation in vitro.
Figure 5: USP44 deubiquitinates Cdc20 in vitro and in vivo.

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Acknowledgements

We thank S. Taylor, H. Yu, W. Earnshaw and J. Jin for gifts of reagents; M. Vidal for providing access to their BioRobot platform; S. Lyman and R. King for communicating unpublished results and assistance with the development of the Taxol screening assay; C. Shamu for access to the ICCB-Longwood screening facilities; S. Reddy for helpful comments throughout the course of the work; and T. Westbrook and A. Smogorzewska for their critical reading of the manuscript. F.S. is a fellow of the Helen Hay Whitney Foundation. M.R. is a Human Frontiers Science Program Long-Term Fellow. The siRNA and ICCB-Longwood resources used were funded in part by a NCI grant (T. Mitchison). M.E.S. is an American Cancer Society Postdoctoral Fellow. X.L.A. is an NIH pre-doctoral fellow. M.W.K. thanks the National Institute of General Medical Sciences for its support for the grant Cell Cycle Regulation. This work was supported by grants from NIH and DOD to S.J.E. and by grants from the NIH to J.W.H. S.J.E. is an investigator of the Howard Hughes Medical Institute.

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Correspondence to Marc W. Kirschner, J. Wade Harper or Stephen J. Elledge.

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This file contains Supplementary Methods, Supplementary Figures 1-11 with Legends, and Supplementary Tables 2 and 3. (PDF 4063 kb)

Supplementary Table 1

This file contains Supplementary Table 1. This file contains detailed information on the Ubiquitin-Proteasome Pathway (UPP) shRNA library. (XLS 526 kb)

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Stegmeier, F., Rape, M., Draviam, V. et al. Anaphase initiation is regulated by antagonistic ubiquitination and deubiquitination activities. Nature 446, 876–881 (2007). https://doi.org/10.1038/nature05694

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