The spindle checkpoint functions of Mad3 and Mad2 depend on a Mad3 KEN box-mediated interaction with Cdc20-anaphase-promoting complex (APC/C).
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.
Wellcome Trust Centre for Cell Biology, Institute of Cell Biology, University of Edinburgh, EH9 3JR, United Kingdom.
Matylda
M
Sczaniecka
Anna
A
Feoktistova
Karen M
KM
May
Jun-Song
JS
Chen
Julie
J
Blyth
Kathleen L
KL
Gould
Kevin G
KG
Hardwick
D000483:Alleles; D000595:Amino Acid Sequence; D064173:Anaphase-Promoting Complex-Cyclosome; D018951:Antigens, CD20 / Q000737:chemistry*; D018797:Cell Cycle Proteins / Q000502:physiology*; D006801:Humans; D064246:Mad2 Proteins; D008954:Models, Biological; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000502:physiology*; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D017434:Protein Structure, Tertiary; D012568:Schizosaccharomyces; D029702:Schizosaccharomyces pombe Proteins / Q000502:physiology*; D017386:Sequence Homology, Amino Acid; D008941:Spindle Apparatus; D043743:Ubiquitin-Protein Ligase Complexes / Q000378:metabolism*
D018951:Antigens, CD20; D018797:Cell Cycle Proteins; D064246:Mad2 Proteins; D009687:Nuclear Proteins; D029702:Schizosaccharomyces pombe Proteins; C577696:mad2 protein, S pombe; C512748:mad3 protein, S pombe; D043743:Ubiquitin-Protein Ligase Complexes; D064173:Anaphase-Promoting Complex-Cyclosome; C584277:BUB1 protein, human; C090464:Bub1 spindle checkpoint protein; D017346:Protein Serine-Threonine Kinases