New Insights into the Role of BubR1 in Mitosis and Beyond - International Review of Cell and Molecular Biology - page 225

3.1. N-terminal domain
Within the N-terminal region, we find several important motifs and protein
subdomains. First, there are two Lys-Glu-Asn (KEN) box motifs (residues
26-28 and 304-306) and three tetratricopeptide repeat (TPR) motifs (res-
idues 50-204). Residues 392-426 encode the Bub3-binding domain (also
called the GLEBS domain). The two KEN boxes are important for
BubR1/Mad3 function in the checkpoint and for binding to Cdc20
( Burton and Solomon, 2007; King et al., 2007 ). The presence of the
KEN-box domain essentially defines BubR1 as the homologue of yeast
MAD3p (which shares the KEN, TPR, and GLEBS domains, but not
the kinase domain). Bub1, by contrast, in both metazoans and yeast shares
the TPR and kinase domains, but lacks the N-terminal KEN domains.
As mentioned earlier, KEN boxes were initially identified as motifs
defining a degron, a peptide signal that leads to the APC/C-dependent
ubiquitination and subsequent degradation of proteins ( Pfleger and
Kirschner, 2000 ). BubR1, however, does not appear to be targeted for deg-
radation by the APC/C Cdc20 prior to anaphase. Instead, the KEN boxes are
critical to BubR1's function in mediating the SAC-dependent inhibition of
the APC/C Cdc20 , possibly by serving as pseudosubstrate inhibitors of
APC/C ( Burton and Solomon, 2007; Chao et al., 2012; King et al.,
2007; Lara-Gonzalez et al., 2011; Tian et al., 2012 ).
The BubR1 TPR repeats are multiple helix-turn-helix motifs packed
together into an extended spiral of antiparallel alpha-helices ( D'Arcy
et al., 2010 ). This motif is important for protein-protein interactions and
is found in a variety of proteins with diverse biological functions
( D'Andrea and Regan, 2003 ). The TPR domains of BubR1 and Bub1 have
been shown to bind to the outer kinetochore protein KNL1 ( Kiyomitsu
et al., 2007, 2011; Krenn et al., 2012 ) and may contribute to the recruitment
of BubR1 to kinetochores (see Section 4.1 ). The TPR domain also contrib-
utes to the binding of BubR1/Mad3 to Cdc20 ( Chao et al., 2012 ).
BubR1/Mad3 protein forms a constitutive complex with Bub3 protein
via the Bub3-binding domain ( Chen, 2002; Hardwick et al., 2000; Larsen
et al., 2007 ). Bub3 is a b -propeller structure that uses its top surface to interact
directly with the Bub3-binding domains of both Bub1 and BubR1/Mad3
( Larsen et al., 2007 ). The Bub3-binding (GLEBS) motif was originally char-
acterized in the nuclear pore complex protein Nup98 and found to be suffi-
cient for binding the mRNA export protein Gle2 (or Rae1) ( Wang et al.,
2001 ). However, there is no evidence that BubR1 interacts in vivo with Gle2.