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. 2 267–277
Correspondence to Hiroaki Kiyokawa: Kiyokawa@northwestern.edu
Abbreviations used in this paper: ACA, anticentromere antibody; APC/C,
anaphase-promoting complex/cyclosome; dsRNA, double-stranded RNA; HECT,
homologous to the E6-AP carboxyl terminus; MCC, mitotic checkpoint complex.
The online version of this article contains supplemental material.
Mitotic progression is controlled by spatiotemporal changes in
protein modifi cations, i.e., phosphorylation mediated by sev-
eral mitotic kinases and ubiquitination mediated by multiple E3
ubiquitin ligases ( Nurse, 2000 ; Gutierrez and Ronai, 2006 ;
Malumbres and Barbacid, 2007 ). The E3 activity of the anaphase-
promoting complex/cyclosome (APC/C), which is sequentially
activated by Cdc20 and Cdh1, plays a central role in coordi-
nating mitotic progression by targeting multiple mitotic regu-
lators to polyubiquitination-dependent degradation ( Nasmyth,
2005 ; Peters, 2006 ). APC/C-Cdc20 is required for degradation
of securin and cyclin B at anaphase onset. Securin keeps sepa-
rase from inducing proteolysis of cohesin, which holds a pair of
sister chromatids together during early mitosis. The spindle
assembly checkpoint delays APC/C-Cdc20 activation until all
chromosomes become aligned at the metaphase plate with
proper spindle attachment ( Musacchio and Hardwick, 2002 ;
Bharadwaj and Yu, 2004 ). Perturbation of this checkpoint re-
sults in chromosome missegregation and aneuploidy. The spin-
dle assembly checkpoint depends on multiprotein complexes
including Mad2, BubR1, and Bub3, known as the mitotic check-
point complex (MCC). The physical assembly of MCC and
its target Cdc20 is regulated according to the status of the spindle –
kinetochore attachment and tension. Among the MCC compo-
nents, Mad2 undergoes very dynamic changes in its conformation
and localization ( Howell et al., 2004 ). Current models sug-
gest that unattached kinetochores are associated stably with
Mad1 and Bub1, whereas Mad2 in differential conforma-
tions dynamically interacts with kinetochore-bound Mad1 and
cytoplasmic Cdc20 ( Nasmyth, 2005 ; Yu, 2006 ), which keeps
APC/C-Cdc20 inactive in the presence of unattached or un-
is required for polyubiquitination and degradation of
securin and cyclin B at anaphase onset. The spindle as-
sembly checkpoint delays APC/C-Cdc20 activation until
all kinetochores attach to mitotic spindles. In this study,
we demonstrate that a HECT (homologous to the E6-AP
carboxyl terminus) ubiquitin ligase, Smurf2, is required
for the spindle checkpoint. Smurf2 localizes to the cen-
trosome, mitotic midbody, and centromeres. Smurf2
depletion or the expression of a catalytically inactive
ctivation of the anaphase-promoting complex/
cyclosome (APC/C) by Cdc20 is critical for the
metaphase – anaphase transition. APC/C-Cdc20
Smurf2 results in misaligned and lagging chromosomes,
premature anaphase onset, and defective cytokinesis.
Smurf2 inactivation prevents nocodazole-treated cells
from accumulating cyclin B and securin and prometa-
phase arrest. The silencing of Cdc20 in Smurf2-depleted
cells restores mitotic accumulation of cyclin B and se-
curin. Smurf2 depletion results in enhanced polyubiqui-
tination and degradation of Mad2, a critical checkpoint
effector. Mad2 is mislocalized in Smurf2-depleted cells,
suggesting that Smurf2 regulates the localization and
stability of Mad2. These data indicate that Smurf2 is a
novel mitotic regulator.
The HECT E3 ligase Smurf2 is required for
Mad2-dependent spindle assembly checkpoint
Evan C. Osmundson , 1,3 Dipankar Ray , 1 Finola E. Moore , 1 Qingshen Gao , 2,4 Gerald H. Thomsen , 5,6
and Hiroaki Kiyokawa 1,2,3
1 Department of Molecular Pharmacology and Biological Chemistry and 2 Robert H. Lurie Comprehensive Cancer Center, Feinberg School of Medicine,
Northwestern University, Chicago, IL 60611
3 Department of Biochemistry and Molecular Genetics, College of Medicine, University of Illinois, Chicago, IL 60607
4 Department of Medicine, Evanston Northwestern Healthcare Research Institute, Evanston, IL 60201
5 Department of Biochemistry and Cell Biology and 6 Center for Developmental Genetics, Stony Brook University, Stony Brook, NY 11794
© 2008 Osmundson 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 2 • 2008 268
ization of Smurf2 are cell cycle regulated, and the E3 activity
of Smurf2 is critical for the spindle assembly checkpoint. The
novel function of Smurf2 suggests that a network of ubiqui-
tination machinery maintains the genomic stability during
Smurf2 is required for mitotic control
during unperturbed cell cycle progression
Pub1p, a fi ssion yeast HECT family E3 ligase, regulates G 2 /M
cell cycle progression by ubiquitinating Cdc25p ( Nefsky and
Beach, 1996 ). The mammalian HECT family includes Smurf1,
Smurf2, AIP4/Itch, and Nedd4 ( Kee and Huibregtse, 2007 ). Our
recent study on the TGF- ? regulation of Cdc25A ubiquitination
( Ray et al., 2005 ) led us to examine whether Smurf proteins,
which are known to modulate TGF- ? signaling ( Zhu et al.,
1999 ; Kavsak et al., 2000 ), were involved in mitotic regulation.
We fi rst examined the levels of Smurf2 protein in HeLa cells
synchronized by a thymidine-aphidicolin protocol ( Fig. 1 a ).
The expression of Smurf2 protein was highest 6 – 8 h after re-
lease, which slightly preceded the peaks of cyclin B1 and Cdc25A
expression around G 2 /M transition. Smurf1 expression was con-
stant throughout the cell cycle (unpublished data). Thus, Smurf2
is a cell cycle – regulated protein that accumulates during late G 2
through early mitosis. We then examined the subcellular local-
ization of Smurf2, which revealed concentrated localization of
Consistent with the key function of Mad2 in the spindle
assembly checkpoint, recent studies using transgenic and knock-
out mouse models suggest that optimal control of Mad2 is criti-
cal for genomic stability and tumor suppression ( Michel et al.,
2001 ; Sotillo et al., 2007 ). Mad2 transcription is known to be
regulated in E2F- and Myc-dependent manners ( Hernando et al.,
2004 ; Menssen et al., 2007 ). Although phosphorylation of Mad2
controls the association of Mad2 with Mad1 and APC/C-Cdc20
( Wassmann et al., 2003 ), it remains obscure whether Mad2 un-
dergoes other posttranslational regulation. In this study, we
demonstrate that a HECT (homologous to the E6-AP carboxyl
terminus) family E3 ubiquitin ligase, Smurf2, plays an essential
role in the spindle assembly checkpoint, regulating the stability
and localization of Mad2. Smurf2 has been known as a negative
regulator of the TGF- ? signaling pathway, targeting receptors
and signaling proteins ( Kavsak et al., 2000 ; Bonni et al., 2001 ;
Stroschein et al., 2001 ; Moren et al., 2005 ). Previous studies
demonstrated that Smurf2 targets TGF- ? receptors to protea-
somal degradation ( Kavsak et al., 2000 ; Di Guglielmo et al.,
2003 ). Smurf2 also has been shown to ubiquitinate Smad2, the
TGF- ? – related transcriptional cofactor SnoN ( Bonni et al., 2001 ),
the GTPase Rap1B ( Schwamborn et al., 2007 ), the RING-H2
protein RNF11 ( Subramaniam et al., 2003 ), the Runt domain
transcription factors Runx2 and Runx3 ( Jin et al., 2004 ; Kaneki
et al., 2006 ), and ? -catenin ( Han et al., 2006 ). Although diverse
transcriptional control by Smurf2-mediated ubiquitination is
well documented, it was unknown that the expression and local-
Figure 1. Smurf2 is a cell cycle – regulated protein localizing specifi cally in the centrosome, mitotic midzone, and midbody. (a) HeLa cells were synchro-
nized by a double-thymidine protocol. At the indicated hours after release from the block, cells were analyzed by immunoblotting for the proteins shown.
(b) Immunofl uorescence microscopy for ? -tubulin (green) and Smurf2 (red) in HeLa cells at interphase and metaphase. Polyclonal anti-Smurf2 antibody was
used for Smurf2 staining. DAPI was used to stain chromosomal DNA (blue). The close-up images are shown with threefold higher magnifi cation. (c) Immuno-
fl uorescent staining for ? -tubulin (green) and Smurf2 (red) in human mammary epithelial MCF-10A cells during mitosis. Bars, 10 μ m.
269SMURF2 IS ESSENTIAL FOR SPINDLE ASSEMBLY CHECKPOINT • Osmundson et al.
To examine whether Smurf2 plays a role in mitotic pro-
gression, we examined the effects of siRNA-mediated Smurf2
depletion on divisions of cultured cell lines. We designed several
siRNAs based on the coding region of human Smurf2 mRNA and
tested the silencing effi ciencies. Of them, siRNAs #1 and #4 as
well as a pool of four siRNAs showed marked down-regulation
of Smurf2 protein in transfected cells ( Fig. 2 a ). Morphological
examinations readily detected several multinucleated HeLa cells
transfected with Smurf2 siRNA #1 but not with control double-
stranded RNAs (dsRNAs; Fig. 2, b and c ). Similar multi-
nucleation was observed in cells transfected with siRNA #4 or the
pooled anti-Smurf2 siRNAs, whereas other siRNAs with poor
knockdown effi ciencies had no effect on nuclear morphology.
Time-lapse microscopy demonstrated that most control HeLa
cells initiated cytokinesis shortly after metaphase and did
not revert from cytokinesis during 5 h of monitoring ( Fig. 2 e
and Video 1, available at http://www.jcb.org/cgi/content/full/
jcb.200801049/DC1). In contrast, a majority of Smurf2 siRNA-
transfected cells did not display well-defi ned cytokinesis after
mitotic rounding up (time 0). Although formation of the cleav-
age furrow was observed, ? 84% of cells with Smurf2 deple-
tion failed cytokinesis, leading to a binucleated state ( Fig. 2 d ).
Similar multinucleation phenotypes were observed in U2OS and
Smurf2 at centrosomes in HeLa cells ( Fig. 1 b ). The specifi city
of Smurf2 polyclonal antibody used for immunofl uorescence
microscopy was verifi ed by immunoblotting with Smurf2-
depleted cells (Fig. S1 a, available at http://www.jcb.org/cgi/
content/full/jcb.200801049/DC1). Smurf2 localized at perinuclear
centrosomes during interphase as well as at centrosomes aligned
bipolarly in metaphase cells, demonstrating colocalization with
a centrosomal marker, ? -tubulin. Similar centrosomal localiza-
tion of Smurf2 was observed in HeLa cells using Smurf2 mono-
clonal antibody and pericentrin antibody, another centrosomal
marker, as well as in U2OS cells transfected with Flag-tagged
Smurf2 (Fig. S1, b and c). We then examined Smurf2 localiza-
tion in MCF-10A cells undergoing mitosis and cytokinesis in
which the protein exhibited a dynamic relocalization pattern.
Smurf2 localized predominantly at centrosomes during meta-
phase, whereas focal signals for Smurf2 were also observed in
noncentrosomal structures in the cytoplasm ( Fig. 1 c ). During
anaphase, a portion of Smurf2 apparently relocalized to the cen-
ter of the spindle midzone, which is rich in microtubules stained
with ? -tubulin antibody. During telophase, Smurf2 was found
concentrated at the midbody in the intercellular bridge. Similar
Smurf2 relocalization during mitosis was observed in HeLa
cells (unpublished data).
Figure 2. Smurf2 silencing inhibits mitotic progression and cytokinesis. (a) Smurf2 levels determined by immunoblotting in HeLa cells (top) and U2OS cells
(bottom) 48 h after transfection with anti – Smurf2 siRNA (siSm2) or nonspecifi c dsRNA (siNS). UN, untransfected control. (b) Morphology of multinucleated
HeLa cells 48 h after transfection with siRNA #1. Hoechst was used for DNA staining. (c) Percentages of multinucleated cells in HeLa cultures at the indi-
cated hours after transfection with siNS (open bars) or Smurf2 siRNA #1 (shaded bars). At least 300 cells were counted per time point per cohort. Mean of
data from two independent experiments are shown. See Fig. S2 a (available at http://www.jcb.org/cgi/content/full/jcb.200801049/DC1) for raw data
from the individual experiments. (d) Percentages of cells that failed cytokinesis in siNS- or siSm2-transfected HeLa cultures quantifi ed from the time-lapse
microscopy. At least 65 mitotic cells per cohort (siNS vs. siSmurf2) were analyzed for the progression of cytokinesis. (e) Representative time-lapse pictures
of siSm2-transfected HeLa cells that showed impaired mitotic progression and failed cytokinesis. Bars: (b) 50 μ m; (e) 10 μ m.
JCB • VOLUME 183 • NUMBER 2 • 2008 270
The localization of Smurf2 at centromeres was observed only
transiently in cells undergoing nocodazole-induced arrest at pro-
metaphase. This localization pattern is consistent with a putative
role for Smurf2 in the spindle assembly checkpoint. We then
determined cellular levels of mitosis regulatory proteins in
Smurf2-depleted and control cells synchronously progressing
into mitosis in the presence of nocodazole ( Fig. 4 b ). Cellular
levels of Smurf2 and the APC/C-Cdc20 substrates securin, cy-
clin B1, and aurora B ( Fang et al., 1999 ) accumulated as control
cells progressed through the G 2 /M transition 8 – 10 h after release
from the thymidine block. Cyclin A2 was markedly down-
regulated during nocodazole-induced prometaphase arrest as
expected ( Fry and Yamano, 2006 ). Strikingly, securin, cyclin B1,
and aurora B failed to accumulate in Smurf2-depleted cells.
In contrast, Smurf2 depletion affected minimally or only mod-
estly the levels of Emi1, cyclin A2, and the APC/C-Cdh1 substrates
Plk1 and Cdc20. These proteins are not direct targets of APC/
C-Cdc20, whereas cyclin B1, securin, and aurora B are well-
characterized substrates. These data suggest that Smurf2 depletion
results in premature activation of APC/C-Cdc20. Flow cytometry
further demonstrated that control cells were arrested with 4N
DNA content 14 h after release from the thymidine block,
whereas 17% of Smurf2-depleted cells had 8N DNA, which is
consistent with binucleation ( Fig. 4 c ). 26 h after release, 63% of
Smurf2-depleted cells exhibited 8N DNA. These data suggest
that Smurf2 depletion resulted in continued cell cycle progres-
sion in the presence of nocodazole without successful cytokine-
sis. To confi rm that Smurf2 depletion permitted inappropriate
APC/C-Cdc20 activity in the presence of nocodazole, we at-
tempted to silence Cdc20 in Smurf2-depleted cells ( Fig. 4 d ).
siRNA-mediated codepletion of Cdc20 and Smurf2 resulted in
restored mitotic accumulation of cyclin B1 and securin (but
not Mad2; Fig. 4 d , fourth row) in synchronized cultures of
nocodazole-treated HeLa cells. Aurora B is also up-regulated mod-
estly by the codepletion. Importantly, the addition of Cdc20
siRNA did not signifi cantly affect the silencing effi cacy of the
Smurf2 siRNA or the levels of cyclin A2 and Plk1. These data
suggest that Smurf2 is required for the spindle assembly check-
point – mediated inhibition of APC/C-Cdc20 and mitotic accu-
mulation of securin and cyclin B1.
Smurf2 is required for stability and proper
localization of Mad2
When the spindle assembly checkpoint is activated, e.g., by no-
codazole, Mad2 localizes in multiprotein complexes at un-
attached or untensed kinetochores, including Mad1 and Bub1
( Bharadwaj and Yu, 2004 ). Cells lacking Mad2 demonstrate a
severely compromised spindle assembly checkpoint ( Michel et al.,
2004 ). Immunoblotting demonstrated that Mad2 was markedly
down-regulated in Smurf2-depleted cells, whereas Mad2 levels
were relatively constant during cell cycle progression of control
cells ( Fig. 4, b and d ). Immunofl uorescence microscopy also
confi rmed that no specifi c localization of Mad2 was detected
at centromeres of Smurf2-depleted cells ( Fig. 4 e ), whereas
localization of BubR1 at centromeres was unaffected ( Fig. 4 f ).
Moreover, Smurf2 depletion down-regulated not only endog-
enous Mad2 but also transfected Mad2 in asynchronous HeLa
MCF-10A cells with Smurf2 depletion (unpublished data). Thus,
Smurf2 is required for successful cytokinesis.
To explore the mechanism of mitotic failure in Smurf2-
depleted cells, we fi rst examined the parameters of chromo-
somal alignment and segregation because cells defective in the
regulation of these events, e.g., depletion of Mad2 or USP44,
demonstrate similar mitotic phenotypes ( Michel et al., 2004 ;
Stegmeier et al., 2007 ). We fi rst examined the morphology of
mitotic control and Smurf2-depleted HeLa cells during mitosis
by staining for the microtubule marker ? -tubulin, Smurf2, and
chromosomal DNA (Fig. S2 b, available at http://www.jcb.org/
cgi/content/full/jcb.200801049/DC1). Of a total of > 600 cells
examined per group, 7.3% and 8.0% in control and Smurf2-
depleted cells, respectively, displayed characteristics of mitosis
(i.e., chromatin condensation and formation of mitotic spin-
dles). Although 4.7% of control cells were found to be in meta-
phase, only 1.9% of Smurf2-depleted cells exhibited typical
metaphase morphology with aligned chromosomes. The de-
crease in the metaphase population suggested that Smurf2 de-
pletion might affect mitotic progression. To further assess the
impact of Smurf2 depletion on chromosomal dynamics during
mitotic progression, we depleted Smurf2 in HeLa cells stably
expressing a GFP – histone H2B fusion protein (GFP-H2B).
Cells were then monitored for chromosome movement by time-
lapse microscopy, which readily showed that Smurf2-depleted
cells initiated anaphase in the presence of misaligned chromo-
somes. Over 75% of Smurf2-depleted cells exhibited misaligned
chromosomes or failed to form a metaphase plate during moni-
tored metaphase – anaphase transition ( Fig. 3, a and b ; and Video 2).
During anaphase, most Smurf2-depleted cells showed defective
segregation such as lagging chromosomes or no appreciable
chromosome segregation ( Fig. 3, c and d ). In contrast, < 10% of
control cells exhibited such defects during mitosis. We also
measured the interval between nuclear envelope breakdown and
the onset of anaphase, focusing on cells that displayed discern-
able elements of chromatin segregation. In control cultures, the
median interval was ? 55 min, whereas that in Smurf2-depleted
cultures was ? 23 min ( Fig. 3, e and f ; and Video 3). These data
indicate that Smurf2 depletion leads to premature anaphase on-
set together with chromosomal alignment and segregation defects.
Thus, Smurf2 seems to play a signifi cant role in the temporal
control of the metaphase – anaphase transition, a process nor-
mally regulated by the spindle assembly checkpoint.
Smurf2 regulates the spindle assembly
Defective chromosome alignment/segregation and premature
anaphase onset in Smurf2-depleted cells are reminiscent of the
phenotypes of impaired spindle assembly checkpoint. To examine
whether Smurf2 is involved in the checkpoint, cells progressing
synchronously through G 2 toward M were treated with the micro-
tubule polymerization inhibitor nocodazole. For examinations of
Smurf2 localization, cells were fi xed as they progressed into pro-
metaphase arrest ( Fig. 4 a ). Interestingly, Smurf2 localized in a focal
fashion and was closely associated with the immuno reac tivities
against human anticentromere antibodies (ACAs), which rec-
ognize several centromere proteins ( Earnshaw et al., 1986 ).
271SMURF2 IS ESSENTIAL FOR SPINDLE ASSEMBLY CHECKPOINT • Osmundson et al.
Figure 3. Smurf2 silencing leads to chromosomal misalignment at metaphase and premature onset of anaphase with defective chromosome segregation.
HeLa cells stably expressing GFP-H2B were transfected with Smurf2 siRNA #1, and chromosomal movement was monitored 24 – 25 h after transfection
by time-lapse fl uorescence microscopy. Quantifi ed data are representative of at least two independent experiments. (a) Defects in chromosomal alignment
in Smurf2-depleted cells at metaphase. Metaphase defects in cells transfected with Smurf2 siRNA (shaded bars) or nonspecifi c dsRNA (siNS, open bars)
were categorized into the two indicated groups. (b) Unaligned chromosomes (*) and lack of metaphase plates (#) during mitosis with Smurf2 depletion.
Arrowheads denote lagging chromosomes. (c) Chromosomal segregation defects in GFP-H2B HeLa cells transfected with Smurf2 siRNA (shaded bars) or
nonspecifi c dsRNA (open bars) at anaphase. (a and c) Data are means ± SEM (error bars) from three independent experiments. (d) Lagging chromosomes
(arrowheads, top row), major segregation defects (arrowheads, middle row), and lack of segregation in GFP-H2B HeLa cells with Smurf2 depletion.
(e) Premature anaphase onset in Smurf2-depleted cells. The metaphase – anaphase transition of GFP-H2B HeLa cells was monitored by time-lapse micros-
copy, and the time from nuclear envelope breakdown (NEBD) until anaphase onset was determined. (f) Representative pictures of the metaphase – anaphase
transition. AO, anaphase onset. Bars, 10 μ m.
JCB • VOLUME 183 • NUMBER 2 • 2008 272
ing a posttranscriptional mechanism. We suspected that Mad2
levels may be determined by ubiquitin-mediated proteasomal
degradation, like many other cell cycle regulators. To examine
whether Mad2 is ubiquitinated, Mad2 was immunoprecipitated
from Smurf2-depleted or control HeLa cells treated with the
proteasomal inhibitor MG132 followed by immunoblotting
for ubiquitin ( Fig. 5 b ). Smurf2 depletion robustly increased
polyubiquitinated forms of Mad2, and proteasomal inhibition
cells (Fig. S3 a, available at http://www.jcb.org/cgi/content/
full/jcb.200801049/DC1). Mad2 down-regulation by Smurf2
depletion was confi rmed in U2OS cells using Smurf2 siRNA #1
and #4 as well as pooled siRNAs (Fig. S3 b), suggesting that this is
not an off-target effect of siRNAs. We then examined whether
Smurf2 siRNA affected Mad2 mRNA levels ( Fig. 5 a ). Despite
the rapid decline of Mad2 protein levels, Mad2 mRNA levels
were essentially unaltered in Smurf2-depleted cells, indicat-
Figure 4. Smurf2 is a novel regulator of the spindle assembly checkpoint. (a) Smurf2 localizes at centromeres when the spindle checkpoint is active.
HeLa cells were synchronized by a double-thymidine protocol (see Materials and methods). Cells were then released into a fresh medium (time 0), and 3 h
later nocodazole was added. Immunofl uorescence microscopy after staining with ACA, anti-Smurf2 antibody, and DAPI for DNA. Cells were fi xed 8.5 h
after release from the thymidine in the presence of nocodazole. (b) Smurf2 depletion results in failed accumulation of APC/C-Cdc20 substrates during
nocodazole-induced metaphase arrest. Immunoblotting was performed for mitotic regulators in HeLa cells transfected with Smurf2 siRNA #1 or nonspecifi c
dsRNA (siNS). After release from the thymidine block, cells were incubated for the indicated hours in the presence of nocodazole. (c) Smurf2-depleted cells
override nocodazole-induced spindle assembly checkpoint, resulting in tetraploidy. Control (siNS) and Smurf2-depleted cells were fi xed at the indicated
times and subjected to fl ow cytometry. (d) Cosilencing of Cdc20 restores mitotic accumulation of cyclin B1 and securin in Smurf2-depleted HeLa cells. Cells
were synchronized and released into nocodazole-containing medium as described in b. (e and f) Smurf2 depletion results in loss of Mad2 from centro-
meres, whereas centromere localization of BubR1 is unaffected. Cells were fi xed 8.5 h after release from the thymidine in the presence of nocodazole, and
immunofl uorescence microscopy was performed. (a, e, and f) The close-up images are shown with threefold higher magnifi cation. Bars, 10 μ m.
273 SMURF2 IS ESSENTIAL FOR SPINDLE ASSEMBLY CHECKPOINT • Osmundson et al.
We then examined whether the forced expression of Mad2
was suffi cient to restore the spindle assembly checkpoint in
Smurf2-depleted cells. Transfection of a Mad2 expression plas-
mid resulted in marked overexpression of Mad2 in cells without
Smurf2 depletion, which did not signifi cantly affect the ex-
pression of securin, cyclin B1, or other mitotic regulators during
synchronous progression into nocodazole-induced arrest ( Fig. 5 d ).
by MG132 further accumulated polyubiquitinated Mad2. We also
found that Smurf2 physically associates with Mad2. Endogenous
Smurf2 and Mad2 coimmunoprecipitated with each other from
extracts of untransfected HeLa cells that progressed from S
phase into mitosis ( Fig. 5 c, and see Fig. S4 for controls). These
data suggest that Smurf2 normally protects Mad2 from poly-
Figure 5. Smurf2 is required for stability and functional localization of Mad2. (a) Smurf2 silencing rapidly down-regulates Mad2 protein but not mRNA.
HeLa cells in asynchronous culture were transfected with Smurf2 siRNA or nonspecifi c dsRNAs (siNS) and harvested at the indicated times after transfec-
tion for Western blotting and RT-PCR. (b) Smurf2 silencing results in increased polyubiquitination of Mad2 followed by proteasomal degradation. HeLa
cells were transfected with a Mad2 expression plasmid and Smurf2 siRNA #1 or control dsRNA. Cells were then treated with 2 μ M MG132 for 4 h and
analyzed by immunoprecipitation (IP) with Mad2 antibody followed by Western blotting using the indicated antibodies. (c) Smurf2 physically interacts with
Mad2. Untransfected HeLa cells were released from synchronization at S phase with double-thymidine treatment and were harvested at the indicated times
for immunoprecipitation followed by Western blotting. Ig, Ig heavy chain; NS, nonspecifi c band. (d) Ectopic expression of Mad2 is unable to restore the
spindle assembly checkpoint in Smurf2-depleted cells. Western blotting for mitotic regulators in HeLa cells transfected with the indicated plasmid and siRNA.
Cells were synchronized with thymidine treatment, released into a nocodazole-containing medium, and harvested at the indicated times after release (see
Fig. 4 b and Materials and methods). (e) Ectopically expressed Mad2 localizes at centromeres in nocodazole-treated control cells but not in Smurf2-
depleted cells. Immunofl uorescence microscopy with anti-Mad2 and ACAs 10 h after release from the thymidine block. The close-up images are shown
with threefold higher magnifi cations. Bars, 10 μ m.
JCB • VOLUME 183 • NUMBER 2 • 2008 274
HeLa cells with an expression plasmid for Smurf2(C716A) and
synchronized them at early S phase with double-thymidine
treatments ( Fig. 6 a ). Cells were then released into a nocodazole-
containing medium. Immunoblotting showed that cells trans-
fected with Smurf2(C716A) failed to accumulate securin with
signifi cant reduction in Mad2 levels. Interestingly, cyclin B1 ac-
cumulation during G 2 /M progression was minimally affected,
which is analogous to previous observations in Mad2 heterozy-
gous cells ( Michel et al., 2004 ). In contrast, transfection with wild-
type Smurf2 affected none of these proteins (Fig. S5, available
This fi nding is consistent with the effects of Smurf2 siRNA,
suggesting that Smurf2(C716A) can act as a dominant-negative
mutant. Furthermore, cells transfected with Smurf2(C716A)
showed a signifi cant increase in cells displaying misaligned
chromosomes, lagging chromosomes during mitotic exit, and
multinucleation ( Fig. 6, b and c ). These data suggest that the E3
ligase activity of Smurf2 is required for proper control of the
spindle assembly checkpoint.
This study has revealed a novel function of the HECT E3 li-
gase Smurf2 in mitosis. During unperturbed mitotic progression,
In Smurf2-depleted cells, transfection of Mad2 resulted in ac-
cumulation of Mad2 to levels comparable with mock-transfected
control cells. However, these cells exhibited almost undetect-
able levels of securin and cyclin B1, indicating that ectopic ex-
pression of Mad2 was insuffi cient to restore the checkpoint.
Cyclin A2, which is known to be degraded even during spindle
assembly checkpoint activation ( Fry and Yamano, 2006 ), was
nearly undetectable in nocodazole-treated cells of all cohorts.
Apparently, the lack of phenotypic rescue was associated with
mislocalization of exogenously expressed Mad2 because most
Mad2 immunoreactivities of nocodazole-treated cells did not
colocalize with the signals from ACAs ( Fig. 5 e ). The mean in-
tensity of Mad2 signals normalized by ACA signals in a cell ex-
pressing exogenous Mad2 in the presence of siSmurf2 was 74%
lower than that in a cell expressing exogenous Mad2 in the pres-
ence of siNS (3.208 ± 0.217 vs. 0.823 ± 0.068, mean ± SEM,
from centromeres examined in eight cells per group). These
data suggest that Smurf2 may regulate not only the stability but
also the localization of Mad2.
We next examined whether the E3 ligase activity of Smurf2
is important for its function to control the spindle assembly
checkpoint. The cysteine-716 residue of Smurf2 is critical for
its E3 ligase activity because Smurf2(C716A) protein is cata-
lytically inactive ( Kavsak et al., 2000 ). Thus, we transfected
Figure 6. Expression of a catalytically inactive Smurf2 impairs the spindle assembly checkpoint with Mad2 destabilization. (a) Immunoblotting for mitotic
regulators in HeLa cells transfected with Flag-tagged Smurf2(C716A) or a control plasmid. Cells were synchronized at S phase with thymidine treatment,
released into a nocodazole-containing medium, and harvested after incubation at the indicated times after release (see Materials and methods). OE,
overexpression. (b) Chromosomal misalignment at metaphase and lagging chromosomes at anaphase observed in cells expressing Smurf2(C716A).
Immunofl uorescence microscopy of cells stained with anti-Flag antibody for exogenously expressed Smurf2, ACA, and DAPI. Cells were harvested 8.5 h
after release from the thymidine block. The second row shows misaligned chromosomes at metaphase, and the bottom row shows lagging chromosomes
at anaphase in cells expressing Smurf2(C716A). Bars, 10 μ m. (c) Percentages of cells with chromosomal defects in control cultures (open bars) and
Smurf2(C716A)-transfected cultures (shaded bars). Data are means ± SEM (error bars) from three independent experiments.
275 SMURF2 IS ESSENTIAL FOR SPINDLE ASSEMBLY CHECKPOINT • Osmundson et al.
hypothesis that a functional interaction of Smurf2 and Mad2
that occurs dynamically between spindle poles/centrosome
and kinetochores may control the spindle assembly check-
point. Of course, it is possible that Smurf2 plays multiple roles
in mitosis and cytokinesis by affecting multiple downstream
targets in addition to Mad2. A previous study showed that
Smad2, 3, and 4 bind to microtubules in the absence of TGF- ? ,
colocalizing with tubulin at the bipolar spindle and midzone
( Dong et al., 2000 ). These Smad proteins can function as a co-
factor of multiple E3 ligases, including SCF (skp1-Cul1-F-box),
APC/C, and Smurf2 ( Fukuchi et al., 2001 ; Ogunjimi et al.,
2005 ; Ray et al., 2005 ). Therefore, the cofactor Smad protein
might complex with Smurf2 alongside microtubules and mod-
ulate Smurf2 activity.
Regulation upstream of the spindle checkpoint compo-
nents has been understudied. The transcriptional control of Mad2
by E2F and Myc was recently identifi ed ( Hernando et al., 2004 );
however, it had been unknown whether cellular levels of Mad2
protein are regulated by ubiquitination. To our knowledge, this
study is the fi rst to demonstrate that Mad2 undergoes poly-
ubiquitination and proteasomal degradation. The experiments
using Smurf2 siRNA and Smurf2(C716A) showed that the E3
ligase activity of Smurf2 is required to prevent Mad2 from poly-
ubiquitination and degradation ( Fig. 7 ). Smurf2 physically as-
sociates with Mad2. Although Smurf2 is likely to affect the
modifi cation and/or conformation of Mad2 at close proximity,
the precise mechanism remains to be determined. A hypotheti-
cal mechanism is that Smurf2 directly modifi es Mad2 by mono-
ubiquitination or Lys63-linked polyubiquitination ( Haglund and
Dikic, 2005 ), and such a modifi cation somehow alters the con-
formation of Mad2 to a stable form and promotes its dynamic
localization at kinetochores critical for surveillance of spindle
integrity. For instance, the chromatin passenger complex pro-
tein survivin undergoes Lys63 polyubiquitination, which is re-
quired for centromere localization of this protein ( Vong et al.,
2005 ). When the level of active Smurf2 is low, Mad2 undergoes
an alternative form of ubiquitination, presumably Lys48-linked
polyubiquitination mediated by an undefi ned E3 ligase, and is
recruited to proteasomal degradation. Another possibility is that
Smurf2 plays an indirect role in the control of Mad2 stability.
Smurf2 may antagonize the action of an unidentifi ed E3 ligase
required for proteasomal degradation of Mad2, possibly by
ubiquitinating and destabilizing the E3 ligase. In this case,
Smurf2 depletion would up-regulate the E3 ligase by stabilization,
leading to enhanced Mad2 polyubiquitination and subsequent
Smurf2 dynamically relocalizes from the centrosome to the
center of the anaphase spindle midzone and fi nally to the mid-
body in the intercellular bridge during cytokinesis. Smurf2 is
also observed at unattached centromeres during spindle assem-
bly checkpoint activation. Smurf2 depletion impairs the spindle
assembly checkpoint, leading to increased misalignment and
missegregation of chromosomes, premature anaphase onset, and
defective cytokinesis. The function of Smurf2 in the spindle as-
sembly checkpoint is mediated at least partly by the stability
and localization of Mad2 because Smurf2 depletion results in
enhanced polyubiquitination and rapid destabilization of Mad2
protein. Mad2 destabilization presumably leads to premature
activation of APC/C-Cdc20, which is associated with impaired
mitotic accumulation of the APC/C-Cdc20 substrates such as
cyclin B1 and securin. The silencing of Cdc20 in Smurf2-
depleted cells restores mitotic accumulation of these proteins,
confi rming the antagonistic function of Smurf2 against APC/C-
Cdc20 activity. Ectopic expression of Mad2 is insuffi cient to
restore the spindle assembly checkpoint in Smurf2-depleted
cells, apparently because Mad2 mislocalizes in the absence of
Smurf2. Collectively, Smurf2 is a novel regulator of the Mad2-
dependent spindle assembly checkpoint.
Mad2 interaction with kinetochore-associated checkpoint
proteins, such as Mad1 and Bub1, is required for the inhibition
of APC/C-Cdc20 until the proper alignment of all mitotic spin-
dles ( Nasmyth, 2005 ; Yu, 2006 ). Mad2, together with BubR1
and Bub3, exhibits rapid movement on and off kinetochores,
transiently interacting with Mad1- and Bub1-associated centro-
meres ( Howell et al., 2004 ). This dynamic regulation of Mad2
is critical for the diffusible inhibitory signal against cytoplasmic
APC/C-Cdc20. This study presents evidence that Smurf2 activ-
ity is required for the normal progression of mitosis as well as
for spindle disruption – mediated mitotic arrest. Similarly, cells
with Mad2 or BubR1 depletion show various mitotic defects (i.e.,
failure in chromosome segregation, premature anaphase onset,
and cytokinesis defects; Taylor and McKeon, 1997 ; Gorbsky et al.,
1998 ; Meraldi et al., 2004 ). Analogous phenotypes have been
observed in cells with depletion of USP44, a deubiquitinating
enzyme that counteracts disassembly of Mad2 – Cdc20 com-
plexes by removing ubiquitin from Cdc20 ( Stegmeier et al., 2007 ).
The binding of Mad2 to Cdc20 requires a substantial conforma-
tional change of Mad2, which Mad1 seems to facilitate at the
kinetochore ( Yu, 2006 ). Although it remains to be elucidated
how Smurf2 regulates the stability and localization of Mad2, it
is tempting to speculate that the E3 ligase activity of Smurf2 is
involved in regulating Mad2 conformation. Smurf2 levels are
highest before the metaphase – anaphase transition, when Smurf2
localizes predominantly at spindle poles/centrosomes. Mad2 also
has been found at spindle poles before anaphase onset, possibly
via microtubule-mediated transit between kinetochores and
spindle poles ( Howell et al., 2000 ; Shah et al., 2004 ). Depletion
of Mad2 and BubR1 results in kinetochore-independent pre-
mature anaphase onset, whereas knockdown of other spindle
checkpoint components (e.g., Bub3 and Bub1) impairs only
kinetochore-dependent checkpoint function, not the timing of
anaphase onset ( Meraldi et al., 2004 ). Our data demonstrating
premature anaphase onset in Smurf2-depleted cells support a
Figure 7. Putative roles of Smurf2 in regulation of the spindle assembly
JCB • VOLUME 183 • NUMBER 2 • 2008 276
For immunoblotting or immunoprecipitation, cells were lysed by sonica-
tion in lysis buffer as described previously ( Tsutsui et al., 1999 ). Densi-
tometric analyses of chemiluminescence signal were performed using a
1D Scientifi c Imaging Systems version 3.6.1 (Kodak) and ScanMaker
Online supplemental material
Video 1 shows impaired mitotic progression, failed cytokinesis, and bi-
nucleation in Smurf2 siRNA (siSmurf2) – transfected HeLa cells demonstrated
by time-lapse phase microscopy. Video 2 shows defective chromosomal
alignment in Smurf2-depleted (siSmurf2) GFP-H2B HeLa cells at metaphase
demonstrated by time-lapse fl uorescence microscopy. Video 3 shows pre-
mature anaphase onset in Smurf2-depleted (siSmurf2) GFP-H2B HeLa cells
demonstrated by time-lapse fl uorescence microscopy. Fig. S1 shows cen-
trosomal localization of endogenous and transfected Smurf2 examined by
multiple antibodies. Fig. S2 shows multinucleation and misaligned chromo-
somes in HeLa cells with Smurf2 depletion. Fig. S3 shows down-regulation
of endogenous and transfected Mad2 in cells with Smurf2 depletion. Fig. S4
shows control immunoblots for Fig. 5 c . Fig. S5 shows that overexpression
of wild-type Smurf2 minimally affects levels of securin, cyclin B1, and
Mad2. Online supplemental material is available at http://www.jcb.org/
We thank Brian Zwecker, Thomas O ’ Grady, Alba Santana, and Suchitra
Prasad for technical help, Chaozhong Zou for reagents, and Toru Naka-
mura, Alisa Katzen, Pradip Raychaudhuri, Elena Pugacheva, Erica Golemis,
Kenji Fukasawa, and the Northwestern Cancer Cell Biology group for help-
This work was supported partly by the National Institutes of Health
(grants CA112282, CA100204, and HD38085 to H. Kiyokawa and grants
CA095221 and CA96986 to Q. Gao), the Department of Defense (grant
DAMD17-01-1-0342 to Q. Gao), the American Cancer Society (grant RSG-
03-048-01 to Q. Gao), and the Searle Leadership Fund and Zell Fund (grants
to H. Kiyokawa).
Submitted: 9 January 2008
Accepted: 10 September 2008
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Materials and methods
Monoclonal antibody against human Smurf2 has been described previ-
ously ( Kavsak et al., 2000 ). Polyclonal Smurf2 antibody was purchased
from Millipore. Antibodies against Mad2 and Cdc20 were purchased
from Santa Cruz Biotechnology, Inc. Anti – aurora B antibody was obtained
from BD Biosciences. Antibodies against ? -tubulin (GTU88), ? -tubulin
(DM1A), BubR1 (8G1), and ? -actin (AC-15) were purchased from Sigma-
Aldrich. Antibodies against securin, Plk1, and Emi1 were purchased from
Invitrogen. Anti – cyclin B1 (Ab-2) and – cyclin A2 (Ab-6) antibodies were
purchased from Neomarkers. Anticentromere CREST autoantiserum was
obtained from Immunovision. FITC-conjugated anti – rabbit Ig and Texas
red – conjugated anti – human Ig were purchased from Vector Laboratories.
Human cervical carcinoma HeLa cells, osteosarcoma U2OS cells, and
mammary epithelial MCF-10A cells were obtained from the American
Type Culture Collection. HeLa and U2OS cells were cultured in Dulbecco ’ s
modifi ed minimum essential medium supplemented with 10% fetal bovine
serum. MCF-10A cells were cultured in Dulbecco ’ s modifi ed minimum
essential medium/F12 medium supplemented with 5% heat-inactivated
horse serum, 10 μ g/ml insulin, 0.5 μ g/ml hydrocortisone, and 20 ng/ml
recombinant human EGF. For the stable expression of GFP-H2B, HeLa cells
were transfected with pBOS-H2B-GFP (Invitrogen; stable cell line provided
by C. Zou, Northwestern University, Evanston, IL). To synchronize cells
with a double-thymidine protocol, cells were treated fi rst with 2 mM thymi-
dine for 18 h, washed once in PBS, and released into fresh media. 9 h
later, thymidine was added to the culture medium to 2 mM, and incuba-
tion was continued for 15 h. Cells were then released into a fresh medium
for synchronous progression of the cell cycle from early S phase. To closely
examine cells progressing into mitosis and the integrity of the spindle
assembly checkpoint, nocodazole was added into the medium 3 h after
release from the second thymidine block to a concentration of 300 nM.
In this scheme, cells started to arrest at prometaphase within 10 h after re-
lease from the second thymidine treatment. To silence Smurf2 and Cdc20
in synchronized cell cultures, cells were transfected with either control or
Smurf2 siRNAs 5 h before the beginning of the fi rst thymidine treatment
and transfected with either control or Cdc20 siRNAs 2 h after the release
of the fi rst thymidine treatment. To determine cell cycle profi les, cells were
fi xed with ice-cold ethanol and stained with propidium iodide as de-
scribed previously ( Tsutsui et al., 1999 ). The sense sequences of the
Smurf2 #1 and #4 siRNAs are 5 ? -GAUGAGAACACUCCAAUUAUU-3 ?
and 5 ? -CAAAGUGGAAUCAGCAUUAUU-3 ? , respectively. The SMARTpool
siRNAs against Smurf2 and Cdc20 and the control dsRNAs were obtained
from Thermo Fisher Scientifi c.
Immunofl uorescence and time-lapse microscopy
For subcellular localization analyses, cells were fi xed with ice-cold metha-
nol for at least 20 min. Alternatively, cells were initially fi xed with 10%
buffered formalin (Sigma-Aldrich) for 5 min and fi xed with ice-cold metha-
nol for 20 min. For centromeric localization of Smurf2 and other antigens,
cells were prepermeabilized with PBS supplemented with 0.5% Triton X-100
and 1 mM MgCl 2 followed by fi xation with 10% buffered formalin or,
alternatively, fi xation with ice-cold methanol as described above. Slide
glasses with fi xed cells were then washed with PBS, permeabilized with
0.2% Triton X-100 on ice for 5 min, and stained with primary antibodies
at 4 ° C for 16 h. Washed slides were then incubated with fl uorescence-
conjugated secondary antibodies at 4 ° C for 1 h. Washed slides were shielded
with coverglasses using Vectashield containing DAPI stain (Vector Labora-
tories) and subjected to microscopic analyses at 25 ° C. For microscopic
studies, we used an inverted fl uorescence microscope (Axiovert 200; Carl
Zeiss, Inc.) with a 40 × NA 0.55 objective lens equipped with a Hg lamp
(X-Cite 120; EXFO Photonic Solutions, Inc.). An AxioVision Digital Imaging
System with a 3D deconvolution camera (Apotome and Axiocam MRm;
Carl Zeiss, Inc.) was used for data acquisition and analyses as well as for
time-lapse analyses of live cells. Time-lapse examinations of GFP-H2B –
expressing HeLa cells for chromatin alignment and segregation and ana-
phase onset were performed as previously described ( Rieder et al., 1994 ;
Meraldi et al., 2004 ).
277 SMURF2 IS ESSENTIAL FOR SPINDLE ASSEMBLY CHECKPOINT • Osmundson et al. Download full-text
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