Directional cell migration is necessary for developmental
morphogenesis, tissue repair and tumour metastasis. A typical
migrating cell has front-rear polarity with a single leading edge at
the front end and a tail at the rear end. Cell polarisation includes
the asymmetrical distribution of signalling molecules and
cytoskeletal components, including actin filaments and microtubules
(Ridley et al., 2003). Although actin and microtubules have distinct
roles, these two cytoskeletal components are coordinated in a
structural or regulatory manner during cell migration (Rodriguez
et al., 2003; Watanabe et al., 2005). Among many regulatory
molecules for cell migration, the Rho-family GTPases, including
Rac1, Cdc42 and RhoA, have crucial roles in cell polarisation and
migration through the regulation of actin filaments, microtubules
and the adhesion apparatus (Burridge and Wennerberg, 2004;
Fukata et al., 2003; Jaffe and Hall, 2005).
During the establishment of polarity in migrating cells, the
temporal capture and stabilisation of some microtubule plus-ends
occur near the actin-enriched leading edges, which are thought
to enable the reorientation of the microtubule-organising centre
and the Golgi complex toward the leading edges. These ensure
that the cells exert biased vesicular transport for directional
migration (Etienne-Manneville, 2004; Kirschner and Mitchison,
1986; Watanabe et al., 2005). Plus-end-tracking proteins (+TIPs)
bind specifically to the growing ends of microtubules and affect
microtubule dynamics (Akhmanova and Steinmetz, 2008). +TIPs
include structurally unrelated protein families and are thought to
connect the Rho-family GTPases to microtubules for the temporal
stabilisation of microtubules, and for the coupling of microtubules
to actin filaments (Gundersen et al., 2004; Watanabe et al., 2005).
For example, activated Rac1/Cdc42 appears to interact with
CLIP-170 (official symbol, CLIP1) through IQGAP1, an effector
of Rac1/Cdc42 and an actin-binding protein, at the leading edges
(Fukata et al., 2002; Watanabe et al., 2004). Although the Rho-
family GTPases and their effectors show asymmetrical
distribution and activity along the front-rear axis of migrating
cells, the typical +TIPs such as EB1 and CLIP-170 accumulate
uniformly at the growing ends of microtubules (Akhmanova and
Steinmetz, 2008). Among the +TIPs, adenomatous polyposis coli
(APC) and CLASPs accumulate asymmetrically in a population
of the growing ends of microtubules near the cortex (Akhmanova
et al., 2001; Bienz, 2002; Mimori-Kiyosue et al., 2005; Nathke
et al., 1996; Watanabe et al., 2004; Reilein and Nelson, 2005;
Wittmann and Waterman-Storer, 2005). Thus, they are probably
involved in a region-specific control of microtubules in migrating
Polarised cell migration is required for various cell behaviours
and functions. Actin and microtubules are coupled structurally
and distributed asymmetrically along the front-rear axis of
migrating cells. CLIP-associating
accumulate near the ends of microtubules at the front of
migrating cells to control microtubule dynamics and cytoskeletal
coupling. Regional inhibition of GSK-3β β is responsible for this
asymmetric distribution of CLASPs. However, it is not known
how GSK-3β β regulates the activity of CLASPs for linkage
between actin and microtubules. Here we identified IQGAP1,
an actin-binding protein, as a novel CLASP-binding protein.
GSK-3β βdirectly phosphorylates CLASP2 at Ser533 and Ser537
within the region responsible for the IQGAP1 binding.
Phosphorylation of CLASP2 results in the dissociation of
CLASP2 from IQGAP1, EB1 and microtubules. At the leading
edges of migrating fibroblasts, CLASP2 near microtubule ends
partially colocalises with IQGAP1. Expression of active GSK-
3β β abrogates the distribution of CLASP2 on microtubules, but
not that of a nonphosphorylatable CLASP2 mutant. The
phosphorylated CLASP2 does not accumulate near the ends of
microtubules at the leading edges. Thus, phosphorylation of
CLASP2 by GSK-3β β appears to control the regional linkage of
microtubules to actin filaments through IQGAP1 for cell
Supplementary material available online at
Key words: GSK-3, IQGAP1, CLASP2, EB1, Microtubule
Phosphorylation of CLASP2 by GSK-3β β regulates its
interaction with IQGAP1, EB1 and microtubules
Takashi Watanabe1,2,*, Jun Noritake1,3,*, Mai Kakeno1,4, Toshinori Matsui1, Takumi Harada1, Shujie Wang1,
Norimichi Itoh1, Kazuhide Sato1, Kenji Matsuzawa1, Akihiro Iwamatsu5, Niels Galjart6and Kozo Kaibuchi1,4,‡
1Department of Cell Pharmacology, Graduate School of Medicine, Nagoya University, 65 Tsurumai, Showa, Nagoya, Aichi 466-8550, Japan
2Institute for Advanced Research, Nagoya University, Furo, Chikusa, Nagoya, Aichi 464-8601, Japan
3Department of Cell Physiology, Division of Membrane Physiology, National Institute for Physiological Sciences, Myodaiji, Okazaki, Aichi 444-8585,
4JST, CREST, 4-1-8 Honcho, Kawaguchi 332-0012, Japan
5Protein Research Network, 1-13-5 Fukuura, Kanazawa, Yokohama, Kanagawa 236-0004, Japan
6Department of Cell Biology and Genetics, Erasmus MC, 3000 DR Rotterdam, Netherlands
*These authors contributed equally to this work
‡Author for correspondence (firstname.lastname@example.org)
Accepted 14 May 2009
Journal of Cell Science 122, 2969-2979 Published by The Company of Biologists 2009
Glycogen synthase kinase (GSK)-3 affects polarised cell
migration through several microtubule-associated proteins,
including +TIPs, such as APC and CLASPs. Phosphorylation of
APC by GSK-3β abrogates its association with microtubules
(Zumbrunn et al., 2001). Near the leading edges where GSK-3β is
inactivated, nonphosphorylated APC appears to preferentially
accumulate at the growing ends of microtubules, and it appears to
be involved in regional control of microtubules (Etienne-Manneville
and Hall, 2003). CLASPs were originally identified as CLIP-170-
interacting proteins and later found to be required for microtubule
stabilisation at the cortical regions of epithelial cells (Akhmanova
et al., 2001; Mimori-Kiyosue et al., 2005). GSK-3β spatially
regulates the binding of CLASPs to microtubules downstream of
Rac1 (Akhmanova et al., 2001; Wittmann and Waterman-Storer,
2005). However, the regulatory mechanism of CLASPs by GSK-
3β is poorly understood because of the lack of extensive in vitro
In light of these observations, we searched for CLASP-interacting
proteins to explore their functions. We identified IQGAP1, an actin-
binding protein, as a novel CLASP-binding protein. Furthermore,
we found that GSK-3β phosphorylated CLASP2 both in vitro and
in vivo, and that this phosphorylation of CLASP2 inhibited its
interaction with IQGAP1, EB1 and microtubules. Thus, GSK-3β
appears to modulate the binding activity of CLASPs to microtubules
and IQGAP1 at specified areas for coupling of microtubules to actin
filaments in migrating cells.
IQGAP1 is a novel CLASP2-interacting protein
To identify CLASP2-interacting proteins, the cytosolic fraction from
porcine brain was applied to affinity beads coated with glutathione
S-transferase (GST) alone, GST-CLASP2-N1 (residues 1-309),
GST-CLASP2-N2 (310-670), GST-CLASP2-M (591-1016) or GST-
CLASP2-C (1017-1294) (Fig. 1A). The bound proteins were eluted
from the column and then resolved by SDS-PAGE. Several proteins
with molecular masses of about 400 kDa (p400), 300 kDa (p300),
190 kDa (p190), 130 kDa (p130), 95 kDa (p95), 85 kDa (p85) and
82 kDa (p82) were specifically detected in the eluate from the GST-
CLASP2-N2 affinity column (Fig. 1B). A protein with a molecular
mass of ~170 kDa (p170) was observed in the eluate from the GST-
CLASP2-C affinity column. No major proteins other than the GST
or its fusion proteins were observed in the eluate from the GST
(Fig. 1B), GST-CLASP2-N1 or GST-CLASP2-M affinity columns
(data not shown). Since the C-terminal region of CLASP2 is known
to interact with CLIP-170, which has a molecular mass of 170 kDa
(Akhmanova et al., 2001), we examined whether p170 was CLIP-
170. An antibody against CLIP-170 recognised p170, indicating
that p170 was indeed CLIP-170 (Fig. 1C). By mass spectrometric
analyses, p300, p190, p95, p85 and p82 were identified as FAM,
IQGAP1, importin β1, phosphofructokinase and muscle-type
phosphofructokinase, respectively. An antibody against IQGAP1
recognised p190 (Fig. 1C). Since IQGAP1 is involved in
microtubule regulation downstream of Rac1 and Cdc42 (Fukata et
al., 2002; Watanabe et al., 2004), we thereafter focused our
investigation on IQGAP1.
CLASPs form a complex with IQGAP1
To examine whether CLASPs interact with IQGAP1 under
physiological conditions, we first tried to precipitate CLASPs from
the lysates of Vero fibroblasts. IQGAP1 was detected in the
immunoprecipitates of two anti-CLASP2 antibodies (Fig. 2A). The
recovery of IQGAP1 in the precipitates was approximately 3%. The
broad reactive bands of CLASP2 might represent other isoforms
and/or its phosphorylation (see below). We also expressed enhanced
GFP (EGFP)-tagged CLASP1α, CLASP2α, and CLASP2γ in
COS7 cells. When immunoprecipitated with an anti-GFP antibody,
endogenous IQGAP1 was coprecipitated with all CLASP constructs
(Fig. 2B). To determine the binding region of CLASP2 to IQGAP1,
the indicated fragments of CLASP2 were fused to EGFP (Fig. 1A)
and expressed in COS7 cells, followed by immunoprecipitation.
Endogenous IQGAP1 was specifically coprecipitated with EGFP-
CLASP2-N2, but not with the other constructs (Fig. 2C), indicating
that the serine/arginine-rich region of CLASP2 is responsible for
its interaction with IQGAP1. CLIP-170 was exclusively precipitated
with EGFP-CLASP2-C (data not shown).
CLASP2 interacts directly with IQGAP1
To examine whether CLASP2 interacts directly with IQGAP1, we
attempted an in vitro binding assay. Maltose-binding protein (MBP)-
fused IQGAP1 fragments were produced, purified (Fig. 3A), and
loaded onto affinity beads coated with GST-CLASP2-N2, because
the serine/arginine-rich region of CLASP2 is sufficient for
interaction with IQGAP1 (Fig. 2C). GST-CLASP2-N2 specifically
interacted with MBP-IQGAP1-C (residues 746-1657) and MBP-
IQGAP1-CT (1503-1657), but not with MBP, MBP-IQGAP1-N (1-
863), or MBP-IQGAP1-GRD (998-1271) (Fig. 3B). Since IQGAP1-
CT also binds to CLIP-170 (Fukata et al., 2002), we constructed
Journal of Cell Science 122 (16)
Fig. 1. Identification of IQGAP1 as a novel interacting molecule with
CLASP2. (A)Schematic of CLASP2. The domain structures of CLASP2 and
its various fragments are represented. CR, conserved region; S/R-rich,
serine/arginine-rich. (B)The cytoplasmic fraction of porcine brain
homogenates was loaded onto beads coated with GST, GST-CLASP2-N-
terminal (GST-CLASP2-N2), or GST-CLASP2-C-terminal (GST-CLASP2-C)
fragment. Aliquots of the eluate were resolved by SDS-PAGE, followed by
silver staining (B) or immunoblotting with anti-IQGAP1 and anti-CLIP-170
antibodies (C). The identified bands are indicated according to their
approximate molecular masses. All results are representative of three
Regulation of CLASPs by phosphorylation
additional deletion fragments and performed in vitro binding assays.
However, we could not distinguish between the CLASP2-binding
region and the CLIP-170-binding region (data not shown). The
binding of MBP-IQGAP1-CT to GST-CLASP2-N2 was dose-
dependent and saturable (Fig. 3C). Scatchard analysis revealed a
single class of affinity binding sites with a Kdof about 480 nM.
Taken together, these results indicate that IQGAP1-CT binds
directly to CLASP2-N2.
Rac1/Cdc42, IQGAP1 and CLASP2 form a complex
Since IQGAP1 is an effector of Rac1 and Cdc42 (Hart et al., 1996;
Kuroda et al., 1996; McCallum et al., 1996), we examined whether
Rac1/Cdc42, IQGAP1 and CLASP2 form a complex. When EGFP-
CLASP2 was immunoprecipitated from the cells expressing EGFP-
CLASP2 and constitutively active Rac1 (Rac1V12), Rac1V12and
IQGAP1 were coimmunoprecipitated (Fig. 3D). Similar results were
obtained with constitutively active Cdc42 (Cdc42V12) instead of
Rac1V12. Constitutively active RhoA (RhoAV14), dominant-negative
Rac1 (Rac1N17), dominant-negative Cdc42 (Cdc42N17) and
coimmunoprecipitated with EGFP-CLASP2. Under these
conditions, CLIP-170 was also precipitated, possibly by its direct
(RhoAN19) were not
association with CLASP2 and/or through IQGAP1. These results
suggest that activated Rac1/Cdc42, IQGAP1 and CLASP2 can form
a complex. The Rho-family GTPases did not significantly affect
the coprecipitation of CLIP-170 or IQGAP1.
GSK-3β phosphorylates CLASP2 at Ser533 and Ser537
GSK-3β can phosphorylate CLASP2 in vitro, and the inhibition
of GSK-3β in epithelial cells impairs the mobility shift of CLASP2
(Wittmann and Waterman-Storer, 2005). Although many putative
consensus sites for phosphorylation by GSK-3β [Ser/Thr-x-x-x-
Ser/Thr (Cohen and Frame, 2001)] are found in CLASP2, especially
in the serine/arginine-rich region, the specific phosphorylation sites
in CLASP2 have not yet been identified.
Fig. 2. Complex formation of CLASPs with IQGAP1. (A)The lysates from
Vero cells were immunoprecipitated with two anti-CLASP2 antibodies.
Endogenous IQGAP1 was precipitated with CLASP2. (B)The indicated
EGFP-CLASP constructs were transfected into COS7 cells and
immunoprecipitated with an anti-GFP antibody. IQGAP1 was coprecipitated
with all examined EGFP-CLASPs (upper panel). (C)EGFP-CLASP2
fragments were transfected into COS7 cells and immunoprecipitated with an
anti-GFP antibody. IQGAP1 was coprecipitated with EGFP-CLASP2-N2. The
expression levels of the different CLASP2 fragments are similar. All results
are representative of three independent experiments.
Fig. 3. Direct interaction of CLASP2 with IQGAP1. (A)Schematic of
IQGAP1. The domain structures of IQGAP1 and its various fragments are
represented. CHD, calponin homology domain; GRD, Ras-GAP-related
domain; IQ motif, calmodulin-binding motif; IQ repeats, IQGAP-specific
repeat motif. (B)Purified MBP-fused IQGAP1 fragments were mixed with the
affinity beads coated with purified GST-CLASP2-N2. Bound MBP fusion
proteins were coeluted with GST fusion proteins by glutathione. The eluates
were subjected to SDS-PAGE, followed by immunoblotting with an anti-MBP
antibody. The bands lower than MBP-IQGAP1-C in the input represent the
degradation products of MBP-IQGAP1-C, which lacks the C-terminus of
IQGAP1. (C)Saturable interaction of purified GST-CLASP2-N2 with MBP-
IQGAP1-CT. The indicated concentrations of MBP-IQGAP1-CT were
incubated with the beads coated with GST-CLASP2-N2 (100 pmol), and the
Kdvalue was calculated by Scatchard analysis. (D)EGFP-CLASP2 and
indicated HA-tagged small GTPases were transfected into COS7 cells. HA-
Rac1V12and HA-Cdc42V12were coprecipitated with EGFP-CLASP2 and
IQGAP1, whereas HA-Rac1N17, HA-Cdc42N17, HA-RhoAN19, and HA-
RhoAV14were not. All results are representative of three independent
To examine the phosphorylation of CLASP2 in vivo, EGFP-
CLASP2-N2 was transfected into COS7 cells with wild-type (WT),
kinase-inactive (KN) or constitutively active (S9A) forms of GSK-
3β and then subjected to SDS-PAGE. The fast and slow migrating
bands of EGFP-CLASP2-N2 were observed without GSK-3β. In
cells expressing GSK-3βWT or S9A, only a slowly migrating band
was evident (Fig. 4A). When the cells were treated with GSK-3
inhibitors (SB216763 and SB415286), the mobility shift of CLASP2
was partially abrogated (Fig. 4B). The mobility shifts were more
evident in 2D-PAGE (supplementary material Fig. S1). Taken
together, our results indicate that CLASP2-N2 is phosphorylated
by GSK-3βin vivo. Except for the serine/arginine-rich region (N2),
no significant mobility shift of other CLASP2 fragments was
observed in SDS-PAGE (data not shown).
Next, purified CLASP2 fragments (N1, N2, M and C) were used
to examine whether GSK-3β directly phosphorylates CLASP2 in
vitro. GSK-3β itself phosphorylated none of these fragments
efficiently (Fig. 4C). GSK-3βgenerally requires prephosphorylation
of its substrates by other kinases (Eldar-Finkelman, 2002). Since
CLASP2 has several putative phosphorylation sites of protein kinase
C (PKC), protein kinase A (PKA), cyclin-dependent kinase 5
(Cdk5), Jun N-terminal kinase (JNK), and casein kinase II (CKII),
we performed in vitro phosphorylation assays with these kinases.
Cdk5 phosphorylated the CLASP2-N2 fragment, but did not
significantly phosphorylate other CLASP2 fragments (Fig. 4C). The
other kinases did not phosphorylate any fragments of CLASP2 under
the same conditions (data not shown). When both Cdk5 and GSK-
3βwere used simultaneously, the phosphorylation level of CLASP2
was significantly increased in comparison to when Cdk5 alone was
used (Fig. 4C), suggesting that Cdk5 functions as a priming kinase,
at least in a cell-free system.
CLASP2 has a consensus motif recognised by Cdk5 at Ser541
in the vicinity of the potential phosphorylation sites of GSK-3β,
Ser533 and Ser537 (Fig. 4D). We produced nonphosphorylatable
constructs of CLASP2-N2 named S533A and S537A, by converting
Ser to Ala, and performed an in vitro phosphorylation assay to
determine the phosphorylation site of CLASP2 by GSK-3β. These
Ala-substituted mutants significantly reduced the phosphorylation
level of CLASP2 (Fig. 4E). Furthermore, a nonphosphorylatable
double mutant of S533 and S537 (2A) reduced the phosphorylation
level more than either single Ala-substituted mutant S533A or
S537A (Fig. 4E). Since the 2A mutant was still phosphorylated to
a small extent in vitro, it appears that GSK-3β can phosphorylate
other minor sites after Cdk5 phosphorylation.
Phosphorylation of CLASP2 inhibits its interaction with
IQGAP1, EB1 and microtubules
We next examined whether the interaction of CLASP2 with
IQGAP1 was affected by CLASP2 phosphorylation. When the
fragment containing both the GSK-3β phosphorylation sites and
the IQGAP1-binding region EGFP-CLASP2-N2 was transfected
into COS7 cells, endogenous IQGAP1 was coimmunoprecipitated
with EGFP-CLASP2-N2 (Fig. 5A). This interaction was decreased
by cotransfection with GSK-3β WT or S9A, but not with KN (Fig.
5A). Similar results were obtained with the use of EGFP-CLASP2
(full-length) instead of the fragment (supplementary material Fig.
S2A). Interestingly, the inhibitory effects of GSK-3β WT and S9A
were more apparent in the cells expressing EGFP-CLASP2-N2 than
in those expressing EGFP-CLASP2. This difference might be
explained by more efficient phosphorylation of EGFP-CLASP2-
N2 in COS7 cells. Treatment with GSK-3 inhibitors restored the
inhibitory effect of GSK-3βWT (supplementary material Fig. S2B).
In addition, inhibition of endogenous GSK-3 activity also increased
the association between endogenous IQGAP1 and CLASP2 in Vero
fibroblasts (supplementary material Fig. S2C). Of note, the GSK-
3 inhibitors attenuated the efficiency of immunoprecipitation. One
possible explanation is that the GSK-3 inhibitors induce the
dephosphorylation of CLASP2 and change its conformational state,
thereby preventing recognition by the antibodies used.
Since EB1 is known to interact with CLASPs and this interaction
is necessary for the regulation of microtubule dynamics (Mimori-
Kiyosue et al., 2005), we examined how it was affected by the
Journal of Cell Science 122 (16)
Fig. 4. Phosphorylation of CLASP2 at Ser533 and Ser537 by GSK-3β.
(A)COS7 cells were transfected with EGFP-CLASP2-N2 and the indicated
GSK-3β construct. Each lysate was subjected to SDS-PAGE, followed by
immunoblot with an anti-GFP antibody. The arrow and arrowhead indicate fast
and slow migrating bands of EGFP-CLASP2-N2, respectively. (B)The cells
expressing EGFP-CLASP2-N2 and GSK-3β WT were cultured in the presence
of GSK-3 inhibitors (10μM SB216763, 50μM SB415286) or control vehicle
(DMSO) for 1 hour. Each lysate was subjected to immunoblotting with an
anti-GFP antibody. The arrow and arrowhead indicate fast and slow migrating
bands of EGFP-CLASP2-N2, respectively. (C)A kinase assay was performed
in vitro using purified GST-CLASP2-N2, GSK-3β and Cdk5. Each reaction
mixture was subjected to SDS-PAGE and detected by autoradiography.
Phosphorylation levels of CLASP2 fragments are shown in the bar graph.
(D)Potential phosphorylation sites of GSK-3β and Cdk5. The numbers denote
amino acid positions of CLASP2. The underlined residues represent a
consensus motif recognised by Cdk5. (E)In vitro kinase assay was performed
using purified CLASP2-N2 Ala-substituted mutants, GSK-3β and Cdk5. Each
reaction mixture was subjected to SDS-PAGE and detected by
autoradiography. The phosphorylation levels of CLASP2 WT or mutants are
shown in the bar graph. All results are representative of three independent
Regulation of CLASPs by phosphorylation
coimmunoprecipitated with EGFP-CLASP2-N2 from COS7 cells
(Fig. 5B). This association was also inhibited in the presence of
GSK-3β WT or S9A (Fig. 5B). Since IQGAP1 and EB1 bind to
the same region within CLASP2, we performed a competition assay.
Increasing amounts of purified EB1 did not appear to interfere with
the association of MBP-IQGAP1-CT with GST-CLASP2-N2
(supplementary material Fig. S2D), indicating that EB1 and
IQGAP1 are able to simultaneously associate with CLASP2.
We also examined the binding of CLASP2 to microtubules in
vitro. Purified CLASP2-N2-2 (residues 263-581) was sedimented
with microtubules (Fig. 5C). IQGAP1 was sedimented in the
of CLASP2. Endogenous EB1 was
presence of CLASP2-N2-2, but not in the absence of CLASP2-N2-
2 (Fig. 5C), suggesting that IQGAP1 binds to microtubules through
CLASP2, and that CLASP2 can simultaneously bind to IQGAP1
and microtubules. Furthermore, the association of CLASP2 with
microtubules was impaired after the phosphorylation by GSK-3β
(Fig. 5D). Thus, GSK-3β can modulate the binding ability of
CLASP2 to microtubules as well as to EB1 and IQGAP1.
To further examine the role of CLASP2 phosphorylation at
Ser533 and Ser537 in the interaction with IQGAP1 and EB1, we
used the nonphosphorylatable EGFP-CLASP2-N2 mutant (EGFP-
CLASP2-N2-2A; EGFP-fused CLASP2-N2 harbouring two Ala
substitutions at Ser533 and Ser537). In the presence of GSK-3β
KN, both EGFP-CLASP2-N2 and EGFP-CLASP2-N2-2A
associated with purified GST-IQGAP1-CT and GST-EB1 (Fig. 6A).
The recovery to GST-EB1 was greater than that to GST-IQGAP1-
CT, suggesting that CLASP2 binds to EB1 with a higher affinity.
When GSK-3β WT was transfected, EGFP-CLASP2-N2 did not
associate with either GST-IQGAP1-CT or GST-EB1, whereas
EGFP-CLASP2-N2-2A did (Fig. 6A), suggesting that Ala
substitutions at Ser533 and Ser537 can be resistant to the inhibitory
effect of GSK-3β. We also attempted to produce a phosphomimic
EGFP-CLASP2-N2 mutant, in which Ser533 and Ser537 were
replaced by Asp or Glu, but found that these mutants still bound
GST-IQGAP1-CT and GST-EB1 (data not shown), indicating that
these substitutions cannot mimic the phosphorylation state and/or
were not sufficient. Taken together, our results indicate that GSK-
3β negatively regulates the interaction of CLASP2 with IQGAP1
and EB1 through the phosphorylation at Ser533 and Ser537.
CLASP2 distributes asymmetrically in migrating cells
To further understand the regulation of CLASP2 by GSK-3β, we
examined the subcellular localisation of CLASP2 and IQGAP1 in
Vero fibroblasts. IQGAP1 localised to the leading edges where
microtubules were targeted (Fig. 6B), whereas CLASP2
accumulated near the plus-ends of a subset of microtubules toward
the leading edges in Vero cells. Microtubule-associated CLASP2
partially colocalised with EB1 as well as microtubule lattices behind
the EB1 signal (Fig. 6C), essentially as described previously
(Akhmanova et al., 2001; Fukata et al., 2002; Wittmann and
Waterman-Storer, 2005). IQGAP1 partially colocalised with
CLASP2 at the leading edges (Fig. 6B). EB1 accumulated at the
plus-ends of microtubules, not only near the leading edges, but also
throughout the wider cortical region and central region; however,
CLASP2 was distributed asymmetrically to the ends of microtubules
(Mimori-Kiyosue et al., 2005; Wittmann and Waterman-Storer,
2005). Transfection of GSK-3β S9A induced the delocalisation of
EGFP-CLASP2 both from microtubules (Wittmann and Waterman-
Storer, 2005) and EB1 (Fig. 6C), which prevented partial
colocalisation of CLASP2 with IQGAP1 at the leading edges (Fig.
6C). However, GSK-3β KN did not affect the localisation of
CLASP2 significantly (supplementary material Fig. S3A). The
inhibitory effect of GSK-3β S9A was negated by treatment of the
cells with GSK-3 inhibitors (SB216763 and SB415286) (Fig. 6C;
and data not shown), but not by treatment with the vehicle alone
(supplementary material Fig. S3B). In fact, these inhibitors enhanced
the accumulation of CLASP2 on microtubules. Furthermore, when
endogenous GSK-3 was eliminated by RNAi, CLASP2 on
microtubules was more apparent throughout the cells
(supplementary material Fig. S3C,D), similarly to the cells treated
with GSK-3 inhibitors. The effect of the depletion was moderate
in the comparison with that of the inhibitors. This might be because
Fig. 5. Impaired association of phosphorylated CLASP2 with IQGAP1, EB1,
and microtubules. (A)COS7 cells were transfected with EGFP-CLASP2-N2
and the indicated GSK-3β construct. When immunoprecipitated with an anti-
GFP antibody, IQGAP1 was coprecipitated with EGFP-CLASP2-N2, whereas
this interaction was abrogated in the presence of active GSK-3β. (B)EGFP-
CLASP2-N2 and the indicated GSK-3β construct were transfected into COS7
cells, and immunoprecipitated with an anti-GFP antibody. EB1 was
coimmunoprecipitated with EGFP-CLASP2-N2, whereas this association was
inhibited by active GSK-3β. The ratio of the coprecipitates (mean ± s.d.) to
precipitated CLASP2 is shown in the bar graph on the right. (C)Purified
CLASP2-N2-2 (aa 263-581) and MBP-IQGAP1-CT were designed to examine
their association with in vitro polymerised microtubules. His-CLASP2-N2-2
was sedimented in a microtubule-dependent manner. IQGAP1 was
cosedimented with microtubules only in the presence of CLASP2. The upper
and lower panels show the results of silver staining and immunoblotting with
an anti-His antibody, respectively. (D)Phosphorylated His-CLASP2-N2-2 was
not sedimented with microtubules. Phosphorylated His-CLASP2-N2-2
(asterisk) migrated more slowly than its nonphosphorylated form. All results
are representative of more than three independent experiments.
the depletion of GSK-3 was incomplete, and mechanisms in
addition to GSK-3-mediated phosphorylation contributed to the
regulation of CLASP2-microtubule
nonphosphorylatable mutant, EGFP-CLASP2-2A, showed a similar
localisation with EGFP-CLASP2, whereas GSK-3βS9A had a lesser
effect on EGFP-CLASP2-2A in comparison with the wild type (Fig.
6C,D). These observations, taken together with the in vitro results
(Fig. 5C,D), indicate that GSK-3β impairs the localisation of
CLASP2 along microtubules by phosphorylating CLASP2 at Ser533
To further examine the role of CLASP2 phosphorylation by GSK-
3β, we raised a rabbit polyclonal antibody that specifically
recognises the phosphorylated CLASP2 at Ser533 and Ser537 (anti-
S533/S537-P antibody). The specificity of this antibody was
examined by immunoblot analysis (Fig. 7A). A fixed amount (2
pmol) of GST-CLASP2-N2 containing increasing amounts of the
phosphorylated form was loaded on the gel. The anti-S533/S537-
P antibody recognised the phosphorylated CLASP2 in a dose-
dependent manner, indicating that the antibody specifically
recognises the phosphorylated CLASP2 at Ser533 and Ser537. We
next examined whether GSK-3β could phosphorylate CLASP2 at
Ser533 and Ser537 in COS7 and Vero cells. Transfection of GSK-
3β WT or S9A induced the phosphorylation of EGFP-CLASP2-N2
at Ser533 and Ser537, whereas the active GSK-3β failed to induce
the phosphorylation of EGFP-CLASP2-N2-2A (Fig. 7B). Treatment
of the cells with GSK-3 inhibitors partially abolished the GSK-3β-
Journal of Cell Science 122 (16)
Fig. 6. Effect of GSK-3β on the distribution and
binding of CLASP2. (A)EGFP-CLASP2-N2-WT
or EGFP-CLASP2-N2-2A mutant was transfected
into COS7 cells. The lysates were subjected to
pull down with GST-IQGAP1-CT or GST-EB1.
The 2A mutant was precipitated with GST-
IQGAP1-CT or GST-EB1. The active GSK-3β
WT inhibited the association of CLASP2-N2-WT,
but not that of the CLASP2-N2-2A mutant, with
IQGAP1 and EB1. (B)Vero cells were stained
with anti-IQGAP1 (red) and anti-CLASP2 (green)
antibodies. These images were merged with the
staining of the anti-tubulin antibody (blue).
(C)Vero cells transfected with EGFP-CLASP2
were stained with anti-EB1 or anti-IQGAP1,
together with anti-tubulin antibodies (blue). Green
represents EGFP-CLASP2 and red indicates EB1
or IQGAP1. Insets in panels are magnified in the
right panels. Scale bars: 10μm. (D)Vero cells
were transfected with EGFP-CLASP2-2A,
followed by immunostaining with anti-EB1 (red)
and anti-tubulin (blue) antibodies. Insets in panels
are magnified in the right panels. Scale bars:
10μm. The cells cotransfected with GSK-3β S9A
are indicated by S9A and those treated with the
GSK-3 inhibitor SB216763 at 10μM for 1 hour
before fixation are labelled SB21. All results are
representative of more than three independent
Regulation of CLASPs by phosphorylation
induced phosphorylation of EGFP-CLASP2-N2 in COS7 cells (Fig.
7B). The anti-S533/537-Pantibody could recognise the
phosphorylation of endogenous CLASP2 at Ser533 and Ser537 in
Vero cells, and the immunoreactivity was also abrogated by
treatment with GSK-3βinhibitors (Fig. 7C), indicating that CLASP2
is phosphorylated at Ser533 and Ser537 by GSK-3β under
In Vero fibroblasts, the immunoreactivity of the anti-S533/S537-
P antibody was mainly detected at the perinuclear region, where it
colocalised with the fluorescence of EGFP-CLASP2 (Fig. 7D).
Treatment with a GSK-3βinhibitor abolished the immunoreactivity
at the perinuclear region. However, the GSK-3 inhibitor did not
apparently affect the fine dot-like immunofluorescence. In addition,
this immunofluorescence showed negligible colocalisation with the
fluorescence of EGFP-CLASP2, especially at the leading edges (Fig.
7D). These results suggest that phosphorylated CLASP2 stays at
the perinuclear region, but not on the ends and lattices of
microtubules at the leading edges. However, we cannot exclude the
possibility that with the exception of localisation in the perinuclear
region, the immunofluorescence of the anti-S533/537-P antibody
is background staining.
Finally, to examine the functional significance of the interaction
between IQGAP1 and CLASP2 in cell migration, we used the
Boyden chamber assay. Endogenous IQGAP1 or CLASP2 was
depleted by two independent siRNAs (Fig. 8A). The transfection
of each siRNA impaired the serum-stimulated cell motility from
the upper to the basal membrane (Fig. 8B). The two siRNAs showed
similar effects on cell motility. The inhibitory effect of siRNA to
IQGAP1 was almost completely rescued by the expression of RNAi-
resistant (RNAiR)-full length IQGAP1, but not that of RNAiR-
IQGAP1-ΔCT lacking the CLASP2-binding region (Fig. 8B). This
indicates that the linkage of CLASP2 to IQGAP1 is required for
effective cell migration.
Taken together, these results suggest that GSK-3β dynamically
and negatively regulates the binding of CLASP2 to EB1 and
microtubules for asymmetrical distribution on microtubules by
Fig. 7. Distribution of phosphorylated CLASP2 in cells.
(A)Purified GST-CLASP2-N2 (2 pmol) containing the indicated
amounts of GST-CLASP2-N2 phosphorylated by GSK-3β was
subjected to SDS-PAGE, followed by immunoblot analysis with
anti-S533/537-P (upper panel) or anti-GST antibodies (lower
panel). (B)COS7 cells were transfected with EGFP-CLASP2-
N2 and the indicated GSK-3β construct. In the right panels, the
cells were treated with GSK-3 inhibitors (SB216763 and
SB415286) or control vehicle (DMSO). Each lysate was
subjected to SDS-PAGE and immunoblot analysis with anti-
S533/S537-P (upper panel) and anti-GFP antibodies (lower
panel). The same lysates were used in Fig. 4. (C)Vero cells were
treated with DMSO, 10μM SB216763 or 50μM SB415286 for
1 hour. The lysates were blotted with an anti-CLASP2 (upper
panel) and anti-S533/537-P antibodies (lower panel). (D)Vero
cells expressing EGFP-CLASP2 were stained with an anti-
S533/S537-P antibody (P-CLASP2; red). The immunoreactivity
of this antibody did not show significant colocalisation with
EGFP-CLAPS2 signals (green), except in the perinuclear region
(arrow). The cells were treated with a GSK-3 inhibitor (10μM
SB216763) for 1 hour before fixation (middle row). The
immunoreactivity of the anti-S533/537-P antibody (red) was
compared with EB1 (green) and tubulin (blue) in the lowest row.
Insets in merged images are magnified in the right panels. Scale
bars: 10μm. All results are representative of three independent
phosphorylation, and that nonphosphorylated CLASP2 on
microtubules is allowed to associate with IQGAP1 for the coupling
of microtubules to actin filaments at the front of migrating cells.
Connection of microtubule ends to actin filaments through
Here, we identified IQGAP1, an effector of Rac1 and Cdc42, as a
novel CLASP-interacting molecule. IQGAP1 colocalises with actin
filaments and partially overlaps with the plus-ends of microtubules
at the leading edges of migrating cells (Fukata et al., 2002). We
have proposed that activated IQGAP1 captures and stabilises CLIP-
170, accumulating only at the plus-ends of microtubules near the
specialised cortices such as the leading edges, where it coordinates
with APC anchored to actin filaments through IQGAP1 (Watanabe
et al., 2005). Consistently, the depletion of IQGAP1 or APC by
RNAi prevents not only proper actin meshwork formation, but also
stabilisation of the plus-ends of microtubules, which is reflected by
the immobilisation of EGFP-CLIP-170 at the leading edges
(Watanabe et al., 2004). Since EB1 does not interfere with the
binding of IQGAP1 to CLASP2 (supplementary material Fig. S2D),
it appears that CLASP2 on the ends of microtubules also participates
in the connection to actin filaments through IQGAP1 (Fig. 9). Our
recent ultrastructural analysis revealed that IQGAP1 predominantly
localises beside actin filaments and at the crossings of actin
filaments beneath the substratum-facing plasma membrane
(Watanabe et al., 2008). In addition, IQGAP1 sometimes localises
to the intersection of microtubule ends and actin filaments. Thus,
IQGAP1 appears to be responsible for initial contacts of the plus-
ends of microtubules with actin filaments at the leading edges (Fig.
Linkage of CLASP2 to IQGAP1 near microtubule ends
CLASP2 accumulates at the plus-ends of microtubules as well as
the lattices behind their plus-ends (Akhmanova et al., 2001; Mimori-
Kiyosue et al., 2005; Wittmann and Waterman-Storer, 2005). Since
the depletion of IQGAP1 did not affect the localisation of CLASP2
on microtubules (supplementary material Fig. S4), IQGAP1 might
not be involved in the process of localisation of CLASP2 to
microtubules. We also found that IQGAP1 was cosedimented with
polymerised microtubules in the presence but not the absence of
CLASP2 (Fig. 5), suggesting that CLASP2 can mediate the
interaction of IQGAP1 with microtubules. Of note, IQGAP1 often
localises beside microtubules growing along actin filaments beneath
the substratum-facing plasma membrane (supplementary material
Fig. S4) (Watanabe et al., 2008), suggesting that IQGAP1 can
connect CLASP2 to actin filaments not only at the plus-ends of
microtubules, but also at the microtubule lattices (Fig. 9).
Microtubules and CLASPs consistently undergo retrograde flow
with actin filaments in the lamellipodia (Salmon et al., 2002;
Tsvetkov et al., 2007; Wittmann and Waterman-Storer, 2001). Thus,
the temporal association of IQGAP1 with CLASPs on the ends and
lattices of microtubules may have a crucial role in efficient coupling
of microtubules in the wider region to actin filaments (Fig. 9).
Possible roles of IQGAP1-mediated cytoskeletal linkages in
CLASP2 is required for persistent motility, whereas CLIP-170 is
not (Akhmanova et al., 2005; Drabek et al., 2006). Truncated APC,
lacking the C-terminal region that is responsible for the linkage to
microtubules, abrogates polarised migration (Barth et al., 2008;
McCartney and Nathke, 2008). We previously reported that the
depletion of IQGAP1 or APC impairs cell migration by preventing
the proper formation of the actin meshwork (Watanabe et al., 2004).
We consistently found that either IQGAP1-depleted or CLASP2-
depleted Vero fibroblasts show cell motility defects in the Boyden
chamber assay (Fig. 8). The deficiency in the IQGAP1-depleted
cells was rescued by the expression of full-length IQGAP1, but not
by the expression of IQGAP1-ΔCT. Although IQGAP1-CT includes
the binding region for CLASP2, CLIP-170 and APC, the association
of IQGAP1 with CLASP2 and/or APC is probably required for cell
migration. The interaction of IQGAP1 with CLIP-170 might
contribute modestly to cell migration (Fig. 9). However, further
analysis will be required to reveal the functional significance of
each individual complex in cell motility.
Phosphorylation of CLASP2 by GSK-3β
CLASPs accumulate asymmetrically near the growing plus-ends of
microtubules toward the leading edges (Akhmanova et al., 2001;
Mimori-Kiyosue et al., 2005; Wittmann and Waterman-Storer,
2005). EB1 is necessary for the recruitment of CLASPs to the plus-
ends of microtubules, whereas CLIP-170 is not absolutely required
(Mimori-Kiyosue et al., 2005). We found that GSK-3β
phosphorylated CLASP2 at Ser533 and Ser537 within the
serine/arginine-rich region (Figs 4 and 7). These phosphorylation
sites are mapped proximal to the conserved EB1-binding motif
Journal of Cell Science 122 (16)
Fig. 8. Requirement of the CLASP2-binding region of IQGAP1 for cell
migration. (A)Vero cells were transfected with the indicated siRNA to
IQGAP1 or CLASP2. 48 hours after transfection, the lysates were
immunoblotted with anti-IQGAP1, anti-CLASP2 and control anti-moesin
antibodies. Each siRNA knocked down its target protein. (B)Vero cells
transfected with either the indicated siRNA or siRNA along with indicated
RNAiR-IQGAP1 were subjected to the Boyden chamber assay. The cells were
allowed to migrate for 4 hours. The depletion of either IQGAP1 or CLASP2
significantly impaired cell migration from the upper to the lower membrane.
The inhibitory effect of IQGAP1 depletion was rescued by the expression of
full-length RNAiR-IQGAP1, but not by expression of RNAiR-IQGAP1-ΔCT
lacking the CLASP2-binding region. All results are representative of more
than three independent experiments.
Regulation of CLASPs by phosphorylation
(Galjart, 2005). The expression of active GSK-3β attenuated the
binding of CLASP2 to EB1 and abrogated the accumulation of
CLASP2 at both the ends and lattices of microtubules (Figs 6 and
7). The middle portion of CLASP2 bound directly to polymerised
microtubules in vitro, but the phosphorylated form of CLASP2 did
not (Fig. 5). The C-terminal region of CLASPs interacts with CLIP-
170 and LL5β (Akhmanova et al., 2001; Lansbergen et al., 2006).
Wittmann and Waterman-Storer (Wittmann and Waterman-Storer,
2005) have proposed that the C-terminal region is not involved in
the regulation of CLASPs by GSK-3β. Consistent with these
observations, GSK-3β did not phosphorylate the C-terminal region
of CLASP2 either in vitro or in vivo (Fig. 4; and data not shown).
The interaction of CLASPs with CLIP-170 or LL5β might not be
controlled by GSK-3β. Collectively, GSK-3β negatively controls
the association of CLASP2 with microtubules as well as EB1
through the phosphorylation of CLASP2 at Ser533 and Ser537 for
the asymmetrical distribution of CLASPs on microtubules in
polarised migrating cells (Fig. 9).
During the course of revision of this paper, Kumar and colleagues
(Kumar et al., 2009) published a paper that partially overlaps with
this study. They show that a CLASP2 mutant with Ala substitutions
at the GSK-3 phosphorylation sites, including the sites we examined,
is resistant to the actions of active GSK-3β based on localisation
on microtubules. However, phosphomimic mutations impaired
binding to polymerised microtubules and EB1 (Kumar et al., 2009).
We also show that the CLASP2-2A mutant localises near the ends
of microtubules, even in the presence of active GSK-3β (Fig. 6D),
whereas phosphorylated CLASP2 does not bind to EB1 and
microtubules (Fig. 5B,D). Furthermore, Kumar and co-workers
found that expression of active GSK-3β destabilises lamella
microtubules by disrupting lateral microtubule interactions with the
cell cortex. Since active GSK-3β disrupts complex formation
between IQGAP1 and CLASP2 (Fig. 5A), that complex probably
contributes to the cytoskeletal linkage at the cell cortex (Fig. 9).
In summary, GSK-3β phosphorylates CLASP2 and controls the
asymmetrical distribution of CLASP2 on microtubules. At the
leading edges, where GSK-3β is inactivated, nonphosphorylated
CLASP2 on microtubules links the microtubules to actin filaments
through direct interaction with IQGAP1 for polarised cell migration.
IQGAP1 probably has a central role in connecting microtubules to
actin filaments, serving as a guiding mechanism through several
+TIPs at the leading edge.
Fig. 9. Regulation of CLASP2 by GSK-3β. A working model
summarising the roles of GSK-3β in regulating CLASP activity.
At the leading edges of migrating cells, GSK-3β is inactivated,
which leads to local accumulation of nonphosphorylated
CLASP2 (red box). Nonphosphorylated CLASP2 is able to
interact with EB1 and microtubules for asymmetrical distribution
in cells. Nonphosphorylated CLASP2 on the ends and lattices of
microtubules associates with IQGAP1 for initial contact of
microtubules with actin filaments, together with CLIP-170. In
addition, CLASP2 on microtubule lattices binds to IQGAP1 for
more efficient coupling of microtubules to actin filaments (see
text for more detail).
Materials and Methods
Antibodies and reagents
Anti-IQGAP1 monoclonal antibody and anti-GFP antibody were purchased from
ZYMED (San Francisco, CA) and Roche (Mannheim, Germany), respectively.
pEGFP-C1 was purchased from Clontech Laboratories (Palo Alto, CA). GSK-3
inhibitors (SB216763 and SB415286) were from Tocris Cookson (Ellisville, MO).
Recombinant His-tagged Cdk5 and GST-tagged p35 and recombinant His-tagged
GSK-3β were from Upstate (Lake Placid, NY). The anti-IQGAP1 rabbit polyclonal
antibody has been described elsewhere (Wang et al., 2007). Anti-CLASP2 antibodies
were raised against GST-CLASP2-C and produced as described previously
(Akhmanova et al., 2001). siRNA sequences are as follows. IQGAP1#1 5?-
UGCCAUGGAUGAGAUUGGA-3?; IQGAP1#2 5?-GUUCUACGGGAAGUA -
AUU GUU-3? (designed to 3?UTR region); CLASP2#1 5?-GUUCAGAAAG -
CCCUUGAUG-3?; CLASP2#2 5?-GACAUACAUGGGUCUUAGA-3?; scrambled
5?-CAGUCGCGU UUGCGACUGG-3?. These siRNAs with dTdT overhangs at each
3? terminus were obtained from Greiner-Japan (Tokyo, Japan). siRNA to GSK-3
was purchased from Sigma: GSK-3α#1, SASI_Hs01_00245062; GSK-3α#2,
SASI_Hs01_00192105. RNAiR-IQGAP1 and its ΔCT were described previously
(Watanabe et al., 2004).
To obtain constructs of CLASP2, we defined the KIAA0627 cDNA spanning from
position 92 to 3976 (Kazusa DNA Research Institute; Chiba, Japan) as CLASP2, and
subcloned the corresponding cDNA fragments into pGEX-4T-1 and pEGFP-C1
vectors. We used the partial KIAA0627 cDNA as CLASP2γin Fig. 2B. For our amino
acid numbering, 533, 537, and 541 correspond to 563, 567, and 571, respectively, in
KIAA0627. The constructs of pMal-IQGAP1 fragments and pEF-Bos small GTPases
were produced as described elsewhere (Fukata et al., 2002). Tags were fused to the
N-termini of proteins of interest. The mutants CLASP2-S533A, -S537A, and
-S533A/S537A were generated with a site-directed mutagenesis kit (Stratagene, La
Jolla, CA). For microtubule sedimentation assays, the region spanning residues 263
to 581 of CLASP2γ(termed CLASP2-N2-2) was subcloned into the pRSET-C1 vector.
Control MBP and MBP-IQGAP1-CT without stop codons were subcloned into the
pET52 vector (Merck, Darmstadt, Germany). pCGN-GSK-3β, pCGN-GSK-3β S9A
and pCGN-GSK-3β kinase-inactive form (K85N; KN) were provided by Akira
Kikuchi (Hiroshima University, Japan).
Preparation of recombinant proteins
The expression and purification of GST and MBP fusion proteins were performed
as described (Fukata et al., 2002). For microtubule sedimentation assays,
His-CLASP2-N2-2 was purified with Ni-NTA resin according to the
manufacturer’s protocols (Qiagen, Hilden, Germany). Control MBP and MBP-
IQGAP1-CT were purified by sequential passages through two resins, amylose
resin and Ni-NTA.
GST-CLASP2-N2 and GST-CLASP2-C affinity column
The affinity column chromatography was performed as described previously (Kuroda
et al., 1996). Briefly, the cytosolic fraction of porcine brain homogenates was loaded
onto glutathione Sepharose 4B (GE Healthcare; Little Chalfont, UK) coated with
GST alone, GST-CLASP2-N2, or GST-CLASP2-C. The columns were washed with
buffer A (20 mM Tris-HCl at pH 8.0, 1 mM dithiothreitol, 1 mM EDTA, 10 mM
p-amidinophenyl-methanesulfonyl fluoride, and 10 mg/ml leupeptin). The proteins
bound to the affinity columns were eluted by buffer A containing 500 mM NaCl and
identified as described elsewhere (Fukata et al., 2002).
In vitro binding assays
Purified MBP-fused IQGAP1 fragments were mixed with affinity beads coated with
GST-CLASP2-N2 in buffer A. The beads were then washed with buffer A, and the
bound proteins were eluted with buffer A containing 10 mM reduced glutathione.
The eluates were subjected to SDS-PAGE, followed by immunoblot analysis using
an anti-MBP antibody. The amount of IQGAP1-CT bounded with GST-CLASP2-N2
was detected in a linear range using serial dilutions of standards by chemiluminescent
detection and estimated with a Densitograph (ATTO, Tokyo, Japan). Purified
IQGAP1-CT was used as the standard for quantification.
Vero cells and COS7 cells were grown in Dulbecco’s modified Eagle’s medium
containing 10% foetal bovine serum (FBS) at 37°C in 5% CO2atmosphere at constant
humidity. Transfection was performed with Lipofectamine 2000 or Lipofectamine
(Invitrogen, Carlsbad, CA) according to the manufacturer’s protocols. Transfection
of siRNA was described previously (Watanabe et al., 2004).
Coimmunoprecipitation of IQGAP1 with CLASP2
The immunoprecipitation assay was performed as described (Fukata et al., 2002). In
brief,subconfluent Vero cells or COS7 cells were harvested and lysed with lysis buffer
B (20 mM Tris-HCl at pH 7.4, 50 mM NaCl, 10 mM p-amidinophenyl-
methanesulfonyl fluoride, 10 mg/ml leupeptin, and 0.5% w/v Triton X-100). The
lysates were mixed with the indicated antibodies and incubated for 1 hour at 4°C.
The immunocomplex was subjected to SDS-PAGE, followed by immunoblotting with
the indicated antibodies.
The kinase reaction for His-GSK-3β was carried out in 50 μl kinase buffer (50 mM
Tris-HCl at pH 7.5, 5 mM MgCl2, 0.3 mM dithiothreitol) containing 100 μM [γ-
32P]ATP, recombinant kinases (20 ng His-GSK-3β and/or 10 ng His-Cdk5 with GST-
p35), and substrate (1 μM GST-CLASP2 protein). After incubation for 30 minutes
at 30°C, the reaction mixtures were boiled in SDS sample buffer and subjected to
SDS-PAGE and silver staining. The radiolabelled bands were visualised with an image
analyser (BAS1500; Fuji, Tokyo, Japan).
Purification and preparation of anti-S533/S537-P antibody
A rabbit polyclonal antibody against CLASP2 phosphorylated at Ser533 and Ser537
(anti-S533/S537-Pantibody) was raised as described previously (Amano et al., 2003).
The phosphopeptide Cys528-Ser-Arg-Glu-Ala-Ser533(-P)-Arg-Glu-Ser-Ser537(-P)-
Arg-Asp-Thr-Ser-Pro542 was chemically synthesised by Biologica (Aichi, Japan) as
an antigen for CLASP2. The antiserum obtained was then affinity-purified against
the respective phosphopeptide.
Microtubule sedimentation assays
The microtubule sedimentation assays was performed with purified proteins as
described previously (Fukata et al., 2002). Porcine tubulin was obtained from
Cytoskeleton (Denver, CO).
Boyden chamber assay
siRNA was transfected into Vero cells with either GFP-GST or RNAiR-IQGAP1.
Cell migration assays were performed using Transwell plates (pore size of 8 μm;
HTS FluoroBlok Insert; Becton Dickinson, Franklin Lakes, NJ). The undersurface
of the membrane was coated with 10 μg/ml fibronectin (Becton Dickinson) diluted
in distilled water at room temperature for 1 hour. The cells were seeded in the
upper chamber (1?104per well) in 500 μl DMEM with 0.1% bovine serum albumin
(BSA). DMEM supplemented with 0.1% BSA and 10% FBS was added into the
lower chamber. The cells were allowed to migrate for 4 hours. After fixation, both
nonmigrated and migrated EGFP-positive cells were counted by EGFP
fluorescence. The ratio of migrated cells to total (migrated + nonmigrated) cells
was calculated. At least 300 EGFP-positive cells were counted in each group
for each experiment. The results were normalised and expressed as a migration
For visualisation of +TIPs and microtubules, the cells were fixed with cold methanol
containing 1 mM EGTA for 20 minutes at –20°C. When necessary, the cells were
postfixed with 3.7% formaldehyde in PBS for 10 minutes and permeabilised with
PBS containing 0.2% Triton X-100 and 1 mg/ml BSA for an additional 10 minutes.
The cells were then stained with the indicated primary antibodies. Cy2-, Cy3-, and
Cy5-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories, West
Grove, PA) were used for immunofluorescence analysis. Fluorescence was examined
using a confocal laser-scanning microscope (Carl Zeiss LSM 510; Carl Zeiss,
Oberkochene, Germany) built around a Zeiss Axio-vert 100M with Plan-
APOCHROMAT (63? NA 1.4 Oil). The images were acquired and processed by
LSM software (Carl Zeiss).
We thank Eisuke Mekada (Osaka University, Japan) for providing
the Vero cells, Akira Kikuchi (Hiroshima University, Japan) for
providing the GSK-3β constructs, and Akira Okamoto (Nagoya
University, Japan) for helpful technical support. We also thank all
members of the Kaibuchi laboratory, especially Mutsuki Amano,
Masaki Fukata, and Yuko Fukata (National Institute for Physiological
Sciences) for helpful discussions, and Kiyoko Murase, Sumi Kamisawa,
Sachi Kozawa, and Takako Ishii for technical and secretarial assistance.
This research was supported in part by Special Coordination Funds for
Promoting Science and Technology (SCF), a Grant-in-Aid for Scientific
Research, the Human Frontier Science Program (HFSP), a Grant-in-
Aid for Creative Scientific Research (JSPS), a Grant-in-Aid for JSPS
Fellows (JSPS), the 21st Century Centre of Excellence (COE) Program
from MEXT, the Global COE Program from MEXT, and the Grant-
in-Aid for CREST (JST). N.G. was supported by grants from the
Netherlands Organisation for Scientific Research (NWO-ALW and
NWO-MW), the Netherlands Ministry of Economic Affairs (BSIK) and
the Dutch Cancer Society (KWF).
Journal of Cell Science 122 (16)
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