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doi: 10.1242/10.1242/jcs.00384
Commentary |
Ontario Cancer Institute, 610 University Avenue, Toronto, Ontario M5G 2M9, Canada
* Author for correspondence (e-mail: jwoodget{at}uhnres.utoronto.ca)
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Summary |
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 Top Summary IntroductionGSK-3 variantsGSK-3 phosphorylation: insights...Physiological roles for tyrosine...The role of GSK-3...GSK-3 and the Hedgehog...Other GSK-3 substratesOther GSK-3-binding proteinsGSK-3 and human diseaseSmall molecule inhibitors of...PerspectivesReferences |
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Key words: Signal transduction, Phosphorylation, Wnt signalling, Insulin signalling, Protein structure-function
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Introduction |
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TopSummaryIntroductionGSK-3 variantsGSK-3 phosphorylation: insights...Physiological roles for tyrosine...The role of GSK-3...GSK-3 and the Hedgehog...Other GSK-3 substratesOther GSK-3-binding proteinsGSK-3 and human diseaseSmall molecule inhibitors of...PerspectivesReferences |
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;
Woodgett and Cohen, 1984). We
now know that, beyond its role in glycogen metabolism, GSK-3 acts as a
downstream regulatory switch that determines the output of numerous signalling
pathways initiated by diverse stimuli (reviewed in
Frame and Cohen, 2001;
Grimes and Jope, 2001;
Woodgett, 2001). The pathways
in which GSK-3 acts as a key regulator, when dysregulated, have been
implicated in the development of human diseases such as diabetes, Alzheimer's
disease, bipolar disorder and cancer. Given its involvement in many pathophysiological processes and diseases, GSK-3 is a tempting therapeutic target. However, its involvement in multiple pathways also raises the issue of selectivity — how might we impact one process while leaving others untouched? We must also assess the full spectrum of GSK-3 functions to avoid later surprises. For example, although inhibition of GSK-3 may be desirable in one context (e.g. in preventing neuronal apoptosis), it could have serious implications for another — for example, it might accelerate hyperplasia by deregulating ß-catenin. That said, the recent development of small molecule inhibitors of GSK-3 has provided new tools for visualizing the cell from the perspective of GSK-3. Since it is highly active in most cell types, regulation of substrate phosphorylation occurs either by inactivation of GSK-3 or by changing substrate accessibility or recognition. Here, we present an overview of the various processes in which GSK-3 plays a key role and describe the current status of knowledge of GSK-3 regulation.
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GSK-3 variants |
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TopSummaryIntroductionGSK-3 variantsGSK-3 phosphorylation: insights...Physiological roles for tyrosine...The role of GSK-3...GSK-3 and the Hedgehog...Other GSK-3 substratesOther GSK-3-binding proteinsGSK-3 and human diseaseSmall molecule inhibitors of...PerspectivesReferences |
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and GSK-3ß
(Woodgett, 1990)
(Fig. 1). GSK-3 has a
mass of 51 kDa, whereas GSK-3ß is a protein of 47 kDa. The difference in
size is due to a glycine-rich extension at the N-terminus of GSK-3.
Although highly homologous within their kinase domains (98% identity), the two
gene products share only 36% identity in the last 76 C-terminal residues
(Woodgett, 1990). Homologues
of GSK-3 exist in all eukaryotes examined to date and display a high degree of
homology; isoforms from species as distant as flies and humans display >90%
sequence similarity within the kinase domain (reviewed in
Ali et al., 2001).
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GSK-3 and GSK-3ß, although structurally similar, are not
functionally identical. This became obvious upon ablation of the GSK-3ß
isoform in mice, which results in an embryonic lethal phenotype
(Hoeflich et al., 2000).
Embryos carrying homozygous deletions of exon 2 of GSK-3ß die around
embryonic day 16 owing to massive liver degeneration caused by extensive
hepatocyte apoptosis. The inability of GSK-3 to rescue the
GSK-3ß-null mice indicates that the degenerative liver phenotype arises
specifically from the loss of the beta isoform. This phenotype is remarkably
similar to that of animals lacking RelA or I-
B kinase 2
(Beg et al., 1995;
Li et al., 1999), components
of the NF-B signalling pathway
(Ghosh and Karin, 2002;
Rothwarf and Karin, 1999).
Regulation of NF-B nuclear import is not disrupted in embryonic
fibroblasts isolated from the GSK-3ß-null mice, which indicates that
GSK-3 affects some other level of NF-B regulation. The specific
target(s) of GSK-3ß in the regulation of NF-B signalling has not
been identified. Other protein kinases can phosphorylate the p65 subunit of
NF-B, increasing transactivation
(Jang et al., 2001;
Wang et al., 2000).
GSK-3ß can phosphorylate the C-terminal domain (residues 354-551) of p65
in vitro (Schwabe and Brenner,
2002), but further studies are required to verify if p65 is a
physiological GSK-3ß substrate and to assess the effect of this
modification. The phenotype of mice lacking GSK-3 has not yet been
reported.
A minor (
15% of total) splice variant of GSK-3ß, GSK-3ß2,
has recently been identified and contains a 13-residue insert within the
kinase domain (Mukai et al.,
2002). Analysis of the in vitro kinase activity of GSK-3ß2
revealed reduced activity towards the microtubule-associated protein tau,
compared with `unspliced' GSK-3ß. An antibody selective for the novel
splice-insertion polypeptide revealed that GSK-3ß2 is localized primarily
to neuronal cell bodies, unlike unspliced GSK-3ß, which is also found in
neuronal processes. It is unclear whether these substrate and subcellular
localization differences can be generalized to other proteins or cells. Given
the location of the insert within a highly conserved sequence, it probably
forms a loop/hook, which might allow differential binding of the splice
variant to scaffolding proteins that then expose the isoform to a distinct
subset of target proteins (Mukai et al.,
2002).
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GSK-3 phosphorylation: insights from crystal structures |
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TopSummaryIntroductionGSK-3 variantsGSK-3 phosphorylation: insights...Physiological roles for tyrosine...The role of GSK-3...GSK-3 and the Hedgehog...Other GSK-3 substratesOther GSK-3-binding proteinsGSK-3 and human diseaseSmall molecule inhibitors of...PerspectivesReferences |
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;
Dajani et al., 2001;
ter Haar et al., 2001). The
structure has provided insight into both its regulation and its preference for
primed, pre-phosphorylated substrates. Protein kinases related to GSK-3, such
as CDK2, p38
and ERK2, require phosphorylation of residues in their
activation loops (T-loops) as a prerequisite for activity
(Bellon et al., 1999;
Brown et al., 1999;
Canagarajah et al., 1997). A
phosphothreonine is used by all three related kinases to align key
ß-strand and -helical domains. The phosphorylation of a T-loop
tyrosine is also required by p38 and ERK2 to open up the catalytic site
for substrate access. The T-loop of GSK-3 is tyrosine phosphorylated at Y216
and Y279 in GSK-3ß and GSK-3, respectively, but not threonine
phosphorylated. Y216/Y279 phosphorylation could play a role in forcing open
the substrate-binding site, but there appear to be no constraints preventing
the open conformation in the unphosphorylated state
(Dajani et al., 2001). Thus,
T-loop tyrosine phosphorylation of GSK-3 might facilitate substrate
phosphorylation but is not strictly required for kinase activity
(Dajani et al., 2001).
GSK-3 has an unusual preference for target proteins that are
pre-phosphorylated at a `priming' residue located C-terminal to the site of
GSK-3 phosphorylation (Fiol et al.,
1987). The consensus sequence for GSK-3 substrates is
Ser/Thr—X—X—X-Ser/Thr-P, where the first Ser or Thr is the
target residue, X is any amino acid (but often Pro), and the last Ser-P/Thr-P
is the site of priming phosphorylation. Although not strictly required,
priming phosphorylation greatly increases the efficiency of substrate
phosphorylation of most GSK-3 substrates by 100-1000-fold
(Thomas et al., 1999). For
example, glycogen synthase, the prototypical primed substrate, requires
priming phosphorylation by casein kinase II (CK2) and then undergoes
sequential multisite phosphorylation by GSK-3
(Fiol et al., 1988;
Fiol et al., 1990).
The function expected for the `missing' phosphothreonine in the GSK-3's T-loop is believed to be replaced by the phosphorylated residue of a primed substrate, which binds to a positively charged pocket comprising R96, R180 and K205 (residue numbers for GSK-3ß). This not only optimizes the orientation of the kinase domains but also places the substrate at the correct position within the catalytic groove for phosphorylation to occur. There are some substrates that lack a priming site. These proteins often display negatively charged residues at or near the priming position that may mimic a phospho-residue.
2) Inhibitory serine phosphorylation
Stimulation of cells with insulin causes inactivation of GSK-3 through a
phosphoinositide 3-kinase (PI 3-kinase)-dependent mechanism. PI-kinase-induced
activation of PKB (also termed Akt) results in PKB phosphorylation of both
GSK-3 isoforms (S9 of GSK-3ß; S21 of GSK-3)
(Cross et al., 1995), which
inhibits GSK-3 activity. This leads to the dephosphorylation of substrates
including glycogen synthase and eukaryotic protein synthesis initiation
factor-2B (eIF-2B), resulting in their functional activation and consequent
increased glycogen and protein synthesis (reviewed in
Cohen et al., 1997).
Numerous other stimuli also lead to inactivation of GSK-3 through S9/S21
phosphorylation, including growth factors such as EGF and PDGF that stimulate
the GSK-3-inactivating kinase p90RSK (also known as MAPKAP-K1)
through MAP kinases (Brady et al.,
1998; Saito et al.,
1994), activators of p70 ribosomal S6 kinase (p70S6K) such as
amino acids, (Armstrong et al.,
2001; Krause et al.,
2002; Terruzzi et al.,
2002), activators of cAMP-activated protein kinase (PKA)
(Fang et al., 2000;
Li et al., 2000;
Tanji et al., 2002) and PKC
activators (Ballou et al.,
2001; Fang et al.,
2002).
The crystal structure of GSK-3 also helps to explain the inhibitory role of
this serine phosphorylation (see Fig.
2A). Phosphorylation of S9/S21 creates a primed pseudosubstrate
that binds intramolecularly to the positively charged pocket mentioned above.
This folding precludes phosphorylation of substrates because the catalytic
groove is occupied. Note that the mechanism of inhibition is competitive. A
consequence of this is that primed substrates, in high enough concentrations,
out-compete the pseudosubstrate and thus become phosphorylated
(Frame et al., 2001). Thus,
although less efficient, unprimed substrates may provide a more accurate
measure of GSK-3 activity in kinase assays performed in vitro. Mutation of
Arg96 to alanine disrupts the pocket of positive charge
(Fig. 2B)
(Frame et al., 2001).
Interestingly, although a peptide of 11 residues that matches the
phosphorylated N-terminus of GSK-3ß peptide-11 competitively inhibits
both primed and unprimed substrates, a truncated version of this peptide
(NTptide-8) inhibits phosphorylation only of primed substrates
(Frame et al., 2001). Thus,
small molecule inhibitors modeled to fit in the positively charged pocket of
the GSK-3 kinase domain could potentially be very effective for selective
inhibition of primed substrates.
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Physiological roles for tyrosine phosphorylation |
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TopSummaryIntroductionGSK-3 variantsGSK-3 phosphorylation: insights...Physiological roles for tyrosine...The role of GSK-3...GSK-3 and the Hedgehog...Other GSK-3 substratesOther GSK-3-binding proteinsGSK-3 and human diseaseSmall molecule inhibitors of...PerspectivesReferences |
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). The kinase(s) responsible has not been identified, although
the tyrosine kinase (Zak1) that phosphorylates the Y216 equivalent is known in
the slime mould (Kim et al.,
1999). Mammalian GSK-3ß expressed in bacteria shows evidence
of autophosphorylation on tyrosine, serine and threonine residues, which
raises the possibility that tyrosine phosphorylation of GSK-3 in mammalian
cells is an autocatalytic event (Wang et
al., 1994). GSK-3 immunoprecipitated from mammalian cells does not
show this behaviour, however (Hughes et
al., 1993).
Although GSK-3 is tyrosine phosphorylated in resting cells, the level may
not be stoichiometric. Apoptotic stimuli such as staurosporine treatment or
neurotrophic factor withdrawal increase GSK-3 activity and tyrosine
phosphorylation in certain neuronal cell lines
(Bhat et al., 2000;
Bijur and Jope, 2001).
Moreover, LPA, through a pathway involving the G proteins
G12 and G13, also increases GSK-3 activity
in neuronal cells at least in part through enhanced tyrosine phosphorylation
of Y216/Y279 (Sayas et al.,
2002). Since treatment of primary neurons with LPA results in
neurite retraction, treatment of a neuroblastoma cell line, Neuro2A, results
in cell rounding (Sayas et al.,
2002), and LPA also causes apoptosis of adult neurons
(Steiner et al., 2000), this
provides further circumstantial evidence for a role for tyrosine
phosphorylation in apoptosis.
Recent work has revealed two possible candidates for kinases that might
tyrosine phosphorylate GSK-3 under these circumstances. Transient increases in
intracellular calcium increase GSK-3-mediated phosphorylation of the
microtubule-associated protein tau, and the increased GSK-3 activity is
attributed to elevated GSK-3 tyrosine phosphorylation
(Hartigan and Johnson, 1999).
The tyrosine kinase that may be responsible for the phosphorylation of GSK-3
is proline-rich tyrosine kinase 2
(PYK2)
, a
calcium-sensitive enzyme. Active, but not kinase-dead, PYK2 increases tyrosine
phosphorylation of GSK-3 both in PYK2-transfected cells and in vitro, and PYK2
and GSK-3ß co-immunoprecipitate in cells transfected with PYK2
(Hartigan et al., 2001). The
Fyn tyrosine kinase is another potential player: insulin treatment of SH-SY5Y
cells for 1 minute causes an increase in association of Fyn with GSK-3ß,
a transient increase in GSK-3ß activity with a concomitant transient
increase in GSK-3ß phosphorylation on Y216 and a transient increase in
tau phosphorylation. The short duration of insulin treatment is crucial for
the observed increase in GSK-3 activity
(Lesort et al., 1999).
Prolonged treatment with insulin, besides the well characterized inhibition of
GSK-3 through serine phosphorylation (discussed above), has also been reported
to inhibit GSK-3 through tyrosine dephosphorylation in Chinese hamster ovary
cells (Murai et al., 1996).
Further characterization of the proposed GSK-3-phosphorylating tyrosine
kinases is required to substantiate a role for them in physiological
processes.
The role of tyrosine phosphorylation of GSK-3 in Dictyostelium is
better understood. Zak1 is essential for activation of GSK-3 during
developmental patterning and is regulated downstream of certain cyclic AMP
receptors involved in chemotaxis (Kim et
al., 1999; Plyte et al.,
1999). Unfortunately, identification of a mammalian orthologue of
Zak1 has so far proven elusive. Kim et al. have recently described an opposing
role for a protein tyrosine phosphatase, which dephosphorylates GSK-3 on
tyrosine residues and results in a prestalk fate
(Kim et al., 2002).
Interestingly, phosphorylation of two tyrosine residues, both residing in the
activation loop of GSK-3, appears to be modulated by cAMP in
Dictyostelium (Kim et al.,
2002). Both of these tyrosine residues, Y214 and Y220, are
conserved in mammals (Y216 and Y219, respectively). Thus, Y219 — in
addition to Y216 — might therefore play a role in GSK-3 regulation in
mammals.
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The role of GSK-3 in the Wnt/ß-catenin pathway |
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TopSummaryIntroductionGSK-3 variantsGSK-3 phosphorylation: insights...Physiological roles for tyrosine...The role of GSK-3...GSK-3 and the Hedgehog...Other GSK-3 substratesOther GSK-3-binding proteinsGSK-3 and human diseaseSmall molecule inhibitors of...PerspectivesReferences |
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;
Polakis, 2000;
Smalley and Dale, 1999).
Members of the Wnt family have been identified in organisms ranging in
complexity from the cnidarian Hydra to humans; at least 19 different Wnts
exist in mammals (Miller,
2002). Severe developmental defects usually occur in mice that
have defective Wnt signalling. One of the pathways regulated by Wnt molecules
is termed the canonical Wnt pathway or the Wnt/ß-catenin pathway
(reviewed in Huelsken and Behrens,
2002; Polakis,
2000; Seidensticker and
Behrens, 2000; Sharpe et al.,
2001) (Fig. 3). Here, we limit discussion of Wnt signalling to this signalling pathway. Wnt
signal transduction ultimately results in the activation of genes regulated by
the T-cell factor (TCF)/lymphoid enhancer factor (LEF) family of architectural
transcription factors (reviewed in Barker
et al., 2000; Brantjes et al.,
2002; Novak and Dedhar,
1999). The effector molecule responsible for activating
TCF/LEF-responsive genes is ß-catenin, which serves as a transactivator
that binds to DNA-bound TCF/LEFs. As in insulin signalling, GSK-3 plays a key
inhibitory role in the Wnt pathway. In unstimulated cells, GSK-3
phosphorylates the N-terminal domain of ß-catenin, thereby targeting it
for ubiquitylation and proteasomal degradation. Exposure of cells to Wnts
leads to inactivation of GSK-3 through an as yet unclear mechanism. The
phosphoprotein Dishevelled is required, after receptor-ligand interaction, to
transduce the signal that results in the inactivation of GSK-3. As a result,
ß-catenin is dephosphorylated (van
Noort et al., 2002) and escapes the ubiquitylation-dependent
destruction machinery. Unphosphorylated ß-catenin accumulates in the
cytoplasm and translocates to the nucleus, where it can associate with the
TCF/LEFs and become a transcriptional transactivator. Mutations in
ß-catenin that prevent its phosphorylation by GSK-3 (gain-of-function
mutations) have been found in cancers of the skin, colon, prostate, liver,
endometrium and ovary (reviewed in
Polakis, 2000). Many of the
components of the Wnt pathway, and their functional roles, were first
identified in studies of the Wnt pathway in Drosophila melanogaster,
which is driven by the Wnt homologue, Wingless
(Dierick and Bejsovec, 1999;
Manoukian and Woodgett, 2002;
Siegfried and Perrimon,
1994). In Drosophila, the GSK-3 homologue is termed
Shaggy (Bourouis et al., 1990)
or Zeste-white3 (Siegfried et al.,
1990).
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Phosphorylation of ß-catenin by GSK-3 occurs in a complex sometimes
referred to as the destruction complex, which consists minimally of the
proteins GSK-3, ß-catenin, axin/conductin and adenomatous polyposis coli
(APC) (Hinoi et al., 2000).
APC is a tumour suppressor protein commonly deleted in familial adenomatous
polyposis and sporadic colorectal cancer
(Polakis, 1997). Axin (and a
related protein known as conductin or axil) harbors several protein-protein
interaction domains and serves as a scaffolding protein that holds together
the elements of the ß-catenin destruction complex. Both Axin and APC are
phosphorylated by GSK-3. Phosphorylation of axin by GSK-3 increases its
stability and binding to ß-catenin
(Ikeda et al., 1998;
Jho et al., 1999;
Yamamoto et al., 1999).
Phosphorylation of APC increases its binding to ß-catenin
(Rubinfeld et al., 1996).
Five groups have recently determined that ß-catenin is also a primed
substrate for GSK-3, with casein kinase I (CKI) acting as the priming kinase
(Amit et al., 2002;
Hagen et al., 2002;
Hagen and Vidal-Puig, 2002;
Liu et al., 2002;
Sakanaka, 2002;
Yanagawa et al., 2002). CKI
phosphorylates S45, which lies four residues C-terminal to three GSK-3 targets
at serines S33, S37 and S41. As a priming kinase, CKI functions as a negative
regulator of Wnt signalling since it promotes GSK-3 function. Such a role
contrasts with that proposed in several previous reports, which identified CKI
as a positive transducer of Wnt signalling
(Gao et al., 2002;
Kishida et al., 2001;
Lee et al., 2001;
McKay et al., 2001). CKI
binds to axin and Dishevelled and phosphorylates not only ß-catenin but
also axin, Dishevelled and APC. Polakis has proposed a model in which CKI can
act as both a positive and a negative regulator of Wnt signalling
(Polakis, 2002). In this
model, CKI plays a role in the destruction complex as the priming kinase for
GSK-3 and is required for transmission of the Wnt signal by assisting in the
activation of Dishevelled, perhaps by increasing its affinity for signalling
intermediates (see below). A novel ankyrin-repeat-containing protein,
Diversin, has been reported to recruit CKI to the destruction complex
(Schwarz-Romond et al.,
2002).
These data raise the possibility that GSK-3 plays only a latent role in
regulation of ß-catenin. For example, if CKI activity is directly
regulated by Wnt signalling, then phosphorylation of S45 would act as the
trigger for subsequent phosphorylation by GSK-3. In this scenario, the
activity of GSK-3 could be totally independent of Wnt regulation. However,
phosphorylation of S45 appears to be constitutive, at least in some cell
types. Although S45 phosphorylation has been reported to decrease upon Wnt
stimulation, the phosphospecific antibodies used in those studies also detect
S41, one of the GSK-3 targets (Amit et al.,
2002). Antibodies selective for phosphoserine 45 do not reveal
changes in stoichiometry in response to Wnt
(Liu et al., 2002). Clearly,
cells have evolved complex mechanisms to titrate ß-catenin levels,
presumably to allow multiple layers of control.
Another interesting player in the regulation of the Wnt pathway, at least
in vertebrates, is a GSK-3-binding protein termed GBP (also known as
FRAT
)
(Farr et al., 2000;
Ferkey and Kimelman, 2002;
Fraser et al., 2002;
Sumoy et al., 1999;
Yost et al., 1998). Binding
of GBP to GSK-3 precludes GSK-3 from binding to axin and thus interferes with
ß-catenin phosphorylation. A small peptide derived from FRAT called
FRAT-tide is sufficient to prevent axin-GSK-3 interaction and prevents
phosphorylation of both axin and ß-catenin
(Thomas et al., 1999).
Comparison of mutations that affect binding of GSK-3 to axin and GBP, as well
as analysis of the crystal structure of a GSK-3-FRAT-tide complex, indicates
that the binding sites on GSK-3 for GBP/FRAT and axin overlap
(Bax et al., 2001;
Ferkey and Kimelman, 2002;
Fraser et al., 2002).
Treatment of Xenopus embryo extracts with CKI
increases binding
of GBP to Dishevelled (Lee et al.,
2001).
GBP also plays a role in the nuclear export of GSK-3
(Franca-Koh et al., 2002). A
mutant of GSK-3 that cannot bind to GBP accumulates in the nucleus. Moreover,
a peptide that interferes with GBP binding to GSK-3 causes endogenous GSK-3 to
accumulate in the nucleus. These findings suggest that GBP regulates access of
GSK-3 to substrates partitioned between nuclear and cytoplasmic compartments.
Since there are two known mammalian GBP homologues
(Freemantle et al., 2002),
each with dynamically regulated expression patterns during development, GBP
could play an important role in modulating GSK-3 function, especially during
development. Rather surprisingly, GBP homologues have not been identified in
Drosophila or C. elegans, which indicates that the protein
is not a core component of canonical Wnt signalling.
A critical aspect of GSK function in the Wnt pathway is that GSK-3 appears
to be insulated from regulators of GSK-3 that lie outside of the Wnt pathway.
For example, insulin signalling leads to inhibition of GSK-3 via serine S9/S21
phosphorylation but does not cause accumulation of ß-catenin. Conversely,
Wnt signalling does not affect insulin signalling
(Ding et al., 2000;
Yuan et al., 1999). How this
insulation occurs is unclear, but it probably stems from the effective
sequestration of a fraction of GSK-3 with axin in the destruction complex.
Note that tissues from mice lacking GSK-3ß do not show evidence of
accumulated ß-catenin even though total GSK-3 levels are reduced by 50%
and there is zero cellular GSK-3ß. Immunoprecipitation of axin from these
tissues reveals that GSK-3ß is simply replaced by GSK-3 (in
wild-type cells, both GSK-3 and GSK-3ß are found bound to axin)
(E. Rubie and J.R.W., unpublished). Since cellular levels of GSK-3 exceed
those of axin, the destruction machinery compensates for the loss of
GSK-3ß by substituting GSK-3.
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GSK-3 and the Hedgehog pathway |
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TopSummaryIntroductionGSK-3 variantsGSK-3 phosphorylation: insights...Physiological roles for tyrosine...The role of GSK-3...GSK-3 and the Hedgehog...Other GSK-3 substratesOther GSK-3-binding proteinsGSK-3 and human diseaseSmall molecule inhibitors of...PerspectivesReferences |
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;
Price and Kalderon, 2002).
The Hh pathway is somewhat similar to the Wnt pathway: both Wnt and Hh are
secreted signalling proteins involved in embryonic patterning that often work
in concert (reviewed in Ingham and
McMahon, 2001). Whereas the Wnt pathway uses ß-catenin to
transduce its signals to the nucleus, the Hh pathway employs a protein called
Cubitus interruptus (Ci) in flies or Gli in mammals
(Ingham and McMahon, 2001). In
the absence of a Hh signal, Ci is targeted for proteolysis. Unlike in Wnt
signalling, proteolysis does not result in total degradation of Ci but instead
processes it from a 155-residue form (Ci155) to a truncated 75-residue amino
acid form (Ci75) that functions as a transcriptional repressor
(Aza-Blanc et al., 1997).
GSK-3, in combination with CKI and the priming kinase PKA, phosphorylates
Ci155 to target it for proteolytic processing in the absence of a Hh signal
(Jia et al., 2002;
Price and Kalderon, 2002).
Activation of Hh signalling results in translocation of full-length Ci155 to
the nucleus, where it activates Hh target genes. As in the Wnt pathway,
several of the molecules involved in Hh signal transduction have been
implicated in cancer (reviewed in Taipale
and Beachy, 2001). Notably, familial germline mutations in one
allele of the Hh receptor, Patched, are associated with an almost complete
penetrance of basal cell carcinoma
(Johnson et al., 1996;
Taipale, 2001). Although GSK-3 phosphorylation of the mammalian homologues of
Ci has yet to be reported, all contain multiple GSK-3 consensus sites next to
PKA sites (Ruis i Altaba, 1999; Jia et
al., 2002). It seems likely, on the basis of the conservation of
the Wnt pathway, that the Gli proteins will soon be confirmed as GSK-3
substrates. |
Other GSK-3 substrates |
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TopSummaryIntroductionGSK-3 variantsGSK-3 phosphorylation: insights...Physiological roles for tyrosine...The role of GSK-3...GSK-3 and the Hedgehog...Other GSK-3 substratesOther GSK-3-binding proteinsGSK-3 and human diseaseSmall molecule inhibitors of...PerspectivesReferences |
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Other GSK-3-binding proteins |
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TopSummaryIntroductionGSK-3 variantsGSK-3 phosphorylation: insights...Physiological roles for tyrosine...The role of GSK-3...GSK-3 and the Hedgehog...Other GSK-3 substratesOther GSK-3-binding proteinsGSK-3 and human diseaseSmall molecule inhibitors of...PerspectivesReferences |
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).
The activation of GSK-3 occurs without changes in its phosphorylation and is
restricted to nuclear GSK-3 and involves an unknown mechanism. In addition, a
yeast two-hybrid screen identified the A-kinase-anchoring protein AKAP220 as a
GSK-3-binding partner (Tanji et al.,
2002). AKAPs are scaffolding proteins, and the interaction between
AKAP220 and GSK-3 occurrs in a complex that also containsPKA and type 1
protein phosphatase. The interaction between AKAP220 and GSK-3 enhances
PKA-mediated inhibition of GSK-3.
Presenilin 1 (PS1) is one of the two mammalian presenilins identified
through association of mutations in these proteins with early-onset familial
Alzheimer's disease. Besides having a role in proteolytic processing of
proteins at the cell membrane such as amyloid precursor protein, presenilin
also binds to ß-catenin and has been implicated in regulating its
cellular levels. PS1 may also function as a scaffolding protein that binds
PKA, GSK-3 and ß-catenin (Palacino
et al., 2001). This novel quaternary complex functions in a
similar manner to the axin complex, using PS1 to couple the priming
phosphorylation by PKA (on S45 of ß-catenin) to subsequent
phosphorylation by GSK-3.
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GSK-3 and human disease |
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TopSummaryIntroductionGSK-3 variantsGSK-3 phosphorylation: insights...Physiological roles for tyrosine...The role of GSK-3...GSK-3 and the Hedgehog...Other GSK-3 substratesOther GSK-3-binding proteinsGSK-3 and human diseaseSmall molecule inhibitors of...PerspectivesReferences |
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; Maccioni et al.,
2001; Mattson,
2001). The GSK-3 inhibitor lithium
(Stambolic et al., 1996),
used for decades as a therapy for bipolar disorder, also implicates GSK-3
activity in the underlying causes of this disease
(Detera-Wadleigh, 2001;
Gould and Manji, 2002;
Manji et al., 1999). In
support of a role for GSK-3 in mood regulation is the fact that another
mood-stabilizing drug, valproate, also inhibits GSK-3, although probably
through an indirect mechanism (Chen et
al., 1999; De Sarno et al.,
2002). Note that lithium and valproate also affect neuronal
inositol metabolism, and their mood-stabilizing properties might be related to
this effect (Williams, 2002; Hall et al.,
2002; Li et al.,
2002). Apart from a role in neurological pathologies, GSK-3 may
also play a role in the development of non-insulin-dependent diabetes mellitus
(NIDDM), a disease often associated with chronic inhibition of muscle glycogen
synthase (reviewed in Kaidanovich and
Eldar-Finkelman, 2002). |
Small molecule inhibitors of GSK-3 |
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TopSummaryIntroductionGSK-3 variantsGSK-3 phosphorylation: insights...Physiological roles for tyrosine...The role of GSK-3...GSK-3 and the Hedgehog...Other GSK-3 substratesOther GSK-3-binding proteinsGSK-3 and human diseaseSmall molecule inhibitors of...PerspectivesReferences |
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)
— but it may also be relevant for some of the more recently identified
functions of GSK-3, such as its role in activating NF-kB, a well characterized
target for anti-inflammatory agents
(Hoeflich et al., 2000). The
best-characterized inhibitor of GSK-3 is lithium
(Klein and Melton, 1996;
Stambolic et al., 1996).
Although fairly specific for GSK-3, compared with other protein kinases,
lithium also affects other enzymes, and a relatively high dose is required (Ki
is mM) to inhibit GSK-3 activity in cell culture
(Stambolic et al., 1996). The
mode of inhibition is through competition for Mg2+
(Ryves and Harwood, 2001).
Recently, the bivalent form of zinc, which mimics insulin action, has also
been shown to inhibit GSK-3 when used at a concentration of 15 µM in a cell
culture system (Ilouz et al.,
2002). In vitro, another metal ion, beryllium, inhibits GSK-3 to
half maximal activity at a concentration of 6 µM
(Ryves et al., 2002).
Several new GSK-3 inhibitors have recently been developed, most of which
are ATP competitive (Martinez et al.,
2002a; Martinez et al.,
2002b). A list of these drugs, their mode of action and some of
their reported effects in cell culture and patients is shown in
Table 2.
|
There may be a skeleton in the closet, however, for anti-GSK-3 therapeutics. In addition to the problem of its broad range of functions, inhibition of the enzyme could presumably lead to enhanced accumulation of ß-catenin, a known oncogene. To mitigate this complication, drugs that selectively target non-axin-associated GSK-3 would be desirable, especially for chronic diseases, such as diabetes. On the positive side, long-term lithium treatment is not associated with enhanced incidence of cancer, although the lithium-treated patient cohort is not typical of the general population.
|
Perspectives |
|---|
|
TopSummaryIntroductionGSK-3 variantsGSK-3 phosphorylation: insights...Physiological roles for tyrosine...The role of GSK-3...GSK-3 and the Hedgehog...Other GSK-3 substratesOther GSK-3-binding proteinsGSK-3 and human diseaseSmall molecule inhibitors of...PerspectivesReferences |
|---|
Although our understanding of GSK-3 regulation is increasing, there are
still several outstanding issues: (1) putative GSK-3 substrates need to be
verified and classified as to whether they require priming phosphorylation;
(2) proteins that have been identified as GSK-3-interacting proteins require
characterization to identify sites of interaction and mechanisms that
regulating binding; (3) the mechanism of Wnt-mediated inactivation of GSK-3
remains to be established; (4) the basis for the specific requirement for
GSK-3ß in regulating NFB function remains unclear (GSK-3
does not substitute); and (5) the specificity of newly emerging GSK-3
inhibitors must be validated.
GSK-3 has reliably and regularly provided surprises ever since its discovery, and several of these have proven to be new signalling paradigms. There is little reason to doubt that there are more revelations in store, but with enhanced tools, such as selective inhibitors, knockout mice and modified binding proteins, there is hope that the true cellular roles for this multi-talented kinase will be appreciated and its utility as a therapeutic target be realized. But there are certainly easier molecules to study...
|
Acknowledgments |
|---|
|
Footnotes |
|---|
Also known as cell adhesion kinase beta; focal adhesion kinase 2; protein
tyrosine kinase 2 beta, cell adhsion kinase beta, calcium-dependent kinase,
realted adhesion focal tyrosine kinase. 
Frequently rearranged in advanced T-cell lymphocytes
|
References |
|---|
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TopSummaryIntroductionGSK-3 variantsGSK-3 phosphorylation: insights...Physiological roles for tyrosine...The role of GSK-3...GSK-3 and the Hedgehog...Other GSK-3 substratesOther GSK-3-binding proteinsGSK-3 and human diseaseSmall molecule inhibitors of...PerspectivesReferences |
|---|
Ali, A., Hoeflich, K. P. and Woodgett, J. R. (2001). Glycogen synthase kinase-3: properties, functions, and regulation. Chem. Rev. 101,2527 -2540.[CrossRef][Medline]
Amit, S., Hatzubai, A., Birman, Y., Andersen, J. S.,
Ben-Shushan, E., Mann, M., Ben-Neriah, Y. and Alkalay, I.
(2002). Axin-mediated CKI phosphorylation of beta-catenin at Ser
45: a molecular switch for the Wnt pathway. Genes Dev.
16,1066
-1076.
Armstrong, J. L., Bonavaud, S. M., Toole, B. J. and Yeaman, S.
J. (2001). Regulation of glycogen synthesis by amino acids in
cultured human muscle cells. J. Biol. Chem.
276,952
-956.
Aza-Blanc, P., Ramirez-Weber, F. A., Laget, M. P., Schwartz, C. and Kornberg, T. B. (1997). Proteolysis that is inhibited by hedgehog targets Cubitus interruptus protein to the nucleus and converts it to a repressor. Cell 89,1043 -1053.[CrossRef][Medline]
Ballou, L. M., Tian, P. Y., Lin, H. Y., Jiang, Y. P. and Lin, R.
Z. (2001). Dual regulation of glycogen synthase kinase-3beta
by the alpha1A-adrenergic receptor. J. Biol. Chem.
276,40910
-40916.
Barker, N., Morin, P. J. and Clevers, H. (2000). The Yin-Yang of TCF/beta-catenin signaling. Adv. Cancer Res. 77,1 -24.[Medline]
Bax, B., Carter, P. S., Lewis, C., Guy, A. R., Bridges, A., Tanner, R., Pettman, G., Mannix, C., Culbert, A. A., Brown, M. J. et al. (2001). The structure of phosphorylated GSK-3beta complexed with a peptide, FRATtide, that inhibits beta-catenin phosphorylation. Structure (Camb) 9,1143 -1152.[Medline]
Beals, C. R., Sheridan, C. M., Turck, C. W., Gardner, P. and
Crabtree, G. R. (1997). Nuclear export of NF-ATc enhanced by
glycogen synthase kinase-3. Science
275,1930
-1934.
Beg, A. A., Sha, W. C., Bronson, R. T., Ghosh, S. and Baltimore, D. (1995). Embryonic lethality and liver degeneration in mice lacking the RelA component of NF-kappa B. Nature 376,167 -170.[CrossRef][Medline]
Bellon, S., Fitzgibbon, M. J., Fox, T., Hsiao, H. M. and Wilson, K. P. (1999). The structure of phosphorylated p38gamma is monomeric and reveals a conserved activation-loop conformation. Structure Fold. Des. 7,1057 -1065.[Medline]
Benjamin, W. B., Pentyala, S. N., Woodgett, J. R., Hod, Y. and Marshak, D. (1994). ATP citrate-lyase and glycogen synthase kinase-3 beta in 3T3-L1 cells during differentiation into adipocytes. Biochem J. 300,477 -482.
Bhat, R. V., Shanley, J., Correll, M. P., Fieles, W. E., Keith,
R. A., Scott, C. W. and Lee, C. M. (2000). Regulation and
localization of tyrosine216 phosphorylation of glycogen synthase kinase-3beta
in cellular and animal models of neuronal degeneration. Proc. Natl.
Acad. Sci. USA 97,11074
-11079.
Bijur, G. N. and Jope, R. S. (2001).
Proapoptotic stimuli induce nuclear accumulation of glycogen synthase kinase-3
beta. J. Biol. Chem.
276,37436
-37442.
Bourouis, M., Moore, P., Ruel, L., Grau, Y., Heitzler, P. and Simpson, P. (1990). An early embryonic product of the gene shaggy encodes a serine/threonine protein kinase related to the CDC28/cdc2+ subfamily. EMBO J. 9,2877 -2884.[Medline]
Boyle, W. J., Smeal, T., Defize, L. H., Angel, P., Woodgett, J. R., Karin, M. and Hunter, T. (1991). Activation of protein kinase C decreases phosphorylation of c-Jun at sites that negatively regulate its DNA-binding activity. Cell 64,573 -584.[CrossRef][Medline]
Brady, M. J., Bourbonais, F. J. and Saltiel, A. R.
(1998). The activation of glycogen synthase by insulin switches
from kinase inhibition to phosphatase activation during adipogenesis in 3T3-L1
cells. J. Biol. Chem.
273,14063
-14066.
Brantjes, H., Barker, N., van Es, J. and Clevers, H. (2002). TCF: Lady Justice casting the final verdict on the outcome of Wnt signalling. Biol. Chem. 383,255 -261.[CrossRef][Medline]
Breton, J. J. and Chabot-Fletcher, M. C.
(1997). The natural product hymenialdisine inhibits interleukin-8
production in U937 cells by inhibition of nuclear factor-kappaB. J.
Pharmacol. Exp. Ther. 282,459
-466.
Brown, N. R., Noble, M. E., Endicott, J. A. and Johnson, L. N. (1999). The structural basis for specificity of substrate and recruitment peptides for cyclin-dependent kinases. Nat. Cell Biol. 1,438 -443.[CrossRef][Medline]
Canagarajah, B. J., Khokhlatchev, A., Cobb, M. H. and Goldsmith, E. J. (1997). Activation mechanism of the MAP kinase ERK2 by dual phosphorylation. Cell 90,859 -869.[CrossRef][Medline]
Carmichael, J., Sugars, K. L., Bao, Y. and Rubinsztein, D.
C. (2002). GSK-3beta inhibitors prevent cellular
polyglutamine toxicity caused by the Huntington's disease mutation.
J. Biol. Chem. 277,33791
-33798.
Chen, G., Huang, L. D., Jiang, Y. M. and Manji, H. K. (1999). The mood-stabilizing agent valproate inhibits the activity of glycogen synthase kinase-3. J. Neurochem. 72,1327 -1230.[CrossRef][Medline]
Chu, B., Soncin, F., Price, B. D., Stevenson, M. A. and
Calderwood, S. K. (1996). Sequential phosphorylation by
mitogen-activated protein kinase and glycogen synthase kinase 3 represses
transcriptional activation by heat shock factor-1. J. Biol.
Chem. 271,30847
-30857.
Coghlan, M. P., Culbert, A. A., Cross, D. A., Corcoran, S. L., Yates, J. W., Pearce, N. J., Rausch, O. L., Murphy, G. J., Carter, P. S., Roxbee Cox, L. et al. (2000). Selective small molecule inhibitors of glycogen synthase kinase-3 modulate glycogen metabolism and gene transcription. Chem. Biol. 7, 793-803.[CrossRef][Medline]
Cohen, P., Alessi, D. R. and Cross, D. A. (1997). PDK1, one of the missing links in insulin signal transduction? FEBS Lett. 410, 3-10.[CrossRef][Medline]
Cross, D. A., Alessi, D. R., Cohen, P., Andjelkovich, M. and Hemmings, B. A. (1995). Inhibition of glycogen synthase kinase-3 by insulin mediated by protein kinase B. Nature 378,785 -789.[CrossRef][Medline]
Cross, D. A., Culbert, A. A., Chalmers, K. A., Facci, L., Skaper, S. D. and Reith, A. D. (2001). Selective small-molecule inhibitors of glycogen synthase kinase-3 activity protect primary neurones from death. J. Neurochem. 77, 94-102.[Medline]
Dajani, R., Fraser, E., Roe, S. M., Young, N., Good, V., Dale, T. C. and Pearl, L. H. (2001). Crystal structure of glycogen synthase kinase 3 beta: structural basis for phosphate-primed substrate specificity and autoinhibition. Cell 105,721 -732.[CrossRef][Medline]
Damiens, E., Baratte, B., Marie, D., Eisenbrand, G. and Meijer, L. (2001). Anti-mitotic properties of indirubin-3'-monoxime, a CDK/GSK-3 inhibitor: induction of endoreplication following prophase arrest. Oncogene 20,3786 -37897.[CrossRef][Medline]
De Ferrari, G. V. and Inestrosa, N. C. (2000). Wnt signaling function in Alzheimer's disease. Brain Res Rev 33,1 -12.[CrossRef][Medline]
De Sarno, P., Li, X. and Jope, R. S. (2002). Regulation of Akt and glycogen synthase kinase-3beta phosphorylation by sodium valproate and lithium. Neuropharmacology 43,1158 -1164.[CrossRef][Medline]
Dent, P., Campbell, D. G., Hubbard, M. J. and Cohen, P. (1989). Multisite phosphorylation of the glycogen-binding subunit of protein phosphatase-1G by cyclic AMP-dependent protein kinase and glycogen synthase kinase-3. FEBS Lett. 248, 67-72.[CrossRef][Medline]
Dent, P., Lavoinne, A., Nakielny, S., Caudwell, F. B., Watt, P. and Cohen, P. (1990). The molecular mechanism by which insulin stimulates glycogen synthesis in mammalian skeletal muscle. Nature 348,302 -308.[CrossRef][Medline]
Detera-Wadleigh, S. D. (2001). Lithium-related genetics of bipolar disorder. Ann. Med. 33,272 -285.[Medline]
Diehl, J. A., Cheng, M., Roussel, M. F. and Sherr, C. J.
(1998). Glycogen synthase kinase-3beta regulates cyclin D1
proteolysis and subcellular localization. Genes Dev.
12,3499
-3511.
Dierick, H. and Bejsovec, A. (1999). Cellular mechanisms of wingless/Wnt signal transduction. Curr. Top. Dev. Biol. 43,153 -190.[Medline]
