New Insights into the Role of BubR1 in Mitosis and Beyond - International Review of Cell and Molecular Biology - page 234

4.1.2 Molecular aspects
Determining precisely how at the molecular level BubR1 is recruited to
kinetochores remains an area of active investigation. The proteins involved
in BubR1 recruitment include Bub1, Bub3, and the KMN complex subunit
KNL1 (KNL1 goes by many names in the literature, including KNL1 in
C. elegans , Blinkin or AF15q14 in human cells, Spc105 in S. cerevisiae and
Drosophila , and Spc7 in S. pombe ). The activity of the kinases Mps1
( Maciejowski et al., 2010; Millband and Hardwick, 2002 ) and Aurora
B( Ditchfield et al., 2003 ) has also been implicated. Bub3 appears to be a
major cofactor for the recruitment of BubR1 to kinetochores. Not only
is BubR1 (or Mad3) nearly always found bound to Bub3 ( Chen, 2002;
Hardwick et al., 2000; Larsen et al., 2007 ), but BubR1 in both vertebrates
and Drosophila , and Mad3 (in yeast) localize to kinetochores in a Bub3-
dependent manner ( Basu et al., 1998; Harris et al., 2005; Millband and
Hardwick, 2002; Taylor et al., 1998 ). Mutation of the Bub3-binding motif
within BubR1 abolishes its kinetochore recruitment ( Elowe et al., 2010;
Malureanu et al., 2009; Wang et al., 2001 ).
The role of Bub1 in BubR1 recruitment is subtler. Immunodepletion of
Bub1 fromXenopus egg extracts or depletion by siRNA in mammalian cells
reduced, but did not eliminate recruitment of BubR1 to kinetochores
( Boyarchuk et al., 2007; Johnson et al., 2004; Sharp-Baker and Chen,
2001 ). In yeast, Bub1 was reportedly both necessary and sufficient to recruit
Mad3 to kinetochores ( Rischitor et al., 2007 ). Interestingly, the kinase
activity of Bub1 was not required, suggesting that Bub1 is acting as a scaffold
protein ( Johnson et al., 2004; Sharp-Baker and Chen, 2001 ). More recently,
a physical interaction between BubR1 and Bub1, mediated by regions
downstream of their TPR domains, was detected by yeast two-hybrid anal-
ysis ( D'Arcy et al., 2010 ).
The KNL1 subunit of the KMN complex is emerging as a main hub of
recruitment for both Bub1 and BubR1 at kinetochores ( Bolanos-Garcia
et al., 2011; D'Arcy et al., 2010; Kiyomitsu et al., 2007, 2011; Krenn
et al., 2012 ). The TPR domains of BubR1 and Bub1 interact with
KNL1 along peptide sequences called KI motifs.
Two recent independent studies have refined the analysis of BubR1-
KNL1 interaction. Bolanos-Garcia et al. (2011) solved the structure for a
BubR1 fragment residues 57-220 (which encompasses the TPRmotif, but
not the first KEN box nor the Bub3-binding domain) in complex with a
KNL1-derived peptide of 19 residues referred to as the KI2 motif and pre-
viously shown to interact with BubR1 ( Kiyomitsu et al., 2011 ). Point