T H E J O U R N A L O F C E L L B I O L O G Y
The Journal of Cell Biology, Vol. 170, No. 7, September 26, 2005 1047–1055
The Rockefeller University Press$8.00
Aurora A phosphorylation of TACC3/maskin is
required for centrosome-dependent microtubule
assembly in mitosis
Tim L. Noetzel,
and Anthony A. Hyman
David N. Drechsel,
Jordan W. Raff,
Max Planck Institute of Molecular Cell Biology and Genetics (MPI-CBG), 01307 Dresden, Germany
Research Institute of Molecular Pathology, A-1030 Vienna, Austria
The Wellcome Trust/Cancer Research UK Gurdon Institute, Cambridge CB2 1QN, England, UK
entrosomes act as sites of microtubule growth,
but little is known about how the number and
stability of microtubules emanating from a cen-
trosome are controlled during the cell cycle. We studied
the role of the TACC3–XMAP215 complex in this process
by using purified proteins and
tracts. We show that TACC3 forms a one-to-one complex
with and enhances the microtubule-stabilizing activity of
Xenopus laevis egg ex-
XMAP215 in vitro. TACC3 enhances the number of mi-
crotubules emanating from mitotic centrosomes, and
its targeting to centrosomes is regulated by Aurora
A–dependent phosphorylation. We propose that Aurora
A regulation of TACC3 activity defines a centrosome-
specific mechanism for regulation of microtubule poly-
merization in mitosis.
In animal cells, microtubules are nucleated from centrosomes
and grow out with their plus ends that lead into the cytoplasm
(for review see Howard and Hyman, 2003). The number and
stability of microtubule plus ends growing from centrosomes
changes through the cell cycle, which is concomitant with as-
sembly of the mitotic spindle (Hannak et al., 2001; Kline-Smith
and Walczak, 2004; Piehl et al., 2004). One obvious mechanism
controlling microtubule assembly at centrosomes is modulation
of the microtubule nucleation rate (for review see Job et al.,
2003). Indeed, the amount of
-tubulin increases at centrosomes
as cells enter mitosis (Khodjakov and Rieder, 1999; Hannak et
al., 2002). However, another equally plausible mechanism is
modulation of the stability of nucleated plus ends. The stability
of microtubule plus ends is dependent on the rate at which mi-
crotubules interconvert between growing and shrinking (called
the catastrophe rate) and between shrinking and growing (called
the rescue rate; Walker et al., 1988; Howard and Hyman, 2003).
egg extracts have been particularly useful
for studying the regulation of microtubule polymerization from
centrosomes. Because meiotic spindles in
can form without centrosomes by nucleation and stabilization of
microtubules around chromosomes (Heald et al., 1996; Karsenti
and Vernos, 2001; Sampath et al., 2004), it is possible to study
separately the regulation of microtubule polymerization around
mitotic centrosomes or chromosomes. In
the dynamic behavior of microtubules in the cytoplasm is regu-
lated, in large part, by the activity of XMAP215, which is a
member of a conserved family of microtubule-associated pro-
teins (Kinoshita et al., 2002; Gard et al., 2004). XMAP215 both
stimulates the growth rate and opposes catastrophe activity of
MCAK, which is member of the Kin I/kinesin-13
family (Walczak et al., 1996; Tournebize et al., 2000; Noetzel et
al., 2005). Physiological microtubule dynamics can be recon-
stituted in vitro by a mixture of XMAP215 and mitotic cen-
tromere-associated kinesin (MCAK) together with tubulin
(Kinoshita et al., 2001). Although XMAP215 and MCAK
clearly have a central role in determining microtubule dynamics
egg extracts, we know little about how these pro-
teins are regulated during cell cycle progression. However,
these proteins are localized to centrosomes, suggesting that they
could be involved in the regulation of microtubule growth from
centrosomes (Kinoshita et al., 2002; Gard et al., 2004).
A clue as to the possible mechanisms regulating
XMAP215 family proteins at centrosomes came from the
Correspondence to Kazuhisa Kinoshita: email@example.com
Abbreviations used in this paper: D-TACC,
FSG, fish skin gelatin; MCAK, mitotic centromere-associated kinesin; TACC,
transforming acidic coiled coil; TOG, tumor overexpressed gene; WT, wild type.
The online version of this article contains supplemental material.
Drosophila melanogaster TACC;
JCB • VOLUME 170 • NUMBER 7 • 2005 1048
discovery that the
XMAP215 family is associated with D-TACC (
transforming acidic coiled coil;
al., 2001). Mutants in TACC family members reduce microtu-
bule number and prevent the localization of XMAP215 family
proteins to centrosomes or spindles in
and Ohkura, 2001; Lee et al., 2001),
(Bellanger and Gönczy, 2003; Le Bot et al., 2003; Srayko et al.,
2003), yeasts (Usui et al., 2003; Sato et al., 2004), and human
cells (Gergely et al., 2003). Beyond the fact that TACC family
members are required to localize XMAP215 family members to
centrosomes, we know little about the role of TACC in the mod-
ulation of microtubule dynamics and in regulating the activity of
XMAP215. In this study, we investigated these issues by using
purified proteins and
egg extracts. Maskin is a
TACC homologue (Stebbins-Boaz et al., 1999); in this paper, we
TACC3 because of its sequence similarity to the
mammalian TACC3 subfamily (Still et al., 2004). We demon-
strate that a
TACC3 is a cell cycle–dependent regulator
member of the
Cullen and Ohkura, 2001; Lee et
TACC3 forms a complex with and
stimulates the activity of XMAP215
To look at the role of the TACC3–XMAP215 complex in mi-
crotubule assembly, we studied the activity of the complex in
vitro. We first confirmed via immunoprecipitation that native
TACC3 and XMAP215 form a protein complex in
egg extracts (Fig. 1 A). When coexpressed in baculovirus-
infected insect cells, recombinant TACC3 and XMAP215 pro-
XMAP215 in Xenopus egg extracts and in vitro.
(A) Coimmunoprecipitation in Xenopus egg ex-
tracts. Immunoprecipitation (IP) was performed
using control IgG (lane 1) or anti-TACC3 anti-
body (lane 2). The blots were probed with anti-
TACC3 (top) or anti-XMAP215 (bottom). (B)
Coimmunoprecipitation in the extracts of bacu-
lovirus-infected insect cells. Total lysates of
TACC3 baculovirus single infected cells and
TACC3 (lane 1) and XMAP215 virus double in-
fected cells (lane 2) were prepared for immuno-
precipitation. In each cell lysate, immunopre-
cipitation were performed using anti-TACC3
(lanes 3 and 4) or control IgG antibody (lanes
5 and 6). Total cell lysate and immunoprecipi-
tates that were dissolved in sample buffer were
subjected to SDS-PAGE, and the gel was
stained with Coomassie brilliant blue. (C) Frac-
tions from the top and bottom of a continuous
sucrose density gradient centrifugation with pu-
rified proteins. Fractions collected after 3–15%
continuous sucrose gradient centrifugation with
TACC3 alone (top), XMAP215 alone (middle),
or a mixture of TACC3 and XMAP215 (bottom)
were subjected to SDS-PAGE. The gels were
stained with Coomassie brilliant blue. (D) Gel
filtration with purified proteins. TACC3 alone
(top), XMAP215 alone (middle), or a mixture of
TACC3 and XMAP215 (bottom) were ana-
lyzed by gel filtration. Fractions were collected
and subjected to SDS-PAGE, and the gels were
stained with Coomassie brilliant blue.
TACC3 forms a complex with
MCAK. (A) Sedimentation analysis to monitor MCAK-dependent microtubule
destabilization activity. 2.5 ?M GMPCPP microtubules were mixed with 125
nM MCAK (lanes 3–14) or control buffer (lanes 1 and 2) in presence of ATP.
500 nM XMAP215 (lanes 5–14) and increasing concentrations of TACC3
from 250 (lanes 7 and 8), 500 (9 and 10), and 1,000 (11 and 12) to 1,500
nM (13 and 14) or control buffer (5 and 6) were added in reactions. Reac-
tion mixtures were sedimented after a 38-min incubation at 30?C, superna-
tants (S) and pellets (P) were subjected to SDS-PAGE, and the gel was stained
with Coomassie brilliant blue. (B) Sedimentation assay to monitor XMAP215
affinity to microtubules. 2.5 ?M GMPCPP microtubules were mixed with
1,250 nM TACC3 (lanes 1 and 2), 500 nM XMAP215 (3 and 4), or 500
nM XMAP215 in addition to increasing concentrations of TACC3 from 250
(5 and 6), 500 (7 and 8), 750 (9 and 10), and 1,000 (11 and 12) to 1,250
nM (13 and 14). Mixtures were incubated for 38 min at 30?C and sedi-
mented, and supernatants (S) and pellets (P) were analyzed by SDS-PAGE.
TACC3 increases the antagonizing activity of XMAP215 against
CENTROSOMES AND MICROTUBULE DYNAMICS IN M PHASE • KINOSHITA ET AL.1049
teins also form a complex (Fig. 1 B). By mixing purified
recombinant proteins and performing sucrose gradient centri-
fugation as well as gel filtration, we showed that TACC3 and
XMAP215 form a one-to-one complex with a native molecular
366 kD (Fig. 1, C and D).
Using a mixture of these purified proteins, we monitored
the ability of the TACC3–XMAP215 complex to oppose the mi-
crotubule-destabilizing activity of MCAK by using a microtu-
bule sedimentation assay (Desai et al., 1999a). In the presence
of MCAK, microtubules were completely destabilized (Fig. 2 A,
lanes 1 and 2) as reported previously (Desai et al., 1999a). The
addition of 500 nM XMAP215 resulted in a slight reduction of
MCAK’s microtubule-destabilizing activity (Fig. 2 A, lanes 3
and 4). However, XMAP215 that complexed with TACC3 more
efficiently opposed the destabilizing activity of MCAK (Fig.
2 A, lanes 7–14). TACC3 alone had no detectable effect on
MCAK activity (not depicted). One possibility is that TACC3
increases the microtubule-stabilizing activity of XMAP215 by
enhancing the affinity of XMAP215 for microtubules. Indeed,
microtubule sedimentation analyses that were performed in the
absence of MCAK indicated that the TACC3–XMAP215 com-
plex has a higher affinity for microtubules than XMAP215
alone (Fig. 2 B). We have previously shown that XMAP215 op-
poses MCAK in a dose-dependent manner (Kinoshita et al.,
2001). Therefore, we conclude that TACC3 stabilizes microtu-
bules in vitro by forming a complex with XMAP215, increasing
the affinity of XMAP215 for microtubules, and, thus, enhancing
the antagonizing activity against MCAK.
TACC3 is required for mitotic microtubule
assembly around centrosomes by
opposing the activity of MCAK
To confirm that TACC3 regulates microtubule growth under
physiological conditions, we analyzed meiotic spindle assem-
bly in cycling extracts (cycled spindles; Murray, 1991; Desai et
al., 1999b). Specifically, we used antibodies to immunodeplete
egg extracts and examined spindle as-
sembly in the depleted extracts. To our surprise, meiotic spindles
that assembled in the absence of TACC3 were indistinguishable
from spindles that assembled in mock-depleted extracts (Fig. S1,
available at http://jcb.org/cgi/content/full/jcb.200503023/DC1)
despite the depletion of TACC3 to undetectable levels. Under
similar depletion conditions for XMAP215, no spindles were
observed. This was unexpected because TACC mutants have
phenotypes in mitotic systems that were previously studied
(see Introduction; Raff, 2002).
In all systems in which localization has been examined,
TACC and XMAP215 are found at centrosomes, suggesting
that TACC3 could have a centrosome-specific role in mitosis.
Accordingly, we looked specifically at the role of TACC3 in
assembly around centrosomes. (A) Microtubules were visual-
ized by the addition of fluorescently labeled tubulin in control
mock-depleted (left) and TACC3-depleted extracts (right).
Centrosomes were added to interphase extracts (top), and
then the extracts were driven into M phase by the addition of
nondegradable cyclin B (bottom). Bar, 10 ?m. (B) Quantifi-
cation of tubulin fluorescence in A. (C) Immunoblots of de-
pleted extracts. The extracts were immunodepleted with con-
trol IgG (lanes 1 and 2; mock dep) or anti-TACC3 antibody
(lanes 3–6; ? TACC3) with adding back of TACC3 (lanes 5
and 6) or control buffer (lanes 1–4). The blots of interphase
(lanes 1, 3, and 5) and mitotic extracts (lanes 2, 4, and
6) were probed by anti-TACC3 (top; TACC3) and anti-
XMAP215 (bottom; XMAP215). (D) Microtubules assembled
around mitotic centrosomes in immunodepleted extracts with
add back of recombinant proteins. Purified recombinant
TACC3 or control buffer were added in mock-depleted or
TACC3-depleted extracts. Bar, 10 ?m. Insets show immu-
nolocalization of TACC3 on centrosomes. Bar, 1 ?m. (E)
Quantification of tubulin fluorescence in D. (F) Inhibition of
MCAK in TACC3-depleted extracts. Microtubules were as-
sembled around centrosomes in mock-depleted extracts plus
control buffer (left), TACC3-depleted extracts plus control
buffer (middle), or anti-MCAK antibody (right). The addition
of inhibitory MCAK antibody rescued the effect of TACC3
depletion on microtubule assembly around mitotic cen-
trosomes. Bar, 10 ?m. (G) Quantification of tubulin fluores-
cence in F. (B, E, and G) Relative fluorescence intensity of la-
beled tubulin around mitotic centrosomes (within 21.079 ?m
radius) was measured. Values shown are the means plus SD.
TACC3 is required for mitosis-specific microtubule
JCB • VOLUME 170 • NUMBER 7 • 20051050
microtubule growth from mitotic centrosomes. Traditionally,
centrosomal microtubule assembly has been examined in
egg extracts by adding human centrosomes directly to mi-
totic extracts (Belmont et al., 1990). However, the formation of
a mitotic centrosome is likely to be a complex process involving
cell cycle transition. To create mitotic centrosomes, we added
isolated centrosomes to interphase extracts and drove the ex-
tracts into mitotic (M) phase by the addition of a nondegradable
cyclin B (cyclin B
90; Glotzer et al., 1991). In mock-depleted
extracts, microtubule assembly around centrosomes was acti-
vated by the induction of M phase (Fig. 3 A, left). In TACC3-
depleted extracts, the microtubule number around centrosomes
was decreased to 13% of that in control extracts (Fig. 3 A, right;
and B). Importantly, adding back recombinant TACC3 protein
to depleted extracts increased microtubule assembly from mi-
totic centrosomes, returning it to the levels that were found in
control extracts (Fig. 3, C–E). In contrast to M phase–induced
extracts, no detectable effect of TACC3 depletion was observed
on microtubule assembly in interphase extracts (Fig. 3 A, top).
We confirmed that TACC3 was localized to these mitotic cen-
trosomes in mock-depleted and TACC3 add-back extracts but
not in TACC3-depleted extracts (Fig. 3 D, insets). We conclude
that TACC3 is required specifically for microtubule assembly
around mitotic centrosomes in
The in vitro characterization of TACC3 activity sug-
gested the possibility that in the absence of TACC3, microtu-
bules cannot grow from mitotic centrosomes because of high
MCAK activity. To test this idea, we examined the effect of
MCAK inhibition on microtubule growth in TACC3-depleted
extracts. We found that the addition of inhibitory MCAK anti-
bodies to TACC3-depleted extracts stimulated the growth of
microtubules from mitotic centrosomes (Fig. 3, F and G). This
led to the conclusion that the likely function of TACC3 is to
antagonize the microtubule destabilization by MCAK, pre-
sumably by increasing XMAP215 activity on centrosomes in
M phase but not in interphase.
Aurora A–phosphorylated TACC3 is
enriched at mitotic centrosomes
The results argue that TACC3 activity at centrosomes is specifi-
cally required for M phase microtubule growth and poses the
question as to whether cell cycle–specific modulation of TACC3
activity could regulate the growth of microtubules from cen-
trosomes. Experiments in
gested a role of the mitotic kinase Aurora A in TACC3 localiza-
tion to centrosomes (Giet et al., 2002; Bellanger and Gönczy,
2003; Le Bot et al., 2003) but could not distinguish between a
specific role of the kinase in modulating TACC3 activity at cen-
trosomes and a more general role in regulating centrosome as-
sembly and maturation. Therefore, we decided to investigate the
specific role of Aurora A in the regulation of TACC3 localiza-
tion and activity. TACC3 has three consensus Aurora A sites
(Cheeseman et al., 2002) that are conserved between
(Ser33, Ser620, and Ser626) and humans (Ser34, Ser552, and
Ser558; Fig. 4 A). Recent experiments have shown that TACC3
is an in vitro substrate of Aurora A in
creau et al., 2005) and humans (Tien et al., 2004). We confirmed
(at Ser626; Pas-
by mass spectrometry that
phorylated by Aurora A at these three consensus sites (Fig. S2,
available at http://jcb.org/cgi/content/full/jcb.200503023/DC1).
Furthermore, we mutated these three conserved sites to alanine
and showed that the incorporation of labeled
mutated protein (3A mutant; TACC3-3A) is reduced to 3% com-
pared with the wild-type (WT) protein (TACC3-WT; Fig. 4 B).
We raised phosphospecific antibodies to the three conserved Au-
rora A sites (Fig. 4 C). Because of the limitations of cytology in
systems and in order to use RNA interference as a con-
trol for antibody specificity, we examined the localization of
phosphorylated TACC3 in human tissue culture cells. Interest-
ingly, phospho-TACC3, which was stained by antibodies against
; Ser558 in humans), localized specifically
to mitotic centrosomes, whereas the general population of
TACC3, which was stained by polyclonal antibodies against the
fragment of TACC3 protein (73–265 amino acids), localized
throughout the mitotic spindle as previously reported (Fig. 5 A;
Gergely et al., 2000, 2003). siRNA of TACC3 greatly reduced
TACC3 is indeed phos-
P into the alanine-
vitro. (A) Consensus sequences for Aurora A phosphorylation in Xenopus
TACC3/maskin. Yellow boxes indicate conserved domains among the
TACC family, whereas green boxes are the domains that are highly con-
served with TACC3 homologues only. The 3A mutant protein (TACC3-3A)
has mutations of alanine substitution on three serine residues (Ser33,
Ser620, and Ser626) in the consensus sequences of Aurora A phosphory-
lation. Numbers represent the positions for amino acid residues of Aurora
A phosphorylation target sites in the amino acid sequence of TACC3. (B)
Aurora A kinase assay with recombinant WT versus alanine mutant
TACC3 proteins. WT or 3A mutant TACC3 was incubated with or without
recombinant Aurora A (?/? Aurora A) in the presence of ??[32P] ATP
(see Materials and methods). The reaction mixture was loaded onto SDS-
PAGE, and the gel was stained with Coomassie brilliant blue (CBB; lanes
1–4). The incorporation of 32P into TACC3 in the gel was measured by au-
toradiography (32P; lanes 5–8). (C) Characterization of phosphospecific
antibodies. TACC3-WT was incubated with or without Aurora A (?/?
Aurora A), and the reaction mixture was loaded onto SDS-PAGE. The blots
were probed with anti-TACC3 antibody (lanes 1 and 2), antiphospho-Ser33
(lanes 3 and 4), antiphospho-Ser620 (lanes 5 and 6), and antiphospho-
Ser626 (lanes 7 and 8).
Determination of Aurora A phosphorylation sites of TACC3 in
CENTROSOMES AND MICROTUBULE DYNAMICS IN M PHASE • KINOSHITA ET AL.1051
the staining of phospho-TACC3 as well as that of nonspecific
TACC3 (Fig. 5 B). Phospho-TACC3 antibodies recognized a
130 kD molecular mass specifically in mitosis-arrested
tissue culture cells (Fig. 5 C) as well as a band of the same mo-
lecular mass that disappeared after the immunodepletion of ei-
ther TACC3 (see Fig. 6 D) or Aurora A (Fig. S3, available at
egg extracts in mitosis. Therefore, we conclude that TACC3
protein on centrosomes is specifically phosphorylated during M
phase by Aurora A.
Phosphorylation of TACC3 by Aurora A
is required for its targeting to mitotic
To test whether phosphorylation is required to target TACC3 to
centrosomes, we examined the localization of the nonphos-
phorylatable mutant of TACC3 in
adding back WT (TACC3-WT) versus alanine-mutated TACC3
(TACC3-3A) to the depleted extracts (Fig. 6 D), we found that
centrosomes contained 10% of TACC3-3A compared with
TACC3-WT (Fig. 6 A, insets; and B). We confirmed that the
TACC3-3A mutant could still interact with XMAP215 both
in vitro and in
egg extracts (Fig. S4, available at
more, TACC3-3A still enhanced the affinity of XMAP215
for microtubules (Fig. 6 E). Importantly, TACC3-3A cannot
target XMAP215 to mitotic centrosomes as efficiently as
TACC3-WT (Fig. S5, available at http://jcb.org/cgi/content/
egg extracts. By
full/jcb.200503023/DC1), and this argues that TACC3 phos-
phorylation by Aurora A is required for efficient centrosomal
localization of the TACC3–XMAP215 complex in M phase.
To test whether targeting of TACC3 to centrosomes is
required for its activity, we assayed centrosome-mediated micro-
tubule assembly in TACC3-depleted extracts that were reconsti-
tuted with either WT or alanine-mutated TACC3. Compared with
the WT protein, the alanine-mutated TACC3 could only poorly
rescue the effect of TACC3 depletion (Fig. 6, A and C). We con-
firmed that, after addition to the extract, TACC3-WT was phos-
phorylated, whereas TACC3-3A was not (Fig. 6 D). Thus, the
targeting of TACC3 to centrosomes by Aurora A phosphoryla-
tion is required to stimulate microtubule growth from mitotic cen-
trosomes. We were unable to detect any effect of Aurora A phos-
phorylation of TACC3-WT on its ability to enhance the affinity
of XMAP215 for microtubules (Fig. 6 E). This implies that the
likely function of Aurora A phosphorylation of TACC3 is target-
ing of the TACC3–XMAP215 complex to mitotic centrosomes
rather than directly regulating its activity toward microtubules.
Activation of microtubule assembly by
the TACC3–XMAP215 complex on
Previous studies have shown that the combination of
XMAP215 and XKCM1/MCAK is essential to promote the dy-
namic properties of mitotic microtubule assembly (Kinoshita et
enriched at mitotic centrosomes. (A) Immuno-
localization of TACC3 and phospho-TACC3.
Deconvolved images of human tissue culture
cells stained for DNA (blue)/microtubules
(MTs; green), TACC3, and phospho-TACC3
(P-TACC3; stained by antiphospho-Ser626 an-
tibodies) in different cell cycle stages. Bars,
10 ?m. (B) Immunolocalization of TACC3 and
phospho-TACC3 in siRNA-treated cells. De-
convolved images of either control or TACC3
RNA interference–treated cells stained for
TACC3 (top), and phospho-TACC3 (P-TACC3;
bottom). Arrows indicate misaligned chromo-
somes that are indicative of the TACC3 RNA
interference phenotype. Bar, 10 ?m. (C) Phos-
phorylation of TACC3 is mitosis specific in hu-
man tissue culture cells. The TACC3 antibody
detects a protein of ?130 kD in extracts pre-
pared from either an asynchronous culture of
HeLa S3 cells (lane 1; I) or cells arrested in mi-
tosis by nocodazole treatment (lane 2; M).
The phosphospecific TACC3 antibody (an-
tiphospho-Ser626) recognizes a band with the
same molecular mass size in mitotic cells (lane
4) but not in asynchronously cultured cells
(lane 3). The blot probed by ?-tubulin antibody
is a loading control (lane 5, asynchronous cul-
tured cells; lane 6, mitotic arrested cells).
Aurora A phosphorylated TACC3 is
JCB • VOLUME 170 • NUMBER 7 • 20051052
al., 2001; Noetzel et al., 2005). We show that in vitro TACC3
and XMAP215 form a one-to-one complex that enhances
the ability of XMAP215 to oppose the inhibitory activity of
MCAK by increasing the affinity of XMAP215 for the micro-
tubule lattice. Therefore, TACC3 appears to have a specific
role in modulating the dynamic behavior of microtubules by
modifying the activity of XMAP215.
Microtubule growth from centrosomes or chromosomes re-
quires the presence of XMAP215 (Tournebize et al., 2000). Be-
meiotic spindle assembly is apparently unaffected
in the absence of TACC3, it appears that TACC3 is not required
for the global activity of XMAP215. Why is TACC3 required
specifically for XMAP215 activity at mitotic centrosomes? Our
data suggest that targeting of the TACC3–XMAP215 complex to
mitotic centrosomes overcomes the high microtubule-destabiliz-
ing activity of MCAK at centrosomes. Consistent with this idea,
MCAK is localized to centrosomes in many systems (Walczak et
al., 1996; Oegema et al., 2001), and colonic and hepatic tumor
overexpressed gene (TOG)/TOG protein, which is a human
XMAP215 orthologue, is required to protect spindle microtu-
bules from MCAK activity at centrosomes (Holmfeldt et al.,
2004). We propose that in the absence of TACC3, XMAP215 is
still able to counteract the activity of cytoplasmic MCAK.
MCAK localization to centrosomes, however, generates an envi-
ronment in which plus end growth is not favored, and XMAP215
cannot stabilize nascent nucleated plus ends. This could either be
because MCAK activity at centrosomes is enhanced or because
of an increased concentration of MCAK at centrosomes. Tar-
geting of TACC3–XMAP215 would enhance the activity of
XMAP215 at centrosomes, stabilizing nascent plus ends and al-
lowing them to grow from centrosomes (Fig. 7). This provides an
explanation for the seemingly paradoxical observation that a plus
end stabilizer localizes to centrosomes where microtubules are at-
tached via their minus ends (Gard et al., 2004; Gräf et al., 2004).
The centrosome-specific role of TACC3 could explain
why the phenotype of RNA interference of TACC family
members in tissue culture cells is not very severe. The chro-
matin-mediated pathway of spindle assembly obscures cen-
trosome-specific effects on microtubule growth. Interestingly,
embryos in which mitotic spindle assembly is
dominated by its centrosomes, the removal of TACC results in
severe microtubule-based phenotypes (Bellanger and Gönczy,
2003; Le Bot et al., 2003; Srayko et al., 2003).
Aurora A regulates centrosomal
microtubule assembly in mitosis
The targeting of TACC3 to centrosomes defines a potential
mechanism for regulating the polymerization of microtubules
from centrosomes in mitosis. Previous data in other systems has
shown that Aurora A is required to target TACC to centrosomes
(Giet et al., 2002; Bellanger and Gönczy, 2003; Le Bot et al.,
2003). However, the specific role of TACC3 phosphorylation
has been unclear, as Aurora A has general roles in both cen-
trosome assembly and cell cycle progression (Hannak et al.,
2001; Giet et al., 2002; Hirota et al., 2003). Our data show
that targeting of TACC3 to centrosomes specifically requires
for centrosomal localization of TACC3 and its microtubule-
stimulating activity. (A) Localization of WT versus phosphoryla-
tion mutant of TACC3 on mitotic centrosomes. Microtubules
around mitotic centrosomes were visualized by the addition of
fluorescent tubulin in add-back experiments. Bar, 10 ?m. Insets
show immunolocalization of TACC3 on centrosomes in immuno-
depleted extracts with add back of WT versus the phosphoryla-
tion mutant of TACC3. Bar, 1 ?m. (B) Quantification of immuno-
staining of TACC3 on centrosomes in A. Relative fluorescence
intensity of TACC3 staining on centrosomes (within 1.079 ?m ra-
dius) was measured. (C) Quantification of tubulin fluorescence in
A. Relative fluorescence intensity of labeled tubulin around mi-
totic centrosomes (within 21.079 ?m radius) was measured. (B
and C) Values shown are the means plus SD. (D) Immunoblots of
depleted extracts in add-back experiments. Mitotic extracts were
immunodepleted with control IgG (lane 1; mock dep) or anti-
TACC3 antibody (lanes 2–4; ? TACC3) with adding back of
control buffer (lanes 1 and 2; ? buffer), TACC3-WT (lane 3; ?
TACC3-WT), or phosphorylation mutant TACC3 (lanes 4; ?
TACC3-3A). (E) Sedimentation assay to monitor XMAP215 affin-
ity to microtubules in the presence of WT or phosphorylation mu-
tant TACC3. 2.5 ?M GMPCPP microtubules were mixed with
0.5 ?M XMAP215 alone (lanes 1, 2, 7, and 8), 0.5 ?M
XMAP215 ? 1.0 ?m TACC3-WT (lanes 3, 4, 9, and 10), or 0.5
?M XMAP215 ? 1.0 ?m TACC3-3A (lanes 5, 6, 11, and 12) in
the absence (lanes 1–6) or presence (lanes 7–12) of Aurora A.
Mixtures were incubated for 38 min at 30?C and sedimented,
and supernatants (S) and pellets (P) were subjected to SDS-
PAGE. The gels were stained with Coomassie brilliant blue (top;
CBB staining) or transferred onto nitrocellurose membrane for im-
munoblotting (IB) using the phosphospecific TACC3 antibody
Phosphorylation of TACC3 by Aurora A is required
CENTROSOMES AND MICROTUBULE DYNAMICS IN M PHASE • KINOSHITA ET AL.1053
Aurora A phosphorylation. Furthermore, targeting is essential
for TACC activity at centrosomes. Because Aurora A activity
increases as cells enter M phase, this would account for the in-
crease in microtubule polymerization that was observed as cen-
trosomes mature during the cell cycle. Consistent with this idea,
the protein level of both TACC3 and Aurora A as well as the ki-
nase activity of Aurora A are all highly cell cycle regulated,
reaching a peak during mitosis in human tissue culture cells (Bis-
choff et al., 1998; Gergely et al., 2003). In
the increase of Aurora A kinase activity may be sufficient to trig-
ger the activation of centrosomal microtubule polymerization in
mitosis. Thus, the phosphorylation of TACC3 provides a mech-
anism for Aurora A to specifically modulate the growth of
microtubules from centrosomes. However, Aurora A regulates
processes other than microtubule assembly at centrosomes. Iden-
tification of the other Aurora A substrates will be essential for
understanding the mechanisms of mitotic spindle assembly.
Xenopus egg extracts,
Materials and methods
Expression and purification of recombinant proteins
Full-length Aurora A cDNA was subcloned into pET21 vector (CLON-
TECH Laboratories, Inc.) for Xenopus Aurora A expression in bacteria
(pET21 Aurora A; a gift from J. Swedlow, University of Dundee, Dundee,
UK). TACC3 was expressed both in bacteria and insect cells. Full-length
TACC3 cDNA was subcloned into pET30 vector (CLONTECH Laborato-
ries, Inc.) for expression in bacteria (pET30a maskin; a gift from J. Rich-
ter, University of Massachusetts, Worcester, MA). The mutations with an
alanine substitution of Aurora A target sites were introduced by PCR.
Bacterially expressed His-tagged proteins were purified by using Ni–
nitrilotriacetic acid (QIAGEN). For the expression of TACC3 in insect
cells, we used the Bac-to-bac baculovirus expression system (Invitrogen).
Full-length TACC3 cDNA was subcloned into pFastbac vector for the
preparation of bacmid DNA (NH2-terminal His6 tagged). TACC3 bacu-
lovirus-infected expresSF? cells (Protein Sciences) were pelleted after a
42-h incubation at 27?C and were resuspended in ice-cold lysis buffer (50
mM Hepes, pH 7.5, 0.1% Triton X-100, 200 mM NaCl, 10 mM CaCl2,
25 U/ml benzonase [Novagen], 10 ?g/ml nocodazole [Sigma-Aldrich],
and 1? protease inhibitor mix). The inhibitor mix consisted of 10 ?g/ml
antipain-HCl, 10 ?g/ml APMSF, 6 ?g/ml chymostatin, 0.5 ?g/ml leu-
peptin, 2 ?g/ml aprotinin, 0.7 ?g/ml pepstatin, and 3.6 ?g/ml E64 (all
protease inhibitors were purchased from Sigma-Aldrich except for E64,
which was obtained from BIOMOL Research Laboratories, Inc.). The re-
suspended pellets were dounced 20 times on ice using a Dounce homog-
enizer. The lysate was cleared by centrifugation, loaded onto a column
(HiTrap Q FF; GE Healthcare), and equilibrated in 20 mM anion ex-
change buffer (Tris, BisTris-propane, and HCl), pH 7.5. The peak con-
taining full-length TACC3 was supplemented with 3 mM imidazole
(Sigma-Aldrich) and loaded onto a HiTrap chelating HP column (GE
Healthcare) that was equilibrated in binding buffer (25 mM Tris-HCl, pH
8.0, 300 mM NaCl, 3 mM imidazole, and 20% glycerol). The column
was washed stepwise with binding buffer supplemented with 5 mM ATP,
1.5 M NaCl, and 30 and 60 mM imidazole and was eluted with 300
mM imidazole. The peak containing full-length TACC3 was pooled and
desalted with a column (NAP25; GE Healthcare) into 20 mM anion
buffer, including 330 mM NaCl, 1 mM DTT, 0.1? protease inhibitor
mix, and 10% glycerol. The recombinant XMAP215 and XKCM1/XlM-
CAK were expressed in expresSF? insect cells (Protein Sciences) and
were purified as previously described (Tournebize et al., 2000; Kino-
shita et al., 2001). All of the purified proteins were aliquoted, frozen in
liquid nitrogen, and stored at ?80?C. Protein concentration was de-
termined by using the Bradford assay and the molecular extinction coeffi-
cient at OD280nm.
Immunoprecipitation, sucrose density gradient centrifugation,
and gel filtration
For immunoprecipitation using insect cell lysates, protein A–Sepharose
beads that bound control rabbit IgG or anti-TACC3 antibodies were incu-
bated with lysate from insect cells that were infected either with TACC3
baculovirus or with both TACC3 and XMAP215 baculovirus. Eluted immu-
noprecipitates were separated by SDS-PAGE and stained by Coomassie
Sucrose gradients (3–15%) were poured as step gradients (five
steps of equal volume) in 1? PBS, 0.1% Tween 20, 1 mM DTT, and
0.2? protease inhibitor mix. Gradients were incubated at 4?C for 12 h
to allow diffusion into a continuous gradient. Equal concentrations of
TACC3 and XMAP215 were mixed in vitro, incubated for 10 min on ice,
diluted into gradient buffer to a final concentration of 1% glycerol,
loaded onto the gradient, and spun for 6.5 h at 4?C and 120,000 g.
Gradients were fractionated from the top by hand with a cut pipette tip.
Fractions were analyzed by 4–12% SDS-PAGE (NuPage; Invitrogen) and
stained by Coomassie brilliant blue. Standard proteins with known sedi-
mentation values were run in parallel and scanned, band intensities
were quantified, and peak fractions were assigned. Standard curves of
peak fractions versus sedimentation coefficient were used to estimate the
S value of proteins. Standard proteins that were used in sucrose gradients
are listed as follows (sedimentation values are indicated in parentheses):
chymotrypsinogen (2.6 S); ovalbumin (3.5 S); BSA (4.6 S); aldolase
(7.3 S); and catalase (11.3 S).
Gel filtration chromatography was performed using a column (model
G5000PWXL; Tosoh) in 1? PBS, 0.1% Tween 20, 1 mM DTT, 5% glycerol,
and 0.2? protease inhibitor mix. The column was calibrated with stan-
dards of known Stokes radii. Standard curves of peak fractions versus the
logarithm of the Stokes radii were used to determine the Stokes radii of pro-
teins. Fractions were separated by 4–12% SDS-PAGE (NuPage; Invitrogen)
and stained by Coomassie brilliant blue. Standard proteins that were used
in gel filtration chromatography are listed as follows (Stokes radii are indi-
cated in parentheses): ribonuclease A (1.64 nm); chymotrypsinogen
(2.09 nm); ovalbumin (3.05 nm); BSA (3.55 nm); aldolase (4.81 nm);
catalase (5.22 nm); ferritin (6.1 nm); and thyroglobin (8.5 nm)
Depolymerization assay and sedimentation analysis of stabilized
Sedimentation analysis of the depolymerization of GMPCPP-stabilized mi-
crotubules was performed as described previously (Desai et al., 1999a).
2.5 ?M of prepolymerized GMPCPP microtubules were added to the reac-
tion mix that was supplemented with 125 nM MCAK or control buffer in 1?
BRB80 (80 mM Pipes, pH 6.8, 1 mM MgCl2, and 1 mM EGTA) containing
125 mM KCl and 1.5 mM Mg-ATP. 500 nM XMAP215 and increasing
amounts of TACC3 or control buffer were added to reactions. Reactions
were sedimentated after 38 min at 30?C through a glycerol cushion. Equiv-
alent amounts of the supernatant and pellets were analyzed by 4–12% SDS-
PAGE followed by Coomassie brilliant blue staining. For sedimentation
XKCM1/MCAK (red) localized on centrosomes generates an environment
in which plus end growth of microtubules (green) is not favored, and
XMAP215/TOG (light blue) cannot stabilize nascent nucleated plus ends
(left). Aurora A phosphorylation of TACC3/maskin targets the TACC3–
XMAP215 complex (dark blue) to centrosomes. The targeting enhances
the activity of XMAP215 at centrosomes, stabilizing nascent plus ends
and allowing microtubules to grow from centrosomes (right). The encir-
cled P (yellow) represents the serine residues that were phosphorylated
by Aurora A in TACC3.
A model of centrosomal microtubule assembly in M phase.
JCB • VOLUME 170 • NUMBER 7 • 20051054
assays to monitor the affinity of XMAP215 to microtubules, 2.5 ?m of prepo-
lymerized GMPCPP microtubules were incubated with 500 nM XMAP215 or
control buffer in 1? BRB80 containing 125 nM KCl in the presence of
TACC3 or control buffer. In each condition, we confirmed that all proteins
were in the supernatant after sedimentation without GMPCPP microtubules.
Preparation of Xenopus egg extracts and immunodepletion
Xenopus egg extracts were prepared as described previously (Murray,
1991; Desai et al., 1999b). Interphase extracts were prepared by the ad-
dition of calcium to cytostatic factor extracts. Cycloheximide was added to
100 ?g/ml to avoid translation of cyclin B messenger in extracts and to
arrest them in interphase. To generate mitotic extracts, a purified, nonde-
gradable cyclin B fragment (cyclin B ?90; a gift from H. Funabiki, The
Rockefeller University, New York, NY; Glotzer et al., 1991) was added to
25 ?g/ml to interphase extracts (Vorlaufer and Peters, 1998; Funabiki
and Murray, 2000). Immunodepletion in Xenopus egg extracts was per-
formed as described previously (Funabiki and Murray, 2000). All of the
immunodepletion experiments were performed in the presence of 100
?g/ml cycloheximde in extracts to block translation, as maskin has been
reported to be a negative regulator of translational machinery during oocyte
maturation (Stebbins-Boaz et al., 1999).
Antibodies and immunoblotting
Phosphospecific antibodies were raised and purified against phosphopep-
tides, including phosphoserine in the consensus target sites of Aurora A phos-
phorylation in Xenopus TACC3 (P-Ser33; QTTGRPphospho-SILRPSQ and
P-Ser620; CNSFKEphopho-SVLRKQ and P-Ser626; VLRKQphopho-SLYLKFC).
Anti–Xenopus TACC3 antibodies were raised and purified against the GST
fusion protein that contained an NH2-terminal fragment of TACC3 (aa
7–208). Anti–human TACC3 antibodies were previously described (Gergely
et al., 2000). Anti–Aurora A and -XMAP215 antibodies were raised and pu-
rified against COOH-terminal peptides (Aurora A, CKNSQLKKKDEPLPGAQ;
and XMAP215, CNIDDLKKRLERIKSSRK) as described previously (Field et al.,
1998). Anti–?-tubulin antibodies (DM1A) were purchased from Sigma-
Aldrich. Anti–NH2-terminal fragments of XlMCAK (XKCM1-NT) antibodies
were gifts from C. Walczak (Indiana University, Bloomington, IN; Walczak
et al., 1996). Immunoblotting was performed by using ECL (GE Healthcare).
For quantification of phospho-TACC3 signal, the signal intensities on the blot
were measured by image software (Scion Corp.).
Microtubule assembly and localization assays using Xenopus egg extracts
Recycled and fluorescently labeled tubulins (rhodamine- or Cy3-labeled)
were prepared from porcine instead of bovine brain as previously de-
scribed (Hyman et al., 1991; Ashford et al., 1998). Centrosomes were
purified from human KE37 cells as described previously (Moudjou and
Bornens, 1998). Xenopus interphase egg extracts that were supplemented
with centrosomes (5 ? 106/ml final) and fluorescently labeled tubulin
(0.5–1 ?M final) to visualize microtubules were incubated with or without
cyclin B ?90 for 20–30 min at RT. A part of the reactions was saved in
SDS-PAGE sample buffer for immunoblotting analysis. For microtubule as-
sembly assay, the extracts were fixed with extract fix (60% glycerol, 1?
Marc’s modified Ringer’s solution, 1 ?g/ml Hoechst 33342, and 10%
formaldehyde; Desai et al., 1999b) and squashed onto coverslips. For the
immunofluorescence of TACC3 on centrosomes, extracts were treated with
20 ?M nocodazole, fixed with fixation buffer (10% glycerol, 1? BRB80,
0.1% Triton X-100, and 5% formaldehyde), and spun down through glyc-
erol cusion (30% glycerol in 1? BRB80) onto coverslips. The samples on
coverslips were postfixed in ?20?C methanol and processed for immuno-
fluorescence as described previously (Desai et al., 1999b). The images
were collected using a wide-field microscope (Axioplan 2; Carl Zeiss Mi-
croImaging, Inc.) that was equipped with plan-Apochromat 100? NA
1.40 objective lens and a digital camera (model C4742-95; Hamamatsu).
For quantification of fluorescence intensity, images were acquired by using
a plan-Neofluor 16? NA 0.50 for the fluorescence signal of tubulin and a
plan-Apochromat 100? NA 1.40 for the signal of TACC3 staining. The in-
tegrated pixel intensities within the circle (21.079 ?m for tubulin; 1.079
?m for centrosomal TACC3) around centrosomes were calculated by using
Metamorph software (Universal Imaging Corp.). All of the measured values
were corrected by the subtraction of background signal in the same field.
In vitro protein kinase assay and mass spectometric analysis
0.4 mg/ml of bacterially expressed TACC3 was incubated with or without
0.02 mg/ml Aurora A in kinase buffer (25 mM Hepes, pH 7.7, 100 mM
KCl, 5 mM MgCl2, 50 mM sucrose, and 0.1% ?-mercaptoethanol) in the
presence of 0.1 mM ATP containing ?-[32P] ATP for 20 min at 30?C. The
reactions were stopped by the addition of SDS-PAGE sample buffer, and
the samples were analyzed by SDS-PAGE. The incorporation of 32P into
the TACC3 in the gel was detected by autoradiography by using a phos-
phoimager (model BAS-1800II; Fujifilm). Mass spectometric analysis for
identification of in vitro Aurora A phosphorylation sites of TACC3 was
performed as described previously (Kraft et al., 2003).
Cell culture and RNA interference
HeLa cells were grown in DME containing 10% serum supplemented with
nonessential amino acids (GIBCO BRL) and were grown using standard
procedures. For RNA interference, HeLa cells were seeded onto glass cov-
erslips 16 h before transfection. Cells that were grown to ?40% conflu-
ency were transfected for 6 h using the OligofectAMINE reagent (Invitro-
gen) and to a final concentration of 20 nM siRNA against either TACC3
(purchased from Ambion; Gergely et al., 2003) or a control siRNA (con-
trol siRNA#1; Ambion) according to the manufacturer’s recommendations.
After transfection, cells were incubated in fresh media for 72 h before
analysis by immunofluorescence.
HeLa S3 cells were grown in suspension in MEM that was modified
for suspension cultures (Biochrom) containing 10% serum supplemented
with nonessential amino acids (Biochrom) by using standard procedures.
Cells were arrested in mitosis by the addition of 90 ng/ml nocodazole
(Sigma-Aldrich) 20 h before harvesting and were shown to be ?95% mi-
totic as judged by DAPI staining.
Immunofluorescence and microscopy for culture cells
Transfected HeLa cells that were grown on coverslips were fixed and per-
meabilized in methanol at ?20?C for 8 min. Cells were washed with PBS
and incubated 10 min in PBS containing 0.2% fish skin gelatin (PBS-FSG;
Sigma-Aldrich) to prevent nonspecific binding of antibodies. Cells were in-
cubated 20 min with primary antibody. Antibodies that were used were
anti–?-tubulin (DM1A; Sigma-Aldrich), anti-TACC3 (Gergely et al., 2000),
and antiphospho-TACC3 (anti–P-Ser626 in this study) diluted to 1 ?g ml?1
in PBS-FSG. After washing in PBS, cells were incubated for 20 min at 37?C
in 1 ?g ml?1 (in PBS-FSG) of donkey secondary antibodies that were la-
beled with either Texas red, FITC, or Cy5 (Jackson ImmunoResearch Labo-
ratories) and were washed in PBS before mounting in the presence of 1 ?g
ml–1 DAPI to visualize chromatin. Three-dimensional datasets were acquired
on an imaging system (DeltaVision; Applied Precision) that was equipped
with a microscope (model IX70; Olympus), a camera (CoolSNAP; Roper
Scientific), and a 100? NA 1.4 plan-Apochromat objective. Images were
computationally deconvolved by using the SoftWork software package
(Applied Precision) and were shown as two-dimensional projections.
Online supplemental material
Fig. S1 shows spindles assembled in mock-depleted versus TACC3-
depleted Xenopus egg extracts. Fig. S2 shows in vitro phosphorylation
sites in TACC3/maskin as determined by mass spectrometry. Fig. S3 shows
TACC3 phosphorylation in Aurora A–depleted extracts. Fig. S4 shows the
characterization of phosphorylation mutant TACC3 in Xenopus egg ex-
tracts and in vitro. Fig. S5 shows centrosomal localization of XMAP215-
GFP in Xenopus egg extracts. Online supplemental material is available at
We are grateful to Jason Swedlow and Joan Ruderman (Harvard Medical
School, Boston, MA) for cDNA of Aurora A/Eg2; Joel Richter for cDNA of
Xenopus TACC3/maskin; Hironori Funabiki for purified cyclin B ?90; Claire
Walczak for anti-XKCM1/MCAK antibodies; Heino Andreas (MPI-CBG) for
help with the frog colony; Andrej Pozniakovski (MPI-CBG) for plasmid con-
struction of the alanine-mutated TACC3; and Arshad Desai (University of Cali-
fornia, San Diego, San Diego, CA), Hironori Funabiki, and Martin Srayko
(MPI-CBG) for stimulating discussion and comments on the manuscript.
K. Kinoshita was supported by a research fellowship from the Uehara
Memorial Foundation and a grant from the Deutsche Forschungsgemeinschaft
priority program Molecular Motors. L. Pelletier was supported by a postdoc-
toral fellowship from the Human Frontier Science Program.
Submitted: 4 March 2005
Accepted: 19 August 2005
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