Department of Biochemistry, School of Medical Sciences, University of
Bristol, Bristol BS8 1TD, UK
Present address: Department of Zoology, University of Oxford, South Parks
Road, Oxford OX1 3PS, UK
* Author for correspondence (e-mail: james.wakefield{at}zoo.ox.ac.uk)
Accepted 14 November 2002
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Summary |
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We demonstrate that lithium and two structurally distinct inhibitors of GSK-3 promote defects in microtubule length and chromosomal alignment during prometaphase. Treated cells contain mono-oriented chromosomes concentrated at the plus ends of astral microtubules, which are longer than in untreated cells. Live microscopy of cells expressing Histone-2B-GFP confirms that the inhibition of GSK-3 suppresses mitotic chromosome movement and leads to a prometaphase-like arrest. We propose that GSK-3 is regulated in a temporal and spatial manner during mitosis and, through controlling microtubule dynamics, plays an important role in chromosomal alignment on the metaphase plate.
Key words: GSK-3, Mitotic spindle, Microtubule, PKB
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Introduction |
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Several studies have also pointed towards a role for GSK-3 in the
regulation of microtubule dynamics during interphase. In neuronal cells, GSK-3
is able to phosphorylate a number of microtubule-associated proteins, such as
MAP2C, MAP1B and Tau (Lovestone et al.,
1996; Goold et al.,
1999
; Sanchez et al.,
2000
). Phosphorylation of these proteins by GSK-3 decreases their
ability to stabilise microtubules
(Lovestone et al., 1996
;
Wagner et al., 1996
;
Utton et al., 1997
).
Microtubule dynamics need to be exquisitely controlled during mitosis in
order to produce a spindle apparatus capable of successfully segregating
chromosomes. As cells enter mitosis, microtubule nucleation from the
centrosomes dramatically increases and microtubules become more 10-100 times
more dynamic (for a review, see Inoue and
Salmon, 1995; Compton,
2000
). The rapidly growing and shrinking microtubules are captured
and stabilised by chromosomes, allowing formation of a bi-polar mitotic
spindle. These changes in microtubule dynamics occur concomitantly with the
phosphorylation of many proteins, through the activation of a number of
mitotic kinases, such as cdc2 kinase, polo kinase and aurora kinase (for a
review, see Cassimeris, 1999
;
Nigg, 2001
).
We sought to examine a possible role for GSK-3 in regulating microtubule stability during mitosis in cultured mammalian cells. Here, we report that GSK-3 is phosphorylated at the minus ends of the mitotic spindle, where active PKB is also localised. Treatment of HeLa cells with inhibitors of GSK-3 leads to an increase in the length of mitotic microtubules and defects in chromosome congression on the metaphase plate, suggesting that GSK-3 is involved in regulating the balance of microtubule dynamics during mitosis.
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Materials and Methods |
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Microtubule sedimentation experiments
Mitotic cells were briefly washed in Buffer A (50 mM Hepes pH 7.4, 50 mM
KCl, 1 mM EDTA, 1 mM EGTA, 1 mM MgCl2), shaken off and centrifuged
at 1000 g. The cell pellet was resuspended in Buffer B [Buffer
A + 0.5% Triton X-100, 1 mM NaF, 1 mM PMSF and 1 µM protease inhibitors
(pepstatin, leupeptin and antipain)] before centrifugation at 14,000
g for 5 minutes. The clarified supernatant was then
centrifuged for 10 minutes at 100,000 g, followed by a further
40 minutes at 100,000 g. Dithiothreitol (DTT) and GTP were
added to the high speed supernatant (1 mM final concentration), which was then
divided into two equal aliquots. The supernatants were warmed to 37°C for
5 minutes to allow polymerisation to initiate, and then taxol was added to a
final concentration of 10 µM. In control supernatants, buffer without taxol
was added. Extracts were left at 37°C for a further 10 minutes, and then
transferred to ice for 10 minutes. The supernatants were then layered onto an
equal volume cushion of Buffer A with 40% sucrose, before centrifugation at
100,000 g for 40 minutes. Equal fractions of supernatant and
pellet were resuspended in protein sample buffer and subjected to
SDS-PAGE.
Immunofluorescence microscopy and live cell imaging
Cells were fixed either with 4% paraformaldehyde for 20 minutes or in
methanol (-20°C) for 5 minutes (for -tubulin and phospho-PKB)
before being permeabilised and blocked in PBS plus 0.1% Triton with 3% BSA for
45 minutes. When visualising GSK-3, coverslips were briefly washed in MTSB
(100 mM PIPES pH 6.8, 1 mM EGTA, 5 mM MgCl2) before incubation at
37°C for 1 minute with MTSB + 0.5% Triton. Cells were then fixed as
described above. Primary antibody incubations were carried out for 1 hour at
room temperature. The following primary antibodies were used: GSK-3 (Upstate
Biotechnology, USA), Phospho-GSK-3
/ß (Ser21/9) (Cell Signalling
Technologies, NEB, USA), PKB (Oncogene, Germany), phospho-PKB (T308)
(Biosource, USA),
-tubulin, DM1A and
-tubulin, GTU-88 (Sigma).
Staining was visualised using the appropriate secondary antibodies (Alexa 488
and Alexa 568; Amersham Bioscience, UK). Coverslips were mounted on slides
with Vectashield mounting medium containing DAPI (Vector Labs, USA). Images
were collected with a Leica SP2 Laser Scanning Confocal Microscope and the
images processed with Adobe Photoshop 5. All movies of Histone2B-GFP dynamics
were acquired using an Olympus/TILL Photonics imaging system. Images were
acquired with a PLAPO 100x, NA 1.40, oil immersion objective;
illumination was provided by a 150W Xenon lamp controlled by a Polychrome IV
monochromator coupled by a Quartz light guide to an Olympus IX-70 microscope.
Emission filters for GFP/Cy3 were from Chroma (Brattleboro, VT). Images were
captured with a TILL IMAGO SVGA camera controlled by TILL visION v.4.0
software and processed using Image J
(http://rsb.info.nih.gov/ij),
QuickTime Pro v. 5.0 (Apple, Cupertino, CA) and Adobe Photoshop v6.0 (Adobe
Systems, San Jose, CA). Live cells were cultured in 35 mm glass-bottomed
dishes (Mat-Tek Co, USA), mounted in MEM (Gibco-BRL) supplemented with 5%
serum and imaged at 37°C with the microscope enclosed in a heated Perspex
box (Solent Scientific, Portsmouth, UK). Images were taken every 3 or 4
seconds using a 50 millisecond exposure. A total of approximately 20 untreated
and GSK-3 inhibitor treated mitotic cells were imaged. All untreated cells
were followed until chromosome decondensation to ensure they were able to exit
mitosis. To measure chromosome movements, distances were calculated from the
kinetochore region of the mitotic chromosome (seen as a constricted, less
fluorescent area in the images) to the centre of the metaphase plate. Maximal
chromosome velocities and trajectories were measured using a manual particle
tracking macro plug-in to Scion Image kindly provided by Jens Rietdorf of the
Advanced Light Microscopy Facility at EMBL Heidelberg.
SDS-PAGE and western blotting
Antibody detection was performed using either enhanced chemiluminescence
(Amersham Bioscience, UK), or the Supersignal kit (Pierce, USA) according to
the manufacturer's instructions. Primary antibodies were used at 1:1000
dilution. Horseradish-peroxidase-conjugated anti-rabbit IgG secondary
antibodies were obtained from Sigma and used at 1:10,000 dilution for the
GSK3ß (Transduction Laboratories, USA) and tubulin antibody, and at
1:2000 for the phospho-specific antibodies. Blots were stripped using Pierce
Restore Western reagent for 30 minutes before re-probing with subsequent
antibodies.
Online supplemental material
The movies show chromosomal movements in prometaphase HeLa cells stably
expressing Histone-2B-GFP. 600 images were acquired at 3 second intervals
(total imaging time of 30 minutes) and converted into movies using QuickTime
Pro v.5.0. QuickTime movies were assembled to play back at 10 frames per
second. Movie 1, an untreated cell; Movie 2, a cell treated with 30 µM
SB-415286 for 60 minutes prior to imaging.
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Results |
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GSK-3 is phosphorylated during mitosis and is present at centrosomes
and spindle poles
To investigate the phosphorylation status of GSK-3 in mitotic cells, we
western blotted cell lysates with a phosphospecific antibody that recognises
both phosphoserine-21 of GSK-3 and phosphoserine-9 of GSK-3ß. In
lysates from unsynchronised cells, GSK-3 is constitutively active and
possessed a very low basal level of phosphorylation
(Fig. 2A). However, there was a
marked increase in GSK-3 phosphorylation in extracts derived from cells
synchronised in mitosis using either nocodazole
(Fig. 2A) or a double thymidine
block (data not shown). We next examined whether the phosphorylation of GSK-3
was spatially regulated during the HeLa cell cycle. Confocal microscopy
revealed that during interphase, phospho-GSK-3 was diffusely distributed
throughout the cell (Fig. 2B).
In contrast, we observed a striking accumulation of phospho-GSK-3 at the
centrosomes upon entry into mitosis. As the cells progressed from prophase to
metaphase, the intensity of phospho-GSK-3 immunoreactivity increased and
extended to the surrounding minus ends of the mitotic spindle
(Fig. 2B). As cells exited
mitosis the level of phospho-GSK-3 on the spindles returned to basal levels,
although weak centrosome staining was seen until late telophase
(Fig. 2B). Similar results were
obtained using HEK293 cells (data not shown). Co-staining cells for GSK-3 and
phospho-GSK-3 confirmed that GSK-3 was present along the length of the
spindle, while phospho-GSK-3 staining was seen only at the centrosomes and
spindle poles (Fig. 2C). The
staining was specific to this antibody; we did not observe centrosomal and/or
spindle pole staining with a number of other phosphospecific antisera (e.g.
phospho-Histone H3; data not shown). To see if the accumulation of
phospho-GSK-3 was dependent on microtubules, we treated cells with 1 µM
nocodazole before fixing and staining (Fig.
2D). We found that although phospho-GSK-3 could still be detected
at centrosomes in cells lacking microtubules, the intensity of staining was
greatly reduced compared with that in untreated cells, suggesting that GSK-3
can associate both with centrosomes and with spindle microtubules.
|
PKB is phosphorylated during mitosis and is present at the
centrosome
As GSK-3 is phosphorylated on the inhibitory serine by the upstream kinase
PKB (Cross et al., 1995), we
next asked whether PKB is also regulated during mitosis. PKB is phosphorylated
on two specific sites, Thr308 and Ser473
(Alessi and Cohen, 1998
).
Thr308 is phosphorylated by an upstream kinase, PDK1 (which is itself
activated by phosphoinositide-lipids), leading to activation of PKB
(Alessi et al., 1997
;
Alessi and Cohen, 1998
).
Activity is further increased by phosphorylation on Ser473 although the
kinase(s) involved are not yet fully defined. We used antibodies specific for
PKB phosphorylated on Thr308 to determine its activity either in interphase or
during mitosis. By western blotting HeLa cell lysates we found that
phosphorylation of PKB on Thr308 was greatly increased in cells synchronised
in mitosis when compared with that in non-synchronised cells, which strongly
suggested that PKB is activated during mitosis
(Fig. 3A).
|
We next examined the regulation of PKB phosphorylation in HeLa cells during the cell cycle by confocal microscopy. PKB was weakly localised to the centrosomes throughout mitosis (Fig. 3B). However, using anti-phospho-PKB (Thr308) antibodies we found that the phosphorylation of the centrosomal PKB rapidly increased as the cells entered mitosis (Fig. 3C). Phosphorylation on Thr308 was initiated during prophase, steadily increased until metaphase, and then returned to basal levels following sister chromosome separation. Immunostaining of HEK293 cells produced similar results (data not shown). Thus PKB, like GSK-3, appears to be regulated in a spatio-temporal manner during mitosis.
Inhibition of GSK-3 causes defects in astral microtubule length and
chromosome alignment
To determine whether the accumulation of phosphorylated GSK-3 at the minus
ends of the mitotic spindle was indicative of a role for GSK-3 in the
regulation of microtubules in cell division, we sought to inhibit GSK-3 with
either of two structurally distinct and selective inhibitors of GSK-3,
SB-216763 and SB-415286 (Coghlan et al.,
2000). We incubated cycling HeLa cells with inhibitors for 90
minutes and then fixed and stained with anti-tubulin antibodies and DAPI, to
visualise microtubules and DNA, respectively.
Clear and specific defects in chromosomal alignment were observed as a result of these treatments (Fig. 4). Bi-polar mitotic spindle assembly did not appear to be greatly affected; however, many spindles contained extended astral microtubules emanating from the centrosomes (Fig. 4B-D). In some cases, additional long microtubules were formed from one pole, which appeared to force the centre of the spindles away from the centre of the cell (Fig. 4C). Careful examination of the position of chromosomes on these metaphase spindles revealed that, in approximately half of the metaphase-like cells, many chromosomes were not correctly aligned (Table 1; Fig. 4B-D). These chromosomes were able to attach to microtubules but remained mono-oriented and displaced from the main spindle. In some examples many of the chromosomes were mono-oriented, and seemed to cluster around one spindle pole (Fig. 4B,C). In addition, we saw many examples where chromosomes were present close to both poles (Fig. 4D).
|
|
To see if the perturbation in spindle dynamics was transient, we incubated cells with SB-415286 for 16 hours before fixation. Overnight treatment with the GSK-3 inhibitor increased the proportion of cells in mitosis and the defects in chromosome alignment and spindle structure described above were more prominent (Fig. 4G). Cells exhibited bipolar spindles containing astral arrays of microtubules that were more dense and much longer than in untreated cells. Furthermore, these poles contained many mono-oriented chromosomes concentrated at the ends of the extended astral microtubules. Similar results were obtained using HEK293 cells (data not shown).
Lithium is a well established inhibitor of GSK-3
(Klein and Melton, 1996;
Stambolic et al., 1996
).
Importantly, incubation of HeLa cells with concentrations of LiCl known to
inhibit GSK-3 (20-40 mM) brought about the same phenotypic effects as either
SB-216763 or SB-415286 (Fig.
4E; Table 1).
The presence of long astral microtubules in cells in which GSK-3 has been
inhibited is suggestive of a disruption of mitotic microtubule dynamics.
Indeed, the above observations are similar to those reported for cells treated
with low doses of the microtubule-stabilising drug taxol
(Ault et al., 1991;
Jordan et al., 1996
). We
confirmed these results by treating HeLa cells with 1 nM taxol for 90 minutes.
We found that a proportion of cells showed chromosomal displacement phenotypes
similar to those observed with the GSK-3 inhibitors
(Fig. 4F). The above results
strongly argue that GSK-3 activity is required in mitosis to correctly align
chromosomes on the metaphase plate. As GSK-3 is normally inactive at the
spindle poles through phosphorylation, the inhibitors must be affecting the
non-phosphorylated and active GSK-3 that is both present along the main body
of the spindle as well as the GSK-3 present in the cytosol.
Inhibition of GSK-3 during mitosis does not affect centrosome
separation but does disrupt mitotic chromosome movement
As the abnormally long microtubule asters seen in cells treated with GSK-3
inhibitors seemed to emanate predominantly from one pole, we examined whether
this was due to a perturbation in microtubule dynamics or as a result of a
failure to separate duplicated centrosomes
(Khodjakov et al., 2000). To
distinguish between these possibilities we treated HeLa cells
(Fig. 5) or HEK293 cells (not
shown) with the GSK-3 inhibitor SB-415286 for 90 minutes and stained them with
antibodies to gamma-tubulin. In cells containing mono-oriented chromosomes two
dots were seen on opposite sides of the metaphase plate
(Fig. 5). Identical results
were obtained using SB-216763 (data not shown). Thus we conclude centrosome
duplication and separation are not affected by inhibiting GSK-3.
|
If the inhibitors affect microtubule dynamics during mitosis, they would be
expected to disrupt the oscillations of the chromosomes as they congress on
the metaphase plate. To address this, we captured images of chromosomes
throughout mitosis using a HeLa cell line stably expressing Histone 2B-GFP
(Kand et al., 1998) and an
Olympus/TILL Photonics imaging system (see Materials and Methods). By imaging
of untreated cells, we were able to observe fast reversible oscillations
associated with mono-oriented chromosomes, as well as general chromosome
movements (Fig. 6A; see also
Movie 1,
http://jcs.biologists.org/supplemental).
The cell shown in the time sequence in Fig.
6A shows chromosome congression on the metaphase plate (0-600
seconds), with subsequent separation (1200-1500 seconds); both daughter cells
subsequently underwent chromosome decondensation (not shown). The arrow in
panel `300 sec' shows the position of a chromosome undergoing significant
movement. This is shown in more detail in the zoomed images (279-458s, the
arrow marks the same starting position in all frames) and is clearly seen in
the associated time-lapse sequence (Movie 1). The maximum chromosome velocity
observed in untreated cells was 5.8 µm min-1. Imaging of cells
treated with either of the GSK-3 inhibitors for 60 minutes revealed that
chromosome movements were highly suppressed; the maximal chromosome velocity
was reduced to 1.0 µm.min-1
[Fig. 6B (using SB-415286); see
Movie 2,
http://jcs.biologists.org/supplemental].
Notably, cells treated with GSK-3 inhibitors for 90 minutes were not seen to
enter anaphase up to 60 minutes after the start of imaging (120 minutes in the
continued presence of the inhibitor, not shown). Similar results were obtained
from time-lapse imaging of more than 20 cells in the presence or absence of
the inhibitors.
|
Particle tracking of these data clearly shows the absence of movement in SB-415286 treated cells (Fig. 7A,C) compared with that in control cells (Fig. 7B,D). Tracks of two dynamic chromosomes were superimposed onto the first image of each time sequence (Fig. 7A,B). Fig. 7C and D show the total distance moved (including movements away from as well as towards the metaphase plate) by these highlighted chromosome over time. The severe reduction in chromosome movement seen in cells treated with GSK-3 inhibitors was further analysed by measuring the distance between the oscillating chromosomes and the centre of the metaphase plate against time (Fig. 7E,F). Whereas chromosomes from untreated prometaphase cells showed several rapid oscillations followed by alignment on the metaphase plate (Fig. 7E), mono-oriented chromosomes in treated cells failed to show similar movement (Fig. 7F). As microtubules are the force behind chromosome congression, these results suggest that inhibiting GSK-3 affects chromosome oscillations and alignment on the metaphase plate through a suppression of normal microtubule dynamics.
|
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Discussion |
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We suggest that the cell-cycle-dependent accumulation of phospho-PKB at the centrosomes and of phospho-GSK-3 at the centrosomes and spindle poles is indicative of a role for these proteins at these sites during mitosis. Thus the active PKB associated with the centrosomes is likely to be capable of phosphorylating the GSK-3 present on the centrosome and nearby spindle microtubules. This would result in the generation of phospho-GSK-3 at centrosomes and spindle poles, while leaving GSK-3 active elsewhere in the cell.
Inhibition of GSK-3 activity during mitosis affects astral
microtubule length and chromosome dynamics
To investigate the role of active GSK-3 during mitosis, we sought to
inhibit GSK-3 throughout the cell using specific inhibitors. One advantage of
probing protein function using inhibitors is that behaviour of cells in which
GSK-3 has been inactivated can be assessed within minutes. Other methods such
as direct gene knockouts and siRNA are subject to the plasticity of biological
systems. The phenotypes analysed are usually the result of much greater
lengths of time without normal levels of the protein of interest and, as such,
the primary defects in cellular function can be extremely difficult to
interpret. In addition, as some proteins, such as GSK-3, fulfil many different
cellular roles, reducing the amount of protein using these methods can result
in the accumulation of numerous phenotypes within a cell. One caveat with
inhibitor studies, however, is the specificity of the compounds used.
Recently, Leclerc and co-workers have investigated the effect on the cell
cycle of a compound that inhibits CDK1
(Leclerc et al., 2001).
Interestingly, this compound was also noted to inhibit GSK-3
(Leclerc et al., 2001
).
However, incubation of unsynchronised cells with this inhibitor resulted in a
G2 arrest and the absence of metaphase figures
(Damiens et al., 2001
).
Furthermore, treatment of mitotic cells led to premature exit from mitosis
without cytokinesis, resulting in aneuploidy and endoreplication
(Damiens et al., 2001
).
Therefore, it is extremely unlikely that the effects on spindle microtubules
and chromosome alignment we observe in cells treated with three distinct GSK-3
inhibitors (SB-216763, SB-415286 and lithium) are a result of inhibiting
CDK1.
By accurately inhibiting GSK-3 in cells about to enter mitosis we can assess the function of this kinase at this time of the cell cycle. As GSK-3 is normally inactive at the spindle poles through phosphorylation, the inhibitors must be affecting both the non-phosphorylated and active GSK-3 that is present along the main body of the spindle as well as the GSK-3 that is free in the cytosol. Treatment of mitotic cells with the GSK-3 inhibitors causes an increase in the length of the astral microtubules, such that some contain bi-polar spindles that are pushed away from the centre of the cell. It also promotes the accumulation of mono-oriented chromosomes on bi-polar spindles. One possible interpretation of these results is that inhibition of GSK-3 activity during mitosis causes a perturbation of normal microtubule dynamics that leads to defective congression of chromosomes on the metaphase plate. This hypothesis is further strengthened by our observations of live HeLa cells expressing Histone-2B-GFP. The frequent transitions between microtubule growth and microtubule shrinkage that occur during mitosis are responsible for causing the chromosome oscillations normally found in prometaphase cells. Importantly, we find that these rapid, reversible chromosome oscillations are inhibited in cells in which GSK-3 is inhibited. Chromosome movements still occur, but do so more slowly, while many of the mono-oriented chromosomes fail to align on the metaphase plate, even after many hours. These cells contain unattached kinetochores and, as such, remain in mitosis presumably through the activation of the spindle checkpoint.
This phenotype is reminiscent of cells treated with low doses of drugs that
interfere with microtubule dynamics and we have confirmed that similar
chromosome displacements occur when HeLa cells are treated with nanomolar
concentrations of taxol. In addition, a recently identified
microtubule-interacting drug, noscapine, leads to a mitotic arrest phenotype
that is similar to the one seen when GSK-3 is inhibited
(Zhou et al., 2002). However,
while noscapine inhibits the rate of microtubule catastrophe without affecting
astral microtubules and microtubule polymerisation, the GSK-3 inhibitors (like
taxol) cause an increase in astral microtubule length and number. These
results suggest that inhibiting GSK-3 promotes stabilisation of microtubules.
This is wholly consistent with several studies which show that GSK-3
destabilises microtubules in interphase cells
(Lovestone et al., 1996
;
Goold et al., 1999
;
Krylova et al., 2000
;
Zumbrunn et al., 2001
). It is
therefore likely that GSK-3 is acting in a similar manner during mitosis, and
that by globally inhibiting the kinase, microtubules become stabilised.
The role of spatially regulating GSK-3 during mitosis
We have shown that GSK-3 is present on the mitotic spindle where it
presumably acts to phosphorylate target proteins. However, in normal cells
there appears to be a phosphorylated and inactive fraction of GSK-3 at the
centrosome and spindle poles. Why should GSK-3 be inactivated here during
mitosis? We propose that this spatial regulation of GSK-3 along the spindle
normally contributes towards differences in microtubule dynamics between those
microtubules near to the centrosomes and those in other areas of the cell.
During mitosis, active PKB would inactivate GSK-3 in the vicinity of the
centrosomes. This would contribute towards the stabilisation of microtubules
in this area of the cell, allowing centrosomes to become the dominant site of
microtubule growth. Conversely, GSK-3 would remain active along the main body
of the spindle, de-stabilising microtubules further away from the poles, and
contributing towards the highly dynamic search for chromosomes by
microtubules. The use of GSK-3 inhibitors during mitosis would mimic the
effect of phosphorylation of GSK-3 but would act throughout the cell, not just
at the spindle poles. As a result, this global inhibition of GSK-3 would lead
to stabilisation of all microtubules, not just those close to the
centrosome.
The substrates for phosphorylation by GSK-3 that lead to destabilisation of
spindle microtubules are not known at present. In interphase cells, GSK-3 has
been reported to phosphorylate Tau and other MAPs
(Lovestone et al., 1996;
Goold et al., 1999
). However,
these MAPs are predominantly expressed in neurons and are unlikely to be
important in regulating microtubule stability in other cell types. GSK-3 is
also able to phosphorylate APC. Recently APC has been found to bind to
microtubules both in vitro and in vivo, increasing their stability
(Zumbrunn et al., 2001
).
Phosphorylation of APC by GSK-3 decreases the interaction between APC and
microtubules, making microtubules less stable
(Zumbrunn et al., 2001
).
Furthermore, mutations in APC cause defects in chromosome segregation
(Kaplan et al., 2001
;
Fodde et al., 2001
). Another
possible target for GSK-3 is the microtubule-associated protein CLASP2
(Akhmanova et al., 2001). CLASP2 associates with CLIP-115 and CLIP-170,
cytoplasmic linker proteins that specifically associate with the ends of
microtubules. Inhibition of GSK-3 increases the accumulation of CLASP2 at the
plus end of microtubules, again leading to their stabilisation (Akhmanova et
al., 2001). Whether CLASP2 has a role in mitosis has not yet been tested.
However, by uncovering a previously unknown role for GSK-3 in chromosomal
alignment and mitosis, identifying the substrates for GSK-3 involved in
spindle microtubule assembly is now an important goal.
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Acknowledgments |
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Footnotes |
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