Lithium inhibits glycogen synthase kinase-3 activity and mimics
Wingless signalling in intact cells
Vuk Stambolic*†Laurent Ruel* and James R. Woodgett*†
Background: Exposing eukaryotic cells to lithium ions (Li+) during development
has marked effects on cell fate and organization. The phenotypic consequences
of Li+treatment on Xenopus embryos and sporulating Dictyostelium are similar
to the effects of inhibition or disruption, respectively, of a highly conserved
protein serine/threonine kinase, glycogen synthase kinase-3 (GSK-3). In
Drosophila, the GSK-3 homologue is encoded by zw3sgg, a segment-polarity
gene involved in embryogenesis that acts downstream of wg. In higher
eukaryotes, GSK-3 has been implicated in signal transduction pathways
downstream of phosphoinositide 3-kinase and mitogen-activated protein kinases.
Results: We investigated the effect of Li+on the activity of the GSK-3 family. At
physiological doses, Li+inhibits the activity of human GSK-3? and Drosophila
Zw3Sgg, but has no effect on other protein kinases. The effect of Li+on GSK-3 is
reversible in vitro. Treatment of cells with Li+inhibits GSK-3-dependent
phosphorylation of the microtubule-associated protein Tau. Li+treatment of
Drosophila S2 cells and rat PC12 cells induces accumulation of cytoplasmic
Armadillo/?-catenin, demonstrating that Li+can mimic Wingless signalling in
intact cells, consistent with its inhibition of GSK-3.
Conclusions: Li+acts as a specific inhibitor of the GSK-3 family of protein
kinases in vitro and in intact cells, and mimics Wingless signalling. This reveals a
possible molecular mechanism of Li+action on development and differentiation.
Glycogen synthase kinase-3 (GSK-3) is a highly conserved
serine/threonine protein kinase implicated in cell-fate
determination and hormonal signalling . Unlike many
protein kinases, GSK-3 is highly active in resting cells and
is primarily regulated by inactivation. Thus, insulin and
growth factors inhibit GSK-3 by inducing phosphorylation
of an amino-terminal serine residue  via activation of
the extracellular signal-regulated kinase (ERK) cascade
and MAP kinase-activated kinase (MAPKAP) kinase-1 ,
or via phosphoinositide 3-kinase (PI 3-kinase)-dependent
activation of protein kinase B (PKB) . A number of
putative GSK-3 substrates have been identified, including
glycogen synthase , the c-Jun component of the AP-1
transcription factor [6,7], the microtubule-associated
protein Tau  and the adenomatous polyposis coli (APC)
gene product .
The Drosophila GSK-3 homologue, Zeste-white3Shaggy
(Zw3Sgg), is an essential component of the Wingless
signalling pathway. Disruption of the kinase causes defects
in segmental organization and cell-fate determination
[10–13]. Genetic epistasis experiments have led to a model
in which Wingless signalling acts via Dishevelled to
suppress the activity of Zw3Sgg[10,11,14], resulting in the
cytoplasmic accumulation of Armadillo (a homologue of
mammalian ?-catenin) . Studies on dorso-ventral axis
formation during Xenopus embryogenesis revealed a similar
requirement for GSK-3 inactivation in Xwnt-8 signalling
and ?-catenin accumulation. For example, expression of
dominant-negative GSK-3 mutants in the ventral side of the
embryo induces formation of an ectopic dorsal axis [16,17],
an effect similar to that induced by ectopic expression of
Xwnt-8, Dishevelled, ?-catenin and LEF-1 [18–22]; these
mutants also rescue the UV-irradiation-mediated ventraliza-
tion of embryos . In Dictyostelium, the GSK-3 homologue
GSK-A also plays a central role in cell-fate determination;
disruption of GSK-A leads to the disproportionate differen-
tiation of stalk cells at the expense of spore cells .
The consequences of GSK-3 inactivation in these systems
have striking parallels with the phenotypic effects of
lithium ions (Li+). In isolated rat adipocytes, Li+specifically
activates glycogen synthase and mimics insulin action on
glycogen deposition . In Xenopus, Li+is a potent modi-
fier of mesoderm induction that can induce a secondary
body axis on the ventral side of the embryo [25,26] and can
rescue UV-ventralized embryos . In Dictyostelium, Li+
influences cell-fate determination during differentiation,
inducing an almost complete redifferentiation of prespore
cells into prestalk cells [27–29]. Li+has recently been
shown to inhibit bacterially expressed GSK-3 activity in
Addresses: *Ontario Cancer Institute, 610
University Avenue, Toronto, Ontario M5G 2M9,
Canada. †Department of Medical Biophysics,
University of Toronto, Toronto, Ontario M5G 2M9,
Correspondence: James R. Woodgett
Received: 2 September 1996
Revised: 11 October 1996
Accepted: 18 October 1996
Current Biology 1996, Vol 6 No 12:1664–1668
© Current Biology Ltd ISSN 0960-9822
Research Paper Lithium-mediated inhibition of GSK-3 Stambolic et al. 1665
vitro . Here, we demonstrate that physiologically effec-
tive concentrations of Li+specifically inhibit all members of
the GSK-3 family both in vitro and in intact cells; this raises
the possibility that GSK-3 is an important physiological
target of Li+action. We also present evidence that Li+
mimics Wingless signalling in intact cells.
Li+inhibits GSK-3 activity in vitro
To determine whether Li+modulates GSK-3 activity, we
tested the ability of purified rat GSK-3? and Zw3Sgg to
modify a GSK-3-specific peptide substrate, phospho-GS1,
in the presence of LiCl or KCl. Li+caused a dose-depen-
dent inhibition of both protein kinases with a half-maximal
effect occurring at 1–2 mM, whereas equal or greater con-
centrations of K+had no effect on GSK-3 activity (Fig. 1a).
Li+had no effect on the activity of other serine kinases
tested, including SAPK/JNK (Fig. 1b) and MAPK/ERK
(data not shown). Furthermore, Li+had no effect on the
activation of p42 and p44 MAP kinases (ERK2 and ERK1)
by phorbol 12-myristate 13-acetate (PMA) and nerve
growth factor (NGF) in PC12 cells, indicating that the ion
does not affect MEK, Raf or Trk protein kinases (Fig. 1c).
The effect of Li+on GSK-3 is reversible
To determine whether the effects of Li+on GSK-3 could
be measured in intact cells, haemagglutinin (HA)-tagged
human GSK-3? and GSK-3?S9A (a mutant in which the
inhibitory phosphorylation site, Ser 9, was replaced by
alanine ) were expressed in COS 1 cells by transient
transfection. The GSK-3 activity in immunoprecipitates
from cells treated with 10 mM Li+was not significantly
different from that from untreated cells (Fig. 2). However,
when LiCl was added to immunoprecipitates from
untreated cells, the activities of GSK-3? and GSK-3?S9A
were both inhibited. The inhibition could be largely
reversed by subsequent washing of the immunoprecipitates
(Fig. 2), indicating that the effect of Li+on GSK-3 is direct,
and that post-translational modifications are not necessary.
Li+inhibits GSK-3 in intact cells
The reversible nature of the inhibition precluded
assessment of the effect of Li+on GSK-3 in cells by
methods that depend on purification. We therefore exam-
ined the in situ phosphorylation of the Tau protein in cells.
GSK-3 phosphorylates several residues on Tau, including
Ser 202. Phosphorylation of Ser 202 creates an epitope rec-
ognized by the monoclonal antibody AT8, and co-transfec-
tion of plasmids encoding GSK-3 and Tau induces AT8
reactivity in COS cells . GSK-3-dependent phosphory-
lation of Tau was assessed in COS 1 cells that co-
expressed HA–GSK-3? and Tau1N4R and were treated
with various concentrations of Li+for 30 minutes. Li+
treatment reduced the AT8 immunoreactivity in a dose-
dependent manner (Fig. 3a, AT8 blot, lanes 4–10), without
altering the Tau levels, as judged by parallel immunoblot-
ting with the Tau.1 monoclonal antibody, the epitope of
which is independent of Ser 202 phosphorylation (Fig. 3a,
Tau.1 blot, lanes 4–10)  . In contrast, K+had no effect
on AT8 reactivity (Fig. 3a, lanes 11–17). Li+did not alter
expression of GSK-3, as judged by immunoblotting with
the 12CA5 monoclonal antibody (Fig. 3a, anti-HA blot,
lanes 1–17). Tau phosphorylation was also used to assess
the effect of Li+on GSK-3?S9A and GSK-3? (Fig. 3b). Li+
inhibited both forms of the kinase (Fig. 3b, AT8 blot),
without altering the levels of Tau (Fig. 3b, Tau.1 blot), or of
GSK-3?S9A and GSK-3? (data not shown). Although GSK-
3?S9A was slightly less sensitive to Li+than GSK-3? (Fig.
3c), this mutant was still effectively inhibited by Li+. These
data show that Li+inhibits both mammalian isoforms of
Li+inhibits the activity of GSK-3? and Zw3Sgg,
but does not affect the activity of SAPK or the
activation of p42 and p44 MAPK. (a) GSK-3?
activity in the presence of Li+and K+(white and
black circles, respectively); Zw3Sggactivity in
the presence of Li+and K+(white and black
squares, respectively). See Materials and
methods for details. Activities are expressed as
the percentage of those of the untreated
controls (100 % is equivalent to 0.1?mol
phosphate transferred per min per mg). (b) The
activity of SAPK in immunoprecipitates from
U937 cells treated with 5?g ml–1anisomycin
or 200 mM sorbitol for 30 min before lysis.
Following immunoprecipitation, the
immunocomplexes were treated with 10 mM
Li+(black bars) or 10 mM K+(white bars) and
their activities toward soluble GST–cJun5–89
(residues 5–89 of cJun tagged with a
glutathione-S-transferase epitope) were
measured in the presence of each ion. SAPK
activity is expressed as the percentage of that
of the untreated control (100 % is equivalent to
0.8 ?molphosphate per min per mg). (c)
Cytoplasmic extracts from serum-starved PC12
cells treated as indicated were separated by
SDS–PAGE and immunoblotted using a
phosphotyrosine-specific anti-MAPK antibody.
The cells were treated with 160 nM PMA or
50 ng ml–1NGF (Vector Biosystems). The sizes
of the molecular weight markers are shown.
Kinase activity (% control)
SAPK activity (% control)
20 mM LiCI
1666 Current Biology 1996, Vol 6 No 12
GSK-3 and, in the case of GSK-3?, inhibition is indepen-
dent of the Ser 9.
Li+mimics Wingless signalling in intact cells
In intact Drosophila cells, Zw3Sggis thought to act down-
stream of the diffusible factor Wingless, with Wingless inac-
tivating Zw3Sgg, resulting in an increase in the levels of
Armadillo. If this model is correct, inhibition of Zw3Sggby
Li+would be expected to mimic the Wingless signal, even
in the absence of the key component of the Wingless recep-
tor, Frizzled. To test this hypothesis, S2 cells, which do not
express Frizzled , were incubated in the presence of Li+
and the levels of cytoplasmic Armadillo were determined
by immunoblotting (Fig. 4). Li+induced time-dependent
accumulation of cytoplasmic Armadillo (Fig. 4a, lanes 2 and
4) without altering the level of Zw3Sgg; treatment with K+
did not affect Armadillo levels (Fig. 4a, lanes 3 and 5). The
effect of Li+on the level of cytoplasmic Armadillo is com-
parable to the effect of overexpression of Dishevelled from
plasmid pPAC-Dsh in S2 cells (Fig. 4, lanes 6 and 7).
SDS–PAGE analysis showed that Li+treatment specifically
caused the accumulation of a faster-migrating form of
Armadillo (Fig. 4a, lower arrow). Induction of this form —
which may represent hypophosphorylated protein — has
previously been found after exposure to Wingless .
In vertebrates, expression of the Wingless homologue
Wnt-1 results in increased cytoplasmic levels of ?-catenin
. In Xenopus, expression of the dominant-negative
mutant of GSK-3 also results in cytoplasmic accumulation
of ?-catenin . To determine whether this role of GSK-
3 was conserved in mammalian cells, we examined the
effect of Li+on ?-catenin levels in PC12 cells. In contrast
to the K+control (Fig. 4b, lanes 3 and 5), Li+induced the
accumulation of cytoplasmic ?-catenin (Fig. 4b, lanes 2
and 4) and did not effect the levels of GSK-3? and GSK-
3? (Fig. 4b). Taken together, these data indicate that
Inhibition of GSK-3 by Li+is reversible and is unaffected by mutation of
an inhibitory phosphorylation site. GSK-3? (white bars) and GSK-
3?S9A (black bars) were immunoprecipitated (IP) from transfected
COS 1 cells pretreated with or without 10 mM LiCl, as indicated;
activities were measured as described in Materials and methods.
Where noted, two extra washes of the immunoprecipitates were
performed with 500 ?l of kinase buffer.
Kinase activity (% control)
Li+ on cells?
Li+ IP treatment?
Li+inhibits GSK-3-dependent phosphorylation of Tau in intact cells.
(a) Inhibition of GSK-3? by Li+but not K+. GSK-3? activity in cells
cotransfected with plasmids encoding GSK-3? and Tau1N4R was
assessed by measuring AT8 immunoreactivity following incubation with
the indicated concentrations of Li+(lanes 4–10) and K+(lanes 11–17).
The Tau.1 and anti-HA immunoblots are controls for Tau1N4R and HA-
GSK-3? expression, respectively. (b) Inhibition of GSK-3?S9A and
GSK-3? by Li+; activity was assessed as in (a). (c) Scanning
densitometry of AT8 blots in (a,b): cells expressed Tau1N4R and GSK-
3? and were treated withLi+(open circles) orK+(filled circles), or
expressed Tau1N4R and GSK-3?S9A and were treated withLi+(open
diamonds) orLi+(open squares). Densities are normalized to those of
– – – – – – –
– – – – – – –
– – – – – – –
0.1 0.20.5 1
– – –
– – –
2 5 10
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
– – –
1234 5 6 789 10 11 12 13
Density (% control)
102345678 9 10
Li+mimics Wingless/Wnt signalling by specifically inhibit-
ing the activity of GSK-3 family members.
The effects of Li+on invertebrate embryogenesis are well
established. Li+is an effective inhibitor of inositol phos-
phatase (IMPase), and phosphoinositides accumulate in
Li+-treated cells. However, in Xenopus, a potent chemical
inhibitor of IMPase does not phenocopy the effects of Li+
on formation of the dorso-ventral axis , strongly sug-
gesting that Li+exerts its effects through other mecha-
nisms. The remarkable similarities between the effects of
Li+on Xenopus embryogenesis, Dictyostelium sporulation
and glycogen synthesis, and the known effects of inhibi-
tion/disruption of GSK-3, suggest that this kinase, or a reg-
ulator of it, is an appealing candidate for mediating Li+
action. We have shown that Li+specifically inhibits GSK-3
family members in vitro and in intact cells, and that Li+can
mimic the actions of Wnt/Wingless on ?-catenin/Armadillo
in mammalian and Drosophila cells. These data support
and strengthen the hypothesis, recently proposed by Klein
and Melton , that inhibition of GSK-3 may contribute
to the mechanism of action of Li+. Indeed, the extraordi-
nary degree of cross-species similarity between the effects
of Li+and the consequences of disruption of GSK-3 func-
tion implies that dominant cellular action of the ion may
be to inhibit GSK-3. Assessment of the relative impor-
tance of the inhibition of GSK-3 to physiological effects of
Li+will require the generation of Li+-insensitive mutants,
which, upon expression in cells or embryos, should confer
The mechanism underlying the stabilization of ?-catenin
by inhibition of GSK-3 is unknown. Yost et al.  have
suggested that ?-catenin is directly phosphorylated by
GSK-3, consistent with the finding that phosphorylation
of Armadillo is decreased in Zw3sggmutant Drosophila
embryos  and that, after treatment with Li+, the
migration of Zw3sggis increased on gels (Fig. 4a). Phos-
phorylation of ?-catenin/Armadillo may destabilize the
protein and limit its interaction with LEF-1-like factors.
Because overexpression of either LEF-1 or ?-catenin
causes duplication of the dorsal axis in Xenopus [18,22], the
concentrations of these proteins appear to be critical.
However, in our hands, neither ?-catenin nor Armadillo is
a good in vivo substrate for GSK-3 or Zw3sgg, respectively
(V.S. and J.R.W., unpublished observations). The recent
demonstration that the APC protein is directly phosphory-
lated by GSK-3  and is linked to catenin stabilization
, raises the possibility that phosphorylated APC targets
?-catenin for phosphorylation by a distinct protein kinase,
which leads to its degradation. In addition, the intriguing
possibility that inhibition of GSK-3 contributes to the
therapeutic effects of Li+in bipolar/manic-depressive dis-
order deserves further investigation.
Materials and methods
Materials, cell culture and transfections
All reagents were purchased from Sigma Chemical Co. unless indicated
otherwise. COS 1, U937 and PC12 cell lines were cultured in DMEM
without sodium pyruvate, containing 10 % FBS (Gibco-BRL), penicillin
and streptomycin. S2 cells were cultured in Schneider’s medium (Gibco-
BRL) containing 10 % FBS, penicillin and streptomycin. EcoRI fragments
encoding HA–GSK-3?, HA–GSK-3?S9A and GSK-3?  were sub-
cloned into the mammalian expression vector pMT2. pSG5/Tau1N4R
was a gift from B. Anderton. The Drosophila dishevelled cDNA (pdc2.6;
a gift from J. Axelrod and N. Perrimon) was subcloned into the Drosophila
expression vector pPAC, generating pPAC-Dsh. COS 1 and S2 cells
were transfected using the calcium phosphate procedure.
For western-blot analysis of Tau phosphorylation, transfected COS 1
cells were lyzed in a MES-based buffer as described  and the lysates
were analyzed. For western-blot analysis of MAPK, Armadillo, ?-catenin,
GSK-3 and Zw3Sgg, S2 or PC12 cells, as appropriate, were lyzed by
incubation on ice for 15 min in a hypotonic lysis buffer containing 50 mM
Tris (pH 7.4), 0.5 mM NaF, 100?M Na-vanadate, 1 mM benzamidine
and 5 ?g ml–1leupeptin; the insoluble fraction was removed by centrifu-
gation and the supernatant was analyzed. Cell lysates were normalized
for total protein, SDS–PAGE sample buffer was added, and samples
were separated by 7.5 % SDS–PAGE (Armadillo and ?-catenin blots) or
12.5 % SDS–PAGE (MAPK, GSK-3, Zw3Sggand Tau blots). Separated
proteins were transferred to PVDF membranes (NEN–Dupont), which
were blocked and probed with the appropriate antibodies. Antibody AT8
was from Innogenetics, Belgium; the Tau.1 antibody from B. Anderton;
Research Paper Lithium-mediated inhibition of GSK-3 Stambolic et al.1667
Li+mimics Wingless/Wnt signalling in intact
cells. (a) Cytoplasmic extracts from S2 cells
treated with 10 mM LiCl or 10 mM KCl, or
transfected with the indicated constructs, were
separated by SDS–PAGE. Samples were
transferred to a PVDF membrane which was
probed with the monoclonal antibodies 7A1
(anti-Armadillo) and 2G2C5 (anti-Zw3Sgg). The
sizes of the molecular weight markers (in kDa)
are indicated. (b) Cytoplasmic extracts from rat
PC12 cells treated with 10 mM LiCl or 10 mM
KCl were similarly probed with monoclonal
antibodies against ?-catenin or GSK-3? and
2 hours 4 hours
2 hours 4 hours
1668 Current Biology 1996, Vol 6 No 12 Download full-text
the anti-MAPK antibody from New England Biolabs; anti-?-catenin from
Transdiction Laboratories; 4G1E11 and 2G2C5 from M. Bourouis; and
7A1 from M. Peifer. Bound immunoglobulins were detected using
enhanced chemiluminescence (NEN–Dupont). HA-tagged proteins were
immunoprecipitated from the ‘Gentle-Soft’ buffer  lysates with the
monoclonal 12CA5 antibody and normalized for total protein as
described . SAPK was immunoprecipitated with a polyclonal anti-
SAPK antiserum  using the same procedure.
Protein purification and kinase assays
For GSK-3 assays, we used rat GSK-3? and histidine-tagged
Drosophila Zw3Sggpurified from baculovirus-infected Sf9 cells , or
immunoprecipitates from transfected COS 1 cells, as described .
The activities of the GSK-3 proteins towards the phospho-GS1
peptide substrate and of SAPK toward GST–cJun5–89 were analyzed
as described previously [3,38].
We thank M. Peifer, B. Anderton and M. Bourouis for antibodies; J. Axelrod
and N. Perrimon for dishevelled cDNA; S. Plyte, A. Manoukian and L. Rubie
for advice and the Toronto Signalling Network for feedback. This work was
funded by an MRC (Canada) grant to J.R.W. L.R. is an EMBO fellow.
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