New Insights into the Role of BubR1 in Mitosis and Beyond - International Review of Cell and Molecular Biology - page 235

mutations within the KI2 motif or within the BubR1 fragment blocked
the binding of the peptide to BubR1 ( Bolanos-Garcia et al., 2011 ).
Another study, however, reported that a KNL1 mutant lacking the KI2
motif did not affect BubR1 kinetochore recruitment ( Krenn et al., 2012 ),
and that BubR1 mutants carrying multiple alanine substitutions (W125A,
L128A, C132A, D137A) in the KNL1 binding site were still recruited to
kinetochores even though their interaction with KNL1 was disrupted.
These studies suggest that although the TPR domain of BubR1 does indeed
interact with KNL1, this interaction is not sufficient, and possibly not even
necessary, for kinetochore recruitment. Rather, it seems likely that addi-
tional contacts, either direct or indirect, between BubR1 and another region
of KNL1 are required.
Two obvious candidates for mediating this interaction are Bub1 and
Bub3, both of which interact themselves with KNL1. Bub1, like BubR1,
makes contacts between its TPR domain and a KI motif in KNL1 ( Krenn
et al., 2012 ). Three recent studies in budding and fission yeast and HeLa
cells have implicated a region of KNL1 containing MELT peptide motifs
in the binding of Bub1 and Bub3 ( London et al., 2012; Shepperd et al.,
2012; Yamagishi et al., 2012 ). Moreover, phosphorylation of threonine
residues in these motifs by Mps1 kinase was necessary for Bub1 and
Bub3 recruitment to kinetochores. There is for the moment no direct evi-
dence that phosphorylation of MELTmotifs is involved in the recruitment
of BubR1 in metazoans. It is also possible that BubR1 interacts with other
KMN network proteins. Consistent with this, in Drosophila spc105 (KNL1)
mutant embryos, the levels of EGFP-BubR1 at the kinetochore are
reduced only about 30% ( Schittenhelm et al., 2009 ). Moreover, yeast
two-hybrid data show that Drosophila Bub1 interacts with the Mis12 com-
plex component Nsl1, thereby providing alternative pathways for Bub1
and consequently BubR1 recruitment at kinetochores ( Schittenhelm
et al., 2009 ).
Phosphorylation of kinetochore proteins by Aurora B may also influence
BubR1 recruitment. Inhibiting Aurora B in HeLa cells by the specific chem-
ical inhibitor ZM447439 or by siRNA depletion profoundly reduce kinet-
ochore levels of BubR1 ( Ditchfield et al., 2003; Lens et al., 2003 ). How
Aurora B regulates BubR1 recruitment is still not clear. One possibility is
that Aurora B phosphorylates BubR1 directly, as the Aurora
B orthologue Ipl1p in budding yeast phosphorylates Mad3p at several resi-
dues ( King et al., 2007 ). In vertebrate cells, Aurora B acts early in mitosis to
recruit Mps1 to kinetochores ( Saurin et al., 2011 ). Therefore, the effect of