New Insights into the Role of BubR1 in Mitosis and Beyond - International Review of Cell and Molecular Biology - page 237

KNL1-bound BubR1-Bub3 is the source of the BubR1 assembled into the
MCC. However, for the moment almost nothing is known about the
dynamics or mechanism of this handover.
4.2.1 BubR1 and the structure of the MCC
BubR1 is the key subunit of the MCC. In vitro , the BubR1-Cdc20 complex
alone is a potent inhibitor of the APC/C ( Fang, 2002; Tang et al., 2001 ).
Bub3, though necessary for the association of BubR1/Mad3with unattached
kinetochores, does not seem necessary for the inhibition of the APC/C in
either metazoans or yeast ( Fang, 2002; Sczaniecka et al., 2008; Tang et al.,
2001 ). And within BubR1, the two KEN boxes and the TPR domain play
critical roles in MCC assembly and function. The N-terminal KEN box
(called here KEN1) and the TPR domain constitute the Cdc20-binding
region of BubR1/Mad3 ( Chao et al., 2012 ). A second binding site between
residues 525 and 700 of BubR1, allowing binding of BubR1 to Cdc20 inde-
pendently of Mad2, has been reported ( Davenport et al., 2006; Malureanu
et al., 2009 ), but does not appear to be relevant to the assembly of the
MCC or the functionality of the SAC ( Elowe et al., 2010; Lara-Gonzalez
et al., 2011 ). Mutations affecting the integrity of the TPR domain were
unable to bind Cdc20 and assemble the MCC, and therefore were SAC
defective, even though the mutant proteins were still recruited correctly
to unattached kinetochores ( Lara-Gonzalez et al., 2011 ).
The importance of the two KEN boxes to SAC function was first dem-
onstrated in budding and fission yeast in three papers ( Burton and Solomon,
2007; King et al., 2007; Sczaniecka et al., 2008 ). These studies found that
mutation of either of the two Mad3 KEN boxes led to elimination of the
checkpoint in vivo . The N-terminal KEN1 box moreover was absolutely
required for the assembly of the Mad3-Cdc20 complex as part of the
MCC. Recalling that KEN motifs were first identified as degron signals
( Pfleger and Kirschner, 2000 ), Burton and Solomon went on to show that
Mad3, via its KEN1 motif, competed with a bona fide KEN-box containing
APC/C substrate for binding to Cdc20 in vitro . They proposed a model
to explain how the MCC inhibited the APC/C by behaving like a
pseudosubstrate, blocking the ability of APC/C cdc20 to recognize the
degron motifs of Cyclin B and Securin. The two KEN boxes of BubR1
were shown to be equally critical to the SAC function in mammals and flies
( Elowe et al., 2010; Lara-Gonzalez et al., 2011; Malureanu et al., 2009;
Rahmani et al., 2009 ), and again KEN1, but not KEN2, seems to be
required for assembly of the MCC. The MCC assembled from BubR1