Lithium inhibits glycogen synthase kinase-3 activity and mimics wingless signalling in intact cells.
ABSTRACT 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.
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 beta 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/beta-catenin, demonstrating that Li+ can mimic Wingless signalling in intact cells, consistent with its inhibition of GSK-3.
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.
- SourceAvailable from: André O von Bueren[Show abstract] [Hide abstract]
ABSTRACT: TP53 mutations confer subgroup specific poor survival for children with medulloblastoma. We hypothesized that WNT activation which is associated with improved survival for such children abrogates TP53 related radioresistance and can be used to sensitize TP53 mutant tumors for radiation. We examined the subgroup-specific role of TP53 mutations in a cohort of 314 patients treated with radiation. TP53 wild-type or mutant human medulloblastoma cell-lines and normal neural stem cells were used to test radioresistance of TP53 mutations and the radiosensitizing effect of WNT activation on tumors and the developing brain. Children with WNT/TP53 mutant medulloblastoma had higher 5-year survival than those with SHH/TP53 mutant tumours (100% and 36.6%¿±¿8.7%, respectively (p¿<¿0.001)). Introduction of TP53 mutation into medulloblastoma cells induced radioresistance (survival fractions at 2Gy (SF2) of 89%¿±¿2% vs. 57.4%¿±¿1.8% (p¿<¿0.01)). In contrast, ß-catenin mutation sensitized TP53 mutant cells to radiation (p¿<¿0.05). Lithium, an activator of the WNT pathway, sensitized TP53 mutant medulloblastoma to radiation (SF2 of 43.5%¿±¿1.5% in lithium treated cells vs. 56.6¿±¿3% (p¿<¿0.01)) accompanied by increased number of ¿H2AX foci. Normal neural stem cells were protected from lithium induced radiation damage (SF2 of 33%¿±¿8% for lithium treated cells vs. 27%¿±¿3% for untreated controls (p¿=¿0.05). Poor survival of patients with TP53 mutant medulloblastoma may be related to radiation resistance. Since constitutive activation of the WNT pathway by lithium sensitizes TP53 mutant medulloblastoma cells and protect normal neural stem cells from radiation, this oral drug may represent an attractive novel therapy for high-risk medulloblastomas.Acta neuropathologica communications. 12/2014; 2(1):3.
- [Show abstract] [Hide abstract]
ABSTRACT: Suicide is one of the leading causes of death in the United States, yet it remains difficult to understand the mechanistic provocations and to intervene therapeutically. Stress is recognized as a frequent precursor to suicide. Psychological stress is well established to cause activation of the inflammatory response, including causing neuroinflammation, an increase of inflammatory molecules in the central nervous system (CNS). Neuroinflammation is increasingly recognized as affecting many aspects of CNS functions and behaviors. In particular, much evidence demonstrates that inflammatory markers are elevated in traits that have been linked to suicidal behavior, including aggression, impulsivity and depression. Lithium is recognized as significantly reducing suicidal behavior, is anti-inflammatory and diminishes aggression, impulsivity and depression traits, each of which is associated with elevated inflammation. The anti-inflammatory effects of lithium result from its inhibition of glycogen synthase kinase-3 (GSK3). GSK3 has been demonstrated to strongly promote inflammation, aggressive behavior in rodents and depression-like behaviors in rodents, whereas regulation of impulsivity by GSK3 has not yet been investigated. Altogether, evidence is building supporting the hypothesis that stress activates GSK3, which in turn promotes inflammation, and that inflammation is linked to behaviors associated with suicide, including particularly aggression, impulsivity and depression. Further investigation of these links may provide a clearer understanding of the causes of suicidal behavior and provide leads for the development of effective preventative interventions, which may include inhibitors of GSK3.Translational psychiatry. 12/2014; 4:e488.
- [Show abstract] [Hide abstract]
ABSTRACT: What is known and objectiveThe current pharmacotherapeutic treatment of major depressive disorder (MDD) generally takes weeks to be effective. As the molecular action of these drugs is immediate, the mechanistic basis for this lag is unclear. A drug that has a more rapid onset of action would be a major therapeutic advance and also be a useful comparator to provide valuable mechanistic insight into the disorder and its treatment.CommentRecent evidence suggests that ketamine produces rapid-onset antidepressant action. Important questions are as follows: is it specific or coincidental to other effects; is there a dose–response relationship; and is the mechanism related to that of current antidepressants. NMDA receptor antagonism is unlikely the explanation for ketamine's antidepressant action.What is new and conclusionIt is not an exaggeration to state that the new findings, if validated, might produce a revolution in understanding and treating depressive disorders.Journal of Clinical Pharmacy and Therapeutics 12/2014; · 2.10 Impact Factor
1664 Research Paper
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
1666Current 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
– – –
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
– – –
1234 5 6 78 9 10 11 12 13
Density (% control)
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 hours4 hours
2 hours 4 hours
1668Current Biology 1996, Vol 6 No 12
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.
1. Plyte S, Hughes K, Nikolakaki E, Pulverer B, Woodgett JR: Glycogen
synthase kinase-3: functions in oncogenesis and development.
Biochim Biophys Acta1992, 1114:147–162.
2. Sutherland C, Leighton I, Cohen P:Inactivation of glycogen synthase
kinase-3? by MAP kinase-activated protein kinase-1 (RSK-2) and
p70 S6 kinase; new kinase connections in insulin and growth factor
signalling.Biochem J 1993, 296:15–19.
3. Stambolic V, Woodgett JR: Mitogen inactivation of glycogen
synthase kinase-3? in intact cells via serine 9 phosphorylation.
Biochem J 1994, 303:701–704.
4. Cross DAE, Alessi DR, Cohen P, Andjelkovich M, Hemmings BA:
Inhibition of glycogen synthase kinase-3 by insulin mediated by
protein kinase B. Nature 1995, 378:785–789.
5. Hemmings BA, Yellowlees D, Kernohan JC, Cohen P: Purification of
glycogen synthase kinase 3 from rabbit skeletal muscle:
copurification with the activating factor (FA) of the Mg-ATP-
dependent protein phosphatase. Eur J Biochem 1982,
6. Nikolakaki E, Coffer P, Hemelsoet R, Woodgett JR, Defize, LHK:
Glycogen synthase kinase-3 phosphorylates Jun-family members
in vitro and negatively regulates their transactivating potential in
intact cells. Oncogene 1993, 8:833–840.
7. de Groot RP, Auwerx J, Bourouis M, Sassone-Corsi P: Negative
regulation of Jun/AP-1: conserved function of glycogen synthase
kinase 3 and the Drosophila kinase shaggy. Oncogene 1993,
8. Hanger DP, Hughes K, Woodgett JR, Brion J-P, Anderton BH:
Glycogen synthase kinase-3 induces Alzheimer’s disease-like
phosphorylation of tau: generation of paired helical filament
epitopes and neuronal localisation of the kinase. Neurosci Letts
9. Rubinfeld B, Albert I, Porfiri E, Fiol C, Munemitsu S, Polakis P: Binding
of GSK-3? to the APC-?-catenin complex and regulation of
complex assembly. Science 1996, 272:1023–1026.
10. Siegfried E, Chou TB, Perrimon N: Wingless signalling acts through
zeste-white 3, the Drosophila homolog of glycogen synthase
kinase-3, to regulate engrailed and establish cell fate. Cell 1992,
11. Siegfried E, Wilder EL, Perrimon N: Components of wingless
signalling in Drosophila.Nature 1994, 367:76–80.
12. Bourouis M, Moore P, Ruel L, Grau Y, Heitzler P, Simpson P: An early
embryonic product of the gene shaggy encodes a serine/threonine
protein kinase related to the CDC28/cdc2+subfamily. EMBO J
13. Ruel L, Bourouis M, Heitzler P, Pantesco V, Simpson P: Drosophila
shaggy kinase and rat glycogen synthase kinase-3 have conserved
activities and act downstream of Notch. Nature 1993, 362:557–560.
14. Noordermeer J, Klingensmith J, Perrimon N, Nusse R: dishevelled and
armadillo act in the wingless signalling pathway inDrosophila.
Nature 1994, 367:80–83.
15. Peifer M, Sweeton D, Casey M, Wieschaus E:wingless signal and
Zeste-white 3 kinase trigger opposing changes in the intracellular
distribution of Armadillo. Development 1994, 120:369–380.
16. He X, Saint-Jeannet J-P, Woodgett JR, Varmus HE, Dawid IB: Glycogen
synthase kinase 3 and dorsoventral patterning in Xenopus
embryos. Nature 1995, 374:617–622.
17. Pierce SB, Kimelman D: Regulation of Spemann organizer formation
by the intracellular kinase Xgsk-3.Development1995, 121:755–765.
18. Behrens J, von Kries JP, Kühl M, Bruhn L, Wedlich D, Grosschedl R,
Birchmeier W: Functional interaction of beta-catenin with the
transcription factor LEF-1. Nature 1996, 382:638–642.
19. Sokol S, Christian JL, Moon RT, Melton DA: Injected Wnt RNA induces
a complete body axis in Xenopus embryos.Cell 1991, 67:741–752.
20. Sokol SY: Mesoderm formation in Xenopus ectodermal explants
overexpressing Xwnt8: evidence for a cooperating signal reaching
the animal pole by gastrulation.Development 1993,
21. Sokol SY, Klingensmith J, Perrimon N, Itoh K: Dorsalizing and
neuralizing properties of Xdsh, a maternally expressed Xenopus
homolog of dishevelled. Development 1995, 121:1637–1647.
22. Funayama N, Fagotto F, McCrea P, Gumbiner BM: Embryonic axis
induction by the armadillo repeat domain of beta-catenin: evidence
for intracellular signalling. J Cell Biol1995, 128:959–968.
23. Harwood AJ, Plyte SE, Woodgett J, Strutt, H, Kay RR: Glycogen
synthase kinase 3 (GSK-3) regulates cell fate in Dictyostelium. Cell
24. Cheng K, Creacy S, Larner J: ‘Insulin-like’ effects of lithium ion on
isolated rat adipocytes II. Specific activation of glycogen synthase.
Mol Cell Biochem1983, 56:183–189.
25. Kao KR, Masui Y, Elinson RP: Lithium induced respecification of
pattern in Xenopus laevis embryos.Nature 1986, 322:371–373.
26. Kao KR, Elinson RP: Dorsalization of mesoderm induction by lithium.
Dev Biol 1989, 132:81–90.
27. Maeda Y: Influence of ionic conditions on cell differentiation and
morphogenesis of the cellular slime molds. Dev Growth
Differentiation 1970, 12:217-226.
28. Sakai Y: Cell type conversion in isolated prestalk and prespore
fragments of the cellular slime mold Dictyostelium discoideum. Dev
Growth Differentiation1973, 15:11–19.
29. Van Lookeren Campagne MM, Wang M, Spek W, Peters D, Schaap P:
Lithium respecifies cyclic AMP-induced cell-type specific gene
expression in Dictyostelium. Dev Gen1988, 9:589–596.
30. Klein P, Melton D: A molecular mechanism for the effect of lithium
on development. Proc Natl Acad Sci USA 1996, 93:8455–8459.
31. Hughes K, Nikolakaki E, Plyte SE, Totty NF, Woodgett JR: Modulation
of the glycogen synthase kinase-3 family by tyrosine
phosphorylation. EMBO J 1993, 12:803–808.
32. Lovestone S, Latimer D, Anderton BH, Hanger D, Gallo J-M, Marquardt,
et al.: Alzheimer’s disease-like phosphorylation of tau is regulated
by glycogen synthase kinase-3 activity in cultured mammalian cells.
Curr Biol 1994, 4:1077–1086.
33. Bhanot P, Brink M, Harryman Samos C, Hsieh J-C, Wang Y, Macke, JP,
et al.: A new member of the frizzled family from Drosophila
functions as a Wingless receptor. Nature 1996, 382:225–230.
34. Papkoff J, Rubinfeld B, Polakis P: Wnt-1 regulates free pools of
catenins and stabilizes APC-catenin complexes.Mol Cell Biol 1996,
35. Yost C, Torres M, Miller JR, Huang E, Kimelman D, Moon RT: The axis-
inducing activity, stability, and subcellular distribution of ?-catenin
is regulated inXenopus embryos by glycogen synthase kinase 3.
Genes Dev 1996, 10:1443–1454.
36. Peifer M, Pai LM, Casey M: Phosphorylation of the Drosophila
adherens junction protein Armadillo: roles for wingless signal and
zeste-white 3 kinase. Dev Biol 1994, 166:543–556.
37. Woodgett JR: Study of protein phosphorylation in cell lines.In Cell
Lines in Neurobiology: A Practical Approach. Edited by Wood J.
Oxford: IRL Press; 1992:133–159.
38. Dai T, Rubie EA, Franklin CC, Kraft A, Gillespie DAF, Avruch J, et al.:
SAP kinases bind directly to the ? domain of c-Jun in resting cells:
implications for repression of c-Jun function. Oncogene 1995,