New Insights into the Role of BubR1 in Mitosis and Beyond - International Review of Cell and Molecular Biology - page 239

precisely the same position as does free Cdc20. This suggests that MCC may
also inhibit the APC/C by a noncompetitive mechanism affecting the active
site, blocking recognition of both KEN-box and D-box substrates. Perhaps,
the KEN2 domain is involved in this process.
Finally, binding of BubR1/Mad3 to Cdc20 helps convert Cdc20 into an
APC/C substrate itself, targeting it for degradation ( King et al., 2007;
Nilsson et al., 2008 ) and providing yet one more potential layer of regulation
of the APC/C.

4.2.2 BubR1 kinase activity and the SAC: Is the kinase already ?

A major difference between yeast Mad3 and most metazoan BubR1s is the
presence of a kinase domain in the latter. Some studies suggested that the
kinase domain of BubR1 was not required for the inhibition of the APC/C
( Fang, 2002; Tang et al., 2001 ) and the SAC function ( Chen, 2002; Elowe
et al., 2007 ). But other reports found that the kinase-dead (KD) BubR1 pro-
tein did not completely restore the checkpoint function ( Kops et al., 2004;
Mao et al., 2003 ). More recently, another group reported that BubR1 kinase
activity is dispensable for short-duration SAC delay of anaphase, per se but
may be important for prolonged nocodazole-induced mitotic arrest
( Malureanu et al., 2009 ).
In Drosophila ,a bubR1-KD allele was reported to sustain a functional
SAC for up to 30 min in otherwise unperturbed in larval neuroblasts (where
the normal duration of mitosis from NEB to anaphase is just 9 min). How-
ever, bubR1-KD cells treated with the spindle poison colchicine for an hour
did show evidence of higher rates of mitotic exit ( Rahmani et al., 2009 ).
These results suggest that the kinase activity in flies may be needed, but only
for long-term maintenance of metaphase arrest.
Suijkerbuijk et al. (2012a) reported very recently that most vertebrate
(but notably not Drosophila ) BubR1 is likely to be a pseudokinase. Despite
retaining three catalytic residues essential for a conventional kinase (K795,
D882, and D911), most vertebrate BubR1s seem to have lost several other
residues normally conserved in functional kinases. The authors also provided
evidence that the K795Rmutation commonly used to generate a “KD” ver-
sion of BubR1 was inadvertently destabilizing the whole protein. The
defects in SAC and abnormal chromosome attachment (see Section 3.2 )
found in studies employing this mutant were therefore likely to be, at least
partially, and possibly entirely, a consequence of overall destabilization of
BubR1, rather than a specific consequence of knocking out the kinase activ-
ity. To reassess the mitotic contribution of the putative kinase activity, the

“ dead ”