T H E J O U R N A L O F C E L L B I O L O G Y
The Rockefeller University Press $30.00
J. Cell Biol. Vol. 183 No. 4 667–680
Correspondence to T. Yen: email@example.com
Abbreviations used in this paper: ACA, anticentromere antibody; APC/C, ana-
phase-promoting complex/cyclosome; CENP, centromere protein; KD, kinase
dead; MCC, mitotic checkpoint complex; NEB, nuclear envelope breakdown.
The online version of this article contains supplemental material.
BubR1 protein kinase is an essential component of the mitotic
checkpoint in metazoans ( Cahill et al., 1998 ; Taylor et al., 1998 ),
where one of its roles is to act as a mechanosensor that monitors
the microtubule attachment status of kinetochores through its
interaction with the kinesin-like motor centromere protein (CENP)
E ( Schaar et al., 1997 ; Chan et al., 1999 ; Yao et al., 2000 ; McEwen
et al., 2001 ; Mao et al., 2003 , 2005 ). In addition to its check-
point roles at the kinetochore, BubR1 is part of the mitotic check-
point complex (MCC) that acts downstream of the kinetochore
by directly inhibiting the anaphase-promoting complex/cyclo-
some (APC/C; Sudakin et al., 2001 ; Braunstein et al., 2007 ;
Musaro et al., 2008 ). Separate from its checkpoint functions,
BubR1 has been shown to be essential for proper kinetochore
microtubule attachments ( Lampson and Kapoor, 2005 ). Recently,
BubR1 was reported to associate with adenomatous polyposis
coli, and its kinase activity regulated the ability of the adenoma-
tous polyposis coli – EB1 complex to establish kinetochore/
microtubule attachments ( Kaplan et al., 2001 ; Zhang et al., 2007 ).
BubR1 is hyperphosphorylated in mitosis ( Chan et al.,
1999 ; Taylor et al., 2001 ), but its signifi cance to kinetochore
attachments and checkpoint regulation was not known. Recent
studies have shown that human BubR1 can be phosphorylated
by Plk1, and these modifi cations are critical for stable kineto-
chore attachments ( Elowe et al., 2007 ; Matsumura et al., 2007 ).
Similarly, Xenopus laevis BubR1 is a substrate of Plx1, and the
phosphorylation generates the 3F3/2 phosphoepitope that is im-
portant for the checkpoint in egg extracts ( Wong and Fang, 2007 ).
We report the identifi cation of four new mitosis-specifi c phos-
phorylation sites that are not targets of Plk1 or aurora B kinases.
Their functional importance was examined with phosphospe-
cifi c antibodies and various phosphomutants. We demonstrate
that the phosphorylation of S670 and S1043 at kinetochores is
sensitive to loss of microtubule attachments but not to tension.
This contrasts with the response of the Plk1 S676 phosphoryla-
tion that is sensitive to tension ( Elowe et al., 2007 ). Cells ex-
pressing phosphodefective (S to A) and phosphomimic (S to D)
BubR1 mutants were delayed in metaphase because of defective
phorylated in mitosis on four residues that differ from sites
recently reported to be phosphorylated by Plk1 (Elowe, S.,
S. Hummer, A. Uldschmid, X. Li, and E.A. Nigg. 2007.
Genes Dev. 21:2205 – 2219; Matsumura, S., F. Toyoshima,
and E. Nishida. 2007. J. Biol. Chem. 282:15217 – 15227).
S670, the most conserved residue, is phosphorylated at
kinetochores at the onset of mitosis and dephosphorylated
before anaphase onset. Unlike the Plk1-dependent S676
phosphorylation, S670 phosphorylation is sensitive to
ubR1 kinase is essential for the mitotic checkpoint
and also for kinetochores to establish microtubule
attachments. In this study, we report that BubR1 is phos-
microtubule attachments but not to kinetochore tension.
Functionally, phosphorylation of S670 is essential for error
correction and for kinetochores with end-on attachments to
establish tension. Furthermore, in vitro data suggest
that the phosphorylation status of BubR1 is important
for checkpoint inhibition of the anaphase-promoting com-
plex/cyclosome. Finally, RNA interference experiments show
that Mps1 is a major but not the exclusive kinase that
specifi es BubR1 phosphorylation in vivo. The combined
data suggest that BubR1 may be an effector of multiple ki-
nases that are involved in discrete aspects of kinetochore
attachments and checkpoint regulation.
Phosphorylation sites in BubR1 that regulate
kinetochore attachment, tension, and mitotic exit
Haomin Huang , 1 James Hittle , 1 Francesca Zappacosta , 2 Roland S. Annan , 2 Avram Hershko , 3 and Timothy J. Yen 1
1 Fox Chase Cancer Center, Philadelphia, PA 19111
2 GlaxoSmithKline, King of Prussia, PA 19406
3 Unit of Biochemistry, Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, Haifa 31096, Israel
© 2008 Huang et al. This article is distributed under the terms of an Attribution–
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tion date (see http://www.jcb.org/misc/terms.shtml). After six months it is available under a
Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license,
as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).
JCB • VOLUME 183 • NUMBER 4 • 2008 668
lacked pS670 and pS1043 staining even though BubR1 was still
detectable. Thus, S670 and S1043 are phosphorylated at kineto-
chores upon mitotic entry and are dephosphorylated at the onset
of anaphase ( Fig. 1 A and Fig. S2 C).
Examination of the phosphorylation status of BubR1 in
lysates that were harvested at various times after cells were re-
leased from a nocodazole block showed that S543, S670, and
S1043 were gradually dephosphorylated as cells exited mitosis
when compared with S435, which was completely dephosphor-
ylated by the time cyclin B1 was degraded (Fig. S2 E). We noted
that the kinetics of S670 and S1043 dephosphorylation, as de-
termined by blots, differed from the abrupt loss of phospho-
signal as determined by staining. This difference was attributed
to the fact that the cytosolic pool of BubR1 was extracted
from cells before fi xing and staining. When extraction was
omitted, pS670 signal was detected in the cytosol of early/
midanaphase cells (Fig. S2 F). As chromosomes were absent
from lysates used for blots, the phosphosignals likely refl ect
the cytosolic pool of BubR1.
We next tested whether the variable intensity of pS670 stain-
ing at kinetochores of prometaphase and early metaphase cells
was caused simply by changes in the amount of BubR1 or
refl ected differences in the phosphorylation state of BubR1.
We determined the relative BubR1 levels among individual kineto-
chores within a cell by normalizing the signals to the kineto-
chore with the highest intensity (100%) as described previously
( Hoffman et al., 2001 ; Feng et al., 2006 ; Liu et al., 2006 ; H. Huang
et al., 2007 ). The relative pS670 intensities among the identi-
cal set of kinetochores were determined by normalizing to the
strongest phospho-BubR1 signal (this comparison was indepen-
dent of the BubR1 normalization). By comparing the ratio of
the normalized intensities of pS670 with BubR1, it was possible
to determine whether the differences in BubR1 phosphorylation
were caused simply by changes in total BubR1 levels or actual
changes in its phosphorylation state ( Fig. 1 C ). A ratio of 1 indi-
cates that the phosphorylation was directly related to the amount
of BubR1 at the kinetochore. Ratios < 1 would indicate that
BubR1 was dephosphorylated at kinetochores, as shown in late
metaphase and early anaphase. Kinetochores in prometaphase tend
to exhibit stronger BubR1 phosphorylation (ratio > 1). The dis-
tribution pattern for early metaphase kinetochores was broader, and
there was a clear shift toward the dephosphorylated state. Thus,
the changes in BubR1 phosphorylation in prometaphase and meta-
phase probably refl ect differences in kinetochore attachments.
We next compared the pS670/BubR1 ratios in cells that
were treated with nocodazole and taxol to suppress microtubule
dynamics. This produced unattached kinetochores that are Mad1
positive as well as bipolar-attached kinetochores that lack de-
tectable Mad1 staining but fail to generate full tension ( Fig. 1 B ).
pS670/BubR1 ratios were increased at unattached kinetochores,
as was seen at many of the kinetochores in normal prometa-
phase ( Fig. 1 C ) and when microtubules were completely de-
polymerized after treatment with a high dose of nocodazole. BubR1
phosphorylation was reduced at many of the attached but ten-
sionless kinetochores. Thus, S670 is phosphorylated and de-
phosphorylated at kinetochores in response to the absence and
presence of microtubule attachments, respectively ( Fig. 1 C ).
kinetochore attachments that failed to generate proper levels of
tension. Furthermore, analysis of these phosphomutants suggests
that phosphorylation of S670 is critical for error correction at
kinetochores. Injection of phospho-BubR1 antibodies also de-
layed cells at metaphase because kinetochores failed to generate
proper levels of tension.
Using a cell-free system that recapitulated the checkpoint
events that lie downstream of the kinetochore ( Sudakin et al.,
2001 ; Braunstein et al., 2007 ), we found that the addition of
phospho-S670 (pS670) antibodies prolonged the inhibition of the
APC/C. Thus, the phosphorylation status of BubR1 may be a
critical determinant of checkpoint activity. Finally, we show that
Mps1 is a major upstream kinase of all four phosphorylation sites
in vivo. Combining our data with others suggests that multiple
kinases regulate BubR1 to facilitate proper kinetochore attach-
ments and checkpoint signaling.
BubR1 is differentially phosphorylated at
attached and unattached kinetochores
BubR1 was immunopurifi ed from extracts prepared from asynchro-
nous and nocodazole-blocked HeLa cells. Mass spectrometry
(Fig. S1, A and B, available at http://www.jcb.org/cgi/content/
full/jcb.200805163/DC1) identifi ed four major signals that cor-
responded to phosphoserines (S453, S543, S670, and S1043).
A minor peak at S676 was also identifi ed that was one of several
sites (S676, T792, and T1008) that were recently reported to
be phosphorylated by Plk1 ( Elowe et al., 2007 ; Matsumura et al.,
2007 ). Of the new phosphoresidues, S670 was conserved from
Drosophila melanogaster to humans, whereas the others exhib-
ited variable degrees of conservation among different species
(Fig. S1 C). Phosphoantibodies were raised against the four phos-
phorylation sites. Western blots of mitotic lysates treated and
untreated with ? protein phosphatase showed that all four anti-
bodies were phosphospecifi c (Fig. S2 A). Phosphospecifi city
of the pS670 and pS1043 antibodies was further confi rmed as
the signals obtained with blots were eliminated with phospho-
peptide but not with the unphosphorylated peptide (Fig. S2 B).
In all subsequent experiments, unphosphopeptides were used to
ensure phosphospecifi city. Only the pS670 and pS1043 anti-
bodies did not exhibit strong cross-reactivity with other phospho-
proteins in Western blots of whole cell lysates, which allowed
their use in immunocytochemistry.
Immunofl uorescence staining showed that both pS670 and
pS1043 antibodies produced identical patterns ( Fig. 1 A and
Fig. S2 C). Staining was sensitive to phosphopeptide but not to
the unphosphorylated peptide (Fig. S2 D). To assess the phos-
phorylation status of BubR1 at kinetochores during different
stages of mitosis, cells were costained with antibodies to detect
pS670 and S1043 (rabbit) and total BubR1 (rat; Fig. 1 A and
Fig. S2 C). BubR1 was detected at kinetochores as early as pro-
phase, but pS670 and pS1043 staining at kinetochores did not
appear until after nuclear envelope breakdown (NEB) when cells
entered mitosis. Phosphostaining remained detectable at kineto-
chores from prometaphase to metaphase. Some cells that were
presumably more advanced in metaphase and in early anaphase
669PHOSPHO - B UB R1 REGULATION OF KINETOCHORE FUNCTIONS • Huang et al.
Figure 1. BubR1 is differentially phosphorylated at attached and unattached kinetochores. (A) HeLa cells were stained for pS670, BubR1, and DAPI. The
arrows indicate kinetochores. Background signal is caused by incomplete extraction of the cytosol. (B) HeLa cells were blocked in mitosis with a low dose of
nocodazole (20 ng/ml) and stained for pS670, BubR1, Mad1, ACA, and DAPI. (C) Intensities of pS670 and BubR1 signals at individual kinetochores were
quantitated, and the values were normalized to the kinetochore with the highest intensity (100%). The normalized values of pS670 and BubR1 for each
kinetochore were used to obtain a ratio of pS670/total BubR1 for each kinetochore and plotted. An example of differential intensities of pS670 relative to
BubR1 at metaphase is presented. Kinetochores a – c showed weaker signals relative to d and e for pS670, whereas the same kinetochores, a ’ – e ’ , showed
equal signal intensities for BubR1. The dashed line indicates the position of the ratio of 1. Pro, prophase; Prometa, prometaphase; Meta, metaphase; Ana,
anaphase; misaligned and aligned, monopolar and bipolar attachments, respectively.
JCB • VOLUME 183 • NUMBER 4 • 2008 670
vestigated by characterizing a series of phosphodefective (S to A)
and phosphomimic (S to D) BubR1 mutants at a single or at all
four sites. The BubR1 mutants were tagged with either 3 × Flag or
GST and cloned into vectors with an H2B-GFP expression cas-
sette. RNAi-resistant alleles of the wild-type and BubR1 mutants
were generated to facilitate experiments that included RNAi
knockdown of endogenous BubR1 ( Fig. 2 B and Fig. S3 A, avail-
able at http://www.jcb.org/cgi/content/full/jcb.200805163/DC1).
We fi rst established that all of the mutants along with wild-type
These changes do not appear to be sensitive to the lack of ten-
sion as reported for the pS676 residue ( Elowe et al., 2007 ).
In vivo characterization of
As BubR1 contributes to attachment as well as checkpoint-
monitoring functions at the kinetochore, the changes in pS670
might refl ect either or both of these activities. The functional sig-
nifi cance of the mitotic phosphorylation sites in BubR1 was in-
Figure 2. Phospho-BubR1 mutants localize to kinetochores in cells depleted of endogenous BubR1. (A and B) HeLa cells depleted of endogenous BubR1
by siRNA were transfected with a vector that coexpressed 3 × Flag BubR1 RNAi (RNAi-resistant alleles) and H2B-GFP and were stained for Flag, hSgo2, and
DAPI (A), or the lysates were probed for CENP-F, BubR1, and Flag (B). Western blots show siRNA-depleted endogenous BubR1 but not the 3 × Flag-BubR1.
UN, untransfected cell lysate; WT, wild type.
671PHOSPHO - B UB R1 REGULATION OF KINETOCHORE FUNCTIONS • Huang et al.
wild-type BubR1 entered anaphase on average 50 min after
NEB, but some took as long as 120 min ( Fig. 3 B , left). The ma-
jority (80%) of the cells entered anaphase ? 20 min after reach-
ing metaphase. These times are similar to cells transfected with
H2B-GFP alone ( Fig. 3 B , middle). The majority of the cells
expressing the QA mutant took ? 100 min from NEB to ana-
phase, but cells that were delayed for > 250 min were also seen
BubR1 were able to localize to kinetochores in mitotic cells
( Fig. 2 A and Fig. S3, B and C). We used time-lapse video-
microscopy to track the fates of transfected cells that ex-
Initial experiments focused on testing the quadruple phos-
phodefective (QA) and phosphomimic (QD) mutants in the pres-
ence of endogenous BubR1 ( Fig. 3 A ). Cells expressing the
Figure 3. BubR1 phosphomutants delay the metaphase to anaphase transition. (A) HeLa cells released from a G1/S block were injected with 3 × Flag
constructs, and H2B-GFP-expressing cells were monitored by time-lapse microscopy (hours:minutes). Select frames from videos of cells injected with wild-
type (WT) BubR1 and the quadruple phosphomutants (QA and QD) are shown. The arrow points to lagging chromosomes. (B) The videos were analyzed
frame by frame, and the behavior of chromosomes was used to determine the percentage of cells from NEB to anaphase onset (left), from metaphase to
anaphase onset (middle), and the frequencies of lagging chromosomes (right). For wild type, n = 20; QA, n = 27; QD, n = 44; vector, n = 22. (C) HeLa
cells depleted of endogenous BubR1 by siRNA were released from a G1/S block and were injected with 3 × Flag – wild-type BubR1, KD, S670A, and S670D
phosphomutant and empty vector. The cells that expressed H2B-GFP were monitored by time lapse and were quantitated as in B. For wild type, n = 30;
S670A, n = 24; S670D, n = 21; KD, n = 35. Vec, control vector. (B and C) Error bars indicate the highest and lowest values within a dataset.
JCB • VOLUME 183 • NUMBER 4 • 2008 672
In contrast to the QA and S670A mutants, virtually all
( > 98%) of the attached kinetochores examined in the QD and
S670D mutants had end-on attachments ( Fig. 4, A and C ). Nev-
ertheless, the attachments were also defective, as they failed to
generate tension ( Fig. 4 B ). The failure of end-on attachments
to generate tension is reminiscent of kinetochores depleted of
CENP-E ( Fig. 4, B – D ; Yao et al., 2000 ; McEwen et al., 2001 ) or
when microtubule dynamics are dampened pharmacologically.
The combined data suggest that phosphorylation of S670 is
critically important for kinetochores to resolve aberrant attach-
ments. However, phosphorylation is also important for end-on
attachments to generate tension.
Phospho-BubR1 antibodies delay anaphase
onset in vivo and in vitro
The in vivo signifi cance of the pS670 and pS1043 residues was
independently investigated by injecting a panel of phospho- and
nonphospho-BubR1 antibodies into cells. HeLa cells released
from a double thymidine block were injected several hours
before they were scheduled to enter mitosis. Cells were either
fi xed for staining or were monitored by time-lapse microscopy.
We fi rst determined that the various BubR1 antibodies were
concentrated at kinetochores in the injected cells that had en-
tered mitosis (Fig. S4 A, available at http://www.jcb.org/cgi/
content/full/jcb.200805163/DC1). As shown previously ( Chan
et al., 1999 ; Shannon et al., 2002 ), cells injected with plain
BubR1 antibodies prematurely exited mitosis before chromosomes
achieved metaphase alignment. In support of our ( Chan et al.,
1999 ) and others ’ RNAi experiments ( Meraldi et al., 2004 ), the
BubR1 antibody – injected cells exited mitosis ? 10 min earlier
than cells injected with nonimmune IgG. In contrast, cells injected
with either the pS670 or pS1043 antibodies exhibited a meta-
phase delay of between 30 and 100 min before entering ana-
phase ( Fig. 5, A and B ).
We were able to rule out nonspecifi c steric effects by the
injected pS1043 antibodies as an explanation for the metaphase
delay. We had fortuitously raised a nonphosphoantibody (T1042)
that spanned the same epitope used to raise the pS1043 anti-
bodies. Despite the fact that the injected T1042 antibodies were
concentrated at kinetochores (Fig. S4 A), they did not interfere
with chromosome alignment or with mitotic progression ( Fig. 5,
A and B ). Given that the peptides used to raise antibodies be-
tween T1042 and pS1043 were virtually identical, the clear dif-
ference in their in vivo response strongly suggests that the
effects of the pS1043 antibodies were phosphospecifi c.
We attempted to detect spindle or kinetochore defects in
the phospho-BubR1 antibody – injected cells to identify a cause
for the metaphase delay. Antibody injections did not interfere
with the formation of a bipolar spindle or the accumulation of
Mad1 and CENP-E at kinetochores (Fig. S4 C). Furthermore,
the injected antibodies did not interfere with normal kineto-
chore attachments as determined by their stability to cold treat-
ment and the end-on nature of the microtubule attachments
( Fig. 5 C ). Consistent with the behavior of the phosphomutants,
the interkinetochore distances in the cells injected with pS670
antibody (1.0 ± 0.1 μ m) were reduced relative to the control
(2.0 ± 0.2 μ m).
( Fig. 3 B , left). The QA mutant took approximately the same
amount of time as wild-type and QD cells to align their chromo-
somes even though the metaphase plates were qualitatively not
as tight as cells expressing wild type or the QD mutant ( Fig. 3 A ).
Compared with wild type, QA-expressing cells were delayed at
metaphase for 20 – 100 min ( Fig. 3 B , middle). The metaphase de-
lay was likely caused by aberrant attachments, as > 60% of the
QA cells that entered anaphase exhibited lagging chromosomes
as compared with ? 5% seen in wild-type BubR1-expressing
cells ( Fig. 3 B , right). The majority (70%) of the cells that ex-
pressed the QD mutant also exhibited a metaphase delay ( Fig. 3 B ,
left and middle). However, the frequency of lagging chromo-
somes exhibited by the QD mutant was threefold lower than the
QA mutant ( Fig. 3 B , right).
We next characterized the single phosphodefective (S670A)
and phosphomimic (S670D) mutants in cells whose endogenous
BubR1 were depleted by siRNA. This residue was selected be-
cause it was conserved among all vertebrate species examined.
Time-lapse experiments showed that depletion of BubR1 accel-
erated cells into anaphase as previously described ( Meraldi
et al., 2004 ). Transfected wild-type BubR1 rescued the BubR1-
depleted cells, as the kinetics of mitotic progression was restored
to that seen in cells transfected with vector alone ( Fig. 3 C , left
and middle). Cells expressing either the S670A or S670D mu-
tants were able to achieve metaphase alignment with normal
kinetics but were delayed by 20 to > 150 min from entering ana-
phase ( Fig. 3 C , left and middle). Over 90% of the cells depleted
of BubR1 exhibited lagging chromosomes upon entry into ana-
phase ( Fig. 3 C , right). The S670A mutant consistently failed to
reduce the frequency of lagging chromosomes to that obtained
with wild type and the S670D mutant. This suggests that the
failure to phosphorylate S670 generates aberrant attachments
that are not resolved when cells enter anaphase ( Fig. 4 ).
We discovered that the kinase-dead (KD) BubR1 mutant
( Elowe et al., 2007 ; Matsumura et al., 2007 ) exhibited a mixed
phenotype when compared with the phospho-BubR1 mutants
( Fig. 3 C , left and middle). As with wild-type BubR1, the KD
mutant restored mitotic progression to the BubR1-depleted cells,
but unlike the phospho-BubR1 mutants, they did not exhibit a
pronounced delay. Nevertheless, the high frequency of lagging
chromosomes in anaphase indicated the presence of defective
attachments that were not resolved at the time of exit. The mixed
phenotype may be best explained if the KD mutant affected both
kinetochore attachments and mitotic checkpoint control.
We used cold treatment to directly assess the integrity of the
kinetochore attachments in BubR1-depleted cells that expressed
the various transfected BubR1 mutants ( Fig. 4 ). All of the attach-
ments (100%) examined in wild-type – transfected cells exhibited
proper bipolar end-on microtubule attachments that were under
full tension ( Fig. 4, A – C ). As predicted, 13% and 11% of all attach-
ments in the QA and the S670A mutants, respectively, exhibited
defects that included lateral, monotelic, syntelic, and others ( Fig. 4,
A and C ). These defects were very similar to those seen in cells
expressing the KD BubR1 mutant. Although lack of tension gener-
ated by these defective attachments is likely responsible for the
transient metaphase delay, it is noteworthy that kinetochores that
established end-on connections also failed to generate tension.
673 PHOSPHO - B UB R1 REGULATION OF KINETOCHORE FUNCTIONS • Huang et al.
Figure 4. Phosphorylation of S670 is required for error correction and kinetochore tension. (A) Synchronized HeLa cells depleted of BubR1 by siRNA were
injected with various GST/BubR1 constructs and treated with MG132 to prevent mitotic exit. Coverslips were chilled on ice for 10 min, extracted, fi xed,
and stained for GST, ACA, tubulin, and DAPI. Tubulin and ACA signals were deconvolved to visualize microtubule attachments at individual bioriented
kinetochores that are shown in the insets. (B) Interkinetochore distances were measured between pairs of ACA foci with end-on attachments (50 < n < 100).
Black bars represent the mean. (C) Histogram depicting the relative frequency of end-on and aberrant attachments ( n > 50) in cells expressing various
BubR1 constructs. Error bars indicate the range for each dataset. (D) HeLa cells depleted of CENP-E were cold treated and stained for ACA and tubulin to
depict end-on attachments and kinetochore tension. WT, wild type.
JCB • VOLUME 183 • NUMBER 4 • 2008 674
in vitro, as addition of the phospho-BubR1 antibodies into
mitotic HeLa lysates interfered with the ability of ? protein
phosphatase to dephosphorylate endogenous BubR1 ( Fig. 6 A ).
We tested whether the ability of the pS670 and pS1043
antibodies to delay anaphase onset might be caused by the pres-
ervation of these phosphoresidues. This possibility was confi rmed
Figure 5. Phospho-BubR1 antibodies delay anaphase onset in vivo. (A) Select frames from time-lapse videos of HeLa H2B-GFP that were injected with the
indicated antibodies. (B) Videos were analyzed frame by frame to determine the percentage of cells from NEB to anaphase onset. For pS670, n = 20;
pS1043, n = 20; T1042, n = 30; BubR1, n = 12; control IgG, n = 23. Error bars represent the highest and lowest values for each timepoint. (C) Cells
injected with nonimmune and pS670 IgG were cold treated and stained for ACA and tubulin to visualize kinetochore attachments (insets).
675 PHOSPHO - B UB R1 REGULATION OF KINETOCHORE FUNCTIONS • Huang et al.
the endogenous substrates for APC/C ( Fig. 6 B ). Addition of
different amounts of pS670 antibodies to the extracts prolonged
the repression of APC/C activity in a dose-dependent fashion
( Fig. 6 C ). Furthermore, the effects of the phospho-BubR1 anti-
bodies were neutralized if the antibodies were preincubated briefl y
with a 1.7-fold excess of phosphopeptide. APC/C activation
was not delayed when similar amounts of the T1042 BubR1
antibody were added to the extract. Furthermore, the addition of
plain BubR1 antibodies accelerated the onset of APC/C activity
( Fig. 6 C ). Thus, the responses of the extracts to the pS670, T1042,
and plain BubR1 antibodies were similar to the response of cells
that were injected with the same set of antibodies.
The level of pS670 in the extracts was reduced by 30 min
of incubation and continued to decline ( Fig. 6 D ). At 90 min, the
level of Cdc27 phosphorylation that is specifi ed in large part by
Cdk1 ( Rudner and Murray, 2000 ; Kraft et al., 2003 ; J.Y. Huang et al.,
2007 ) was reduced. Addition of pS670 antibodies delayed the
dephosphorylation rate of Cdc27 relative to controls. Based on
these in vitro studies, one explanation for how the injected phospho-
BubR1 antibodies delayed metaphase is that they preserved
the phosphorylation of BubR1, and this prolonged its ability to
inhibit the APC/C. Similarly, the phosphomimic BubR1 mutants
These experiments do not exclude other possibilities in which
the phosphoantibody blocks the interaction of p670 BubR1 with
critical factors important for generating kinetochore tension.
The metaphase delay exhibited by the phospho-BubR1
mutants can be ascribed to defective kinetochore attachments.
However, the delay may also be caused by the fact that BubR1
can act downstream of the kinetochore as an inhibitor of the
APC/C. We and recently others ( Sudakin et al., 2001 ; Braunstein
et al., 2007 ) have shown that BubR1 is part of the MCC that
binds to and potently inhibits the APC/C in mitotic HeLa cells.
Using a cell-free system (extracts prepared from nocodazole-
arrested HeLa cells) that recapitulates the mitotic checkpoint
events that lie downstream of the kinetochore ( Braunstein et al.,
2007 ), we tested whether addition of the phospho-BubR1 anti-
bodies altered the kinetics of checkpoint inhibition. As shown
previously ( Braunstein et al., 2007 ), control extracts incubated
at 30 ° C remained in a checkpoint-arrested state for a brief pe-
riod before inhibition of APC/C is relieved, as is evident by the
degradation of endogenous cyclin B1 (or securin; Fig. 6 B and
not depicted). As has been shown, the degradation was depen-
dent on the APC/C, as the addition of peptide containing the
wild-type destruction box of cyclin B1 effectively competed with
Figure 6. Phospho-BubR1 antibodies prolong the lag in the APC/C activation in cell extracts. (A) Mitotic extracts were incubated with ? protein phospha-
tase (PPase) in the presence of pS670 or nonimmune IgG for 0, 30, 60, and 120 min. Membranes were probed for BubR1 to see changes in the mobility
of BubR1 (top) and pS670 status (bottom). (B) Mitotic extracts were incubated with and without the N terminus of cyclin B1 (CycB), and samples were taken
at various times and probed for endogenous cyclin B1. WT-N, wild-type cyclin B1 N-terminus fragment. (C) Extracts were incubated in the presence of the
indicated antibodies (Abs) for various times, and the level of endogenous cyclin B1 was determined. 1 × = 0.8 μ M of antibody or phosphopeptide. (D) Cell
extracts with and without pS670 antibody were probed to evaluate the phosphorylation status of BubR1 (pS670) and Cdc27 (based on mobility shifts).
JCB • VOLUME 183 • NUMBER 4 • 2008 676
sensitive to kinetochore tension. Interestingly, this is opposite
of the increased phosphorylation of S676 that was recently re-
ported to result from the loss of tension ( Elowe et al., 2007 ).
Given that the sites reported here are not targets of Plk1, the dif-
ference in the phosphorylation response to loss of tension sug-
gests that BubR1 is differentially phosphorylated depending on
the status of microtubule attachment and tension.
Phosphorylation of S670 appears to be important for error
correction, as kinetochores containing the S670A mutant accu-
mulated aberrant attachments at an ? 15-fold higher frequency
than wild type. This conclusion is strengthened by the fact that
the S670D phosphomimic mutant is able to prevent the accumu-
lation of defective attachments as effectively as wild-type BubR1.
The inability to correct these defective attachments by the S670A
mutant explains the high incidence of lagging chromosomes in
anaphase. As the attachment defects of the S670A mutant were
similar to the KD mutant, the phosphorylation of S670 may act
through its kinase domain ( Kaplan et al., 2001 ; Matsumura et al.,
2007 ; Zhang et al., 2007 ). The defective attachments reported
here are similar to those reported for the Plk1 phosphodefective
mutants ( Elowe et al., 2007 ; Matsumura et al., 2007 ). Thus, it is
possible that phosphorylation of BubR1 at S670 affects its
could have contributed to the metaphase delay by extending the
actions of the cytosolic inhibitor of the APC/C in addition to their
effects on kinetochore attachments.
Mps1 is required to phosphorylate BubR1
Recent studies showed that BubR1 is phosphorylated by Plk1
( Elowe et al., 2007 ; Matsumura et al., 2007 ) and aurora B
( Ditchfi eld et al., 2003 ; Hauf et al., 2003 ). Western blots using
the four phosphopeptide antibodies showed that none of them
were affected in mitotic cells treated with the aurora kinase
inhibitor hesperadin. Furthermore, treatment of cells with rosco-
vitine, a Cdk inhibitor, or staurosporine did not affect these
phosphorylation sites (Fig. S5, A and B, available at http://www
.jcb.org/cgi/content/full/jcb.200805163/DC1). pS670 levels were
unaffected in mitotic cells that were depleted of aurora B or Plk1
by siRNA (Fig. S5, C – E). In cells that were depleted of > 90%
of their Mps1 kinase, phosphorylation of S435, S543, S670, and
S1043 was reduced to ? 25% of control levels ( Fig. 7 ). Mps1 is
a major but not the exclusive kinase that specifi es BubR1 phos-
phorylation in vivo.
BubR1 is hyperphosphorylated in mitosis, and some of the sites
depend on aurora B and Plk1 kinases ( Ditchfi eld et al., 2003 ;
Elowe et al., 2007 ; King et al., 2007 ; Lenart et al., 2007 ;
Matsumura et al., 2007 ). Although the sites that are dependent
on aurora B remain to be identifi ed, three candidate Plk1 sites
were recently reported in human BubR1 ( Elowe et al., 2007 ;
Matsumura et al., 2007 ; Wong and Fang, 2007 ). However, only one
(S676) of the three sites was directly confi rmed to exist in vivo.
We have identifi ed and confi rmed with phosphoantibodies the
existence of four additional mitosis-specifi c phosphorylation
sites (S435, S543, S670, and S1043) that are not dependent on
aurora B or Plk1 kinases. Instead, Mps1 appears to be a major,
but not the only, kinase responsible for these phosphorylations
in vivo. Whether it directly or indirectly phosphorylates BubR1
will require in vitro experiments. The contribution of Cdk1 is
unclear despite the fact that the addition of Cdk inhibitor to mi-
totic cells failed to inhibit phosphorylation of BubR1. It is possi-
ble that Cdk1 is important for phosphorylating BubR1 at the
onset of mitosis and is not essential for maintaining the phos-
phorylated state. Finally, it is also possible that some of the phos-
phorylation sites may be caused by BubR1 autokinase activity.
Immunofl uorescence staining with pS670 and pS1043
antibodies showed that they fi rst appeared at kinetochores at the
onset of mitosis and were dephosphorylated at late metaphase
and in early anaphase. Quantitative analysis showed that changes
in the levels of pS670 at kinetochores were not caused solely by
the fl uctuations in BubR1 levels ( Hoffman et al., 2001 ). Thus,
phosphorylation of S670 (and pS1043) indeed increased in re-
sponse to unoccupied microtubule-binding sites as seen during
prometaphase. Upon microtubule attachment, S670 is de-
phosphorylated such that it is no longer detectable by late meta-
phase when kinetochores are fully saturated with microtubules.
We found that dephosphorylation of pS670 was not directly
Figure 7. Mps1 is required to phosphorylate BubR1 in cells. Cells trans-
fected with control, and Mps1 siRNA were released from a double thymi-
dine block. MG132 was added 8 h later, and mitotic cells were harvested
2 h later by shakeoff. Lysates were probed with all four phospho-BubR1
antibodies. Two different amounts of control lysates were used, so it was
possible to directly compare the band intensities with Mps1-depleted lysate.
CENP-F, BubR1, and cyclin B1 were used as loading controls.
677 PHOSPHO - B UB R1 REGULATION OF KINETOCHORE FUNCTIONS • Huang et al.
defective attachments suggests that these phosphorylation sites
are not essential for BubR1 ’ s ability to delay mitotic exit. Indeed,
the QA or QD mutants did not interfere with the cells ’ ability to
become arrested in mitosis for a prolonged period ( > 6 h) in re-
sponse to nocodazole (unpublished data). The reason why the
phosphomutants were only able to mount a transient delay in
response to the attachment defects may be because the amount
of wait anaphase signal generated from these kinetochores is
not as high as the level generated by unattached kinetochores.
This is supported by the fact that Mad1 (and thus Mad2) check-
point protein is no longer present at the kinetochores that have
established microtubule attachments. In the case of nocodazole
treatment, Mad1 and Mad2 are recruited back to the kineto-
chores, and the full complement of checkpoint proteins is avail-
able to generate a robust level of wait anaphase signal. Similar
arguments were used to explain why cells depleted of CENP-F
and hSgo2 only delay transiently despite the presence of numerous
aberrant attachments ( Feng et al., 2006 ; H. Huang et al., 2007 ).
The behavior of the BubR1 KD mutant was complex. As
mentioned in the Results, we believe that kinase activity is re-
quired for error correction. However, unlike the phosphomutants
that exhibited a transient metaphase delay, the KD mutant exited
mitosis with kinetics similar to wild-type cells. As the anaphase
cells exhibited a high frequency of lagging chromosomes, the
KD mutant exited mitosis not because it had repaired its attach-
ment defects but most likely because of its inability to maintain
the delay. The possibilities are that the KD mutant prevented the
defective kinetochores from generating a wait anaphase signal
and that it may also have failed to sustain the checkpoint signal-
ing events that are required to inhibit the APC/C. Others have
reported that the kinase activity of BubR1 is not essential for
checkpoint activity ( Tang et al., 2001 ; Chen, 2002 ; Harris et al.,
2005 ; Rancati et al., 2005 ; Kiyomitsu et al., 2007 ; Malmanche
et al., 2007 ). Although this may be true for nocodazole treatment,
the large number of unattached kinetochores could collectively
generate suffi cient amounts of signal to sustain a checkpoint de-
lay. In contrast, subtle kinetochore defects (as seen here) that may
not generate robust wait anaphase signals may depend more criti-
cally on BubR1 kinase activity. Indeed, our original claim that
kinase activity was important for the mitotic checkpoint was
based on the inability of a BubR1 KD mutant to delay cells that
had only a few unattached kinetochores as a result of treatment
with a low dose of nocodazole ( Chan et al., 1999 ). The impor-
tance of BubR1 kinase activity in the spindle checkpoint was also
reported in Xenopus , where mutants that lacked kinase activity
failed to sustain a checkpoint arrest ( Wong and Fang, 2007 ).
Analysis of cytosolic extracts prepared from cells released
from a nocodazole block for different times revealed that only
S435 was completely dephosphorylated with the same kinetics
as cyclin B1 degradation. The signifi cance of the S435 site to
kinetochore attachments or checkpoint regulation remains to be
investigated. The phosphorylation of the remaining three sites
gradually declined as cells exited mitosis. By staining unextracted
cells, we confi rmed that phosphorylation of at least S670 was
detectable in the cytosol of early anaphase cells. The reduced
level of these phosphorylations in anaphase cells suggests that
there may be a threshold that maintains the mitotic state.
kinase activity or the ability of the other sites to be phos-
phorylated by Plk1. However, it remains to be seen whether the
phosphorylation sites identifi ed in this study regulate BubR1
Mechanistically, phosphorylation of BubR1 may be re-
quired for error detection and correction. As long as end-on at-
tachments are not made, BubR1 remains phosphorylated so that
the error correction system remains active. The kinetochore lo-
calization of MCAK (mitotic centromere – associated kinesin),
aurora B, and hSgo2, proteins thought to partly contribute to the
error correction system ( Desai et al., 1999 ; Hauf et al., 2003 ;
Andrews et al., 2004 ; Kline-Smith and Walczak, 2004 ; Lan et al.,
2004 ; Cimini et al., 2006 ; H. Huang et al., 2007 ), were not notice-
ably affected by the phospho-BubR1 mutants (unpublished data).
As the frequency of attachment defects exhibited by the phospho-
BubR1 mutants seems to be higher than that reported for cells
depleted of MCAK, other systems may also be affected. More
recent data that link BubR1 with blinkin, a subunit of the Mis12
complex ( Kline et al., 2006 ; Kiyomitsu et al., 2007 ), suggest
another explanation for the origin of the defective attachments
seen in the phosho-BubR1 mutants.
Our experiments also showed that BubR1 is required by
kinetochores that have established end-on microtubule attach-
ments to develop tension. This was made evident by the fact that
both of the S670A and S670D mutants were unable to generate
normal levels of tension at kinetochores that established end-on
attachments. Whether phosphorylation of S670 is essential for
generating tension is diffi cult to say, as the S670D mutant was
as equally defective as the S670A mutant. One possibility is that
the aspartic acid (D) cannot functionally substitute for a phospho-
serine, as in a case where S670 has to undergo cycles of phosphor-
ylation and dephosphorylation. Conceptually, the S670 residue
may be critical for BubR1 to stimulate downstream kinetochore
components to generate tension. As the tension defect exhibited
by the S670 mutants is very similar to that seen when CENP-E
is depleted from kinetochores ( Yao et al., 2000 ; McEwen et al.,
2001 ), it is possible that BubR1 regulates CENP-E ’ s ability to
generate tension after microtubules have established end-on con-
nections. Although the frequency of lagging chromosomes ex-
hibited by the S670D mutant is low, we cannot exclude the
possibility that the failure to generate tension might result in chromo-
some nondisjunction, which would not be detected by conven-
tional fl uorescence microscopy.
Our results suggest that BubR1 phosphorylation at S670
acts in error correction and in promoting tension to end-on at-
tachments. It is noteworthy that immuno-EM data showed that
BubR1 is localized primarily to the outer kinetochore plate in
metaphase cells, but in prometaphase cells it was also found to
occupy a second zone that was situated near the inner plate
( Jablonski et al., 1998 ). It is possible that the two functions as-
cribed for phospho-BubR1 refl ect discrete functional domains
that are spatially separated within the kinetochore. The loss of
the inner BubR1 labeling in metaphase cells could also refl ect
the reduction of BubR1 that is seen at the light level when micro-
The fact that cells expressing the BubR1 phosphomutants
are able to induce a transient metaphase delay in the presence of
JCB • VOLUME 183 • NUMBER 4 • 2008 678
length BubR1 cDNA was cloned into pENTR (Gateway; Invitrogen) to facili-
tate transfer into Destination vectors by in vitro recombination reactions.
A QuikChange Site-Directed Mutagenesis kit (Agilent Technologies) was
used to mutate phosphorylation sites as well as to introduce RNAi-resistant
alleles of BubR1. To make phosphospecifi c antibodies, phosphopeptides
were coupled to keyhole limpet hemocyanin and used to immunize rabbits
(Babco). Serum was passed through a phosphopeptide affi nity column. The
eluted antibodies that contain a mixture of phospho- and nonphospho-
antibodies were passed through a nonphosphopeptide column. The fl ow
through was tested for phosphospecifi city.
Cell culture and RNAi
HeLa cells were grown in DME + 10% FBS in a humidifi ed incubator at 37 ° C.
Nocodazole was used at 20-nM (low) and 60-nM (high) fi nal concentrations.
BubR1 siRNA, CAGGAACAACCTCATTCTAAA, was obtained from
QIAGEN. siRNAs were diluted in serum-free OptiMEM and HiPerfect
(QIAGEN) as per the manufacturer ’ s instructions and added to cells so that
the fi nal concentration of siRNA was 20 nM. 24 – 36 h after transfection,
cells were fi xed and stained or lysed in SDS sample buffer.
Cell synchronization and microinjection
Cells were synchronized at the G1/S boundary by a double thymidine
block. Cells were grown in the presence of 2 mM thymidine for 15 h,
washed, and released into fresh medium for 9 h, and thymidine was added
for another 15 h. 5 – 6 h after thymidine release, 2 – 4 mg/ml of antibodies
in PBS was injected into the cytoplasm of cells. Cells were either fi xed sev-
eral hours later for immunofl uorescence staining or monitored by time-lapse
microscopy. To analyze BubR1 mutants in cells depleted of endogenous
BubR1, siRNAs were transfected at the time of the fi rst thymidine block.
DNA was injected as described previously ( Hagting et al., 2002 ; Di Fiore
and Pines, 2007 ) into the nucleus of cells the next day, before the second
round of thymidine was added. Plasmid DNA for injections was diluted in
PBS into a fi nal concentration of 50 – 75 ng/ μ l. Injections were performed
with a semiautomated microinjector (model 5242; Eppendorf) attached to
an inverted microscope (Eclipse TE300; Nikon). The cells were plated onto
no. 1.5 coverslips (18 × 18 mm), and the injection area was scribed with
a diamond pen. All cells ( ? 300 – 400) within the scribed loop were
injected, and viability was > 95% at 2 – 3 h after injection.
Cells were fi xed for 7 min in freshly prepared 3.5% paraformaldehyde/
PBS, pH 6.9, extracted in KB (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, and
0.1% BSA) with 0.2% Triton X-100 for 5 min at room temperature, and
rinsed in KB. In some cases, cells were preextracted for 90 s before fi xing.
Primary and secondary antibodies were diluted in KB and added to cover-
slips for 30 min at 37 ° C in a humidifi ed chamber. Commercial antibodies
to tubulin (Sigma-Aldrich), GST (Cell Signaling Technology), Flag (Sigma-
Aldrich), and Plk1 (Santa Cruz Biotechnology, Inc.) were used. Human anti-
centromere antibody (ACA) serum was provided by J.B. Rattner (University
of Alberta, Calgary, Canada). Antibodies to human BubR1, CENP-E, and
Mad1 were obtained from our laboratory ( Liao et al., 1994 ; Chan et al.,
1998 ; Jablonski et al., 1998 ; Campbell et al., 2001 ). Antibodies were
used at a fi nal concentration of 0.5 – 1.0 μ g/ml. Secondary antibodies con-
jugated to Alexa Fluor 488, 555, and 647 (Invitrogen) were used at 1 μ g/ml.
Images were visualized with a 100 × /1.4 NA objective attached to an
inverted microscope (Eclipse TE2000S; Nikon), and 0.25 – 1- μ m image
stacks were captured with a charge-coupled device camera (Photometrics
Cascade 512F; Roper Scientifi c). Raw images were analyzed with Meta-
Morph software (MDS Analytical Technologies). Images are presented as
maximum projections and quantitated as previously described ( Hoffman
et al., 2001 ). Deconvolution was conducted with AutoQuant (Media Cyber-
netics). All image fi les were reformatted as TIFF fi les, and Photoshop
(Adobe) was used to assemble the fi gures. For time-lapse studies, HeLa or
HeLa – GFP-H2B was plated onto No. 1.5 coverslips (18 × 18 mm) in Hepes-
buffered medium and imaged with either an Eclipse TE300 or TE2000S in-
verted microscope. Images were captured every 5 – 10 min overnight at
37 ° C and processed with ImagePro Plus software (Media Cybernetics).
Online supplemental material
Fig. S1 shows the identifi cation of BubR1 phosphorylation sites by mass
spectrometry. Fig. S2 shows the specifi city of BubR1 phosphoantibodies
and phosphorylation states of BubR1 in vivo. Fig. S3 shows that phos-
pho-BubR1 mutants do not disrupt Mad1 and CENP-E localization at
kinetochores. Fig. S4 shows that injected antibodies are concentrated
at kinetochores, and antibody injections do not affect formation of the
spindle and localization of Mad1 and CENP-E at kinetochores. Fig. S5
We showed that the addition of phospho-BubR1 antibodies
to checkpoint-arrested extracts delayed the activation of the
APC/C. These extracts were made from HeLa cells that were
arrested in mitosis with nocodazole. In the absence of chromo-
somes, APC/C is reactivated after a 30-min lag. During the lag,
APC/C is inhibited by the checkpoint, as shown recently by as-
sociation of the MCC with the APC/C ( Braunstein et al., 2007 ).
This was further reinforced in this study, as the addition of
BubR1 antibodies, the same ones that abrogated the checkpoint
in cells, accelerated the activation of APC/C. The ability of the
pS670 antibodies to extend the lag in a dose-dependent manner
suggests that the phosphorylation state of BubR1 in the extracts
is a critical determinant of APC/C inhibitory activity. Although
there are many explanations for how the phosphoantibodies act,
the most straightforward explanation for which we have evi-
dence is that they preserve the phosphorylation state of BubR1.
As the majority of BubR1 in the extracts is associated with the
MCC ( Sudakin et al., 2001 ), we believe that phosphorylation of
BubR1 is important for MCC inhibitory functions. Indeed, the
pS670 levels are lowered when APC/C is reactivated. These
in vitro fi ndings can be used to explain how the injected pS670
antibodies and the S670D phosphomutant delayed cells in mitosis.
In addition to their actions at the kinetochore, the antibodies and
phosphomimic mutant can prolong the inhibition of the APC/C
as part of the MCC.
The newly identifi ed phosphorylation sites in BubR1 add
to the complexity of its regulation. The fact that BubR1 is phos-
phorylated (directly or indirectly) by multiple kinases such as
Plk1, aurora B, and Mps1 suggests that it is an effector of multi-
ple upstream events. Therefore, BubR1 may integrate and co-
ordinate many of the early events that are required to capture and
establish end-on microtubule attachments to later events such as
the generation of tension and silencing of the checkpoint signal-
ing. In addition, phosphorylation of the cytosolic pool of BubR1
appears to be critically important for regulating mitotic exit.
Materials and methods
BubR1 was immunopurifi ed from asynchronous and mitotically arrested
HeLa cells with BubR1 antibodies that were coupled to protein A beads.
Samples were separated on a precast 4 – 12% denaturing gel (Novex) and
stained with Colloidal blue (Invitrogen). Bands corresponding to BubR1
were excised, reduced, alkylated, and digested with trypsin in situ as de-
scribed previously ( Joyal et al., 1997 ). An aliquot of each tryptic digest was
concentrated on a pipette (C18 ZipTip; Millipore), eluted with 2:1 metha-
nol/ammonium hydroxide (30% vol/vol), and loaded into a nanospray
needle for analysis by precursor ion-scanning mass spectrometry. Precursor
ion spectra for m/z 79, a selective marker for phosphopeptides, were re-
corded on a triple quadrupole mass spectrometer (API 3000; Sciex)
equipped with a nanoelectrospray source and operated in the negative-ion
mode ( Gomez et al., 2007 ). Phosphopeptides identifi ed by precursor ion
scanning were sequenced by liquid chromatography tandem mass spec-
trometry. Peptides were loaded on a trap cartridge and backfl ushed at
300 nl/min to a 75- μ m inner diameter 15-cm column (C18 Zorbax; Agilent
Technologies) or to a 75- μ m inner diameter 15-cm column (PepMap C18;
Applied Biosystems) using an acetonitrile/water containing 0.1% formic acid
gradient. The mass spectrometer was set to perform full-time tandem mass
spectrometry on precursor ions selected for each target phosphopeptide.
DNA and antibodies
BubR1 was PCR amplifi ed from a cDNA library (marathon-ready cDNA;
Clontech Laboratories, Inc.) and confi rmed by sequence analysis. The full-
679 PHOSPHO - B UB R1 REGULATION OF KINETOCHORE FUNCTIONS • Huang et al.
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tion. Online supplemental material is available at http://www.jcb.org/
We would like to acknowledge expert services provided by B. Connor, the
core facilities at Fox Chase Cancer Center that include the Laboratory Animal
Facility, Hybridoma facilities, and the DNA synthesis and sequencing facilities.
We also gratefully acknowledge S.T. Liu for insightful discussions and J. Jackson
and P. Huang (GlaxoSmithKline, Collegeville, PA) for providing support to raise
the BubR1 phosphoantibodies.
This work was supported by grants from the Leukemia and Lymphoma
Society, the National Institutes of Health (GM86877 and GM44762), a core
grant (CA06927), an appropriation from the Commonwealth of Pennsylvania,
and the Greenberg Fund. H. Huang is supported by the Plain and Fancy Fel-
lowship from the Fox Chase Cancer Center Board of Associates.
Submitted: 28 May 2008
Accepted: 21 October 2008
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