GSK-3beta regulates phosphorylation of CRMP-2 and neuronal polarity.
ABSTRACT Neurons are highly polarized and comprised of two structurally and functionally distinct parts, an axon and dendrites. We previously showed that collapsin response mediator protein-2 (CRMP-2) is critical for specifying axon/dendrite fate, possibly by promoting neurite elongation via microtubule assembly. Here, we showed that glycogen synthase kinase-3beta (GSK-3beta) phosphorylated CRMP-2 at Thr-514 and inactivated it. The expression of the nonphosphorylated form of CRMP-2 or inhibition of GSK-3beta induced the formation of multiple axon-like neurites in hippocampal neurons. The expression of constitutively active GSK-3beta impaired neuronal polarization, whereas the nonphosphorylated form of CRMP-2 counteracted the inhibitory effects of GSK-3beta, indicating that GSK-3beta regulates neuronal polarity through the phosphorylation of CRMP-2. Treatment of hippocampal neurons with neurotrophin-3 (NT-3) induced inactivation of GSK-3beta and dephosphorylation of CRMP-2. Knockdown of CRMP-2 inhibited NT-3-induced axon outgrowth. These results suggest that NT-3 decreases phosphorylated CRMP-2 and increases nonphosphorylated active CRMP-2, thereby promoting axon outgrowth.
- [show abstract] [hide abstract]
ABSTRACT: We previously identified Rho-associated protein kinase (Rho-kinase) as a specific effector of Rho. In this study, we identified collapsin response mediator protein-2 (CRMP-2), as a novel Rho-kinase substrate in the brain. CRMP-2 is a neuronal protein whose expression is up-regulated during development. Rho-kinase phosphorylated CRMP-2 at Thr-555 in vitro. We produced an antibody that specifically recognizes CRMP-2 phosphorylated at Thr-555. Using this antibody, we found that Rho-kinase phosphorylated CRMP-2 downstream of Rho in COS7 cells. Phosphorylation of CRMP-2 was observed in chick dorsal root ganglion neurons during lysophosphatidic acid (LPA)-induced growth cone collapse, whereas the phosphorylation was not detected during semaphorin-3A-induced growth cone collapse. Both LPA-induced CRMP-2 phosphorylation and LPA-induced growth cone collapse were inhibited by Rho-kinase inhibitor HA1077 or Y-32885. LPA-induced growth cone collapse was also blocked by a dominant negative form of Rho-kinase. On the other hand, semaphorin-3A-induced growth cone collapse was not inhibited by a dominant negative form of Rho-kinase. Furthermore, overexpression of a mutant CRMP-2 in which Thr-555 was replaced by Ala significantly inhibited LPA-induced growth cone collapse. These results demonstrate the existence of Rho-kinase-dependent and -independent pathways for growth cone collapse and suggest that CRMP-2 phosphorylation by Rho-kinase is involved in the former pathway.Journal of Biological Chemistry 09/2000; 275(31):23973-80. · 4.65 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: Short interfering RNAs (siRNAs) can mediate sequence-specific inhibition of gene expression in mammalian cells. We and others have recently developed expression vector-based systems for synthesizing siRNAs or hairpin siRNAs in mammalian cells. Expression vector-based RNA interference (RNAi) effectively suppresses expression of target genes and is likely to be a powerful tool for analysis of gene function. Here we compare inhibition by vectors expressing hairpin siRNA designs either with different loop sequences connecting the two siRNA strands, or with duplex regions of different lengths. Our results suggest that lengthening the 19-nucleotide duplex region of a relatively ineffective hairpin siRNA can increase inhibition, but increasing the length of an effective 19-nt hairpin siRNA does not increase inhibition. We also demonstrate that hairpin siRNA vectors can be used to inhibit two target genes simultaneously. We have targeted glycogen synthase kinase-3alpha (GSK-3alpha) and GSK-3beta, two related kinases involved in the regulation of a variety of cellular processes and also implicated in the pathogenesis of several human diseases. Inhibition of either GSK-3alpha or GSK-3beta by transfection of hairpin siRNA vectors leads to elevated expression of the GSK-3 target beta-catenin, whereas inhibition of both kinases further increases beta-catenin expression. Our results suggest that vector-based siRNA inhibition may be useful for dissecting the functional roles of GSK-3alpha and GSK-3beta in somatic cells. The ability to inhibit two or more genes simultaneously with hairpin siRNA expression vectors should facilitate studies of gene function in mammalian cells.Molecular Therapy 03/2003; 7(2):228-36. · 7.04 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: As a first step toward elucidating mechanisms involved in the sorting of synaptic vesicle proteins in neurons, we have used immunofluorescence microscopy to determine the distribution of two synaptic vesicle proteins, synapsin I and synaptophysin, in hippocampal neurons developing in culture. In mature cultures, synapsin I and synaptophysin immunoreactivity was concentrated in puncta that were restricted to sites where axons contacted neuronal cell bodies or dendrites. Electron-microscopic immunocytochemistry demonstrated that these puncta corresponded to vesicle-filled axonal varicosities that were exclusively presynaptic. At early stages of development, before cell-cell contact, both synapsin I and synaptophysin were preferentially localized in axons, where they were particularly concentrated in the distal axon and growth cone. In axons that did not contact other cells, immunostaining for these two proteins had a granular appearance, which persisted for at least 7 d, but focal accumulations of vesicles comparable to those seen at sites of synaptic contact were not observed. When neurons contacted one another, numerous puncta of synapsin I and synaptophysin formed within the first week in culture. Double-label immunofluorescence demonstrated that the two vesicle antigens were closely codistributed throughout these stages of development. These observations demonstrate that synaptic vesicle proteins assume a polarized distribution within nerve cells beginning early in development, as soon as the axon can be identified. In contrast, differences in microtubule polarity orientation that distinguish mature axons and dendrites, and that have been proposed to account for the selective sorting of some materials in nerve cells, first appear at a subsequent stage of development. The selective distribution of synaptic vesicle proteins to the axon occurs in isolated cells, independent of interactions with other cells. In contrast, the formation of large clusters of vesicles typical of presynaptic specializations requires contact with an appropriate postsynaptic target. Thus, in cultured hippocampal neurons, the localization of synaptic vesicles in presynaptic specializations is the result of sorting mechanisms intrinsic to individual neurons as well as to mechanisms mediated by cell-cell contact.Journal of Neuroscience 07/1991; 11(6):1617-26. · 6.91 Impact Factor
Cell, Vol. 120, 137–149, January 14, 2005, Copyright ©2005 by Elsevier Inc. DOI 10.1016/j.cell.2004.11.012
GSK-3? Regulates Phosphorylation
of CRMP-2 and Neuronal Polarity
neurite/axon is generated by two mechanisms: the
transport of microtubule polymer and the microtubule
assembly at the plus ends of the microtubules. Both
appear to contribute to axon outgrowth (Baas, 1997).
CRMP-2, which also has been independently identi-
fied as Ulip2/CRMP-62/TOAD-64/DRP-2, is one of at
least five isoforms (Goshima et al., 1995; Arimura et al.,
2004). CRMP-2 is expressed exclusively and highly in
the developing nervous system. Mutations in the
UNC-33 gene, a Caenorhabditis elegans homolog of
CRMPs, lead to severely uncoordinated movement and
abnormalities in the guidance of axons of many neurons
(Hedgecock et al., 1985). Previously, we showed that
neurons, the overexpression of full-length CRMP-2 in-
duces the formation of multiple axons and elongation
of the primary axon, and the dominant-negative form of
CRMP-2 inhibits axon formation (Inagaki et al., 2001).
CRMP-2 shows the ability to convert immature neurites
and preexisting dendrites to axons. Thus, CRMP-2 is
crucial for axon outgrowth and determination of the fate
of the axon and dendrites, thereby establishing and
maintaining neuronal polarity. In addition, we recently
demonstrated that CRMP-2 binds to tubulin heterodi-
mers to promote microtubule assembly, thereby en-
hancing axon elongation and branching (Fukata et al.,
2002). However, the molecular mechanism by which
CRMP-2 is regulated remains unclear.
A ternary complex of PAR-3, PAR-6, and atypical pro-
events from worms to mammals (Etienne-Manneville
and Hall, 2003b), including cultured hippocampal neu-
rons (Shi et al., 2003; Nishimura et al., 2004). The PAR-
6-PAR-3-aPKC complex accumulates at the tip of the
axon, and its polarized localization and aPKC activity
are important for axon specification (Shi et al., 2003;
Nishimura et al., 2004). aPKC can also phosphorylate
GSK-3? and inactivate its kinase activity, and GSK-3?
is important for polarization of migrating fibroblasts
(Etienne-Manneville and Hall, 2003a). However, it re-
mains unknown whether GSK-3? is involved in neuronal
polarity and, if so, what the target substrates of GSK-3?
We report here that GSK-3? phosphorylates CRMP-2
at Thr-514, inactivates the CRMP-2 activity, and partici-
pates in neuronal polarization through CRMP-2. NT-3
and brain-derived neurotrophic factor (BDNF) inhibit
GSK-3? via the phosphatidylinositol-3-kinase (PI3-
kinase)/Akt (also known as PKB) pathway, and thereby
reduce phosphorylation levels of CRMP-2 at Thr-514,
leading to axon elongation and branching.
Takeshi Yoshimura,1Yoji Kawano,1
Nariko Arimura,1Saeko Kawabata,1
Akira Kikuchi,2and Kozo Kaibuchi1,*
1Department of Cell Pharmacology
Graduate School of Medicine
65 Tsurumai, Showa-ku, Nagoya
2Department of Biochemistry
Graduate School of Biomedical Sciences
1-2-3, Kasumi, Minami-ku
Neurons are highly polarized and comprised of two
structurally and functionally distinct parts, an axon and
dendrites. We previously showed that collapsin re-
sponse mediator protein-2 (CRMP-2) is critical for
specifying axon/dendrite fate, possibly by promoting
neurite elongationvia microtubuleassembly. Here,we
showed that glycogen synthase kinase-3? (GSK-3?)
phosphorylated CRMP-2 at Thr-514 and inactivated it.
The expression of the nonphosphorylated form of
CRMP-2or inhibition of GSK-3? induced the formation
neuronal polarization, whereas the nonphosphory-
lated form of CRMP-2 counteracted the inhibitory ef-
fects of GSK-3?, indicating that GSK-3? regulates neu-
ronal polarity through the phosphorylation of CRMP-2.
Treatment of hippocampal neurons with neurotro-
phin-3 (NT-3) induced inactivation of GSK-3? and de-
phosphorylation of CRMP-2. Knockdown of CRMP-2
inhibited NT-3-induced axon outgrowth. These results
suggestthat NT-3decreases phosphorylatedCRMP-2
and increases nonphosphorylated active CRMP-2,
thereby promoting axon outgrowth.
The neuron is one of the most highly polarized cells
known and is comprised of two structurally and func-
tionally distinct parts, an axon and dendrites (Craig and
Banker, 1994). The specification of the axon is thought
to depend on its length relative to the other minor pro-
cesses, which are called immature neurites (Bradke and
Dotti, 2000). Elongation of one of the immature neurites
is necessary for axon specification. Intracellular mecha-
nisms that help to enhance neurite/axon outgrowth evi-
dently require reorganization of cytoskeletons including
occurs in the cell body and the growth cone (Brown
et al., 1992). Formation of the microtubule array in the
GSK-3? Phosphorylates CRMP-2
Ithas beenpreviouslyreportedthat thephosphorylation
of CRMP-2 at Thr-514 by unidentified kinases induces
consensus site for the phosphorylation by GSK-3? is
were cotransfected with GSK-3? wild-type (wt) and
CRMP-2 wt and subjected to immunoblot analysis with
anti-CRMP-2 antibody (Figure 1B). The mobility shift of
CRMP-2 (asterisk) was observed in the cells cotrans-
fected with GSK-3? wt and CRMP-2 wt. We then pro-
duced the antibody that specifically recognized phos-
phorylated CRMP-2 at Thr-514 (anti-pT514 antibody).
In the COS7 cells cotransfected with CRMP-2 wt and
GSK-3? wt, anti-pT514 antibody recognized the upper
bands, which corresponded to the phosphorylated
CRMP-2 at Thr-514. In contrast, the immunoreactive
band was not observed in runs with CRMP-2 T514A
(Thr-514 was replaced by Ala) and GSK-3? wt. Consis-
ity shift of CRMP-2 (asterisk) was not observed in these
cells. These results suggest that GSK-3? phosphorylates
CRMP-2 at Thr-514.
Next, we examined whether GSK-3? phosphorylates
tion of CRMP-2 at Thr-514 was not observed when
GSK-3? alone was added. GSK-3? requires prephos-
phorylation of its substrate (Eldar-Finkelman, 2002).
CRMP-2 has a consensus motif recognized by Cdk5 at
Ser-522 in the vicinity of the phosphorylation site of
GSK-3?, Thr-514. We found that GSK-3? phosphory-
lated CRMP-2 that was prephosphorylated by Cdk5
in vitro. When Cdk5 alone was added, CRMP-2 at Thr-
514 was not phosphorylated. These results indicate that
GSK-3? can phosphorylate CRMP-2 at Thr-514 after
CRMP-2 is phosphorylated by Cdk5.
We then evaluated each phosphorylation site (Thr-
514, Ser-518, and Ser-522). Not only CRMP-2 T514A but
also S518A (Ser-518 was replaced by Ala), S522A (Ser-
522 was replaced by Ala), and AAA (Thr-514, Ser-518,
and Ser-522 were replaced by Ala) blocked the band
shift of CRMP-2 (see Supplemental Figure S1A at http://
body was not observed in runs with T514A, S518A,
S522A, and AAA. It is reported that the phosphorylation
of CRMP-2 at Thr-514 but not Ser-518 or Ser-522 in-
duces the mobility shift of CRMP-2 (Gu et al., 2000).
These results suggest that Ser-518 and Ser-522 are re-
quired for the phosphorylation of CRMP-2 at Thr-514
by GSK-3?. Ser-522 appears to be phosphorylated by
Cdk5 as a priming kinase, and Ser-518 and Thr-514
seem to be phosphorylated by GSK-3? after the phos-
phorylation at Ser-522.
Figure 1. GSK-3? Phosphorylates CRMP-2 at Thr-514
(A)Potentialphosphorylation sitesofCRMP-2by GSK-3?andCdk5.
The numbers denote amino acid positions. The optimal consensus
site for the phosphorylation by GSK-3? is Ser/Thr-Xaa-Xaa-Xaa-
a consensus motif recognized by Cdk5.
(B) COS7 cells were cotransfected with CRMP-2 and GSK-3? mu-
tants. Samples were subjected to immunoblot analysis with anti-
pT514 (top) and anti-CRMP-2 (bottom) antibodies. The mobility shift
(C) Kinase assay was performed using purified CRMP-2, GSK-3?,
and Cdk5 in vitro. Each reaction mixture was subjected to SDS-
PAGE and immunoblot analysis with anti-pT514 (top) and anti-
CRMP-2 (bottom) antibodies.
Nonphosphorylated Pool of CRMP-2 Localizes
in Axonal Growth Cone
In cultured hippocampal neurons, neurons extend sev-
eral minor processes during the first 12–24 hr after plat-
ing (stages 1 and 2; Dotti et al. ). Then, one of the
processes begins to extend rapidly to form an axon,
resulting in the morphological polarization of the neuron
logical features of dendrites (stage 4). By 7 days in vitro
(DIV), the neurons become highly polarized, and the
axon and dendrites continue to mature and subse-
quently develop (stage 5).
We examined whether CRMP-2 is phosphorylated by
Ser/Thr-Xaa-Xaa-Xaa-Ser/Thr (where Xaa represents
any amino acid; Frame and Cohen ; Eldar-Finkel-
man ). Considering the consensus sequences of
GSK-3?, we speculated that CRMP-2 was phosphory-
lated by GSK-3? at Thr-514 (Figure 1A). To examine
whether GSK-3? phosphorylates CRMP-2, COS7 cells
GSK-3? and CRMP-2 in Neuronal Polarity
Figure 2. Nonphosphorylated CRMP-2 Lo-
calized in the Axonal Growth Cone
presence of GSK-3 inhibitors (2 mM LiCl,
5 ?M SB216763, or 25 ?M SB415286), buffer,
2 mM NaCl, or DMSO for 48 hr before TCA
treatment. Each sample was subjected to
SDS-PAGE and immunoblot analysis with
anti-pT514 (top) and anti-CRMP-2 (bottom)
antibodies. The asterisk indicates the mobil-
ity shift of CRMP-2. These results are repre-
sentative of three independent experiments.
(B) Nonphosphorylated pool of CRMP-2 lo-
calized in growth cone of growing axon. Hip-
pocampal neurons were fixed at 3 DIV and
then immunostained with anti-pT514 (Ba and
Bd)and anti-CRMP-2(Bband Be)antibodies.
lated CRMP-2 to total CRMP-2, [Bg]) are
shown. The graph (Bf-2) plots the fluores-
cence intensities of CRMP-2 phosphorylated
at Thr-514 (red) and total CRMP-2 (green) in
the line (Bf, from X to Y). The ratio of fluores-
total CRMP-2) was measured in the indicated
Scale bar, 10 ?m.
The immunoblot analysis with anti-pT514 antibody re-
vealed that CRMP-2 was phosphorylated at Thr-514 in
hippocampal neurons at 3 DIV (more than 75% of neu-
rons at stage 3; Figure 2A). About 30% of CRMP-2 was
constitutively phosphorylated at Thr-514. When hippo-
campal neurons were cultured in the presence of GSK-3
inhibitors (LiCl, SB216763, or SB415286), the phosphor-
ylation levels of CRMP-2 at Thr-514 were decreased.
Cdk inhibitor (olomoucine) also decreased the phos-
phorylation levels of CRMP-2 at Thr-514 (see Supple-
mental Figure S1B on the Cell web site). These results
suggest that GSK-3? phosphorylates CRMP-2 at Thr-
514 after CRMP-2 is phosphorylated by Cdk5 in hippo-
To investigate the spatial distribution of phosphory-
lated CRMP-2 at Thr-514, hippocampal neurons were
fixed at 3 DIV and then immunostained with anti-pT514
(phosphorylated CRMP-2, red) and anti-CRMP-2 anti-
bodies (total CRMP-2, green; Figure 2B). CRMP-2
(green) was enriched in the distal part of the growing
axon as previously reported (Inagaki et al., 2001). Phos-
phorylated CRMP-2 at Thr-514 (red) was enriched in the
distal part of the growing axon without growth cone.
The merged images of CRMP-2 and phosphorylated
CRMP-2 at Thr-514 immunofluorescence enable us to
roughly estimate the phosphorylation levels of CRMP-2.
The merged image in the shaft was yellow, whereas that
in the axonal growth cone was more greenish than that
in the shaft. Thus, the ratio of phosphorylated CRMP-2
to total CRMP-2 in the axonal growth cone was lower
than that in the shaft.
Further, clear evidence was obtained by intensity im-
aging of phosphorylated CRMP-2 and total CRMP-2
(Figure 2B). The intensity imaging was similar in shafts,
Figure 3. Phosphorylated CRMP-2 Lowers Its Activity to Interact with Tubulin
with anti-tubulin antibody (DM1A; [Ab], [Ae], [Ah], and [Ak]). HeLa cells having spindles in mitotic phase were chosen to show the CRMP-2 local-
(B) HeLa cells transfected with GFP-CRMP-2 wt were immunostained with anti-pT514 and tubulin (DM1A) antibodies.
(C) Porcine brain lysate was mixed with glutathione-Sepharose 4B beads coated with purified GST, CRMP-2 wt-GST, or CRMP-2 T514D-
GSK-3? and CRMP-2 in Neuronal Polarity
that of total CRMP-2 in the axonal growth cone. The
intensity of total CRMP-2 and phosphorylated CRMP-2
results indicate that there is a nonphosphorylated
CRMP-2 pool at Thr-514 in the growing axonal growth
cone. We confirmed that GSK-3 inhibitor decreased the
phosphorylation levels of CRMP-2 by immunostaining
with anti-pT514 antibody (Supplemental Figure S1C).
Nonphosphorylated CRMP-2 Promotes Axon
Outgrowth and Induces the Formation
of Multiple Axon-Like Neurites
neurons were transfected with Myc-CRMP-2 wt, T514A,
or T514D and fixed at 3 DIV. CRMP-2 wt enhanced axon
et al., 2002; Figures 4A and 4B). CRMP-2 T514A pro-
moted axon outgrowth and branching more than CRMP-2
wt, whereas CRMP-2 T514D had the weaker activity.
These results are consistent with the observation that
CRMP-2 T514D showed lower activity to interact with
tubulin. CRMP-2 S518A, S522A, and AAA promoted
axon outgrowth as well as CRMP-2 T514A (Supplemen-
tal Figure S2A).
To examine the effect of CRMP-2 mutants on axon
formation, hippocampal neurons transfected with Myc-
CRMP-2 wt, T514A, or T514D were fixed at 6 DIV. To
visualize secondary axons, neurons at 6 DIV are better
later (Inagaki et al., 2001). CRMP-2 wt induced the for-
mation of multiple axon-like neurites as previously re-
ported (Inagaki et al. ; Figure 4D). CRMP-2 T514A
increased the percentage of neurons that had multiple
long neurites and multiple Tau-1-positive neurites (Fig-
ures 4C and 4D). The effect of CRMP-2 T514A on the
formation of multiple axon-like neurites was stronger
than that of CRMP-2 wt, whereas CRMP-2 T514D
showed the weaker activity (Figure 4D). The consistent
results were obtained by using other axonal markers,
such as synapsin I and synaptophysin for axon and
MAP2 for dendrite (Fletcher et al., 1991; Supplemental
Figure S2B). CRMP-2 S518A, S522A, and AAA induced
the formation of multiple axon-like neurites as well as
CRMP-2 T514A (Supplemental Figure S2C).
Phosphorylation of CRMP-2 Lowers its Activity
for Interaction with Tubulin
We recently found that the phosphorylation of CRMP-2
at Thr-555 by Rho-kinase turns off the ability of CRMP-2
to bind tubulin (N. Arimura, C. Menager, Y.K., Y. Fukata,
unpublished data). Here, we tried to prepare CRMP-2
phosphorylated by GSK-3? in vitro but found that the
Then, we produced and characterized a phosphomimic
CRMP-2 mutant. CRMP-2 T514D (Thr-514 was replaced
by Asp) is expected to mimic the phosphorylated form
of CRMP-2 (Kamisoyama et al., 1994; Sweeney et al.,
1994; Bresnick et al., 1995). Because ectopic CRMP-2
was diffusely overexpressed in neurons, it was difficult
to examine the colocalization of ectopic CRMP-2 with
microtubules in detail at axons. Instead, we used HeLa
cells, in which green fluorescent protein (GFP)-tagged
CRMP-2 was uniformly distributed along microtubules
(Fukata et al., 2002). GFP-CRMP-2 wt was clearly local-
ized along the mitotic spindle (N. Arimura, C. Menager,
Y.K., Y. Fukata, M. Amano, Y. Goshima, N. Morone, J.
mental Figure S1D). GFP-CRMP-2 T514A, S518A,
S522A, and AAA were also localized along the mitotic
spindle, whereas GFP-CRMP-2 T514D, S518D, S522D,
and DDD resulted in lower ability to localize along the
mitotic spindle (Figure 3A, Supplemental Figure S1D).
Under the same conditions, phosphorylated CRMP-2
was diffusely distributed throughout the cytoplasm and
not localized along the mitotic spindle (Figure 3B).
Next, we directly compared the binding activity of
CRMP-2 wt and T514D to tubulin in vitro. Porcine brain
wt-GST as previously described (Fukata et al., 2002;
Figure 3C). The stoichiometry of bound tubulin to
CRMP-2 wt was about 0.3, whereas the stoichiometry
of bound tubulin to CRMP-2 T514D was about 0.05. The
than that of CRMP-2 wt. Taken together, these results
suggest that the binding activity of CRMP-2 to tubulin
is decreased by the phosphorylation of CRMP-2 by
GSK-3? Regulates Neuronal Polarity via CRMP-2
Inhibition of GSK-3? results in enhanced neurite growth
in rat cerebellar granule neurons and DRG neurons (Mu-
noz-Montano et al., 1999; Jones et al., 2003). To investi-
gate the effect of inhibition of GSK-3? on axon outgrowth
and formation, hippocampal neurons were cultured in
the presence of GSK-3 inhibitors (LiCl, SB216763, or
SB415286). These inhibitors slightly enhanced axon
elongation (Figure 5A) and branching (data not shown)
at 3 DIV and increased the percentage of neurons that
had multiple long neurites and multiple Tau-1-positive
neurites at 6 DIV (Figures 5B and 5C). The consistent
results were obtained by using other axonal markers,
such as synapsin I and synaptophysin for axon and
MAP2 for dendrite (Supplemental Figure S3A).
To confirm these results, we used hairpin short in-
terfering RNA (siRNA) construct for GSK-3? (Yu et al.,
2003). Previous applications of the same hairpin siRNA
construct in mammalian cells have shown efficient and
specific inhibition of GSK-3? (Yu et al., 2003), and we
GST in vitro. The bound proteins were coeluted with GST fusion proteins by the addition of buffer containing glutathione. Portions of start
samples (top) and eluates (middle) were subjected to SDS-PAGE followed by immunoblot analysis with anti-tubulin antibody (DM1A). CRMP-2
wt-GST and CRMP-2 T514D-GST were immobilized in comparable quantities (bottom; Coomassie brilliant blue staining). These results are
representative of three independent experiments.
Figure 4. Nonphosphorylated CRMP-2 Enhanced Axon Elongation and Branching and Induced the Formation of Multiple Axon-Like Neurites
(A) Hippocampal neurons were transfected with Myc-GST (Aa), Myc-CRMP-2 wt (Ab), T514A (Ac), or T514D (Ad). Neurons were fixed at 3 DIV
and then immunostained with anti-Myc antibody. Scale bar, 100 ?m.
(B) Axon length (Ba) and the number of branch tips per axon (Bb) were measured at 3 DIV neurons transfected with the indicated plasmids.
n ? 50 per experimental condition. Data are means ? SD of triplicate determinations. Asterisks indicate statistical significance (Student’s t
test; *p ? 0.05; **p ? 0.01).
(C) Hippocampal neurons were transfected with Myc-GST (Ca), Myc-CRMP-2 T514A (Cb). Neurons were fixed at 6 DIV and then immunostained
with anti-Myc and Tau-1 antibodies. The enlarged images of the neurites (1, 2, 3) are shown. The neuron transfected with Myc-GST (Ca) had
one Tau-1-positive neurite (1). The neuron transfected with Myc-CRMP-2 T514A (Cb) had multiple Tau-1-positive neurites (1, 2, 3). Scale bar,
(D) The percentages of the cells that had multiple long neurites (Da) and multiple Tau-1-positive neurites (Db) were measured at 6 DIV neurons
transfected with the indicated plasmids. Fifty cells for each plasmid were measured by tracing images of immunofluorescence staining with
anti-Myc antibody. Data are means ? SD of triplicate determinations. Asterisks indicate statistical significance (Student’s t test; *p ? 0.05;
**p ? 0.01).
GSK-3? and CRMP-2 in Neuronal Polarity
obtained the similar result in rat 3Y1 cells (Supplemental
Figure S3B), indicating that the hairpin siRNA construct
for GSK-3? is effective in rat cells. Rat hippocampal
neurons were cotransfected with hairpin siRNA for
GSK-3? and Myc-GST. Inhibition of GSK-3? by hairpin
siRNA enhanced axon elongation at 3 DIV (Figure 5A)
andincreased thepercentageof neuronsthat hadmulti-
ple long neurites and multiple Tau-1-positive neurites
at 6 DIV (Figures 5B and 5C). The consistent results
were obtained by using other axonal markers, such as
synapsin I and synaptophysin for axon and MAP2 for
dendrite (data not shown).
Next, hippocampal neurons were cotransfected with
HA-GSK-3? wt, S9A (Ser-9 was replaced by Ala; consti-
tutively active form), or KD (Lys-85 was replaced by
Met; kinase dead mutant) and Myc-GST. The ectopic
tion (Figures 5D and 5E) and branching (data not shown)
at 3 DIV, whereas GSK-3? KD had no apparent effect.
Most of the cells transfected with Myc-GST (about 75%)
displayed normal polarity with a single neurite, which
was stained with Tau-1 antibody, and some minor pro-
cesses (Nishimura et al., 2004). The remaining (about
25%) neurons expressing Myc-GST remained in stage
2 of development. The expression of GSK-3? wt and
S9A increased the percentage of neurons that have no
Tau-1-positive neurites (Figures 5D and 5E). GSK-3?
S9A showed slightly higher activity than GSK-3? wt in
this capacity. GSK-3? KD had no obvious effect. The
consistent results were obtained by using other axonal
markers, such as synapsin I and synaptophysin for axon
(Supplemental Figure S3C). Further, the effect of GSK-3?
S9A on axon formation was examined at 6 DIV, and the
consistent results were obtained (Supplemental Figure
S3D). These results suggest that the overexpression of
GSK-3? impairs neuronal polarity, presumably by sup-
pressing neurite elongation.
Next, we investigated whether the expression of the
nonphosphorylated form of CRMP-2 compensates for
the GSK-3?-induced defect. The expression of CRMP-2
wt or CRMP-2 T514A counteracted the effects of GSK-3?
S9A on axon outgrowth and neuronal polarity (Figure
5F), but CRMP-2 T514D had no obvious effects (data
not shown). Taken together, these results suggest that
GSK-3? regulates neuronal polarity through the phos-
phorylation of CRMP-2, though we can not neglect the
possibility that GSK-3? is involved in neuronal polarity.
(PIP3) and phosphoinositide-dependent kinase (PDK;
Scheid and Woodgett ). Activated Akt phosphory-
lates GSK-3? at Ser-9 and inactivates its kinase activity
(Grimes and Jope, 2001).
We examined whether NT-3 regulates the phosphory-
lation levels of CRMP-2 (Figure 6A). Hippocampal neu-
rons were stimulated by NT-3 for 3, 10, 30, or 90 min.
A decrease of the phosphorylation levels of CRMP-2
was not observed during first 3 min. After the 10 min
stimulation, the phosphorylation levels of CRMP-2 were
decreased. The decrease was sustained until at least
90 min after the stimulation. BDNF had a similar effect
NT-3 and BDNF increased the phosphorylation levels of
Akt at Ser-473 and GSK-3? at Ser-9, whereas NGF had
no obvious effects on the phosphorylation levels of
tor (wortmannin) inhibited NT-3- and BDNF-induced
decrements of the phosphorylation levels of CRMP-2
and increments of the phosphorylation levels of GSK-3?
and Akt. Because the phosphorylation of GSK-3? at
Ser-9 is known to inactivate GSK-3?, our findings sug-
gest that NT-3 and BDNF decrease the phosphorylation
ylated active CRMP-2 via the PI3-kinase/Akt/GSK-3?
To examine the effect of NT-3 on the spatial distribu-
tion of phosphorylated CRMP-2 at Thr-514, hippocam-
pal neurons were fixed at 3 DIV after the treatment of
(phosphorylated CRMP-2, red) and anti-CRMP-2 anti-
bodies (total CRMP-2, green; Figure 6C). The phosphor-
ylation levels of CRMP-2 at Thr-514 (red) were de-
creased not only in the axonal growth cone but also in
the shaft as compared with nontreated cells (Figures 2B
and 6C). In the cell body, the obvious decrease of the
phosphorylation levels of CRMP-2 was not observed.
We then examined whether CRMP-2 is involved in
NT-3- and BDNF-induced axon outgrowth and branching.
NT-3 and BDNF promoted axon elongation and
branching as previously described (Tucker ; Fig-
ures 6D and 6E). We used siRNA to directly test whether
endogenous CRMP-2 is required for axon elongation
and branching by NT-3 and BDNF (Figure 6E). Expres-
sion of CRMP-2 was markedly inhibited by CRMP-2
siRNA in 10%–20% cells as revealed by immunocyto-
chemistry as described (Nishimura et al. ; data
not shown). Knockdown of CRMP-2 caused a marked
inhibition of NT-3- and BDNF-induced axon elongation
and branching, indicating that CRMP-2 is necessary for
NT-3- and BNDF-induced axon outgrowth and branching.
Taken together, these results suggest that NT-3 de-
creases the phosphorylation levels of CRMP-2 and in-
creases nonphosphorylated active CRMP-2 to promote
axon outgrowth and branching.
NT-3 and BDNF Regulate CRMP-2 Phosphorylation
via the PI3-Kinase/Akt/GSK-3? Pathway
NT-3 and BDNF but not NGF enhance axon elongation
and branching in hippocampal neurons (Ip et al., 1993;
Morfini et al., 1994; Labelle and Leclerc, 2000). GSK-3?
is known to be constitutively active, and its activity can
be inhibited by treatment with NT-3 and BDNF (Huang
and Reichardt, 2003; Segal, 2003). This inhibitory mech-
anism is thought to be mediated by the PI3-kinase/Akt
pathway (Markus et al., 2002; Huang and Reichardt,
2003; Segal, 2003). Several groups, including ours, re-
ported that PI3-kinase inhibitors inhibited axon elonga-
tion (Shi et al., 2003; Menager at al., 2004). PI3-kinase
activates Akt by the phosphorylation of Akt at Thr-308
Phosphorylation of CRMP-2 by GSK-3?
In the present study, we found that CRMP-2 was phos-
phorylated at Thr-514 by GSK-3? in vitro and in vivo
(Figures 1B, 1C, 2A, and 2B; Supplemental Figure S1C).
GSK-3? alone did not phosphorylate CRMP-2 in vitro
Figure 5. GSK-3? Regulates Neuronal Polarity via CRMP-2
(A) Neurons transfected with Myc-GST were cultured in the presence of GSK-3 inhibitors (2 mM LiCl, 5 ?M SB216763 or 25 ?M SB415286),
buffer, 2 mM NaCl, or DMSO for 48 hr before fixation (Aa). Hairpin siRNA construct for GSK-3? or control vector was cotransfected into
neurons with Myc-GST (Ab). Neurons were fixed at 3 DIV and then immunostained with anti-myc antibody to measure axon length. n ? 50
per experimental condition. Data are means ? SD of triplicate determinations. Asterisks indicate statistical significance (Student’s t test; *p ?
0.05; **p ? 0.01).
(B) Neurons transfected with Myc-GST were cultured in the presence of GSK-3 inhibitor (5 ?M SB216763) for 5 days before fixation (Ba).
Neurons were cotransfected with hairpin siRNA for GSK-3? and Myc-GST (Bb). The neurons were fixed at 6 DIV and then immunostained
with anti-Myc and Tau-1 antibodies. The enlarged images of the neurites (1, 2) are shown. The neuron treated with GSK-3 inhibitor (5 ?M
GSK-3? and CRMP-2 in Neuronal Polarity
(Figure 1C). GSK-3? requires a priming phosphate to
phosphorylate its substrates. CRMP-2 has a consensus
motif recognized by Cdk5 at Ser-522 in the vicinity of
Thr-514 (Figure 1A). We found that treatment of Cdk
inhibitor (olomoucine) decreased the phosphorylation
levels of CRMP-2 in COS7 cells (data not shown) and
hippocampal neurons (Supplemental Figure S1B) and
that GSK-3? phosphorylated CRMP-2 that was pre-
phosphorylated by Cdk5 in vitro (Figure 1C). Therefore,
it is likely that Cdk5 phosphorylates CRMP-2 at Ser-
522, whereby GSK-3? phosphorylates CRMP-2 at Thr-
514 in vivo. Ser-518 is also a potential phosphorylation
site of CRMP-2 by GSK-3?.
Phosphomimic CRMP-2 was not colocalized with the
mitotic spindle in vivo (Figure 3A, Supplemental Figure
ciated with the mitotic spindle (Figure 3B). The binding
indicate that the phosphorylation of CRMP-2 at Thr-514
lowers its binding activity to tubulin. Phosphorylated
CRMP-2 at Thr-514 was enriched in the growing axon,
whereas the phosphorylation levels of CRMP-2 were
low in the growth cone (Figure 2B), making it convenient
for CRMP-2 to copolymerize with tubulin dimers into
microtubules in the growth cone (Arimura et al., 2004).
In shafts, the phosphorylation of CRMP-2 at Thr-514
may prevent the copolymerization of CRMP-2 with tu-
bulin dimers into microtubules until reaching the growth
cone, or it may induce the dissociation of CRMP-2 from
microtubules. It may be noted that the phosphorylation
of CRMP-2 at Ser-518 and Ser-522 is implicated in the
formation of degenerating neurites in Alzheimer’s dis-
ease (Gu et al., 2000). Phosphorylated CRMP-2 was
identified as an antigen for 3F4 monoclonal antibody,
which was raised against partially purified paired helical
filaments and labeled neurofibrillary tangles and some
plaque neurites. The level of 3F4 antigen is increased in
disease. These observations raise the possibility that
hyperphosphorylation of CRMP-2 is involved in the de-
induced growth cone collapse (Eickholt et al., 2002).
Uchida et al. (2005) have recently found that Sema3A
induces the phosphorylation of CRMP-2 at Ser-522 by
Cdk5 followed by the phosphorylation at Thr-509 and
Ser-518. Thus, Sema3A appears to increase the phos-
phorylation levels of CRMP-2 at Ser-522 and thereby
promote growth cone collapse.
Neuronal Polarity and GSK-3?
Previously, we found that CRMP-2 is crucial for de-
termining the fate of the axon and dendrites, thereby
establishing and maintaining neuronal polarity (Inagaki
et al., 2001). CRMP-2 appears to be critical for axon
formation by promoting elongation of one of the imma-
ture neurites, which is the future axon and within which
CRMP-2 is enriched. CRMP-2 binds to tubulin hetero-
dimers and promotes microtubule assembly to enhance
axon growth and branching (Fukata et al., 2002). Re-
cently, it has been reported that the polarized distribu-
tions of PAR-3/PAR-6 and aPKC activity are important
for axon specification in hippocampal neurons and that
the PI3-kinase activity at the axon is required for polar-
ized localization of PAR-3 (Shi et al., 2003; Nishimura et
al., 2004). We recently reported that the local contact
of the immature neurites with adhesion molecules such
as laminin induces the rapid production of PIP3at the
tip of the neurite through the action of PI3-kinase and
that PIP3is involved in axon specification, possibly by
stimulating elongation of an immature neurite (Menager
et al., 2004). Elongation of one of the immature neurites
is necessary for axon specification (Bradke and Dotti,
2000). The accompanying paper (Jiang et al., 2005 [this
issue of Cell]) and we have now shown that the expres-
sion of constitutively active GSK-3? suppresses axon
formation and that inhibition of GSK-3? induces the for-
mation of multiple axons in hippocampal neurons (Fig-
ures 5B–5E; Supplemental Figures S3A, S3C, and S3D).
We also found that the inhibitory effect of GSK-3? on
CRMP-2 (Figure 5F). Taken together, these results indi-
cate that GSK-3? regulates neuronal polarity through
the phosphorylation of CRMP-2.
We propose the role of GSK-3? and CRMP-2 in axon
specification as follows (Figure 7). The activation of PI3-
kinase at the selective immature neurite produces PIP3,
thus activating Akt and recruiting the PAR-3/PAR-6/
aPKC complex at the growth cone. Activated Akt and
aPKC inhibit GSK-3? by its phosphorylation, whereby
nonphosphorylated CRMP-2 is increased in the growth
cone. Nonphosphorylated active CRMP-2 promotes mi-
crotubule assembly and Numb-mediated endocytosis
of cell adhesion molecules to enhance elongation of the
immature neurite for axon specification (Fukata et al.,
SB216763) or transfected with hairpin siRNA for GSK-3? had multiple Tau-1-positive neurites (1, 2). Scale bar, 100 ?m.
(C) The percentage of the cells that had multiple long neurites (Ca) and multiple Tau-1-positive neurites (Cb) were measured at 6 DIV neurons
treated with the indicated compounds or transfected with the indicated plasmids. n ? 50 per experimental condition. Data are means ? SD
of triplicate determinations. Asterisks indicate statistical significance (Student’s t test; *p ? 0.05; **p ? 0.01).
(D) Hippocampal neurons were cotransfected with HA-GSK-3? S9A and Myc-GST. Neurons were fixed at 3 DIV and then immunostained with
anti-myc (Da and Dd) and axonal marker Tau-1 (Db and De) antibodies. The merged images (Dc and Df) are shown. Arrows indicate Tau-1-
positive neurites, and arrowheads denote Tau-1-negative neurites in the transfected cells. Scale bar, 50 ?m.
(E) Hippocampal neurons were cotransfected with HA-GSK-3? wt, S9A, or KD and Myc-GST. Neurons were fixed at 3 DIV and then immuno-
stained with anti-myc and axonal marker Tau-1 antibodies. The axon length (Ea) and the percentage of the cells that had no Tau-1-positive
neurites (Eb) were measured. Fifty cells for each plasmid were measured by tracing images of immunofluorescence staining with anti-Myc
antibody. Data are means ? SD of triplicate determinations. Asterisks indicate statistical significance (Student’s t test; **p ? 0.01).
(F) Cotransfection of HA-GSK-3? wt or S9A with CRMP-2 T514A was performed in hippocampal neurons. The axon length (Fa) and the
percentage of the cells that had no Tau-1-positive neurites (Fb) were measured at 3 DIV neurons. n ? 50 per experimental condition. Data
are means ? SD of triplicate determinations. Asterisks indicate statistical significance (Student’s t test; *p ? 0.05; **p ? 0.01).
Figure 6. NT-3 and BDNF Regulate CRMP-2 Phosphorylation via the PI3-Kinase/Akt/GSK-3? Pathway
(A) 3 DIV hippocampal neurons were stimulated with NT-3 for 3, 10, 30, or 90 min after neurons were cultured in neurobasal medium for 2 hr.
The cell lysates were resolved by SDS-PAGE and immunoblotted with anti-pT514 and anti-CRMP-2 polyclonal antibodies. The relative levels
of CRMP-2 phosphorylation at Thr-514 were calculated with those of untreated control cells.
(B) Hippocampal neurons were treated with BDNF, NT-3, or NGF for 30 min after neurons were cultured in neurobasal medium for 2 hr.
Neurons were treated with PI3-kinase inhibitor (100 nM wortmannin) or DMSO during the last 2 hr. Immunoblot analyses were performed with
anti-pT514, anti-CRMP-2 polyclonal, anti-phospho-GSK-3? (Ser-9), anti-GSK-3?, anti-phospho-Akt (Ser-473), and anti-Akt antibodies. The
asterisk shows the mobility shift of CRMP-2 induced by the phosphorylation of CRMP-2 at Thr-514.
(C) NT-3 decreased the phosphorylation levels of CRMP-2 at Thr-514 in the axonal growth cone and the shaft. Hippocampal neurons were
fixed at 3 DIV after the treatment of NT-3 for 30 min and then immunostained with anti-pT514 (Ca) and anti-CRMP-2 (Cb) antibodies. The
merged (Cc) is shown. The fluorescence intensities of 30 cells were measured in growth cones (Cd), shafts (Ce), and cell bodies (Cf). Scale
bar, 50 ?m. Data are means ? SD of triplicate determinations. Asterisks indicate statistical significance (Student’s t test; *p ? 0.05; **p ? 0.01).
GSK-3? and CRMP-2 in Neuronal Polarity
phatase activity toward CRMP-2 to accelerate the de-
phosphorylation of CRMP-2. Further studies are neces-
sary to address these issues.
One can expect that overdosage of NT-3 or BDNF
induces the formation of multiple axons. However, we
found that this was not the case. One possible explana-
tion is that NT-3 and BDNF receptors (TrkC and TrkB)
of NT-3 and BDNF (Frank et al., 1996). Prolonged inhibi-
tion of GSK-3? may be necessary for multiple axon for-
GSK-3? phosphorylates MAP1B and the adenoma-
; Frame and Cohen ). The phosphorylation
of MAP1B by GSK-3? suppresses detyrosination of mi-
crotubules and decreases the numbers of stable micro-
tubules (Goold et al., 1999; Gordon-Weeks and Fischer,
2000). It is possible that NT-3 and BDNF inhibit GSK-3?
and thereby increase the stability of microtubules to
enhance axon outgrowth through the dephosphoryla-
tion of MAP1B. The binding of APC to microtubules
increases microtubule stability, and the interaction of
APC with microtubules is decreased by the phosphory-
lation of APC by GSK-3? (Zumbrunn et al., 2001). Inacti-
vation of GSK-3? by NT-3 and BDNF may induce the
microtubules. Thus, GSK-3? appears to regulate dy-
namics of microtubules through the phosphorylation of
specific microtubule-associated proteins.
Figure 7. Model Schema to Regulate the Phosphorylation of
CRMP-2 by GSK-3?
NT-3, BDNF, and adhesion molecules are thought to activate PI3-
kinase, thereby producing PIP3. PIP3activates Akt via PDK. Acti-
vated Akt phosphorylates and inactivates GSK-3?. The binding ac-
tivity of CRMP-2 to tubulin is decreased by the phosphorylation by
to promote microtubule assembly, thereby enhancing axon elonga-
tion and branching.
2002; Nishimura et al., 2003). The relation between Akt
and PAR-3/PAR-6/aPKC remains to be clarified.
Materials and Chemicals
cDNA encoding human CRMP-2 was obtained using the methods
of Arimura et al. (2000). pCAGGS vector was provided by Dr. M.
OH). Hairpin siRNA for GSK-3? construct, pU6-GSK-3? HP2, was
provided by Dr. David L. Turner (University of Michigan, MI; Yu
et al. ). The following antibodies were used: anti-CRMP-2
monoclonal antibody (C4G), kindly provided by Dr. Y. Ihara (Univer-
sity of Tokyo, Tokyo, Japan); anti-CRMP-2 polyclonal antibody
raised against MBP-CRMP-2; monoclonal anti-?-tubulin (DM1A,
Sigma, St. Louis, MO); monoclonal anti-unique ?-tubulin (TUJ1,
Berkeley Antibody Company, Richmond, CA); polyclonal anti-c-Myc
(A-14,SantaCruz Biotechnology,Inc.,SantaCruz, CA);anti-GSK-3?
(Transduction Laboratories, Lexington, KY); anti-phospho-GSK-3?
(Ser-9; Cell Signaling Technology Inc., Beverly, MA); anti-Akt (New
England BioLabs, Beverly, MA); anti-phospho-Akt (Ser-473) (New
England BioLabs); monoclonal Tau-1 (Chemicon, Temecula, CA);
anti-synapsin I (Calbiochem, San Diego, CA); anti-synaptophysin
(Chemicon); anti-MAP2 (Sigma); and anti-actin (Chemicon) antibod-
ies. Recombinant human NT-3 and BDNF were purchased from
PeproTech EC LTD (London, UK). NGF was from Upstate Biotech-
nology,Inc. (Charlottesville,VA). RecombinantHis-tagged Cdk5and
GST-tagged p35 were from Upstate. GSK-3 inhibitors (SB216763 and
itor (olomoucine) was from Sigma. PI3-kinase inhibitor (wortmannin)
was from Wako (Osaka, Japan).
Inhibition of CRMP-2 Phosphorylation by NT-3
We demonstrated that the phosphorylation of CRMP-2
was suppressed by NT-3 and BDNF (Figures 6A–6C).
Knockdown of CRMP-2 with siRNA inhibited NT-3- and
BDNF-induced axon outgrowth and branching (Figure
6E). These results suggest that NT-3 and BDNF de-
crease the phosphorylation levels of CRMP-2 and pro-
mote axon outgrowth and branching via nonphosphory-
lated CRMP-2. NT-3 and BDNF are thought to activate
PI3-kinase, thereby producing PIP3. PIP3activates Akt
via PDK. Activated Akt phosphorylates and inactivates
GSK-3?. Treatment of hippocampal neurons with NT-3
vation of GSK-3? (Figure 6B). Activated Akt and inacti-
vated GSK-3? are localized in the growth cone of axons
(Eickholt et al., 2002). These findings suggest that NT-3
and BDNF inhibit GSK-3? via the PI3-kinase/Akt path-
way, thereby decreasing the phosphorylation levels of
CRMP-2 at Thr-514 (Figure 7). It is also possible that
NT-3 and BDNF inhibit GSK-3? via the PAR-3/PAR-6/
aPKC pathway, and NT-3 and BDNF increase the phos-
with anti-Myc antibody. Scale bar, 100 ?m.
(E) Hippocampal neurons were cotransfected with CRMP-2 siRNA and Myc-GST and then treated with NT-3 or BDNF. They were fixed at 3
DIV and then immunostained with anti-Myc antibody. Axon length (Ea) and the number of branch tips per axon (Eb) were measured at 3 DIV
neurons. n ? 50 per experimental condition. Data are means ? SD of triplicate determinations. Asterisks indicate statistical significance
(Student’s t test; **p ? 0.01).
CRMP-2 mutants were generated with a site-detected mutagenesis
kit (Stratagene, La Jolla, CA). The cDNA fragments encoding
Carlsbad, CA) to obtain the construct of CRMP-2 tagged with histi-
dine (His) at the N terminus of protein, pB-GEX (rearranged vector
from pGEX; Amersham Pharmacia Biotech, Buckinghamshire, UK)
of protein, pCAGGS-myc, and pEGFP-C1 (Clontech, Palo Alto, CA)
vectors, respectively. pCGN-GSK-3?, pCGN-GSK-3? S9A, and
pCGN-GSK-3? kinase dead (KD; K85N) and pGEX-2T-GSK-3? were
constructed as described by Tanji et al. (2002). All fragments were
confirmed by DNA sequencing.
plemented with B-27 supplement (Invitrogen) and 1 mM glutamine.
Neurons were seeded on coverslips with PDL only for 6 DIV to
visualize secondary axons. Neurons were transfected using a cal-
cium phosphate method before plating to analyze the morphology
(Nishimura et al., 2003; Menager et al., 2004). Neurons were fixed
at 3 DIV or 6 DIV with 3.7% formaldehyde in PBS for 10 min at room
temperature, followed by treatment for 10 min with 0.05% Triton
X-100 on ice and 10% NGS in PBS for 1 hr at room temperature.
Neurons were then immunostained with indicated antibodies and
observed with a confocal laser microscope (LSM510 Carl Zeiss,
Oberkochen, Germany) built around an Axiovert 100 M (Carl Zeiss).
The length of a longest neurite was measured as that of an axon at
3 DIV. The percentage of neurons with the second longest neurite
whose length was longer than a half-length of the longest neurite
was counted as that of multiple long neurites. Neurotrophins were
added to the medium at a concentration of 100 ng/ml after the
transfection. Neurons were cultured for 2 days and were then grown
with fresh neurotrophins (100 ng/ml) for 1 day. For some experi-
ments, neurons were treated with 10% (w/v) TCA. The resulting
Protein Purification and Preparation of Anti-pT514 Antibody
GST- and His-tagged proteins were purified from E. coli on glutathi-
one-Sepharose 4B beads (Amersham) and Ni-NTA agarose (Qiagen,
Hilden, Germany)according tothe manufacturers’protocols. Rabbit
polyclonal antibody against CRMP-2 phosphorylated at Thr-514
(anti-pT514 antibody) was raised as described by Amano et al.
(2003). As the antigen,the phosphopeptide Gly-Cys- Thr509-Pro-Lys-
chemically synthesized by Biologica Co. (Aichi, Japan). The antise-
rum obtained was then affinity purified against the respective phos-
L. Turner (University of Michigan, MI) for their kind gifts of materials;
Drs. Y. Rao (Washington University, MO), Y. Goshima (Yokohama
City University, Kanagawa, Japan), M. Amano, M. Fukata, Y. Fukata,
S. Taya, C. Menager, T. Nishimura, Miss K. Fujii, and Mr. A. Hattori
for helpfuldiscussion; MissK. Yamadafor preparingsome materials
and technical assistance; and Mrs. T. Ishii for secretarial assistance.
This research was supported in part by grants-in-aid for scientific
research from the Ministry of Education, Culture, Sports, Science
Research from MEXT; The 21st Century Centre of Excellence (COE)
Program from MEXT; special coordination funds for promoting Sci-
cal Devices Agency (PMDA).
Culture of COS7 and Rat 3Y1 Cells for Immunoblot Analysis
COS7 and rat 3Y1 cells were seeded on a 60 mm dish in Dulbecco’s
modified Eagle’s medium (DMEM) with 10% fetal bovine serum
(FBS) and cultured overnight at 37?C in an air/5% CO2atmosphere
at constant humidity. Transfections were carried out using lipofec-
tamine reagent (Invitrogen) according to the manufacturer’s proto-
col. Cells were grown in DMEM with 10% FBS for 1 day and then
in DMEM for 1 day. Cells were treated with 10% (w/v) trichloroacetic
acid (TCA). The resulting precipitates were subjected to SDS-PAGE
and immunoblot analysis.
Culture of HeLa Cells for Immunofluorescence Analysis
HeLa cells were seeded on coverslips in DMEM with 10% FBS and
cultured overnight at 37?C in an air/5% CO2atmosphere at constant
humidity. Transfections were carried out using lipofectamine re-
agent. After 2 days’ culture, HeLa cells were fixed with 3.7% formal-
dehyde in phosphate-buffered saline (PBS) for 10 min at room tem-
perature followed by treatment for 10 min with 0.2% Triton X-100
Received: April 12, 2004
Revised: September 4, 2004
Accepted: November 2, 2004
Published: January 13, 2005
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