Chem. Rev. 2001, 101, 2527−2540
2527
Glycogen Synthase Kinase-3: Properties, Functions, and Regulation
Adnan Ali,†,‡ Klaus P. Hoeflich,† and James R. Woodgett*
Division of Experimental Therapeutics, Ontario Cancer Institute, 610-University Avenue, Toronto, Ontario, Canada M5G 2M9
Received December 21, 2000
Contents
I. Introduction
A. Isolation and Characterization of GSK-3
II. Regulation of GSK-3 Activity
III. GSK-3 Homologues
A. Dictyostelium gsk-A
B. Sea Urchin GSK-3
C. Caenorhabditis elegans gsk-3
D. Drosophila Shaggy/Zeste-White3
E. Zebrafish ZGSK-3
F. Xenopus XGSK-3
IV. Mammalian GSK-3
A. Wnt Signaling
B. Inhibition of GSK-3 by GBP/FRAT
C. GSK-3 Phosphorylation of β-Catenin and
APC
V. Regulation of GSK-3 by Other Signaling
Pathways
VI. GSK-3β in Inflammatory Signaling
A. NF-κB Pathway
B. Xenopus rel
C. SCF Ubiquitin Ligase Complex
VII. Small Molecule Inhibitors of GSK-3
A. Lithium
B. Other Inhibitors of GSK-3
VIII. Concluding Remarks
IX. Acknowledgments
X. Note Added in Proof
XI. References
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disorders, and cancer. GSK-3 genes are highly conserved and have been identified in every eukaryote
investigated to date. Studies of GSK-3 homologues
in various organisms have revealed physiological
roles for the enzyme in differentiation, cell fate
determination, and spatial patterning to establish
bilateral embryonic symmetry. GSK-3 plays an important role in at least two signal transductory
systems, namely, the Wnt/wingless and PI’3 kinase
pathways which influence proliferation and cell
survival, respectively. This review focuses on the
biochemical and functional properties of GSK-3 and
discusses recent advances in understanding the
involvement of this unusual enzyme in human pathophysiology. Such insights have uncovered new paradigms in signaling and point to GSK-3 as a potential
therapeutic target.
2534
A. Isolation and Characterization of GSK-3
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GSK-3 was originally identified as one of five
protein kinases that phosphorylate the rate-limiting
enzyme of glycogen synthesis, glycogen synthase
(GS).2-4 Following peptide sequencing of enzyme
purified from skeletal muscle, a screen of a rat brain
cDNA library revealed that GSK-3 is encoded by two
independent genes, GSK-3R and GSK-3β, with molecular weights of 51 and 47 kDa, respectively.1
Purified GSK-3R and GSK-3β exhibit similar biochemical and substrate properties.5 The two genes
display 85% overall sequence identity (see Table 1),
which is even higher in the catalytic domain (93%).
Chromosomal mapping identified the cytological
location of human GSK-3R as 19q13.2, whereas
human GSK-3β maps to 3q13.3.6 The GSK-3β gene
promoter contains several CAAT boxes as well as
positive and negative transcriptional response elements.7 Northern blot analysis has shown that GSK3R and GSK-3β are somewhat variably expressed in
different mammalian tissues, but there is poor correlation between the levels of mRNA and protein.1,8
This may represent differential modes of transcriptional and translational regulation for the two isoforms.
I. Introduction
Glycogen synthase kinase-3 (GSK-3) is a serinethreonine kinase encoded by two isoforms in mammals, termed GSK-3R and GSK-3β.1 Initially GSK-3
was implicated in muscle energy storage and metabolism, but since its cloning, a more generalized
role in cellular regulation has emerged, highlighted
by the wide array of substrates controlled by this
enzyme that includes cytoplasmic proteins and nuclear
transcription factors. GSK-3 targets encompass proteins implicated in Alzheimer’s disease, neurological
* To whom correspondence should be addressed. Phone: 416-9462962. Fax: 16-946-2984. E-mail: jwoodget@uhnres.utoronto.ca.
† A. Ali and K. Hoeflich contributed equally to this review.
‡ Present address: Department of Biological Sciences, 401-Sunset
Avenue, University of Windsor, Windsor, ON, Canada N9B 3P4.
Phone: 519-253-3000, Ext 2730. Fax: 519-971-3609. E-mail:
aali@uwindsor.ca.
II. Regulation of GSK-3 Activity
Besides glycogen synthase, a number of other
GSK-3 substrates have been identified.9,10 These
include translation initiation factor eIF2Bǫ, PKA,
phosphatase subunit RGI, and ATP-citrate lyase (see
Table 2). GSK-3 also phosphorylates several transcription factors such as CREB, c-Jun, c-Myc, c-Myb,
10.1021/cr000110o CCC: $36.00 © 2001 American Chemical Society
Published on Web 07/04/2001
2528 Chemical Reviews, 2001, Vol. 101, No. 8
Adnan Ali received his B.Sc. degree in Biology from the University of
Punjab in 1985 and his M.S. degree in Biology from Quaid-e-Azam
University, Islamabad, in 1987. He received his M.Sc. degree in 1992
and Ph.D. degree in 1995 in Molecular Biology under the supervision of
John Heikkila at the University of Waterloo, Canada. After a postdoctoral
fellowship with Nick Ovsenek (Saskatchewan), he continued his interest
in signal transduction as a fellow with Jim Woodgett. Presently, he is
Assistant Professor at the Department of Biological Sciences, University
of Windsor, Canada. His research interests include the elucidation of roles
that integrin-linked kinase may play in the regulation of PI3′Kinase and
Wnt/wg pathways.
Klaus Hoeflich obtained his B.Sc. (Hons) degree from Simon Fraser
University and is currently working toward his Ph.D. degree with Professor
Jim Woodgett in the Department of Medical Biophysics, University of
Toronto. He was the recipient of the Canadian Institutes of Health Research
(CIHR) Next Generation Award in 2000. His research interests have
focused on the molecular mechanisms of signaling of apoptosis by tumor
necrosis factor via protein kinases; these studies form the basis of his
Ph.D. thesis (mid-2001).
as well as heat shock transcription factor HSF-1 in
vivo11 and in vitro.12 Brain-associated proteins such
as amyloid precursor protein, Tau, and neurofilament
protein are also targets of GSK-3.13 Phosphorylated
Tau has a lower affinity for microtubules, and hyperphosphorylation appears to promote association into
paired helical filaments, a pathological feature of
Alzheimer’s disease.14-16
GSK-3 is a serine/threonine-selective kinase that
recognizes and phosphorylates the consensus sequence SXXXS(P) in certain proteins.17 For some
substrates, like glycogen synthase, phosphorylation
by GSK-3 requires prior phosphorylation of a serine
residue C-terminal to the target site. The specificity
of this priming site has been probed with a synthetic
peptide corresponding to a GSK-3 phosphorylation
site in eIF2Bǫ. Peptides phosphorylated at serine or
Ali et al.
Jim Woodgett is Amgen Professor of Cancer Biology at the University of
Toronto and Head of the Division of Experimental Therapeutics at the
Ontario Cancer Institute/Princess Margaret Hospital in Toronto. His Ph.D.
studies with Philip Cohen (Dundee, Scotland) introduced him to glycogen
synthase kinase-3, and he has spent the last 20 years trying to escape
its clutches (unsuccessfully). His other interests include the PI3′ kinase
and mammalian MAPK pathways, regulation of transcription by phosphorylation, and DNA microarray technology. A laboratory web site explains
his efforts to beat his addiction to protein kinases: http://kinase.uhnres.utoronto.ca
threonine in the priming position were conducive to
subsequent GSK-3 phosphorylation.18 Phosphotyrosine, however, was not a specificity determinant for
GSK-3. Although prior phosphorylation of the consensus site seems to be a common mode by which
GSK-3 targets its substrates, this priming mechanism is not requisite for all substrates. For example,
GSK-3 phosphorylation of β-catenin does not appear
to require priming (see section IV).19 This differential
requirement for substrate interaction may provide a
control mechanism by which the specificity of signal
transduction is influenced by the contemporaneous
activation of other signaling pathways (which modulate the activity of the various priming kinases).
In studies in fruit fly and frog embryos, expression
of kinase-deficient GSK-3 induces phenotypes which
mimic Wnt/wingless (Wg) signaling and result in the
stabilization of β-catenin/armadillo (see sections III
and IV; Figure 1).20-24 This is consistent with a model
in which GSK-3 is constitutively active and negatively regulated by upstream signals. In Drosophila,
the GSK-3 homologue is termed shaggy or zestewhite3 (herein denoted as Zw3sgg). A series of epistasis analyses indicated that Zw3sgg activity is inhibited
by Wg protein, an effect mimicked by expression of
downstream components of Wg signaling, namely,
frizzled-2 (DFz-2) and dishevelled (Dsh).25 The degree
of Zw3sgg kinase activity regulates the phosphorylation of armadillo, which in turn modulates its stability (see below). The molecular mechanism by which
activity of Zw3sgg is regulated by Wg is unclear. Work
in our laboratory has shown that Wg-dependent
inactivation of Zw3sgg is accompanied by serine phosphorylation, although the identity of the relevant
kinase acting on GSK-3 is currently unknown. The
most proximal upstream regulatory protein in this
pathway is dishevelled. This protein is also phosphorylated by a Dsh-associated kinase (DAK). The finding that casein kinase-Iǫ (CK-Iǫ) and casein kinaseII (CK-II) can bind Dsh suggested a possible role for
the casein kinases (and DAK) in the transduction of
Glycogen Synthase Kinase-3
Chemical Reviews, 2001, Vol. 101, No. 8 2529
Table 1. GSK-3 Is Widely Conserved throughout Evolutiona
full-length protein% identity (similarity)
catalytic domain% identity (similarity)
species
homologues
GSK-3R(human)
GSK-3β (human)
GSK-3R (human)
GSK-3β (human)
Homo sapiens
Homo sapiens
Rattus norvegicus
Rattus norvegicus
Mus musculus
Danio rerio
Danio rerio
Xenopus laevis
Ciona intestinalis
Paracentrotus lividus
Drosophila melanogaster
Hydra vulgaris
Caenorhabditis elegans
Petunia hybrida
Nicotiana tabacum
Arabidopsis thaliana
Oryza sativa
Medicago sativa
Dictyostelium discoideum
Schizosaccharomyces pombe
Saccharomyces cerevisiae
Plasmodium flaciparum
Trifolium repens
Ricinus communis
Cicer arietinum
GSK-3R
GSK-3β
GSK-3R
GSK-3β
GSK-3β
ZGSK-3R
ZGSK-3β
XGSK-3β
GSK-3
SUGSK-3
zw3sgg
GSK-3
gsk-3
shaggy
shaggy
ASK/ATK1
OSK
MSK1-3
gskA
Skp1
MCK1/MRK1
GSK-3
GSK-3
shaggy-like
GSK-3
100
76 (84)
93 (93)
76 (84)
76 (84)
80 (86)
77 (84)
75 (83)
80 (87)
71 (83)
71 (81)
66 (80)
77 (87)
69 (82)
62 (76)
62 (76)
63 (77)
62-63 (76)
58 (72)
60 (75)
45-58 (62-76)
53 (74)
71 (84)
74 (88)
66 (77)
83 (89)
100
83 (89)
92 (92)
93 (93)
78 (84)
92 (93)
90 (92)
83 (89)
79 (88)
79 (87)
72 (86)
77 (87)
67 (80)
69 (80)
65 (81)
65 (80)
64-68 (76-79)
61 (73)
64 (78)
53 (68)
54 (72)
68 (81)
70 (84)
71 (82)
100
91 (97)
99 (99)
90 (96)
91 (97)
92 (96)
90 (96)
90 (96)
90 (96)
84 (92)
82 (92)
78 (91)
80 (89)
80 (89)
71 (84)
70 (85)
71 (84)
70-71 (83-84)
69 (85)
58 (73)
54-60 (73-77)
56 (76)
71 (84)
74 (88)
73 (85)
91 (97)
100
91 (96)
99 (99)
100 (100)
91 (97)
99 (99)
91 (97)
91 (97)
87 (94)
85 (94)
80 (92)
80 (89)
81 (89)
73 (85)
71 (87)
71 (85)
70-72 (83-85)
69 (83)
67 (82)
54-57 (72-76)
58 (76)
71 (85)
74 (89)
74 (85)
a
GSK-3 homologues are represented as percentage identity and similarity (in parentheses) over the full-length and catalytic
region of human GSK-3R and GSK-3β proteins. GSK-3 proteins share a remarkable degree of identity within the catalytic domain,
and in vertebrates, the identity over this region is greater than 90%. GSK-3 family members have also been identified in numerous
invertebrates (nematode, fruit fly, ascidians, ticks, sea urchin, and malaria parasite), plants (Arabidopsis, Hydra, garden petunia,
rice, clover, sunflower, alfalfa, chickpea, and tobacco), and fungi (baker’s yeast, fission yeast, and slime mold).
Table 2. Protein Substrates of GSK-3a
substrate
acetyl CoA carboxylase
adenomatous polyposis coli protein
ATP-citrate lyase
axin
β-catenin
C/EBPa
c-Jun
c-Myb
c-Myc
CREB
cyclin D1
eIF-2B translation factor
G subunit of phosphatase 1
glycogen synthase
heat shock factor-1
inhibitor-2
insulin receptor substrate 1
JunD
L-myc
NF-ATc
RII subunit of cAMP-dependent protein kinase
Tau
specific phosphorylation sequence
ref
n.d.
FXVEXTPXCFSRXSSLSSLS
LLNASGSTSTPAPSRTASFSESR
SANDSEQQS330
SDADTLSLT341
SLTDS343
DSGIHSGATTTAPS
TPPPTPVPSP
EEPQTVPEMPGETPPLSPIDMESQER
APVSCLGEHHHCTPSPPVDH
DIWKKFELLPTPPLSPSRRSG
KRREILSRRPSYR
EEVDLACTPTDVRDVDI
DSEELDSRAGSPQLDDIKVF
AIFKPGFSPQPSRRGSSESSEEVY
RPASVPPSPSLSRHSSPHQSEDEE
KEEPPSPPQSP
GLMKIDEPSTPYHSMIGDDDDAYSD
n.d.
SPPLSPIDMETQER
DIWKKFELVPSPPTSPPWGL
n.d.
LREARSRASTPPAAPPS
TPPKSPSAAK
SPVVSGDTSPR
154
155
156
90
19
157
158
159
160
161
162
163
164
161
12
165
166
167
168
169
170
171
a
GSK-3 substrates are listed with the peptide sequences surrounding their phosphorylation sites. GSK-3-targeted serines and
threonines are indicated in bold font. Sites of “priming” prephosphorylation required for GSK-3 phosphorylation (if known) are
underlined. Some substrates have numerous GSK-3 phosphorylation sites in multiple regions and could all not be listed here.
The notation “n.d.” indicates that the phosphorylation sequence is undetermined.
the wingless signal.26,27 Dishevelled lives two functional lives. In addition to modulating the wingless
pathway, it is also a component in regulation of
planar polarity in Drosophila which involves the
JNK/SAPK pathway. Interestingly, DAK dramati-
cally enhances the function of Dsh in the Wnt
pathway while inhibiting in the establishment of
planar polarity. Dsh thus appears to act as a teetertotter in controlling flux through two distinct signaling systems.
2530 Chemical Reviews, 2001, Vol. 101, No. 8
Figure 1. Role of GSK-3 in the Wnt signaling pathway.
In the absence of a Wnt signal, GSK-3 in the cytoplasm
interacts with β-catenin, axin, and APC. GSK-3 phosphorylates these proteins, leading to the Slimb/βTrCP-mediated ubiquitination and proteolytic degradation of β-catenin. Upon binding of Wnt by a seven-transmembrane
domain receptor, dishevelled is activated resulting in the
down regulation of GSK-3 kinase activity. These, and other,
signaling events and interactions stabilize β-catenin, leading to the activation of LEF-1/Tcf-mediated transcription
(see text for further details). Abbreviations: casein kinaseII (CKII), protein kinase C-R (PKCR), dishevelled (Dsh),
GSK-3-binding protein (GBP), protein phosphatase-2A
(PP2A), adenomatous polyposis coli protein (APC).
Under resting conditions, GSK-3/Zw3sgg is highly
phosphorylated at a tyrosine residue located within
the phosphorylation loop proximal to the ATP binding
site (in an analogous position to the activating
threonine and tyrosine phosphorylation sites in
MAPKs).28,29 Constitutive phosphorylation of this
tyrosine is important for kinase activity. The Schizosaccharomyces pombe homologue of GSK-3 termed
Skp1 is also phosphorylated on a tyrosine residue in
the P-loop, and this phosphorylation is required for
its efficient activity.30 In mammalian cells, GSK-3R
is constitutively phosphorylated at tyrosine-279,
GSK-3β at tyrosine-216.28 Dephosphorylation of these
residues is accompanied by kinase inactivation. Mitogens lead to inactivation of GSK-3 (see section V).
Since this effect is reversible by treatment with
serine/threonine-specific phosphatases, mitogeninduced inhibition is believed to be due to increased
serine/threonine phosphorylation of GSK-3 rather
than tyrosine dephosphorylation.31 Indeed, serine 9
of GSK-3β (and serine 21 in GSK-3R) is phosphorylated upon insulin stimulation.32-34 Several protein
kinases target serine 9/21 including PKB/Akt, pp90
Rsk and cyclic AMP-dependent protein kinase (see
section V).33,35-37
Inactivation of GSK-3 by phosphorylation at serine
9/21 or via the Wnt pathway invokes different cellular consequences. The mechanism underlying this
signal-dependent regulation is likely via the existence
of distinct pools of or complexes containing GSK-3.
For example, in cells, cytoplasmic β-catenin is associated with a scaffold protein called axin, which
also binds GSK-3. The population of GSK-3 molecules
Ali et al.
bound to axin is selectively sensitized to Wnt signaling. However, a different pool of GSK-3 appears to
be responsible for transducing the effects of insulin/
PI 3′ kinase signaling. The finding that axin-associated GSK-3 is insensitive to insulin-induced serine
9 phosphorylation but instead is coupled to Wnt
activation further supports this notion.38 Since GSK-3
has multiple substrates, the existence of distinct
pools of responsive kinase molecules may allow
specific responses to certain agonists, leaving other
targets unaffected. This idea is further strengthened
by the finding that inhibition of GSK-3 by certain
Wnt signaling components (such as FRAT-1/GBP, see
section III), leads to the inhibition of GSK-3-induced
phosphorylation of β-catenin without effecting the
phosphorylation of other substrates such as glycogen
synthase.39
In mammalian cells, protein kinase C-like activity
(PKC) has been implicated in Wg effects on GSK-3
activity.40,41 Certain PKCs phosphorylate GSK-3 in
vitro, resulting in inactivation. Wg-induced inactivation of GSK-3 is sensitive to both the PKC inhibitor
Ro31-8220 and prolonged pretreatment of 10T1/2
fibroblasts with phorbol ester, suggesting a role for
PKC in the regulation of GSK-3. In addition, it has
been demonstrated that serum enhances the effect
of certain GSK-3 inhibitors on the accumulation of
cytoplasmic β-catenin through the activation of PKC.42
However, since Wnt-induced accumulation of cytoplasmic β-catenin is only partially inhibited by PKC
inhibitors, PKC likely serves to enhance the effects
of Wnt signaling rather than being directly coupled
to this pathway.
III. GSK-3 Homologues
The transmission of Wnt signal involves a cascade
of events including modification and secretion of Wnt
protein, its binding to cell surface frizzled receptors,
activation of cytosolic intracellular components, and
culminating in transcriptional activation of target
genes in the nucleus. The Wnt signaling pathway is
highly conserved and has been studied in a wide
range of organisms. Likewise, GSK-3 homologues
have been isolated from a number of organisms
(Table 1), and roles have been discerned in early
development as well as in cell fate decisions in
Dictyostelium, Drosophila, Zebrafish, Xenopus, and
mammals.4 Besides its involvement in cell fate and
establishment of bilateral symmetry in animal embryos, other roles for GSK-3 have also been implicated in lower eukaryotes. For example, the GSK-3
homologue in Schizosaccharomyces pombe, skp1, may
play a role in cytokinesis.30
A. Dictyostelium gsk-A
The slime mold Dictyostelium discoideum harbors
a single GSK-3-related gene termed gsk-A that
shares over 70% identity with human GSK-3β.43
Whereas in other systems the regulation of GSK-3
is inhibitory, Dictyostelium GSK-3 has been shown
to be activated. Although gsk-A is not involved in the
early development of Dictyostelium, it plays a critical
role in specifying cell fate in a cAMP-dependent
Glycogen Synthase Kinase-3
process that controls differentiation into spore or
stalk cells.44 During normal development, aggregated
progenitor cells differentiate into three cell types:
prespore, prestalk-A, and prestalk-B. In gskA- mutant cells, differentiation proceeds with a reduction
proportional to the prespore cell population and
enhanced formation of prestalk-B cells. Since extracellular cAMP levels affect the differentiation process
in a similar manner, it was hypothesized that gsk-A
regulates cAMP responses in aggregated cells. Recent
findings indicate that extracellular cAMP interacts
with the cAR3 serpentine receptor to regulate the
activity of a tyrosine kinase, ZAK-1.45-47 In prespore
cells, the activation of ZAK-1 increases tyrosine
phosphorylation and the activity of gsk-A, resulting
in spore cell differentiation, while inhibiting stalk cell
formation. Cells mutant for cAR3 or ZAK-1 exhibit
phenotypes similar to gsk-A null cells. Prestalk cells
carry cell-type-specific cAR4 receptors in addition to
cAR3. In these cells, interaction between the cAMP
and cAR4 leads to the inhibition of gsk-A, resulting
in the development of prestalk cells, while spore
formation is inhibited due to lack of ZAK-1 activation.
One of the targets of cAMP activation of gsk-A is a
Dictyostelium STAT transcription factor.48 GSK-A
phosphorylation of this protein at a serine residue
enhances nuclear export, causing functional inactivation of the STAT.48 The physiological significance of
STAT regulation by GSK-A with respect to cellular
differentiation has yet to be addressed.
B. Sea Urchin GSK-3
Urchin GSK-3 is 88% similar to its vertebrate
homologues (94% in the catalytic domain) and has
been implicated in the establishment of animalvegetal (A-V) axis in early development of sea
urchin. During the embryogenesis, animal cells give
rise to ectodermal tissues whereas vegetal cells carry
a mesodermal and endodermal fate.49 Overexpression
of wild-type GSK-3 results in the animalization of the
embryos by giving rise to ectodermal phenotype.50 By
comparison, inhibition of GSK-3 (by treating embryos
with lithium, see section VI), ectopic expression of
kinase-dead GSK-3 or mutant β-catenin, promotes
vegetal cell fate. This implies that in animalized cells
the constitutive activity of GSK-3 inhibits accumulation of β-catenin, thus promoting an ectodermal fate
for these progenitors. Conversely, in the vegetal
region, inhibition of GSK-3 by developmental cues
induces an accumulation of β-catenin, resulting in
transcriptional regulation of downstream genes required for establishing the endoderm and mesoderm
lineages.47 The similar effects of ectopic expression
of GSK-3 homologues from both Xenopus and sea
urchin illustrates the conserved function of the Wnt
pathway in regulating the specification of bilateral
symmetry in vertebrates and invertebrates.
C. Caenorhabditis elegans gsk-3
C. elegans gsk-3 displays a high degree of similarity
to vertebrate GSK-3 homologue and has been assigned roles in endodermal induction and spindle
orientation.51,52 Initial endodermal induction occurs
Chemical Reviews, 2001, Vol. 101, No. 8 2531
at the four-cell stage when the posterior blastomere
(P2) induces a neighboring blastomere (EMS).53 This
polarization results in two differentiated daughter
cells, E and MS. Eventually, E gives rise to endodermal structures while MS forms the mesoderm. One
of the important genes that controls the endodermal
induction is Pop-1, which has been shown to be a
component of the Wnt signaling pathway. Unlike
LEF-1/Tcf, which acts as transcriptional activator in
the presence of upstream signals in other species,
Pop-1 acts as a repressor for the endoderm induction.
Previous findings have suggested that a Wnt signal
from a P2 cell triggers gsk-3 activation. Activated
gsk-3 then acts positively in the regulation of wrm1/armadillo and, in turn, relieves the Pop-1-mediated
transcriptional repression in the responding cell,
named E. The inhibition of the repressor does not
occur in the EMS cell; therefore, the specification of
endoderm fate is limited to the E cell. While Wnt
signaling in C. elegans does not reflect a typical Wnt
pathway as established for vertebrates, another
β-catenin homologue, Bar-1, interacts with Pop-1,
induces LEF-1/Tcf promoter activity, and is a possible
candidate in the regulation of endodermal induction.54 Like β-catenin in higher vertebrates, Bar-1
contains putative GSK-3 phosphorylation sites in its
N-terminal sequence that are possibly responsible for
the transmission of Wnt signal. In response to a Wnt
signal, gsk-3 also regulates spindle orientation via
the cytoskeleton. However, the genes that regulate
endodermal fate do not appear to play any role in
the cytoskeletal-related processes.52 Hence, it seems
likely that gsk-3 serves as a branching point for
selectively regulating two Wnt-dependent processes
during nematode development.
D. Drosophila Shaggy/Zeste-White3
Drosophila represents the archetypal organism for
understanding the Wnt/wingless pathway. In flies,
the GSK-3 homologue, Zw3sgg, was originally characterized as a segment-polarity.55,56 Mutation of this
gene in Drosophila embryos induces a phenotype in
which the embryos lack most of the ventral denticles
normally present in the anterior region of each
segment. Sequence comparison (90% identity in the
kinase domain) as well as genetic studies demonstrated remarkable similarity between GSK-3/Zw3sgg
in mammals and insects.4,37-59 Zw3sgg is essential for
embryonic patterning as well as in events such as
mesoderm formation and cardiogenesis in Drosophila.60 Using genetic and biochemical approaches,
various components of Wg signaling have been ordered within the pathway. For example, the embryonic phenotypes of armadillo and dishevelled mutants are very similar to the disruption of wingless,
whereas Zw3sgg has a mutant phenotype very similar
to that of embryos in which wingless protein has been
expressed in all cells. These data also support the
notion that the functions of Zw3sgg are antagonized
by Wg signaling. Mutations in Wg and zw3sgg have
opposite effects on cell fate determination and armadillo protein levels.20,61
As described in section IV, the Drosophila wingless
(wg) gene encodes a secreted glycoprotein homologous
2532 Chemical Reviews, 2001, Vol. 101, No. 8
to vertebrate Wnt proteins.62 Wg protein is capable
of inhibiting GSK-3 activity in rat 10T1/2 fibroblasts,
inferring a high degree of conservation in Wnt/Wg
signaling between species.40 Wg has been assigned
distinct roles in processes such as embryonic segmentation and imaginal disk patterning.57,63 In Drosophila cultured cells, frizzled-2 (Dfz-2) was identified
as a Wg receptor with a cysteine-rich extracellular
domain (CRD) followed by seven transmembrane
domains, similar in structure to G-protein-coupled
serpentine receptors (direct evidence for a G-protein
component in Wg signaling has yet to be demonstrated). Other well-studied components of the wingless pathway include dishevelled (dsh), a scaffold
protein termed axin, and armadillo. The armadillo
gene encodes the Drosophila homologue of β-catenin.
As mentioned above, unlike the other components of
the Wg signaling, Zw3sgg plays an inhibitory role.57
Secreted Wg protein is received by neighboring cells,
and a signal transduction cascade is initiated.62 The
mechanism by which the Wg signal is transmitted
across the membrane is incompletely understood. In
addition to interaction of Wg with the extracellular
CRD region of Dfz-2, a single-pass transmembrane
protein, arrow, has also been identified that can
transduce a signal to Dsh.64 In embryos and cultured
cells, Wg signal leads to the hyperphosphorylation
of Dsh protein. “Activated” Dsh acts to block the
function of Zw3Sgg and D-axin (the Drosophila homologue of axin), leading to decreased phosphorylation
of armadillo (see section IV). Axin is a critical element
in the regulation of β-catenin/armadillo levels in
vertebrates.65 D-Axin negatively regulates Wg signaling by suppressing intracellular levels of armadillo
via promotion of phosphorylation of this protein by
Zw3Sgg. D-Axin acts as a scaffold protein and binds
Zw3Sgg, armadillo, and APC (adenomatous polyposis
coli protein), forming a regulatory complex (see
section IV).25,66,67 Wg signaling reduces phosphorylation of armadillo, allowing this protein to accumulate in the cytoplasm, which, in turn, facilitates
interaction with members of the LEF-1/Tcf transcription factor family. In cultured cells armadillo/βcatenin plays two roles. A membrane-associated
population is involved in cell adhesion and participates in the formation of adherens junctions by
binding to E-cadherin and R-catenin.68 This function
has been genetically isolated from the Wg signaling
events using mutants of armadillo that impact adhesion but not the Wg pathway. Only soluble, cytoplasmic armadillo appears to be relevant to Wg responses.69 Another zinc finger protein, teashirt, has
also been implicated in the transmission of wingless
signals.70 Wg signaling promotes the phosphorylation
and nuclear accumulation of teashirt, a process
dependent upon binding of teashirt to the C-terminal
portion of armadillo. Zw3sgg is also associated with
teashirt in a complex, and nuclear levels of teashirt
are influenced by Zw3sgg. Since teashirt expression
is limited to embryonic central segments, it has been
speculated that it regulates trunk formation.71
Ali et al.
nation of maternally derived gene products as well
as activation of the zygotic genome regulates early
embryogenesis.72 Evidence for the involvement of
Wnt pathway during zebrafish development derives
from microinjection experiments that demonstrate a
requirement for the Wnt receptor, frizzled, in the
specification of dorsal mesoderm.73 Coexpression of
GSK-3 reverses the effect of frizzled overexpression.
At the protein level the two zebrafish GSK-3 homologues, ZGSK-3R and ZGSK-3β, exhibit 90% similarity with mammalian and Xenopus homologues.74
F. Xenopus XGSK-3
Transcripts encoding the known components of
Wnt pathway are present in the Xenopus embryo as
maternal products.75-77 After fertilization, cortical
rotation relocalizes these molecules by the movement
of outer cytoplasm with respect to the inner. This
rotation brings the dorsalizing components to the
future dorsal side of the embryo. Thus, by the morula
stage of development the decision for specifying the
dorsal and ventral symmetry has already been established within the developing embryo. The dorsal
side, which eventually gives rise to ectodermal cells,
seems to be relatively abundant in β-catenin and
Dsh.47,78 However, the relative levels of GSK-3 are
low in this region. One of the major advantages in
using Xenopus embryos is that microinjection is
relatively facile due to the size of the oocytes. Using
this approach, misexpression of a dominant-negative
form of GSK-3β in the ventral side of embryo resulted
in the formation of secondary dorsal axis.22 Consistent with this finding, ectopic expression of wild-type
GSK-3 within the dorsal side caused ventralization.
Results from ectopic expression of β-catenin are also
in agreement with the canonical model of Wnt/Wg
pathway.79 These experiments clearly show the importance of the regulation of GSK-3 activity in the
establishment of the embryonic polarity. Inhibition
of GSK-3 in the dorsal region leads to accumulation
of β-catenin, which then can activate the transcription of dorsal-specific genes, while active GSK-3 in
the ventral side destabilizes β-catenin leading to
ventral fates. A similar role for β-catenin during
development is also observed during avian development where bilaterally symmetric distribution of
nuclear β-catenin coincides with the process of gastrulation.80 The involvement of GSK-3 in spatial
determination is consistent with the role of Wnt
signaling in the control of the neural plate and neural
crest formation during the development of the avian
embryonic nervous system.81
In Xenopus oocytes, a novel role of GSK-3 in the
release of cell cycle arrest has recently been uncovered.82 Prior to maturation the arrested oocytes
contain constitutively active GSK-3; progesteronemediated inhibition of this activity appears to contribute to meiotic maturation.
IV. Mammalian GSK-3
E. Zebrafish ZGSK-3
A. Wnt Signaling
A crucial step during early vertebrate development
is establishment of dorso-ventral polarity. A combi-
Studies on the physiological functions of mammalian GSK-3 have taken important cues from the
Glycogen Synthase Kinase-3
role of the proteins in simpler organisms, especially
with respect to its regulation by the Wnt pathway.
In contrast, most of our knowledge of the regulation
of GSK-3 by the phosphatidylinositol (PI) 3′ kinase
pathway has derived from studies in mammals. That
is not to say that this mode of regulation is not
conserved in invertebrates, since Zw3sgg is inactivated
by insulin signaling in a PI3′ kinase-dependent
manner in Drosophila (Ali, A.; Woodgett, J. R.
Manuscript in preparation).
In mammals, the Wnt signaling pathway is not
only important in early embryonic development (see
section III), but also a target for tumorigenesis.83
Inappropriate activation of Wnt signaling has been
observed in various human cancers, including hepatomas, colon carcinomas, melanomas, and uterine and
ovarian cancers. The first member of the Wnt family,
Wnt-1, was originally identified as an oncogene
activated by the insertion of mouse mammary tumor
virus in virus-induced mammary adenocarcinomas.84
In addition to Wnts, the downstream signaling
components β-catenin and FRAT (frequently rearranged in advanced T-cell lymphomas, also known
as GSK-3 binding protein, GBP), both positive effectors of the pathway, have been identified as protooncogenes. The two negative regulators of the pathway, APC (adenomatous polyposis coli) protein and
axin, function as tumor suppressors. Mutations of
these Wnt signaling pathway components have a
similar consequencesaccumulation of β-catenin in
the cytoplasm. Increased cellular levels of β-catenin
induce activation of downstream genes, some of
which, such as cyclin D1,85 WISP-1,86 and c-Myc,87
have been implicated in cancer.
The molecular cascade of events in mammalian
Wnt signaling is very similar to that in simpler
organisms (see section III). In the absence of a Wnt
signal, cytoplasmic β-catenin levels are kept low via
GSK-3 phosphorylation, which targets β-catenin for
ubiquitin-mediated degradation by Slimb/βTrCP (Figure 1).88,89 As in invertebrates, axin also negatively
regulates Wnt signaling and forms a complex with
GSK-3, β-catenin, and APC.90 Binding of axin to
GSK-3 promotes GSK-3-dependent phosphorylation
of both β-catenin and axin, the latter of which is
necessary for axin stability. GSK-3 also phosphorylates APC, and this helps promote binding of APC to
β-catenin.91 This interaction is facilitated by axin,
which associates with APC via a RGS (regulators of
G-protein signaling) domain. Whether the interaction
between APC and axin is critical for Wnt signaling
remains to be established: although deletion of this
domain has a dominant-negative effect on Wnt
signaling in Xenopus, it is dispensable in Drosophila.76,92
In the presence of a Wnt signal, the protein
complex containing GSK-3, axin, APC, and β-catenin
is disrupted (possibly by recruiting the GSK-3 inhibitor GBP/FRAT1) and GSK-3 kinase activity is downregulated. GSK-3 phosphorylation of β-catenin is thus
prevented, leading to accumulation of β-catenin and
subsequent interaction with HMG-box transcription
factors of the LEF-1/Tcf (leukemia enhancer factor-
Chemical Reviews, 2001, Vol. 101, No. 8 2533
1/T cell factor) family and hence activation of specific
target genes.93
B. Inhibition of GSK-3 by GBP/FRAT
A novel GSK-3-binding protein (GBP) was identified in Xenopus, and it functions in vivo to stabilize
β-catenin and is required for the formation of the
endogenous dorsal axis.94 Injection of GBP antisense
RNA prevents correct axis formation, while injection
of GBP mRNA into the ventral region of the embryo
leads to the formation of a second axis and therefore
produces a tadpole with two heads. It has been
shown, both in vivo and in vitro, that GSK-3 cannot
bind GBP and axin simultaneously. When a limiting
amount of GSK-3 is coexpressed with an excess of
axin, addition of GBP reduces the amount of GSK-3
bound to axin.95 Axin does not bind GBP in vitro,
either directly or in the presence of GSK-3, and
moreover, when axin, GSK-3, and GBP are coexpressed at comparable levels, immunoprecipitation
of GBP brings down GSK-3 but not axin, demonstrating that these proteins cannot form a trimolecular complex. Li et al. (1999) detected a quaternary
complex of overexpressed GSK-3, axin, Dsh, and GBP
by immunoprecipitation in mammalian cells, but this
quaternary complex was less stable than either the
GSK-3/axin/Dsh or GSK-3/GBP complexes and possibly represents a transition state.96 GBP has been
demonstrated to inhibit GSK-3-mediated phosphorylation of protein substrates without eliminating the
kinase activity of GSK-3 toward small peptides.
Taken together, this leads to a model in which GBP
functions in part to prevent GSK-3 binding to the
axin/APC/β-catenin complex. However, a molecular
mechanism to explain how GBP itself is regulated
during Wnt signaling remains to be determined.
GBP is related to a mammalian protein termed
frequently rearranged in advanced T-cell lymphoma
1 (FRAT1).97 GBP and FRAT1 share three highly
conserved domains, the most C-terminal of which
interacts with GSK-3. The other two domains may
interact with other proteins, such as Dsh. FRAT1 is
an oncogene that is upregulated during infection with
Moloney murine leukemia virus. FRAT1 expression
confers a selective advantage to tumor cells that
overexpress the oncogenes Myc and Pim1.97 Expression of a 39 amino acid peptide corresponding to
residues 188-226 of FRAT1 (containing the GSK-3
interaction domain) blocks the interaction of GSK-3
with axin and prevents GSK-3-catalyzed phosphorylation of axin and β-catenin, hence allowing accumulation of β-catenin. A second GBP homologue,
FRAT2, mimics the effect of GBP on secondary axis
formation.
GBP homologues have yet to be identified in
Drosophila or Caenorhabditis elegans, and given the
complete sequencing of these genomes, they are
therefore unlikely to exist. This suggests that GBP
may have evolved as a selective component of the
Wnt pathway that acts as a further layer in the
control of axin and GSK-3.
C. GSK-3 Phosphorylation of β-Catenin and APC
GSK-3 can phosphorylate β-catenin on serine and
threonine at positions 33, 37, 41, and 45 (see Table
2534 Chemical Reviews, 2001, Vol. 101, No. 8
2).98 An N-terminally truncated form of β-catenin that
lacks the GSK-3 phosphorylation sites and thereby
circumvents the normal requirement for Wnt signaling is stabilized constitutively in vivo and is not
impaired in its ability to bind E-cadherin, R-catenin,
and LEF-1/Tcf members. Mutations in β-catenin that
impair or block phosphorylation of these sites have
been identified in certain colon cancers (particularly
those in which APC is wild type). These β-catenin
mutants have been useful in delineating the physiological outcome of GSK-3-mediated β-catenin phosphorylation. For instance, when an N-terminally
truncated human β-catenin mutant (DN87-β-catenin)
is placed under the control of the keratin K14
promoter and expression is driven in the basal layer
of the mouse epidermis, these cells become capable
of inducing follicle morphogenesis normally occurring
only in embryogenesis.99 In addition, these mice
develop hair follicle tumors such as trichofolliculomas
and pilomatrichomas, which are often found in
certain human familial polyposis syndromes.100 An
approach relying on Cre recombinase excision of
β-catenin exon 3, which contains the GSK-3-phosphorylated residues, in the intestinal epithelium
provided evidence for intestinal adenomatous polyps
in mice at young ages.101 Interestingly, the intestinal
and colonic tumors in these mice resembled those of
the APCD716 knockout.102
Mammalian studies have also suggested that a
more complex mechanism for the regulation of β-catenin levels by GSK-3 involves APC.91 APC is directly
phosphorylated by GSK-3 via axin, which increases
binding of APC to β-catenin and its subsequent
degradation. β-Catenin contains a critical sequence,
made up of the N-terminal armadillo repeats, that
provides binding sites for the cytoplasmic fragment
of E-cadherin, the 15- and 20-amino acid repeats of
APC, the N-terminal region of LEF-1/Tcf, and a
central domain of axin. E-cadherin, LEF-1/Tcf, and
APC compete for binding to this region. Deletion
mutagenesis indicates that all binding sites are
located in armadillo repeats 3-8 of β-catenin, which
form a tightly packed superhelix.
Xenopus extracts contain an activity that promotes
the binding of β-catenin to axin at low concentrations.103 This activity is inhibited by expression of
RGS domain of axin, suggesting that it is either APC
or APC complexed to other proteins. Also, axinDRGS
(which cannot bind APC) does not bind β-catenin in
extracts. Also, with purified components in vitro, APC
accelerates the binding of β-catenin to axin, thereby
recapitulating the effect of Wnt-stimulated extracts
on binding between the two proteins. Taken together,
these results identify APC as the activity stimulating
the axin-β-catenin interaction. As a caveat, mutation
of a Drosophila APC homologue does not affect Wnt/
Wg function;104 however, additional APC-like molecules exist in flies and there may be some functional
redundancy between them.105
These general results are supported by experiments performed using SW480 colon carcinoma cells,
which contain a truncated nonfunctional APC, showing that endogenous β-catenin is stabilized.106 If APC
is reintroduced in these cells, β-catenin is degraded.
Ali et al.
While endogenous axin (and a related protein conductin) is present in these cells, it is presumably
incapable of regulating β-catenin phosphorylation in
the absence of wild-type APC. In this way, overexpression of the axin-related protein, conductin, can
induce β-catenin degradation in SW480 cells, while
a conductin mutant which lacks the β-catenin binding
domain cannot.67 More recently, SW480 cells have
been successfully utilized to further examine the
functional interaction and stability of various β-catenin mutants.107 These experiments showed that
β-catenin mutants that do not interact with conductin
(and were therefore resistant to degradation induced
by ectopic expression of conductin) were, however,
effectively degraded in the presence of wild-type APC.
A double point mutant of β-catenin that bound to
neither 15- nor 20-amino acid repeats of APC was
still degraded by exogenous wild-type APC or a
fragment of APC containing only the 20-amino acid
and SAMP (for Ser, Ala, Met, and Pro) repeats,
indicating that APC indeed does not need to bind
directly to β-catenin to induce β-catenin degradation.
This is consistent with previous findings that APC
and β-catenin can interact indirectly via conductin/
axin’s binding of the APC SAMP repeats.67 Finally,
only a mutant β-catenin incapable of binding either
the 15- and 20-amino acid repeats of APC or conductin binding was fully stable in the presence of
exogenous wild-type APC. Thus, all possible interactions between β-catenin and APC appear functionally
equivalent in the in vitro assays, namely, the direct
interaction via either the 15- or 20-amino acid repeats
or the indirect interaction via conductin/axin. However, it was found that APC with mutated SAMP
repeats did not induce degradation of β-catenin in
SW480 cells. The stability of β-catenin can thus be
regulated if a degradation complex with conductin
and APC is formed, that is, if β-catenin makes at
least one interaction with either conductin or APC.
However, direct interaction between conductin/axin
and APC is required for this complex to be regulatory.
This is supported by sequence data from human
tumors, which showed that the majority of mutations
in APC result in deletion of the SAMP repeats that
provide an axin interaction domain.108 Many of these
same mutants retain 15- and 20-amino acid repeat
units that allow β-catenin binding. Thus, the tumorigenic potential of mutated APC correlates with the
loss of binding to axin rather than to β-catenin.
V. Regulation of GSK-3 by Other Signaling
Pathways
Glycogen synthase contains four GSK-3-targeted
phosphorylation sites.4 Inhibition of GSK-3 reduces
the level of phosphorylation of glycogen synthase,
which then becomes active for converting glucose into
glycogen. Stimulation of glycogen synthesis by insulin
also involves the dephosphorylation of serine residues
in glycogen synthase.
Several signal-dependent mechanisms have been
proposed to lead to inhibition of GSK-3 without
involvement of the Wnt pathway. Although some
studies support the involvement of mitogen-activated
protein kinase (MAPK) in GSK-3 regulation, other
Glycogen Synthase Kinase-3
Figure 2. Role of GSK-3 in phosphatidylinositol 3-kinase
signaling. Upon receptor tyrosine kinase activation, PI3′
kinase is recruited to the plasma membrane and phosphorylates phosphoinositides at the 3′-position of their inositol
ring. This, in turn, recruits PH-domain-containing proteins
such as PKB and the PDKs. Once phosphorylated by the
PDKs, PKB is activated and phosphorylates GSK-3 leading
to its inhibition. See text for details. Abbreviations: growth
factor (GF), phosphatidylinositol 3-kinase (PI3′K), PI3′Kdependent protein kinase-1/2 (PDK1/2), protein kinase B
(PKB), phosphoinositide (Pi), phosphoinositides(4,5)P2 (PIP2),
phosphoinositides(3,4,5)P3 (PIP3), phosphatase and tensin
homologue (PTEN), integrin-linked kinase (ILK).
data do not. For example, in rat adipocytes EGF
activates MAPK without effecting GSK-3 activity.109
Similarly, the inhibitors of MAPK have little effect
on GSK-3 kinase activity. In vitro, the MAPKAPK-1
(MAPK-activated protein kinase-1)-activated ribosomal S6 kinases p70 (p70S6k) and p90 (p90rsk) inhibit
GSK-3; however, specific inhibitors of these protein
kinases fail to block signal-induced inhibition of
GSK-3 in vivo.33,35
Many of the agonists that lead to suppression of
GSK-3, including mitogens and insulin, do so in a
wortmannin-sensitive manner, implicating PI3′K in
the pathway. One of the most important physiological
mediators of PI3′K signaling is the protein-serine
kinase PKB, also termed AKT, which exists in three
forms in mammalian cells (R, β, and γ). Mitogenic
stimulation results in the phosphorylation of PKBa
at two conserved sites: threonine 308 (T308) and
serine 473 (S473)110,111 These phosphorylation events
are dependent on PI3′ kinase activity and are required for full activation of PKB (see Figure 2). In
vitro, activated PKB phosphorylates serine 21 of
GSK-3R and serine 9 of GSK-b, both modifications
causing inactivation. Antibodies that selectively bind
the phosphorylated sites on both GSK-3 subtypes
have demonstrated a good correlation between PI3′K
activity and phosphorylation of serine 21/9, although
there are exceptions (see below).
Elevation of cyclic AMP within cells causes rapid
activation of cyclic AMP-dependent protein kinase
(PKA). Recently, PKA has been shown to directly
bind and phosphorylate GSK-3 at serines 21 (GSK3R) and 9 (GSK-3β), respectively.36,37 Another enzyme
implicated in GSK-3 inactivation is integrin-linked
kinase (ILK) which, like PKB, is also regulated by
Chemical Reviews, 2001, Vol. 101, No. 8 2535
PI3′K signaling. Human ILK phosphorylates and
inhibits GSK-3 activity in vitro at an as yet undetermined site.112 We have found that expression of
Drosophila DILK induces inactivation of zw3sgg by
inducing serine phosphorylation. In cultured Drosophila cells, insulin induces serine phosphorylation of
zw3sgg, which is enhanced by overexpression of wildtype but not by kinase-deficient forms of DILK (Ali,
A.; Woodgett, J. R. Unpublished data). Whether this
effect of DILK is mediated directly or via a thirdparty protein kinase (such as PKB) is unclear.
Although GSK-3 can be inactivated by at least two
distinct molecular mechanisms within cells, the
relevant pathways appear to be independently insulated from each other to the point that effects on
GSK-3 substrates are dependent upon the pathway
by which the kinase is inactivated. This distinction
is illustrated by genetic studies in Drosophila where
overexpression of activated PKB fails to generate a
naked cuticle phenotype, which is typical of activation
of the wingless pathway or disruption of zw3sgg.113
Furthermore, unlike injection of GSK-3, microinjection of activated PKB does not result in formation of
a secondary axis in Xenopus. These data might
suggest that distinct populations of GSK-3 exist
within cells that are somehow attentive to certain
signals but not others. It is tempting to speculate that
GSK-3 molecules sequestered into the axin/β-catenin
complex have adapted to a specific function that is
entirely independent of the bulk of the GSK-3 molecules within a cell.
VI. GSK-3β in Inflammatory Signaling
A. NF-KB Pathway
The Rel family of proteins represents a large group
of trans-acting transcription factors that have been
implicated in development, differentiation, and oncogenesis. The founding member of the family v-rel
is the oncogene of reticuloendotheliosis virus strain
T, which causes rapid and fatal leukemia in juvenile
birds.114 Cellular counterparts of v-rel have been
cloned in numerous other organisms. The most highly
characterized member of this family is NF-κB, which
plays an important role as a regulator of the immune
response and was first discovered as a constitutively
nuclear transcription factor in mature B cells that
bound to an element in the kappa immunoglobulin
light-chain enhancer. NF-κB is, in fact, a group of
binary complexes of proteins with related promoterbinding and transactivation activities. All members
of this family contain a 300 amino acid N-terminal
DNA-binding and dimerization domain, known as the
Rel-homology domain, and most combinations of NFκB homo- and heterodimers can be found in vivo. The
prototypical NF-κB complex consists of a p65-p50
heterodimer. p65/RelA, RelB, and c-Rel stimulate
transcription, whereas p50/NF-κB1 and p52/NF-κB2
serve primarily to bind DNA. Involvement of NF-κB
in the immune system was confirmed by the discovery that treatment with inflammatory cytokines,
such as tumor necrosis factor-R (TNF-R) and interleukin-1 (IL-1), facilitated its release from cytoplasmic I-κB inhibitor proteins, which resulted in trans-
2536 Chemical Reviews, 2001, Vol. 101, No. 8
location to the nucleus, binding of DNA, and induction
of gene expression. In addition to the regulation of
NF-κB activity at the level of subcellular localization,
new information has emerged regarding the role of
NF-κB phosphorylation in transactivation of the p65/
RelA subunit and proteolytic processing of p105, the
precursor for the p50 subunit.
An important physiological function for NF-κB was
revealed by studies that interfered with NF-κBinduced gene expression (for instance, using actinomycin D or cycloheximide). Treatment of cells with
TNF-R in the presence of such inhibitors potently
induced cell death.115 This effect was traced to an
anti-apoptotic function for NF-κB. Thus, under normal circumstances, TNF-R induces both pro- and
anti-apoptotic pathways. Inhibition of the latter
switches the balance toward cell death. The consequences of suppression of this protective role is most
strikingly manifested in the developing mouse embryo. Mouse embryos lacking the p65 subunit of NFκB die during days 13-15 of gestation due to massive
apoptosis of their developing hepatocytes.116 However, p65-deficient mice that also lack TNF-R or one
of its receptors (TNFR1) are viable.117,118 Embryonic
fibroblast cells from these mice have defects in NFκB activation and heightened sensitivity to TNF-Rmediated apoptosis.119 Several other mutations in
components of the NF-κB pathway also exhibit this
tell-tale phenotype (such as IKKb and T2K, two
protein kinases involved in the phosphorylation of
I-κB).120,121
Unexpectedly, mice lacking GSK-3β suffer from
defects similar to those of mice that are mutant for
essential components of the NF-κB pathway.122 Hence,
GSK-3β-deficient mice are morphologically normal up
to approximately day 12 of embryonic development
but die between days 13.5-14.5 due to massive liver
degeneration and hepatocyte apoptosis. This death
can be prevented by blocking the function of TNF-R.
Embryonic fibroblast cells from GSK-3β mutant mice
are highly sensitive to TNF-R-mediated apoptosis but
not to other apoptotic stimuli. In addition, GSK-3βdeficient fibroblasts show a significant reduction in
NF-κB DNA binding and reporter gene response
following treatment with TNF-R or IL-1.
The mechanism by which GSK-3β impacts NF-κB
activity is not known, but the kinetics of NF-κB
nuclear translocation and the half-life of the regulatory protein I-κB-R is unaffected in GSK-3β mutant
cells (see Figure 3). These data rule out an effect on
the cascade of proteins that culminates in phosphorylation of I-κB and its degradation. Presumably
then, GSK-3β is required for a later step in the
signaling pathway, such as a direct or indirect
requirement for phosphorylation of the NF-κB subunit, p65, or an as yet unidentified transcriptional
co-activator of p65. Whatever the molecular mechanism, it is clear that this effect is specific for GSK3β since GSK-3R is unable to compensate. This is in
stark contrast to the lack of effect of loss of GSK-3β
on the Wnt pathway. GSK-3β mutant mice do not
exhibit overt disregulation of this pathway as might
have been predicted, indicating that the Wnt pathway does not discriminate between these two GSK-3
Ali et al.
Figure 3. Model of NF-κB regulation by GSK-3β. GSK3β is required for NF-κB transactivation independent of
I-κB-R degradation and NF-κB nuclear translocation. See
text for details. Abbreviations: nuclear factor-κB (NF-κB),
tumor necrosis factor (TNF), inhibitor of NF-κB (I-κB).
isoforms. Indeed, to date, NF-κB activation is the only
known differentiator between these two enzymes.
While the finding that GSK-3β is required for NFκB activation was surprising, there are other clues
in the literature.
B. Xenopus rel
Additional support for a connection between GSK-3
and NF-κB during vertebrate development has
emerged from studies of Xenopus laevis development.
The mRNA of at least one Xenopus member of the
rel family, XrelA, is expressed in oocytes and early
embryos. XrelA impacts the dorsoventral patterning
process.123 XrelA induces a ventralizing effect early
in embryonic development and attenuates morphogenetic movements characteristic of dorsal mesoderm. XrelA RNA was also shown to reverse the
strong dorsal axis-promoting effects of a dominant
mutant of Xenopus GSK-3β. This complementation
effect argues in favor of a p65 NF-κB homologue
being downstream of GSK-3β, at least in some
systems.
C. SCF Ubiquitin Ligase Complex
Recently, components of the ubiquitin proteasome
pathway have been implicated as a common link
between the Wnt and NF-κB signal transduction
pathways. Ubiquitin-dependent proteolysis by the
proteasome plays an essential role in a number of
key biological processes, including cell-cycle progression, transcription, and signal transduction.124,125
Frequently the target protein is first marked for
degradation or processing by phosphorylation. The
phosphorylated protein is then recognized and ubiquitinated in a process that requires three proteins:
a ubiquitin-activation enzyme (E1), a ubiquitinconjugating enzyme (E2), and a ubiquitin ligase (E3).
Glycogen Synthase Kinase-3
Ubiquitin is first attached to an E1 protein in an
ATP-dependent reaction to form a high-energy thioester bond. The ubiquitin is then transferred from
the E1 to an E2 enzyme, which functions in conjunction with an E3 protein to link ubiquitin to lysine
residues in the targeted protein. A specific lysine
residue in the conjugated ubiquitin can then attach
to a second ubiquitin, and reiteration of this process
results in the assembly of a polyubiquitin chain. The
polyubiquitinated protein is recognized by the 26S
proteasome and is subsequently degraded. Ubiquitination of both β-catenin and I-κB is targeted by F-box/
WD40 repeat-containing proteins, such as Drosophila
Slimb (supernumerary limbs) and its mammalian
homologue β-TrCP, that are components of a class
of E3 ligases, termed the Skp1/Cullin1/F-box (SCF)
complex.88,126-128
A common structural feature between I-κB and
β-catenin is that phosphorylation occurs on two
closely located serines at positions 32/36 (I-κB-R) and
33/37 (β-catenin). This suggests that Slimb/β-TrCP
recognizes a DSPGXXSP amino acid motif. The association between Slimb/β-TrCP and the substrates
is specific because other F-box proteins neither
interact nor promote ubiquitination. Since phosphorylation and degradation of β-catenin is mediated by
a constitutively active GSK-3β and IkB is inducibly
phosphorylated by IKKs in response to various extracellular stimuli, what then distinguishes I-κB and
β-catenin? Perhaps it is only the kinases that are
responsible for phosphorylation of these substrates:
phosphorylated DSGXXS motifs on these substrates
might be the signal for the common ubiquitination
pathway through Slimb/β-TrCP. Another possible
explanation is the differential use of E2 ubiquitinconjugating enzymes.
Overexpression of a stabilized β-catenin mutant in
which the GSK-3-targeted serines were altered to
alanine induces activation of the NF-κB pathway.129
The proposed mechanism of cross-talk between the
GSK-3/β-catenin and NF-κB pathways has been
suggested to occur via LEF-1/Tcf-dependent upregulation of βTrCP levels and facilitated ubiquitination
of phosphorylated I-κB, since β-catenin signaling
augments NF-κB activation by a constitutively active
IKKb.
VII. Small Molecule Inhibitors of GSK-3
A. Lithium
The alkali metal lithium was first discovered in
1817, and over the ensuing period of time, lithium
has been utilized in various formulations as a remedy
for a multitude of human maladies.130 With the
seminal work of Australian physician/scientist, John
Cade, and subsequent clinical studies by Mogens
Schou in the early 1950s, lithium was introduced as
an effective therapy for manic-depressive illness
(bipolar affective disorder).131,132
More recently, lithium has also been shown to
perturb the development of diverse organisms, including Xenopus, zebrafish, sea urchins, and Dictyostelium (see section III). For instance, in Dictyo-
Chemical Reviews, 2001, Vol. 101, No. 8 2537
stelium, lithium alters cell fate determination, blocking spore cell formation and promoting stalk development.133 In Xenopus embryos, lithium treatment
causes an expansion of dorsal mesoderm, leading to
formation of a second dorsal axis.134 Treatment of sea
urchin animal blastomeres with lithium causes them
to display a morphology resembling that of isolated
vegetal blastomeres.135 Importantly, these effects
have since been shown to be similar to embryonic
patterning defects resulting from either disruption
of the GSK-3 gene or overexpression of inactive forms
of GSK-3 that have a dominant-negative activity.
The developmental and neuropsychiatric effects of
lithium have been attributed to a variety of biochemical processes including modulation of G proteins and
inhibition of inositol monophosphatase (IMPase), an
enzyme important for recycling of myo-inositol in the
phosphatidylinositide (PI) pathway.136 Exposure to
lithium reduces the level of myo-inositol in the rat
brain137 via noncompetitive inhibition of IMPase138
and to a lesser extent inositol polyphosphate-1phosphatase (INPP-1).139 However, an alternative
target of lithium was suggested by the close similarity between lithium action133,140 and the effect of
either GSK-3 disruption in Dictyostelium43 and ectopic expression of Wnt genes in Xenopus embryos.141
While examining lithium-mediated effects on dorsal/
ventral patterning in Xenopus embryos, Klein and
Melton (1996) observed that injection of a potent and
selective IMPase inhibitor (L690,330)142 did not phenocopy lithium. The nature of the connection between
lithium and the Wnt pathway was revealed by the
finding that lithium inhibits the activity of purified
GSK-3R and β.143 This finding was extended to GSK-3
function in intact cells.144 These results directed
attention toward GSK-3 as an important cellular
target for lithium action.
Lithium is highly selective for GSK-3. While one
report suggested that lithium could activate PKB/
AKT,145 this result has not been confirmed. Lithium
has thus been employed as a tool to study the role of
GSK-3 in various cellular processes. Although the
pleiotropic nature of the ion on cells means that if a
lithium effect is observed this may not be mediated
by GSK-3, if lithium has no effect on a process, a role
for GSK-3 can be excluded.
In the case of the reduction in NF-κB activity in
cells lacking GSK-3β (see section V), the results
obtained using fibroblasts lacking functional GSK3β through genetic disruption were reproduced using
lithium, a potent inhibitor of GSK-3. Lithium inhibited the induction of NF-κB transactivation by 70%
relative to a potassium control in HEK293 cells, and
lithium-treated wild-type fibroblasts were sensitized
to TNF-induced killing, similar to GSK-3β null cells.
These results are consonant with those of Beyaert
et al. (1989), who first reported that lithium causes
a dose-dependent enhancement of TNF cytotoxicity
in human and murine cell lines.146 Lithium also
enhanced the in vivo antitumor action of TNF.
Similar results have been obtained when using
interleukin-2 (IL-2), another NF-κB agonist.147 IL-2
in combination with lithium demonstrated a stronger
inhibitory effect on tumor growth than IL-2 alone,
2538 Chemical Reviews, 2001, Vol. 101, No. 8
as determined by reduction of tumor size and prolongation of survival in tumor-bearing mice.
B. Other Inhibitors of GSK-3
Hymenialdisine (HD) and related compounds, such
as debromohymenialdisine (DBH), were originally
purified from the marine sponge Axinella verrucosa
and have recently been identified in a screen for
antiinflammatory compounds. For example, DBH
exhibits antiinflammatory activity in a model of
adjuvant-induced arthritis in the rat.148
HD was evaluated for its effects on the activation
of NF-κB using electrophoretic mobility shift assay
(EMSA) and luciferase reporters under the control
of either the HIV-LTR or IL-8 promoter. Similar to
lithium treatment or genetic disruption of GSK-3β,
DH inhibited NF-κB DNA binding and NF-κBregulated gene expression without affecting I-κB
degradation.149,150 In particular, HD caused a concentration-dependent inhibition of luciferase production regardless of the stimulus used (TNF-R, LPS,
or PMA), and HD-inhibited DNA binding in U937
cells was most evident at 1.0 mM HD, commensurate
with 50% inhibition of TNF-stimulated DNA binding.
The striking similarities between HD effects and
GSK-3β inhibition were noted by Dr. Michel Roberge,
who had been working with HD. Both HD and DBH
have been demonstrated as potent in vitro inhibitors
of GSK-3β (Hoeflich, Roberge, and Woodgett, unpublished observations).151 HD and DBH also interfere
with in vivo phosphorylation of specific proteins by
GSK-3β. In particular, phosphorylation of Tau151 and
β-catenin (Hoeflich, Roberge, and Woodgett, unpublished observations) are completely inhibited by HD
and DBH in vivo. These effects occur at two-orders
of magnitude lower concentrations of HD/DBH than
lithium.
Although HD is also known to inhibit PKC,148 the
selective PKC inhibitor RO 32-0432152 has no effect
on TNF-R-stimulated luciferase reporter activity or
IL-8 production at low concentrations. As expected,
RO 32-0432 only inhibited luciferase production in
response to PMA stimulation (with IC50 values of 0.2
mM), demonstrating the ability of this compound to
inhibit PKC. These data support the idea that the
antiinflammatory activity of HD compounds may be
related to their ability to block the activity of the
transcription factor NF-κB via inhibition of GSK-3β.
Additional ATP competitor molecules specific for
GSK-3 (Ki 10-30 nM) have been recently reported
to stimulate glycogen synthesis in liver cell lines and
to induce transcription of β-catenin-regulated genes
in epithelial cells.153 The availability of these inhibitors will greatly assist dissection of the physiological
processes influenced by GSK-3.
VIII. Concluding Remarks
Since its inauspicious discovery over 20 years ago,
considerable progress has been made in understanding the regulation, signaling mechanisms, and physiological effects of glycogen synthase kinase-3. Progress
was fueled by research using genetically tractable
organisms by many different groups, most of whom
Ali et al.
had no interest in glycogen metabolism! Despite
these advances, much is still unclear. The means by
which cells effectively sequester different populations
of GSK-3 that are responsive to distinct pathways
and that culminate in specific responses is poorly
understood. Some data point to differential requirements for substrate priming. Specifically, some GSK-3
substrates do not require prephosphorylation whereas
others, like glycogen synthase, require prephosphorylation at a serine-threonine just C-terminal to the
GSK-3 site.39 Thus, different substrate phosphorylation requirements might allow GSK-3 to affect one
substrate but not another. This idea is supported by
effects of a 39 amino acid peptide corresponding to
the GSK-3-binding domain of FRAT, which prevents
the GSK-3-catalyzed phosphorylation of axin and
β-catenin but not other substrates, such as glycogen
synthase and eIF2B. Phosphorylation of these latter
targets is independent of axin but instead is dependent on priming prephosphorylation.
Among the targets of GSK-3 regulation is a who’s
who list of transcription factors including β-catenin,
NF-AT, C/EBP, c-Jun, c-Myc, NF-κB, and CREB,
most of which are negatively regulated in mammalian cells. As such, GSK-3 carries significant
responsibility in regulating gene expression, and it
is therefore perhaps not too surprising that its
regulation is complex and redundant. With the recent
development of small molecule inhibitors of the
enzyme, additional targets are sure to be revealed.
More importantly, new biological functions will be
discerned, possibly leading to new therapies. There
is considerable concern about the consequences of
interfering with such a pleiotropic enzyme. Indeed,
predicted effects on β-catenin disregulation raise the
spectre of inhibitors being tumor promoters. However, the history of this enzyme is replete with
surprises, and it would be foolish to discount useful
therapeutic consequences of its careful modulation.
IX. Acknowledgments
We apologize to the authors whose original work
is not included in the references owing to space
limitations. Special thanks to current and former lab
members who have contributed to the work on GSK3, particularly Jing Jin, Elizabeth Rubie, Laurent
Ruel, and Vuk Stambolic. During part of this study,
A.A. was a recipient of an NSERC postdoctoral
fellowship and K.P.H. was supported by a Canadian
Institutes of Health Research (CIHR) studentship.
J.R.W. is supported by grants from the CIHR and
Howard Hughes Medical Institute and is a CIHR
Senior Scientist.
X. Note Added in Proof
Recently, two groups have solved the crystal structure of GSK-3β.172,173 The most striking aspect of the
structure was insight into the mechanism by which
the protein kinase activity is inhibited upon phosphorylation of Serine 9. As mentioned in section II
and Table 2, several GSK-3 targets require prior
phosphorylation to be recognized by GSK-3. When
Serine 9 is phosphorylated, the phosphoserine binds
Glycogen Synthase Kinase-3
to a region within the catalytic lobe that normally
binds the prephosphorylated residue of the substrate,
thus acting as an intramolecular pseudosubstrate
inhibitor. This same conclusion was also arrived at
by biochemical studies in which Arginine 96 in GSK3β lies in the active site as coordinates phosphorylated Serine 9. This mechanism does not impact
substrates that do not require prior phosphorylation
(e.g., β-catenin) and provides a means by which cells
may regulate phosphorylation of some GSK-3 without
affecting others.
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