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J. Biol. Chem., Vol. 283, Issue 34, 23039-23047, August 22, 2008

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The Spindle Checkpoint Functions of Mad3 and Mad2 Depend on a Mad3 KEN Box-mediated Interaction with Cdc20-Anaphase-promoting Complex (APC/C)^*

Matylda Sczaniecka¹, Anna Feoktistova, Karen M. May², Jun-Song Chen, Julie Blyth, Kathleen L. Gould³, and Kevin G. Hardwick⁴

From the Wellcome Trust Centre for Cell Biology, Institute of Cell Biology, University of Edinburgh, EH9 3JR, United Kingdom and the Howard Hughes Medical Institute (HHMI) and Department of Cell and Developmental Biology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232

Mitotic progression is driven by proteolytic destruction of securin and cyclins. These proteins are labeled for destruction by an ubiquitin-protein isopeptide ligase (E3) known as the anaphase-promoting complex or cyclosome (APC/C). The APC/C requires activators (Cdc20 or Cdh1) to efficiently recognize its substrates, which are specified by destruction (D box) and/or KEN box signals. The spindle assembly checkpoint responds to unattached kinetochores and to kinetochores lacking tension, both of which reflect incomplete biorientation of chromosomes, by delaying the onset of anaphase. It does this by inhibiting Cdc20-APC/C. Certain checkpoint proteins interact directly with Cdc20, but it remains unclear how the checkpoint acts to efficiently inhibit Cdc20-APC/C activity. In the fission yeast, Schizosaccharomyces pombe, we find that the Mad3 and Mad2 spindle checkpoint proteins interact stably with the APC/C in mitosis. Mad3 contains two KEN boxes, conserved from yeast Mad3 to human BubR1, and mutation of either of these abrogates the spindle checkpoint. Strikingly, mutation of the N-terminal KEN box abolishes incorporation of Mad3 into the mitotic checkpoint complex (Mad3-Mad2-Slp1 in S. pombe, where Slp1 is the Cdc20 homolog that we will refer to as Cdc20 hereafter) and stable association of both Mad3 and Mad2 with the APC/C. Our findings demonstrate that this Mad3 KEN box is a critical mediator of Cdc20-APC/C inhibition, without which neither Mad3 nor Mad2 can associate with the APC/C or inhibit anaphase onset.

Received for publication, May 9, 2008 , and in revised form, June 12, 2008.

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^* This work was supported by grants from the HHMI (to J. S. C., A. F., and K. L. G.) and the Wellcome Trust (to J. B., K. M., and K. G. H.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains three supplemental figures.

This article was selected as a Paper of the Week.

¹ A graduate student funded by the Darwin Trust of Edinburgh. Present address: Royal (Dick) School of Veterinary Studies, The University of Edinburgh, Easter Bush Veterinary Centre, Roslin, EH25 9RG, UK.

² Funded by an Human Frontier Science Program grant.

³ An Investigator of the HHMI.

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⁴ To whom correspondence should be addressed: Tel.: 44-131-650-7091; Fax: 44-131-650-7037; E-mail: Kevin.Hardwick{at}ed.ac.uk.

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				F. Herzog, I. Primorac, P. Dube, P. Lenart, B. Sander, K. Mechtler, H. Stark, and J.-M. Peters Structure of the Anaphase-Promoting Complex/Cyclosome Interacting with a Mitotic Checkpoint Complex Science, March 13, 2009; 323(5920): 1477 - 1481. [Abstract] [Full Text] [PDF]