shaggy
GSK-3 targets Dishevelled in neurons Dishevelled has been implicated in the regulation of cell fate decisions, cell polarity, and neuronal function. However, the mechanism of
Dishevelled action remains poorly understood. The cellular localization and function of the mouse Dishevelled protein,
DVL-1, has been examined. Endogenous DVL-1 colocalizes with axonal microtubules and sediments with brain microtubules. Expression of DVL-1 protects
stable microtubules from depolymerization by nocodazole in both dividing cells and differentiated neuroblastoma cells. Deletion analyses
reveal that the PDZ domain, but not the DEP domain, of DVL-1 is required for microtubule stabilization. The microtubule stabilizing
function of DVL-1 is mimicked by lithium-mediated inhibition of glycogen synthase kinase-3ß (GSK-3ß) and blocked by expression of
GSK-3ß. These findings suggest that DVL-1, through GSK-3ß, can regulate microtubule dynamics. This new function of DVL-1 in controlling microtubule stability
may have important implications for Dishevelled proteins in regulating cell polarity (Krylova, 2000).
DVL-1 regulates MT stability as expression of DVL-1 increases the level of acetylated MTs and protects MTs from depolymerization by nocodazole in COS and
neuroblastoma 2a cells. Similar results have been observed with microtubule-associated proteins such as Tau, MAP-1B, and MAP-2, which increase MT stability. Thus, DVL-1 acts as a MT stabilizing factor. How does DVL-1 regulate MT stability? Dishevelled proteins contain three conserved domains, a DIX domain also present in Axin; a negative regulator of the WNT signaling pathway; a PDZ domain present in a number of junctional proteins of the PSD-95 and Disc-large family, and a DEP domain present in the Caenorhabditis elegans protein Egl-10 and in Plecktrin. The PDZ domain is required for Armadillo stabilization through the canonical
WNT pathway. In contrast, the DEP domain, but not the PDZ domain, is required for JNK signaling. The PDZ domain is also required for MT stabilization, while deletion of the DIX domain has a weaker
effect. In contrast, deletion of the DEP domain does not affect the MT stabilizing function of DVL-1. The requirement of the PDZ domain suggests that DVL-1 may
be acting through the canonical pathway. Consistent with these findings, DVL-1 stabilizes MTs through the inhibition of GSK-3ß, a downstream component of the
canonical WNT pathway. This mechanism is supported by three findings: (1) expression of GSK-3ß blocks the MT stabilizing activity of DVL-1; (2)
dnGSK-3ß enhances DVL-1 function; (3) lithium, an inhibitor of GSK-3ß, mimics the MT stabilizing activity of DVL-1. Inhibition of
GSK-3ß by DVL-1 could lead to changes in the phosphorylation of microtubule-associated proteins such as Tau, MAP-1B, and MAP-2 that are direct targets of
GSK-3ß. Studies of Tau and MAP-1B show that GSK-3ß-mediated phosphorylation
decreases the ability of Tau to bind to MTs and decreases MT stabilization by MAP-1B. Thus,
inhibition of GSK-3ß by DVL-1 may change the ability of MAPs to stabilize MTs. Although inhibition of GSK-3ß by lithium stabilizes MTs, DVL-1 has a stronger
stabilizing effect than do high levels of lithium. Thus, it is likely that DVL-1 promotes MT stability via inhibition of GSK-3ß, but an alternative pathway may also
contribute to this function of DVL-1. Dishevelled has recently been found to colocalize with actin fibers. More importantly, Dishevelled
has been proposed to influence the actin cytoskeleton through the JNK pathway. Since MT dynamics is influenced by
actin, Dishevelled could regulate MTs indirectly through the JNK pathway by changing the actin cytoskeleton. However, this mechanism seems unlikely, because the DEP
domain of DVL-1, essential for the JNK pathway, is not required for MT stability. Consequently, these data suggest that MT stabilization by DVL-1 is mediated
through GSK-3ß signaling and a parallel, as yet unidentified, pathway (Krylova, 2000).
In maturing neurons, endogenous DVL-1 colocalizes with axonal MTs that contain high levels of stable MTs. More importantly, DVL-1 increases the level of stable
MTs in differentiated NB2a neuroblastoma cells and protects axonal MTs against nocodazole treatment. These findings suggest that neuronal DVL-1 may stabilize
axonal MTs in vivo. Consistent with this view, a higher proportion of endogenous DVL-1 becomes associated with MTs from adult brain samples, which are
enriched in stable MTs. Moreover, DVL-1 sediments along with cold stable MT fractions; these fractions represent the most stable pool of MTs. Thus, the localization of DVL-1 to
MTs correlates with increased MT stability in maturing axons. Further studies are needed to address the in vivo role of DVL-1 in microtubule dynamics in developing
neurons (Krylova, 2000).
Alzheimer's disease is associated with increased production and
aggregation of amyloid-ß (Aß) peptides. Aß peptides are derived
from the amyloid precursor protein (APP) by sequential proteolysis,
catalysed by the aspartyl protease BACE, followed by
presenilin-dependent gamma-secretase cleavage. Presenilin interacts
with nicastrin, APH-1 and PEN-2, all of which are
required for gamma-secretase function. Presenilins also interact
with alpha-catenin, ß-catenin and glycogen synthase kinase-3ß
(GSK-3ß), but a functional role for these proteins in
gamma-secretase activity has not been established. Therapeutic concentrations of lithium, a GSK-3 inhibitor,
block the production of Aß peptides by interfering with APP
cleavage at the gamma-secretase step, but do not inhibit Notch processing.
Importantly, lithium also blocks the accumulation of Aß
peptides in the brains of mice that overproduce APP. The target of
lithium in this setting is GSK-3alpha, which is required for maximal
processing of APP. Since GSK-3 also phosphorylates tau protein,
the principal component of neurofibrillary tangles, inhibition of
GSK-3alpha offers a new approach to reduce the formation of both
amyloid plaques and neurofibrillary tangles, two pathological
hallmarks of Alzheimer's disease (Phiel, 2003).
In summary, this study shows that GSK-3alpha facilitates APP processing
and that lithium inhibits the generation of Aß peptides through
inhibition of GSK-3alpha. In support of this conclusion: (1) lithium
reduces Aß production in cultured cells and in the brains of
mice that overproduce Aß peptides; (2) kenpaullone, an alternative
GSK-3alpha inhibitor, also inhibits Aß production; (3) RNAi-mediated
depletion of GSK-3alpha reduces Aß production, and (4) moderate
overexpression of GSK-3alpha increases Aß production. Lithium inhibits the GSK-3-mediated phosphorylation of tau, which, in its
hyperphosphorylated state, is the main component of neurofibrillary
tangles. Thus, GSK-3alpha offers an attractive target for pharmacological agents aimed at reducing the formation of amyloid
plaques and neurofibrillary tangles, the pathological hallmarks of
Alzheimer's disease. Lithium also protects neurons from proapoptotic
stimuli and could therefore reduce neuronal cell death
associated with Alzheimer's disease. Lithium has been used for
more than 50 years to treat bipolar disorder, but has a narrow
therapeutic window and a higher frequency of side effects in older
patients. Thus, although lithium might be considered for the
prevention of Alzheimer's disease, especially in younger patients
with FAD mutations or Down's syndrome, new agents that specifically target GSK-3alpha may prove to be valuable in the treatment of
Alzheimer's disease (Phiel, 2003).
Other GSK-3ß interactions Notch receptors modulate transcriptional targets following the proteolytic release of the Notch intracellular domain (NotchIC). Phosphorylated forms of NotchIC have been identified within the nucleus and have been associated with CSL members, as well as correlated with regions of the receptor that are required for activity. Genetic studies have suggested that Shaggy, the Drosophila homolog of glycogen synthase kinase-3ß (GSK3ß) may act as a positive modulator of the Notch signaling. GSK3ß is a serine/threonine kinase and is a component of the Wnt/wingless signaling cascade. GSK3ß is able to bind and phosphorylate Notch1IC in vitro, and attenuation of GSK3ß activity reduces phosphorylation of NotchIC in vivo. Functionally, ligand-activated signaling through the endogenous Notch1 receptor is reduced in GSK3ß null fibroblasts, implying a positive role for GSK3ß in mammalian Notch signaling. As a possible mechanistic explanation of the effect of GSK3ß on Notch signaling, it was observed that inhibition of GSK3ß shortens the half-life of Notch1IC. Conversely, activated GSK3ß reduces the quantity of Notch1IC that was degraded by the proteasome. These studies reveal that GSK3ß modulates Notch1 signaling, possibly through direct phosphorylation of the intracellular domain of Notch, and that the activity of GSK3ß protects the intracellular domain from proteasome degradation (Foltz, 2002).
Wnt and its intracellular effector ß-catenin regulate developmental and oncogenic processes. Using expression cloning to identify novel components of the Wnt pathway, casein kinase Iepsilon (CKIepsilon) has been identified.
CKIepsilon mimics Wnt in inducing a secondary axis in Xenopus, stabilizing ß-catenin, and stimulating gene transcription in cells. Inhibition of endogenous CKIepsilon by kinase-defective CKIepsilon or CKIepsilon
antisense-oligonucleotides attenuates Wnt signaling. CKIepsilon is in a complex with axin and other downstream components of the Wnt pathway, including Dishevelled. CKIepsilon appears to be a positive regulator of the pathway and a link between upstream signals and the complexes that regulate ß-catenin (Sakanaka, 1999).
Some of the downstream molecules in the Wnt pathway have been shown to
form complexes containing negative regulators (GSK-3ß, axin, and
adenomatous polyposis coli tumor suppressor protein) and a positive
regulator (ß-catenin) in vivo. The
possibility that CKIepsilon is also in a complex with these molecules was examined. Axin, a negative regulator of the Wnt pathway, binds to
CKIepsilon. Endogenous CKIepsilon coimmunoprecipitates with
overexpressed axin. Because the C-terminal domain is unique for CKIepsilon/delta isoforms, a test was performed to see whether deletion of this
domain alters activity. Binding of axin to DeltaC-CKIepsilon
and DeltaC-KN-CKIepsilon (KN stands for Kinase Negative) is much reduced compared to wild-type CKIepsilon and
KN-CKIepsilon. These results suggest that the
C-terminal domain of CKIepsilon is important for its interaction with axin,
which may be the reason that DeltaC-CKIepsilon and CKIalpha did not activate
the Wnt pathway in either Xenopus or mammalian cells. To further study the complex of CKIepsilon with axin and GSK-3ß, it was
shown that GSK-3ß coimmunoprecipitates with CKIepsilon, but much less
with DeltaC-CKIepsilon, and only in the presence of axin. This finding suggests that CKIepsilon is a positive regulatory molecule in the Wnt pathway and its interaction through its
C terminus with the axin-GSK-3ß complex is likely to be important for
its activity. Furthermore, endogenous
CKIepsilon has been detected in the complex with overexpressed Dvl3, an
upstream molecule of the axin-GSK-3ß complex (Sakanaka, 1999).
Heat shock transcription factor 1 (HSF-1) activates the transcription of heat shock genes in eukaryotes. Under normal physiological growth conditions, HSF-1 is a monomer. Its transcriptional activity is repressed by constitutive phosphorylation. Upon activation, HSF-1 forms trimers, acquires DNA binding activity, increases transcriptional activity, and appears as punctate granules in the nucleus. In this study, using bromouridine incorporation and confocal laser microscopy, it has been demonstrated that newly synthesized pre-mRNAs colocalize to the HSF-1 punctate granules after heat shock, suggesting that these granules are sites of transcription. Evidence that glycogen synthase kinase 3ß (GSK-3ß) and extracellular signal-regulated kinase mitogen-activated protein kinase (ERK MAPK) participate in the down regulation of HSF-1 transcriptional activity. Transient increases in the expression of GSK-3ß facilitate the disappearance of HSF-1 punctate granules and reduce hsp-70 transcription after heat shock. ERK is shown to be the priming kinase for GSK-3ß. Taken together, these results indicate that GSK-3ß and ERK MAPK facilitate the inactivation of activated HSF-1 after heat shock by dispersing HSF-1 from the sites of transcription (He, 1998).
GSK-3 consists of two isoforms: GSK-3alpha (51 kDa) and GSK-3ß (46 kDa). It was first identified as an enzymatic activity that phosphorylates and inactivates glycogen synthase. A second role of GSK-3
was found when studies showed that inhibition of phosphatase type I activity is relieved when GSK-3
phosphorylates phosphatase inhibitor 2. At least 15 other substrates have been reported to be
phosphorylated by GSK-3, including the transcription factors c-Jun, JunD, c-myb, c-myc, L-myc,
CREB, and NF-AT, most of which become inactivated when phosphorylated by GSK-3.
GSK-3 tends to phosphorylate serine/threonine residues located next to a proline which, in turn, is near
another serine residue that has been prephosphorylated by some other protein kinase (referred to as
priming kinase). GSK-3 is constitutively active and, as a result, suppresses many of its
substrates under normal physiological growth conditions. It appears that the activity of HSF-1 can be down regulated by protein kinases that are activated by
diverse signal transduction pathways. The ERK MAPK pathway is activated during cell growth and
development by multiple signaling pathways, which in turn are activated by growth factor receptors,
G protein-coupled receptors, ceramide production, and a protein kinase C-dependent pathway. Recent evidence has suggested that ERK MAPK activation by heat
shock may be through ceramide activation of protein kinase Raf-1. The pathways leading to
GSK-3ß regulation are complex. GSK-3ß activity is down regulated by PKB/Akt, p70S6K (see Drosophila RPS6-p70-protein kinase), or p90rsk as
a result of phosphorylation on serine residues. Activation of PKB/Akt leads to
increased cell survival, as is the case with activation of ERK. The ability of ERK to mediate cell
survival is dependent on the activation of transcription factors such as Elk1 and repair of damaged
proteins. The ability of PKB/Akt to mediate cell survival is likely to be dependent on downstream
effectors such as p70S6K and protein translation, activation of FRAP/TOR, and inhibition of GSK-3ß. Interestingly, heat shock stimulates the activity of GSK-3ß and ERK MAPK. The increase in
GSK-3ß activity may occur through its phosphorylation on a tyrosine residue by an unknown tyrosine
kinase. Thus, it appears that when activated, HSF-1 reduces the expression of most other genes and
must be inactivated in a timely manner for cell proliferation to continue. The cell has developed an
elegant mechanism for doing this, since some of the enzymes that control cell proliferation are capable
of inactivating HSF-1 (He, 1998 and references).
Activation of the Ras/Raf/ERK pathway extends the half-life of the Myc protein and thus enhances
the accumulation of Myc activity. Investigated were two N-terminal phosphorylation sites in
Myc, Thr 58 and Ser 62, known to be regulated by mitogen stimulation. Phosphorylation of these
two residues is critical for determining the stability of Myc. Phosphorylation of Ser 62 is required for Ras-induced stabilization of
Myc, likely mediated through the action of ERK. Conversely, phosphorylation of Thr 58, likely mediated by GSK-3 but dependent
on the prior phosphorylation of Ser 62, is associated with degradation of Myc. Further analysis demonstrates that the Ras-dependent PI-3K pathway is also
critical for controlling Myc protein accumulation, likely through the control of GSK-3 activity. These observations thus define a synergistic role for multiple
Ras-mediated phosphorylation pathways in the control of Myc protein accumulation during the initial stage of cell proliferation (Sears, 2000).
The amino acid sequence surrounding Ser 62 represents a consensus ERK recognition sequence, and evidence has been presented that ERK can
mediate the phosphorylation of Myc at Ser 62. Mutation of Ser 62 prevents mitogen- and Ras-induced stabilization of Myc. Moreover, phosphorylation at Ser 62 is enhanced under conditions
where Myc is stabilized. The importance of Ser 62 in the control of Myc stability is seen in the strict requirement
for the stabilization of Myc by Ras, but seen from work that has demonstrated an impaired transforming function when Ser 62 is altered. In contrast, phosphorylation at Thr 58 coincides with a decreased stability of Myc and mutations that prevent Thr 58 phosphorylation lead to stable Myc
protein. Once again, this coincides with work that has shown that alteration of Thr 58 enhances the transforming activity of Myc and that mutations at this site are common in Myc proteins derived from tumors. Various
lines of work suggest that the GSK-3 protein kinase is most likely responsible for the phosphorylation of Myc at Thr 58. Thr 58 lies within an established
consensus, and GSK-3 has been shown to phosphorylate Thr 58 in Myc in vitro. However,
unlike ERK, which is tightly regulated by cell growth, the level of GSK-3 protein is constant and does not fluctuate with cell growth. Nevertheless, despite the
continual presence of GSK-3 protein, the activity of the kinase is regulated during the initial phase of cell proliferation. In particular,
GSK-3 activity is inhibited through the action of PI-3K/AKT. Thus, as Ras initiates the PI-3K/AKT pathway, GSK-3 activity is held
in check, preventing the phosphorylation of Thr 58. Only when AKT activity declines would GSK-3 then have the capacity to phosphorylate Thr 58 to induce
the degradation of Myc. Thus, Ras activation elicits two responses within the cell that can cooperate to enhance Myc stability: a direct effect of ERK and an
indirect effect of AKT (Sears, 2000 and references therein).
Integrin-linked kinase (ILK) is an ankyrin-repeat containing serine-threonine protein kinase capable of interacting with the
cytoplasmic domains of integrin ß1, ß2, and ß3 subunits. Overexpression of ILK in epithelial cells disrupts
cell-extracellular matrix as well as cell-cell interactions; suppresses suspension-induced apoptosis (also called Anoikis), and
stimulates anchorage-independent cell cycle progression. In addition, ILK induces nuclear translocation of ß-catenin,
where the latter associates with a T cell factor/lymphocyte enhancer-binding factor 1 (TCF/LEF-1) to form an activated
transcription factor. ILK activity is rapidly, but transiently, stimulated upon the attachment of cells to
fibronectin, as well as by insulin, in a phosphoinositide-3-OH kinase [Pi(3)K]-dependent manner. Furthermore,
phosphatidylinositol(3,4,5)trisphosphate specifically stimulates the activity of ILK in vitro, and in addition, membrane
targeted constitutively active Pi(3)K activates ILK in vivo. ILK is an upstream effector of the
Pi(3)K-dependent regulation of both protein kinase B (PKB/AKT) and glycogen synthase kinase 3 (GSK-3). Specifically,
ILK can directly phosphorylate GSK-3 in vitro and when either stably or transiently overexpressed in cells, can inhibit GSK-3
activity, whereas the overexpression of kinase-deficient ILK enhances GSK-3 activity. In addition, kinase-active ILK can
phosphorylate PKB/AKT on serine-473, whereas kinase-deficient ILK severely inhibits endogenous phosphorylation of
PKB/AKT on serine-473, demonstrating that ILK is involved in agonist stimulated, Pi(3)K-dependent, PKB/AKT
activation. ILK is thus a receptor-proximal effector for the Pi(3)K-dependent, extracellular matrix and growth factor
mediated activation of PKB/AKT, and the inhibition of GSK-3 (Delcommenne 1998).
Families bearing mutations in the presenilin 1 (PS1) gene develop Alzheimer's disease. Previous studies have shown that the
Alzheimer-associated mutations in PS1 increase production of amyloid ß protein (Aß1-42). PS1 is shown also
to regulate phosphorylation of the microtubule-associated protein tau. PS1 directly binds tau and a tau kinase, glycogen synthase
kinase 3ß (GSK-3ß). Deletion studies show that both tau and GSK-3ß bind to the same region of PS1, residues
250-298, whereas the binding domain on tau is the microtubule-binding repeat region. The ability of PS1 to bring tau and
GSK-3ß into close proximity suggests that PS1 may regulate the interaction of tau with GSK-3ß. Mutations in PS1 that
cause Alzheimer's disease increase the ability of PS1 to bind GSK-3ß and, correspondingly, increase its tau-directed kinase
activity. It is proposed that the increased association of GSK-3ß with mutant PS1 leads to increased phosphorylation of tau (Takashima, 1998).
WNT-7a induces axonal spreading and branching in developing cerebellar granule neurons. This effect is mediated through
the inhibition of GSK-3ß, a serine/threonine kinase and a component of the WNT pathway. Lithium, an inhibitor of
GSK-3ß, mimics WNT-7a in granule cells. The effect of GSK-3ß inhibition on cytoskeletal
re-organization. Lithium induces axonal spreading and increases growth cone area and perimeter. This effect is associated
with the absence or reduction of stable microtubules in spread areas. Lithium induces the loss of a phosphorylated form of
MAP-1B, a microtubule associated protein involved in axonal outgrowth. Down-regulation of the phosphorylated
MAP-1B, MAP-1B-P, from axonal processes occurs before axonal remodelling is evident. In vitro phosphorylation assays
show that MAP-1B-P is generated by direct phosphorylation of MAP-1B by GSK-3ß. WNT-7a, like lithium, also leads
to loss of MAP-1B-P from spread axons and growth cones. These data suggest that WNT-7a and lithium induce changes in
microtubule dynamics by inhibiting GSK-3ß that in turn lead to changes in the phosphorylation of MAP-1B. These
findings suggest a novel role for GSK-3ß and WNTs in axonal remodelling and identify MAP-1B as a new target for
GSK-3ß and WNT (Lucas, 1998).
Glycogen synthase kinase-3ß (GSK-3ß) has been postulated to mediate the pathogenic effects of tau hyperphosphorylation,
ß-amyloid-induced neurotoxicity and presenilin-1 mutation in Alzheimer's disease. By using the tet-regulated system,
conditional transgenic mice overexpressing GSK-3ß in the brain during adulthood were produced while avoiding perinatal lethality due to embryonic
transgene expression. These mice show decreased levels of nuclear ß-catenin and hyperphosphorylation of the microtubule-associated protein tau in hippocampal neurons, the latter resulting in pretangle-like somatodendritic localization of tau. Neurons displaying somatodendritic localization of tau often show
abnormal morphologies and detachment from the surrounding neuropil. Reactive astrocytosis and microgliosis were also indicative of
neuronal stress and death. This was further confirmed by TUNEL and cleaved caspase-3 immunostaining of dentate gyrus granule cells. These results demonstrate that
in vivo overexpression of GSK-3ß results in neurodegeneration and suggest that these mice can be used as an animal model to study the relevance of GSK-3ß
deregulation to the pathogenesis of Alzheimer's disease (Lucas, 2001).
Somatodendritic tau in Tet/GSK-3ß mice is often associated with the endoplasmic reticulum. Similar results were found in transgenic mice
overexpressing the shortest isoform of tau. In all of these cases, immunodetection was performed with antibodies that recognize phosphorylation or conformation epitopes found in PHF-tau. Interestingly, paired helical filaments in AD brains are often found arising from the endoplasmic reticulum and other membrane structures. It is therefore possible that, in animal models, association of tau with the endoplasmic reticulum represents an early stage in the formation of neurofibrillary lesions. An additional and compatible explanation for the
association of tau with the endoplasmic reticulum might be its interaction with PS-1. PS-1 has been found to bind both tau and GSK-3ß in co-immunoprecipitation
experiments performed on human brain extracts, and PS-1 is located predominantly in the endoplasmic reticulum and in the Golgi apparatus (Lucas, 2001 and references therein).
ß-catenin is a central component of the cadherin cell adhesion complex and plays an essential role in
the Wingless/Wnt signaling pathway. In the current model of this pathway, the amount of ß-catenin
(or its invertebrate homolog Armadillo) is tightly regulated and its steady-state level outside the
cadherin-catenin complex is low in the absence of Wingless/Wnt signal. The
ubiquitin-dependent proteolysis system is involved in the regulation of ß-catenin turnover.
ß-catenin, but not E-cadherin, p120(cas) or alpha-catenin, becomes stabilized when
proteasome-mediated proteolysis is inhibited and this leads to the accumulation of multi-ubiquitinated
forms of ß-catenin. Ubiquitination is inhibited and the protein stabilized with the substitution of the serine residues in
the glycogen synthase kinase 3ß (GSK3ß) phosphorylation consensus motif of ß-catenin. This motif in ß-catenin resembles a
motif in IkappaB (inhibitor of NFkappaB) that is required for the phosphorylation-dependent
degradation of IkappaB via the ubiquitin-proteasome pathway. Ubiquitination of
ß-catenin is greatly reduced in Wnt-expressing cells, providing the first evidence that the
ubiquitin-proteasome degradation pathway may act downstream of GSK3ß in the regulation of
ß-catenin (Aberle, 1997).
ß-catenin, a member of the Armadillo repeat protein family, binds directly to the cytoplasmic domain of E-cadherin, linking it via alpha-catenin to the actin cytoskeleton. A 30-amino acid region within the cytoplasmic domain of E-cadherin, conserved among all classical cadherins, has been shown to be essential for ß-catenin binding. This region harbors several putative casein kinase II (CKII) and glycogen synthase kinase-3ß (GSK-3ß) phosphorylation sites and is highly phosphorylated. In vitro this region is indeed phosphorylated by CKII and GSK-3ß, which results in an increased binding of ß-catenin to E-cadherin. Additionally, in mouse NIH3T3 fibroblasts expression of E-cadherin with mutations in putative CKII sites results in reduced cell-cell contacts. Thus, phosphorylation of the E-cadherin cytoplasmic domain by CKII and GSK-3ß appears to modulate the affinity between ß-catenin and E-cadherin, ultimately modifying the strength of cell-cell adhesion (Lickert, 2000).
E-cadherin is a major adherens junction protein of epithelial cells, with a central role in cell-cell adhesion and cell polarity. Newly synthesized E-cadherin is targeted to the basolateral cell surface. Targeting information in the cytoplasmic tail of E-cadherin was analyzed by utilizing chimeras of E-cadherin fused to the ectodomain of the interleukin-2alpha (IL-2alpha) receptor expressed in Madin-Darby canine kidney and LLC-PK(1) epithelial cells. Chimeras containing the full-length or membrane-proximal half of the E-cadherin cytoplasmic tail were correctly targeted to the basolateral domain. Sequence analysis of the membrane-proximal tail region revealed the presence of a highly conserved dileucine motif, which was analyzed as a putative targeting signal by mutagenesis. Elimination of this motif resulted in the loss of Tac/E-cadherin basolateral localization, pinpointing this dileucine signal as being both necessary and sufficient for basolateral targeting of E-cadherin. Truncation mutants unable to bind beta-catenin were correctly targeted, showing, contrary to current understanding, that beta-catenin is not required for basolateral trafficking. These results also provide evidence that dileucine-mediated targeting is maintained in LLC-PK(1) cells despite the altered polarity of basolateral proteins with tyrosine-based signals in this cell line. These results provide the first direct insights into how E-cadherin is targeted to the basolateral membrane (Miranda, 2001).
Activation of phosphatidylinositide 3'-OH kinase (PI 3-kinase) is implicated in mediating a variety of growth factor-induced responses, among which are the
inactivation of glycogen synthase kinase-3 (GSK-3) and the activation of the serine/threonine protein kinase B (PKB). GSK-3 inactivation occurs through
phosphorylation of Ser-9, and several kinases, such as protein kinase C, mitogen-activated protein kinase-activated protein kinase-1 [p90(Rsk)], p70(S6kinase),
and also PKB have all been shown to phosphorylate this site in vitro. In the light of the many candidates to mediate insulin-induced GSK-3 inactivation, the role of PKB has been investigated by constructing a PKB mutant that exhibits dominant-negative function (inhibition of growth factor-induced activation of PKB at
expression levels similar to wild-type PKB), because currently no such mutant has been reported. The PKB mutant (PKB-CAAX) acts as an efficient
inhibitor of PKB activation and also of insulin-induced GSK-3 regulation. Furthermore, it is shown that PKB and GSK-3 co-immunoprecipitate, indicating a direct
interaction between GSK-3 and PKB. An additional functional consequence of this interaction is implicated by the observation that the oncogenic form of PKB,
gagPKB, induces a cellular relocalization of GSK-3 from the cytosolic to the membrane fraction. These results demonstrate that PKB activation is both necessary and
sufficient for insulin-induced GSK-3 inactivation and establish a linear pathway from insulin receptor to GSK-3. Regulation of GSK-3 by PKB is likely through
direct interaction, since both proteins co-immunoprecipitate. This interaction also results in a translocation of GSK-3 to the membrane in cells expressing transforming
gagPKB (van Weeren 1998).
Dorsal accumulation of ß-catenin in early Xenopus embryos is required for body axis formation. Recent
evidence indicates that ß-catenin is dorsally stabilized by the localized inhibition of the kinase Xgsk-3,
utilizing a novel Wnt ligand-independent mechanism. Using a two-hybrid screen, GBP was identified, a
maternal Xgsk-3-binding protein that is homologous to a T cell protooncogene in three well-conserved
domains. GBP inhibits in vivo phosphorylation by Xgsk-3, and ectopic GBP expression induces an axis
by stabilizing ß-catenin within Xenopus embryos. An in vivo GSK-3 assay was used that takes advantage of the observation that the microtubule-binding protein, tau, is a GSK-3 substrate, and antibodies specific for the GSK-3-phosphorylated
form of tau are available. Oocytes expressing GSK-3 were injected with recombinant tau protein, and 2 hr later, extracts from oocytes were
analyzed by Western blotting using either anti-tau antibodies T14/46 that recognize all
forms of tau or an antibody specific for the GSK-3 phosphorylated form of tau. In the presence of Xgsk-3, tau migrates as a series of
higher molecular weight forms that are absent when Xgsk-3 is not present. The
slower migrating forms are due to phosphorylation by Xgsk-3, as shown with the antibody specific for GSK-3
phosphorylated tau. Coinjection of GBP eliminates the highest molecular weight forms of tau and reduces the level of GSK-3-specific phosphorylation 5-fold,
whereas a GBP mutant has no effect, either on the mobility or GSK-3-specific phosphorylation of tau. These results demonstrate that GBP functions to inhibit the ability of Xgsk-3 to
phosphorylate protein substrates and that this inhibition requires binding to Xgsk-3. Two recently identified proteins, Frat1/FRAT1 and FRAT2, have provided
important insight into the structure and function of GBP. The Frat proteins share three well-conserved regions with GBP
that are predicted to be important for the activity and regulation of the Frats and GBP. Region III,
which is the most highly conserved domain, contains the GSK-3 binding and inhibitory activities. A C-terminal fragment
of GBP, which lacks domains I and II, binds Xgsk-3 and induces an ectopic axis: replacement of two adjacent
residues in region III abrogates both Xgsk-3 binding and axis induction. A fragment of FRAT2 containing
domains II and III binds Xgsk-3 in vivo and induces an ectopic axis, demonstrating that FRAT2 and GBP share the same
function, which is most likely due to the conserved amino acids in domain III. Antisense oligonucleotide depletion of
the maternal GBP mRNA demonstrates that GBP is required for the establishment of the dorsal-ventral axis
in Xenopus embryos. These results define a family of GSK-3-binding proteins with roles in development
and cell proliferation (Yost, 1998).
The Axin-dependent phosphorylation of ß-catenin catalysed by glycogen synthase kinase-3 (GSK3) is inhibited during embryogenesis. This protects ß-catenin
against ubiquitin-dependent proteolysis, leading to its accumulation in the nucleus, where it controls the expression of genes important for development. Frequently
rearranged in advanced T-cell lymphomas 1 (FRAT1) is a mammalian homolog of a GSK3-binding protein (GBP), which appears to play a key role in the correct
establishment of the dorsal-ventral axis in Xenopus laevis. FRATtide (a peptide corresponding to residues 188-226 of FRAT1) binds to
GSK3 and prevents GSK3 from interacting with Axin. FRATtide also blocks the GSK3-catalysed phosphorylation of Axin and ß-catenin, suggesting a potential
mechanism by which GBP could trigger axis formation. In contrast, FRATtide does not suppress GSK3 activity towards other substrates, such as glycogen synthase
and eIF2B, whose phosphorylation is independent of Axin but dependent on a 'priming' phosphorylation. This may explain how the essential cellular functions of
GSK3 can continue, despite the suppression of ß-catenin phosphorylation (Thomas, 1999).
The Frat1 gene was first identified as a proto-oncogene involved in progression of mouse T cell lymphomas. More recently, FRAT/GBP
(GSK-3 ß Binding Protein) family members have been recognized as critical components of the Wnt signal transduction pathway. In an
attempt to gain more insight into the function of Frat1, Frat1-deficient mice were generated in which most of the coding domain was
replaced by a promoterless ß-galactosidase reporter gene. While the pattern of LacZ expression in Frat1
lacZ/+
mice indicates that Frat1 is
expressed in various neural and epithelial tissues, homozygous Frat1lacZ
mice are apparently normal, healthy and fertile. Tissues of
homozygous Frat1lacZ
mice show expression of a second mouse Frat gene, designated Frat3. The Frat1 and Frat3 proteins are structurally
and functionally very similar, since both Frat1 and Frat3 are capable of inducing a secondary axis in Xenopus embryos. The overlapping
expression patterns of Frat1 and Frat3 during murine embryogenesis suggest that the apparent dispensability of Frat1 for proper development may be due to the presence of a second mouse gene encoding a functional Frat protein (Jonkers, 1999).
The Axin-dependent phosphorylation of ß-catenin catalysed by glycogen synthase kinase-3 (GSK3) is inhibited during embryogenesis. This protects ß-catenin against ubiquitin-dependent proteolysis, leading to its accumulation in the nucleus, where it
controls the expression of genes important for development. Frequently rearranged in advanced T-cell lymphomas 1 (FRAT1) is a
mammalian homolog of a GSK3-binding protein (GBP) that appears to play a key role in the correct establishment of the
dorsal-ventral axis in Xenopus laevis. FRATtide (a peptide corresponding to residues 188-226 of FRAT1)
binds to GSK3 and prevents GSK3 from interacting with Axin. FRATtide also blocks the GSK3-catalysed phosphorylation of Axin
and ß-catenin, suggesting a potential mechanism by which GBP could trigger axis formation. In contrast, FRATtide does not
suppress GSK3 activity toward other substrates, such as glycogen synthase and eIF2B, whose phosphorylation is independent of Axin but dependent on a 'priming' phosphorylation. This may explain how the essential cellular functions of GSK3 can continue, despite the suppression of ß-catenin phosphorylation (Thomas, 1999).
The activities of cyclin D-dependent kinases serve to integrate extracellular signaling during G1 phase with the
cell-cycle engine that regulates DNA replication and mitosis. Induction of D-type cyclins and their assembly into
holoenzyme complexes depends on mitogen stimulation. Conversely, the fact that D-type cyclins are labile proteins
guarantees that the subunit pool shrinks rapidly when cells are deprived of mitogens. Phosphorylation of cyclin D1 on
a single threonine residue near the carboxyl terminus (Thr-286) positively regulates proteasomal degradation of D1. Glycogen synthase kinase-3ß (GSK-3ß) phosphorylates cyclin D1 specifically on
Thr-286, thereby triggering rapid cyclin D1 turnover. Because the activity of GSK-3ß can be inhibited by signaling
through a pathway that sequentially involves first Ras, then phosphatidylinositol-3-OH kinase (PI3K: see Drosophila Pi3K92E), and finally protein kinase B
(Akt), the turnover of cyclin D1, like its assembly, is also Ras dependent and, hence, mitogen regulated. In contrast,
Ras mutants defective in PI3K signaling, or constitutively active mitogen-activated protein kinase-kinase (MEK1)
mutants that act downstream of Ras to activate extracellular signal-regulated protein kinases (ERKs), cannot stabilize
cyclin D1. In direct contrast to cyclin D1, which accumulates in the nucleus during G1 phase and exits into the
cytoplasm during S phase, GSK-3ß is predominantly cytoplasmic during G1 phase, but a significant fraction enters
the nucleus during S phase. A highly stable D1 mutant (in which an alanine is substituted for the threonine at position
286 and is refractory to phosphorylation by GSK-3ß) remains in the nucleus throughout the cell cycle.
Overexpression of an active, but not a kinase-defective, form of GSK-3ß in mouse fibroblasts causes a
redistribution of cyclin D1 from the cell nucleus to the cytoplasm. Therefore, phosphorylation and proteolytic turnover
of cyclin D1 and its subcellular localization during the cell division cycle are linked through the action of GSK-3ß (Diehl, 1998).
GSK-3ß-dependent phosphorylation of cyclin D1 at Thr-286 promotes the nuclear-to-cytoplasmic redistribution of cyclin
D1 during S phase of the cell cycle, but how phosphorylation regulates redistribution has not been resolved. For example,
phosphorylation of nuclear cyclin D1 could increase its rate of nuclear export relative to nuclear import; alternatively, phosphorylation of
cytoplasmic cyclin D1 by GSK-3ß could inhibit nuclear import. GSK-3ß-dependent
phosphorylation is shown in this study to promote cyclin D1 nuclear export by facilitating the association of cyclin D1 with the nuclear exportin CRM1. D1-T286A, a cyclin D1 mutant that
cannot be phosphorylated by GSK-3ß, remains nuclear throughout the cell cycle, a consequence of its reduced binding to CRM1. Constitutive
overexpression of the nuclear cyclin D1-T286A in murine fibroblasts results in cellular transformation and promotes tumor growth in immune compromised mice.
Thus, removal of cyclin D1 from the nucleus during S phase appears essential for regulated cell division (Alt, 2000).
Overexpression of cyclin D1 is a common event in various forms of cancer. D1 can be overexpressed
as a result of gene amplification or because it is targeted through chromosomal translocations. However, in certain tumors, high levels of cyclin D1 expression have not been
explained by such mechanisms, and events affecting cyclin D1 turnover might play some role. Although
the p16INK4a-cyclin D1-Rb pathway is disabled in many forms of human cancer, colon carcinomas
provide a conspicuous exception. Inactivation of the adenomatous polyposis coli (APC) tumor
suppressor is the single most common event in colon cancer. APC is a target of
Wnt signaling, and it regulates the proteolytic turnover
of ß-catenin in a manner that depends on phosphorylation of ß-catenin by GSK-3. ß-Catenin mutants that have lost GSK-3 phosphorylation sites remain
constitutively active as coactivators of TCF/LEF-dependent transcription, and such mutations have now
been found in the major fraction of colon cancers that lack mutated APC alleles. The fact that GSK-3 can also regulate cyclin D1 turnover suggests that
deregulation of Wnt signaling in colon cancer may target cyclin D1 in addition to the APC-ß-catenin
complex (Diehl, 1998 and references).
To characterize the contribution of glycogen synthase kinase 3ß (GSK3ß) inactivation to insulin-stimulated glucose metabolism,
wild-type (WT-GSK), catalytically inactive (KM-GSK), and uninhibitable (S9A-GSK) forms of GSK3ß were expressed in
insulin-responsive 3T3-L1 adipocytes using adenovirus technology. WT-GSK, but not KM-GSK, reduces basal and
insulin-stimulated glycogen synthase activity without affecting the stimulation of the enzyme by insulin. S9A-GSK similarly
decreases cellular glycogen synthase activity, but also partially blocks insulin stimulation of the enzyme. S9A-GSK expression also
markedly inhibits insulin stimulation of IRS-1-associated phosphatidylinositol 3-kinase activity, but only weakly inhibits
insulin-stimulated Akt/PKB phosphorylation and glucose uptake, with no effect on GLUT4 translocation. To further evaluate the role
of GSK3ß in insulin signaling, the GSK3ß inhibitor lithium was used to mimic the consequences of insulin-stimulated
GSK3ß inactivation. Although lithium stimulates the incorporation of glucose into glycogen and glycogen synthase enzyme activity,
the inhibitor is without effect on GLUT4 translocation and pp70 S6 kinase. Lithium stimulation of glycogen synthesis is
insensitive to wortmannin, which is consistent with its acting directly on GSK3ß downstream of phosphatidylinositol 3-kinase.
These data support the hypothesis that GSK3ß contributes to insulin regulation of glycogen synthesis, but is not responsible for the
increase in glucose transport (Summers, 1999).
The phosphorylation state of cytoskeletal proteins, including certain microtubule-associated proteins, may influence the development and plasticity of axons and dendrites in the mammalian neuron in response to appropriate extracellular stimuli. In particular, high molecular weight microtubule-associated protein 2, has been implicated in dendrite growth and synaptic plasticity and is thought to be modulated
by phosphorylation and dephosphorylation. Threonines 1620/1623 on the microtubule-associated protein 2 molecule become phosphorylated in vivo and are targets for proline-directed protein kinases in vitro. Using the phosphorylated site-specific antibody 305, the decreased phosphorylation state of high molecular weight
microtubule-associated protein 2 during the development of cultured cerebellar granule neurons is reported. Phosphorylation of high molecular
weight microtubule-associated protein 2 at this site is significantly inhibited by lithium in short-term cultured neurons, which suggests the implication of glycogen synthase kinase-3. In long-term cultured neurons, such phosphorylation is also partially inhibited by PD098059, an inhibitor of extracellular signal-regulated protein kinase activation, which indicates an additional contribution of this kinase to high molecular weight
microtubule-associated protein 2 phosphorylation at this stage. Both in short-term and long-term cultured neurons, okadaic acid augments high molecular weight microtubule-associated protein 2 phosphorylation at this site through the inhibition of protein
phosphatases 1 and/or 2A. Finally, glutamate receptor activation leads to a dephosphorylation of high molecular weight microtubule-associated protein 2 at this site that can also be effectively prevented by okadaic acid. These results are consistent with the participation of glycogen synthase kinase-3, extracellular signal-regulated protein kinases and protein phosphatases 1 and 2A, in the regulation of microtubule-associated protein 2 phosphorylation within living neurons, which may be modulated by extracellular signals like the neurotransmitter glutamate (Sanchez Martin, 1998).
Membrane-bounded organelles (MBOs) are delivered to different domains in neurons by fast axonal transport. The importance of kinesin for fast antero grade transport is well established, but mechanisms for
regulating kinesin-based motility are largely unknown. Biochemical and in vivo
evidence is provided that kinesin light chains (KLCs) interact with and are in vivo substrates for glycogen synthase kinase 3 (GSK3). Active GSK3 inhibits anterograde, but not retrograde, transport in squid axoplasm and
reduces the amount of kinesin bound to MBOs. Kinesin microtubule binding and microtubule-stimulated ATPase activities are unaffected by GSK3 phosphorylation of KLCs. Active GSK3 was also localized preferentially to regions known
to be sites of membrane delivery. These data suggest that GSK3 can regulate fast anterograde axonal transport and targeting of cargos to
specific subcellular domains in neurons (Morfini, 2002).
Pathologic alterations in the microtubule-associated protein tau have been implicated in a number of neurodegenerative disorders, including Alzheimer's disease (AD), progressive supranuclear palsy (PSP), and frontotemporal dementia (FTD). Tau overexpression, in combination with phosphorylation by the Drosophila Shaggy exacerbates neurodegeneration induced by tau overexpression alone, leading to neurofibrillary pathology in the fly. Furthermore, manipulation of other Wingless signaling molecules downstream from Shaggy demonstrates that components of the Wnt signaling pathway modulate neurodegeneration induced by tau pathology in vivo but suggest that tau phosphorylation by GSK-3 differs from canonical Wnt effects on ß-catenin stability and TCF activity. The genetic system that has been established provides a powerful reagent for identification of novel modifiers of tau-induced neurodegeneration that may serve as future therapeutic targets (Jackson, 2002).
GSK-3ß, by virtue of its role as a major tau kinase, its formation of a complex with tau in the microtubule fraction from
bovine brain, and its colocalization with phosphorylated tau during development is a leading candidate for initiating pathologic tau hyperphosphorylation. The data presented here demonstrate that hyperphosphorylation of tau by GSK-3ß accelerates neurodegeneration and causes fibrillary tau-immunoreactive inclusions in vivo, confirming previous in vitro studies (Jackson, 2002).
Autophosphorylation-triggered ubiquitination has been proposed to be the major pathway regulating cyclin E protein abundance: phosphorylation of cyclin E on T380 by its associated CDK allows binding to the receptor subunit, Fbw7, of the SCFFbw7 ubiquitin ligase. This model has been tested in vivo; it has been found to be an inadequate representation of the pathways that regulate cyclin E degradation. Assembly of cyclin E into cyclin E-Cdk2 complexes is shown (1) to be required in vivo for turnover by the Fbw7 pathway, (2) Cdk2 activity is required for cyclin E turnover in vivo because it phosphorylates S384, (3) phosphorylation of T380 in vivo does not require Cdk2 and is mediated primarily by GSK3, and (4) two additional phosphorylation sites, T62 and S372, are also required for turnover. Thus, cyclin E turnover is controlled by multiple biological inputs and cannot be understood in terms of autophosphorylation alone (Welcker, 2003).
The phenotypic changes of increased motility and invasiveness of cancer cells are reminiscent of the epithelial-mesenchymal transition (EMT) that occurs during embryonic development. Snail, a zinc-finger transcription factor, triggers this process by repressing E-cadherin expression; however, the mechanisms that regulate Snail remain elusive. This study found that Snail is highly unstable, with a short half-life about 25 min. GSK-3beta binds to and phosphorylates Snail at two consensus motifs to dually regulate the function of this protein. Phosphorylation of the first motif regulates its beta-Trcp-mediated ubiquitination, whereas phosphorylation of the second motif controls its subcellular localization. A variant of Snail (Snail-6SA), which abolishes these phosphorylations, is much more stable and resides exclusively in the nucleus to induce EMT. Furthermore, inhibition of GSK-3beta results in the upregulation of Snail and downregulation of E-cadherin in vivo. Thus, Snail and GSK-3beta together function as a molecular switch for many signalling pathways that lead to EMT (B. P. Zhou, 2004).
The activity of glycogen synthase kinase-3 (GSK-3) is necessary for the maintenance of the epithelial architecture. Pharmacological inhibition of its activity or reducing its expression using small interfering RNAs in normal breast and skin epithelial cells results in a reduction of E-cadherin expression and a more mesenchymal morphology, both of which are features associated with an epithelial-mesenchymal transition (EMT). Importantly, GSK-3 inhibition also stimulates the transcription of Snail, a repressor of E-cadherin and an inducer of the EMT. NFkappaB was identified as a transcription factor inhibited by GSK-3 in epithelial cells that is relevant for Snail expression. These findings indicate that epithelial cells must sustain activation of a specific kinase to impede a mesenchymal transition (Bachelder, 2005).
The Disrupted in Schizophrenia 1 (DISC1) gene is disrupted by a balanced chromosomal translocation (1; 11) (q42; q14.3) in a Scottish family with a high incidence of major depression, schizophrenia, and bipolar disorder. Subsequent studies provided indications that DISC1 plays a role in brain development. This study demonstrates that suppression of DISC1 expression reduces neural progenitor proliferation, leading to premature cell cycle exit and differentiation. Several lines of evidence suggest that DISC1 mediates this function by regulating GSK3β. First, DISC1 inhibits GSK3β activity through direct physical interaction, which reduces β-catenin phosphorylation and stabilizes β-catenin. Importantly, expression of stabilized β-catenin overrides the impairment of progenitor proliferation caused by DISC1 loss of function. Furthermore, GSK3 inhibitors normalize progenitor proliferation and behavioral defects caused by DISC1 loss of function. Together, these results implicate DISC1 in GSK3β/β-catenin signaling pathways and provide a framework for understanding how alterations in this pathway may contribute to the etiology of psychiatric disorders (Mao, 2009).
A circadian clock located in the suprachiasmatic nucleus (SCN) regulates the period of physiological and behavioral rhythms to approximately 24 h. Lithium can lengthen the period of circadian rhythms in most organisms although little is known about the underlying mechanism. In the present study, Drosophila Shaggy ortholog glycogen synthase kinase-3 (GSK-3) protein expression was examined in the SCN after lithium treatment. When locomotor activity was assessed, an association was found between the effect of lithium and the period of circadian oscillation as well as the level of GSK-3 protein expression. The decreased expression of GSK-3 and increased expression of phosphorylated GSK-3 (pGSK-3) resulted in an antiphasic circadian rhythm between the two in the SCN of lithium-treated mice housed under both light-dark and constant dark conditions. The enzyme activity of GSK-3 in the SCN was low when the level of pGSK-3 protein was high, as examined by immunoblotting analysis. Thus, GSK-3 enzyme activity has a correlation with the expression of GSK-3 protein in the SCN. Although both GSK-3 and pGSK-3 proteins are also expressed in the arcuate nucleus, lithium did not affect their expression. Based on the association that was found between lengthened circadian period and GSK-3 protein and GSK-3 activity in the SCN, it is suggested that GSK-3 plays a role in regulating the period of the mammalian circadian pacemaker (Iwahan, 2004).
Cryptochrome 1 and 2 act as essential components of the central and peripheral circadian clocks for generation of circadian rhythms in mammals. Mouse cryptochrome 2 (mCRY2) is phosphorylated at Ser-557 in the liver, a well characterized peripheral clock tissue. The Ser-557-phosphorylated form accumulates in the liver during the night in parallel with mCRY2 protein, and the phosphorylated form reaches its maximal level at late night, preceding the peak-time of the protein abundance by approximately 4 h in both light-dark cycle and constant dark conditions. The Ser-557-phosphorylated form of mCRY2 is localized in the nucleus, whereas mCRY2 protein is located in both the cytoplasm and nucleus. Importantly, phosphorylation of mCRY2 at Ser-557 allows subsequent phosphorylation at Ser-553 by glycogen synthase kinase-3beta (GSK-3beta), resulting in efficient degradation of mCRY2 by a proteasome pathway. As assessed by phosphorylation of GSK-3beta at Ser-9, which negatively regulates the kinase activity, GSK-3beta exhibits a circadian rhythm in its activity with a peak from late night to early morning when Ser-557 of mCRY2 is highly phosphorylated. Altogether, the present study demonstrates an important role of sequential phosphorylation at Ser-557/Ser-553 for destabilization of mCRY2 and illustrates a model that the circadian regulation of mCRY2 phosphorylation contributes to rhythmic degradation of mCRY2 protein (Harada, 2005).
The differentiation of preadipocytes into adipocytes requires the suppression of canonical Wnt signaling, which appears to involve a peroxisome proliferator-activated receptor gamma (PPARgamma)-associated targeting of ß-catenin to the proteasome. In fact, sustained activation of ß-catenin by expression of Wnt1 or Wnt 10b in preadipocytes blocks adipogenesis by inhibiting PPARgamma-associated gene expression. The mechanisms regulating the balance between ß-catenin and PPARgamma signaling that determines whether mouse fibroblasts differentiate into adipocytes has been investigated. Specifically, it has been shown that activation of PPARgamma by exposure of Swiss mouse fibroblasts to troglitazone stimulates the degradation of ß-catenin, which depends on glycogen synthase kinase (GSK) 3ß activity. Mutation of serine 37 (a target of GSK3ß) to an alanine renders ß-catenin resistant to the degradatory action of PPARgamma. Ectopic expression of the GSK3ß phosphorylation-defective S37A-ß-catenin in Swiss mouse fibroblasts expressing PPARgamma stimulates the canonical Wnt signaling pathway without blocking their troglitazone-dependent differentiation into lipid-laden cells. Analysis of protein expression in these cells, however, shows that S37A-ß-catenin inhibits a select set of adipogenic genes because adiponectin expression is completely blocked, but FABP4/aP2 expression is unaffected. Furthermore, the mutant ß-catenin appears to have no affect on the ability of PPARgamma to bind to or transactivate a PPAR response element. The S37A-ß-catenin-associated inhibition of adiponectin expression coincides with an extensive decrease in the abundance of C/EBPalpha in the nuclei of the differentiated mouse fibroblasts. Taken together, these data suggest that GSKß is a key regulator of the balance between ß-catenin and PPARgamma activity and that activation of canonical Wnt signaling downstream of PPARgamma blocks expression of a select subset of adipogenic genes (Liu, 2004).
Axon-dendrite polarity is a cardinal feature of neuronal morphology essential for information flow. A differential distribution of GSK-3ß activity is found in the axon versus the dendrites. A constitutively active GSK-3ß mutant inhibits axon formation, whereas multiple axons formed from a single neuron when GSK-3ß activity is reduced by pharmacological inhibitors, a peptide inhibitor, or siRNAs. An active mechanism for maintaining neuronal polarity was revealed by the conversion of preexisting dendrites into axons upon GSK-3 inhibition. Biochemical and functional data show that the Akt kinase and the PTEN phosphatase are upstream of GSK-3ß in determining neuronal polarity. These results demonstrate that there are active mechanisms for maintaining as well as establishing neuronal polarity, indicate that GSK-3ß relays signaling from Akt and PTEN to play critical roles in neuronal polarity, and suggest that application of GSK-3ß inhibitors can be a novel approach to promote generation of new axons after neural injuries (Jiang, 2005).
Serine (Ser) 9 in GSK-3ß can be regulated by multiple kinases, among which Akt is differentially localized in axons. To test for GSK-3ß regulation by the candidate upstream molecules, the same inhibitors for PI3K as those used previously for studies of neuronal polarity were used. LY294002 significantly inhibits Akt phosphorylation at Ser 473 and GSK-3ß phosphorylation at Ser 9. LY294002 does not affect GSK-3ß phosphorylation at Tyr216. In addition to the biochemical evidences, the distribution of GSK-3ß and pGSK-3ß was examined. LY294002 reduces the ratio of pGSK-3ß over GSK-3ß. aPKC is another candidate GSK-3ß regulator, but GSK-3ß phosphorylation is not affected by the aPKC inhibitor bisindolylmaleimide I (Bis), or by the MAPK inhibitor U0126 or the PKA inhibitor KT5720 (Jiang, 2005).
To test for the functional significance of Akt, attempts were made to examine the effect of inhibiting Akt by using short hairpin RNAs (shRNAs), but they caused neuronal death. The effect of increasing Akt activity on neuronal polarity was examined by using Myr-Akt, a constitutively active Akt derived by fusion with the myristoylation signal of Src. The wild-type Akt does not affect neuronal polarity, whereas Myr-Akt causes the formation of multiple axons (Jiang, 2005).
Because GSK-3ß S9A should not be phosphorylated by Akt after the substation of Ser 9, it was possible to test for the relationship between Akt and GSK-3ß by cotransfection of GSK-3ß S9A and Myr-Akt. GSK-3ß S9A could partially, but not completely, reverse the effect of Myr-Akt on the formation of multiple axons. These results are consistent with the idea of Akt being upstream of GSK-3ß. The partial reversal could result from either incomplete overlap in GSK-3ß S9A and Myr-Akt transfection or the possibility that GSK-3ß constitutes a part, but not all, of the output for Akt (Jiang, 2005).
Detailed examination indicates that Akt plays multiple roles in axon and dendrite development. Myr-Akt increases the number of neurites per neuron both in neurons forming multiple axons, and in neurons with single axons. Myr-Akt increases both the numbers of axons and dendrites from the normal total of five neurites to eight. When the number of axons is increased in Myr-Akt neurons, axon length is shorter than normal, perhaps due to limited materials for an increased number of axons. When the number of axons is not increased in Myr-Akt neurons, the single axon is longer than normal, indicating that Akt promotes axon outgrowth. The length of dendrites is not affected by Myr-Akt (Jiang, 2005).
Only the effect of increasing axon number is shared between Akt activation and GSK-3ß inactivation. GSK-3ß is therefore only one downstream component of Akt, specifically mediating the function of Akt in axon-dendrite polarity, but not in neurite number or axon length (Jiang, 2005).
PTEN is a lipid and protein phosphatase that functions in opposition to PI3K by dephosphorylating the lipid product of PI3K, phosphatidylinositol 3,4,5-trisphosphate (PIP3) . The role of PTEN was examined further and the functional relationship of PTEN and GSK-3ß was characterized. Neuronal polarity is lost upon PTEN overexpression. Detailed analysis indicates that PTEN transfection inhibits axon formation without affecting dendrite formation. The inactive PTEN control (PTEN G129R) does not affect neuronal polarity (Jiang, 2005).
To investigate the role of endogenous PTEN, PTEN siRNA, a small inhibitor RNA (siRNA) construct designed specifically for PTEN, was used, and PTEN SsiRNA, a scrambled control. PTEN siRNA, but not PTEN SsiRNA, reduces PTEN protein levels in hippocampal neurons. PTEN siRNA increases the number of axons at the expense of dendrites. When the level of PTEN protein was examined, the level of PTEN was reduced by more than 50% in PTEN siRNA neurons with multiple axons, whereas PTEN was reduced less than 20% in PTEN siRNA neurons with single axons. Significant reduction of PTEN expression therefore correlates with the formation of multiple axons (Jiang, 2005).
To test for a functional relationship between PTEN and GSK-3ß, two kinds of experiments were performed. The first was to transfect neurons with PTEN and also treat them with SB415286. SB415286 almost completely reverses the effect of PTEN overexpression on axon-dendrite polarity, resulting in multiple axons at the expense of dendrites. The second kind of experiment was to cotransfect PTEN siRNA and GSK-3ß S9A. The effect of PTEN siRNA on multiple axon formation could be inhibited by an S0A mutant of GSK-3ß in which Ser 9 was replaced with alanine. These results indicate that GSK-3ß is downstream of PTEN in axon-dendrite polarity formation because GSK-3ß manipulations dominate over PTEN manipulations (Jiang, 2005).
One role of PTEN does not seem to be downstream of GSK-3ß. When neurons are cotransfected with PTEN siRNA and GSK-3ß S9A, GSK-3ß S9A could not eliminate axons, suggesting that either PTEN siRNA can antagonize the effect of GSK-3ß S9A in axon formation or that GSK-3ß S9A has to act in a PTEN-dependent pathway to inhibit axon formation. In the latter scenario, GSK-3ß activation could be upstream of PTEN in inhibiting axon formation (Jiang, 2005).
Neurons are highly polarized and comprised of two structurally and functionally distinct parts, an axon and dendrites. Collapsin response mediator protein-2 (CRMP-2: Drosophila homolog Collapsin Response Mediator Protein) is critical for specifying axon/dendrite fate, possibly by promoting neurite elongation via microtubule assembly. GSK-3ß phosphorylates CRMP-2 at Thr-514 and inactivates it. The expression of the nonphosphorylated form of CRMP-2 or inhibition of GSK-3ß induces the formation of multiple axon-like neurites in hippocampal neurons. The expression of constitutively active GSK-3ß impairs neuronal polarization, whereas the nonphosphorylated form of CRMP-2 counteracts the inhibitory effects of GSK-3ß, indicating that GSK-3ß regulates neuronal polarity through the phosphorylation of CRMP-2. Treatment of hippocampal neurons with neurotrophin-3 (NT-3) induces inactivation of GSK-3ß and dephosphorylation of CRMP-2. Knockdown of CRMP-2 inhibits NT-3-induced axon outgrowth. These results suggest that NT-3 decreases phosphorylated CRMP-2 and increases nonphosphorylated active CRMP-2, thereby promoting axon outgrowth (Yoshimura, 2005).
CRMP-2, which has also been identified as Ulip2/CRMP-62/TOAD-64/DRP-2, is one of at least five isoforms. CRMP-2 is expressed exclusively and highly in the developing nervous system. Mutations in the UNC-33 gene, a Caenorhabditis elegans homolog of CRMPs, led to severely uncoordinated movement and abnormalities in the guidance of axons of many neurons. Overexpression of full-length CRMP-2 induces the formation of multiple axons and elongation of the primary axon, and the dominant-negative form of CRMP-2 inhibits axon formation. CRMP-2 shows the ability to convert immature neurites and preexisting dendrites to axons. Thus, CRMP-2 is crucial for axon outgrowth and determination of the fate of the axon and dendrites, thereby establishing and maintaining neuronal polarity. In addition, CRMP-2 binds to tubulin heterodimers to promote microtubule assembly, thereby enhancing axon elongation and branching (Yoshimura, 2005).
A ternary complex of PAR-3, PAR-6, and atypical protein kinase C (aPKC) functions in various cell polarization events from worms to mammals, including cultured hippocampal neurons. The PAR-6-PAR-3-aPKC complex accumulates at the tip of the axon, and its polarized localization and aPKC activity are important for axon specification. aPKC can also phosphorylate GSK-3ß and inactivate its kinase activity, and GSK-3ß is important for polarization of migrating fibroblasts.
This study reports that GSK-3ß phosphorylates CRMP-2 at Thr-514, inactivates the CRMP-2 activity, and participates in neuronal polarization through CRMP-2. NT-3 and brain-derived neurotrophic factor inhibit GSK-3ß via the phosphatidylinositol-3-kinase (PI3-kinase)/Akt (also known as PKB) pathway, and thereby reduce phosphorylation levels of CRMP-2 at Thr-514, leading to axon elongation and branching (Yoshimura, 2005).
A model is presented of the role of GSK-3 and CRMP-2 in axon specification. The activation of PI3-kinase at the selective immature neurite produces PIP3, thus activating Akt and recruiting the PAR-3/PAR-6/aPKC complex at the growth cone. Activated Akt and aPKC inhibit GSK-3 by its phosphorylation, whereby nonphosphorylated CRMP-2 is increased in the growth cone. Nonphosphorylated active CRMP-2 promotes microtubule assembly and Numb-mediated endocytosis of cell adhesion molecules to enhance elongation of the immature neurite for axon specification. The relation between Akt and PAR-3/PAR-6/aPKC remains to be clarified (Yoshimura, 2005).
An essential step during the development of hippocampal neurons is the polarised outgrowth of a single axon. Recently, it has been suggested that inhibition of GSK-3ß via Akt/PKB-dependent phosphorylation of Ser9, specifically at the tip of the presumptive axon, is required for selective axonal outgrowth. This study reports that, by using neurons from double knock-in mice in which Ser9 and Ser21 of the two GSK-3ß isoforms have been replaced by Ala, polarity develops independently of phosphorylation at these sites. Nevertheless, global inhibition of GSK-3ß disturbs polarity development by leading to the formation of multiple axon-like processes in both control and knock-in neurons. This unpolarised outgrowth is accompanied by the symmetric delivery of membrane components to all neurites. Finally, the APC protein accumulates at the tip of one neurite before and during axon elongation, but global inhibition of GSK-3ß leads to APC protein accumulation in all neurites. It is concluded that GSK-3ß inhibition promotes the development of neuronal polarity, but that this is not mediated by Akt/PKB-dependent phosphorylation (Gartner, 2006).
Glycogen synthase kinase-3β (GSK-3β) is thought to mediate morphological responses to a variety of extracellular signals. Surprisingly, no gross morphological deficits were found in nervous system development in GSK-3β null mice. Therefore a short hairpin RNA (shRNA) was designed that targeted both GSK-3 isoforms. Strong knockdown of both GSK-3α and β markedly reduced axon growth in dissociated cultures and slice preparations. Then the role of different GSK-3 substrates were assessed in regulating axon morphology. Elimination of activity toward primed substrates only using the GSK-3 R96A mutant was associated with a defect in axon polarity (axon branching) compared to an overall reduction in axon growth induced by a kinase-dead mutant. Consistent with this finding, moderate reduction of GSK-3 activity by pharmacological inhibitors induced axon branching and was associated primarily with effects on primed substrates. These results suggest that GSK-3 is a downstream convergent point for many axon growth regulatory pathways and that differential regulation of primed versus all GSK-3 substrates is associated with a specific morphological outcome (Kim, 2006).
Suppression of glycogen synthase kinase 3 (GSK3) activity in neurons yields pleiotropic outcomes, causing both axon growth promotion and inhibition. Previous studies have suggested that specific GSK3 substrates, such as adenomatous polyposis coli (APC) and collapsin response mediator protein 2 (CRMP2), support axon growth by regulating the stability of axonal microtubules (MTs), but the substrate(s) and mechanisms conveying axon growth inhibition remain elusive. This study shows that CLIP (cytoplasmic linker protein)-associated protein (CLASP), originally identified as a MT plus end-binding protein, displays both plus end-binding and lattice-binding activities in nerve growth cones, and reveal that the two MT-binding activities regulate axon growth in an opposing manner: The lattice-binding activity mediates axon growth inhibition induced by suppression of GSK3 activity via preventing MT protrusion into the growth cone periphery, whereas the plus end-binding property supports axon extension via stabilizing the growing ends of axonal MTs. A model is proposed in which CLASP transduces GSK3 activity levels to differentially control axon growth by coordinating the stability and configuration of growth cone MTs (Hur, 2011).
Many substrates of GSK3 harbor multiple consecutive GSK3 sites (S/T-X-X-X-S/T) that are sequentially phosphorylated by GSK3. Extensive biochemical approaches have confirmed that two clusters of such sites in CLASP2 are phosphorylated by GSK3 and that complete phosphorylation of all GSK3 sites disrupts both the plus end-tracking and the lattice-binding activities of CLASP2. However, phosphorylation of serine residues in only one of the two GSK3 motifs specifically abrogates the lattice-binding activity, while retaining the plus end-binding activity. It was also confirmed that introducing phosphomimetic mutations into only one of the two GSK3 motifs in resCLASP2-FL (resCLASP2-5xS/D) specifically prevented lattice-binding activity while preserving the plus end-tracking activity. Consistent with the lack of lattice-binding activity, resCLASP2-5xS/D did not inhibit growth cone MT protrusion into the growth cone periphery and failed to reverse the axon growth promotion induced by siCLASP2. Importantly, when the endogenous CLASP2 was replaced with the resCLASP2-5xS/D mutant, siGSK3 was unable to inhibit axon growth, providing ample evidence that the attenuation of axon growth induced by GSK3 depletion requires the lattice-binding activity of CLASP2 (Hur, 2011).
The expansion of the mammalian cerebral cortex is safeguarded by a concerted balance between amplification and neuronal differentiation of intermediate progenitors (IPs). Nonetheless, the molecular controls governing these processes remain unclear. This study found that the scaffold protein Axin is a critical regulator that determines the IP population size and ultimately the number of neurons during neurogenesis in the developing cerebral cortex. The increase of the IP pool is mediated by the interaction between Axin and GSK-3 in the cytoplasmic compartments of the progenitors. Importantly, as development proceeds, Axin becomes enriched in the nucleus to trigger neuronal differentiation via beta-catenin activation. The nuclear localization of Axin and hence the switch of IPs from proliferative to differentiative status are strictly controlled by the Cdk5-dependent phosphorylation of Axin at Thr485. The results demonstrate an important Axin-dependent regulatory mechanism in neurogenesis, providing potential insights into the evolutionary expansion of the cerebral cortex (Fang, 2013).
The fate decision of NPCs between amplification and differentiation
controls the number of neurons produced during brain
development and ultimately determines brain size. However, it
is unclear how the NPCs make this fundamental choice. This study shows that the subcellular localization of a signaling scaffold
protein, Axin, defines the activation of specific signaling
networks in NPCs, thereby determining the amplification or
neuronal differentiation of NPCs during embryonic development. Cytoplasmic Axin in NPCs enhances IP generation,
which ultimately leads to increased neuron production, whereas
nuclear Axin in IPs promotes neuronal differentiation. Intriguingly,
the Cdk5-dependent phosphorylation of Axin facilitates
the nuclear accumulation of the protein, thereby functioning as
a 'brake' to prevent the overproduction of IPs and induce
neuronal differentiation (Fang, 2013).
The expansion of cortical surface
may result from increased numbers of neuroepithelial (NE) cells
and radial glial cells (RGs) or from an amplified IP pool. NE/RG augmentation evidently controls the global enlargement of cortical surface. The amplification of a subset of RGs expressing
the transcription factor Cux2 was recently suggested to
facilitate upper-layer neuron expansion (Franco, 2012).
However, there is a lack of experimental evidence indicating
whether IP amplification also substantially contributes to the
expansion of upper-layer cortical neurons and the cerebral
cortex. Nonetheless, upper-layer neurons are generated during
mid- and late neurogenesis, at which
time IPs play the primary role in neuron production. Moreover,
the enlargement of IP-residing SVZ is temporally correlated
with the increased number of upper-layer neurons and expanded
cortical surface. Therefore, it is tempting to
speculate that the amplification of IPs during mid- and late
corticogenesis has facilitated the evolutionary expansion of the
cerebral cortex. The present findings demonstrate that increasing
Axin levels during midcorticogenesis, which leads to
the transient amplification of IPs without affecting the RG pool,
is sufficient to expand the surface of the neocortex. Previous studies show that Axin expression is tightly regulated
by different posttranslational modifications including deubiquitination, SUMOylation,
methylation, and phosphorylation, which increase the stability of Axin; meanwhile, polyubiquitination and poly-ADP-ribosylation lead to its degradation. Thus, the adaptive
evolution of the Axin gene that regulates its posttranslational
modifications and hence its expression level might be involved
in the evolutionary expansion of the cerebral cortex (Fang, 2013).
To ensure the development of a cerebral cortex of the proper
size, the amplification and neuronal differentiation of IPs need to
be precisely controlled. A reduced number of IPs due to precocious
depletion of NEs/RGs or inhibition
of IP generation/proliferation ultimately lead
to the generation of fewer cortical neurons, resulting in a smaller
cortex - a characteristic feature of human microcephalic syndromes (Fang, 2013).
In contrast, the overexpansion of IPs generates an excessive number of neurons, which is associated with macrocephaly and autism. The current findings demonstrate that Axin strictly controls the process of indirect neurogenesis to ensure the production of a proper
number of neurons. Although cytoplasmic Axin simultaneously
maintains the RG pool and promotes IP amplification to sustain
rapid and long-lasting neuron production, subsequent enrichment
of Axin in the nuclei of IP daughter cells triggers neuronal differentiation
and prevents the overexpansion of IPs. In addition, the results demonstrate
that Cdk5-mediated phosphorylation regulates
the nucleocytoplasmic shuttling of Axin, thereby controlling the
switching of NPCs from proliferative to differentiation status (Fang, 2013).
The findings show that Axin phosphorylation in IPs triggers
neuronal differentiation in a rostrolateral-high to caudo-medial-low
gradient correlated with the spatial gradient of neurogenesis. Thus, the gradient of Axin
phosphorylation may provide a quantitative tool for evaluating the
temporal and spatial gradient of IP differentiation into neurons.
Importantly, nuclear Axin phosphorylation is rapidly induced in
IP daughter cells in the G1 phase, which is the stage when progenitor
cells actively respond to neurogenic signals; this suggests that the timing of Axin phosphorylation-dependent IP differentiation is regulated by diffusible
extracellular signals. Therefore, understanding
how Axin phosphorylation is regulated in IPs by extracellular cues
and niches should shed new light on the molecular basis underlying
the gradient-specific differentiation of IPs (Fang, 2013).
The findings also highlight the importance of Cdk5 in embryonic
neurogenesis. Although Cdk5 plays critical roles in neuronal
development and is implicated in the neurogenesis of cultured neural stem cells,
it remains unclear whether Cdk5 regulates embryonic neurogenesis.
The current findings provide in vivo evidence that Cdk5 is required
for the neuronal differentiation of IPs, at least in part through
phosphorylating Axin. Intriguingly, although cdk5/ cortices
exhibited an accumulation of IPs and reduced neuron production
during early-mid neurogenesis, the brain size of
these mutant mice remained unchanged by the end of neurogenesis. This may be due to the compensatory increase of neuron production from the expanded pool of
IPs during the mid-to-late neurogenesis stages. Therefore, elucidating
how Cdk5 is involved in different stages of neurogenesis
may provide insights into the molecular control of neuronal number and subtypes (Fang, 2013).
Several factors that regulate the generation and amplification of
IPs have been identified. Nonetheless, key
questions remain open: how RGs determine to differentiate into
IPs instead of neurons, how RG-to-IP transition and IP differentiation
are coordinated, and how IP amplification and differentiation
are balanced. The present results show that the interaction
between cytoplasmic Axin and GSK-3β maintains the RG pool
and promotes IP production. The signaling mechanisms
underlying the action of Axin-GSK-3β interaction require
further investigation. It is hypothesized that Axin regulates IP differentiation
from RGs via various molecular mechanisms. First,
the Axin-GSK-3β complex may reduce the level of Notch receptor
or β-catenin, leading to the suppression of Notch- and Wnt-mediated
signaling, respectively. Given that Axin and
GSK-3β can associate with the centrosome and mitotic spindle, Axin-GSK-3β interaction may also modulate cleavage plane orientation.
Furthermore, Axin-GSK-3β can interact with and affect the
microtubule-binding activity of adenomatous polyposis coli
(APC), which is required for establishing
the apical-basal polarity and asymmetric division of RGs. Finally, interaction with Axin can cause GSK-3β inhibition, which may enhance IP amplification through the activation of Shh signaling (Fang, 2013).
The timing of IPs to undergo cell-cycle exit balances the proliferative
and neurogenic divisions of IPs and switches the RG-to-
IP transition to the neuronal differentiation of IPs. This study shows that the interaction between Axin and β-catenin in the nucleus
switches the division of IPs from proliferative to neurogenic by
enhancing the neurogenic transcriptional activity of β-catenin. Indeed, Axin and β-catenin are required for the signal transduction of Wnt,
RA, and TGF-β, which
triggers and promotes neuronal differentiation. Thus, Axin in
the nucleus may serve to transduce and converge multiple
neurogenic signaling pathways to β-catenin during neurogenesis.
However, the mechanism by which nuclear Axin enhances
the transcriptional activity of β-catenin requires further investigation.
Given that β-catenin exerts its transcriptional regulation of
target genes through association with T cell factor/lymphoid
enhancer factor (Tcf/Lef), it is hypothesized that nuclear Axin
facilitates β-catenin/Tcf/Lef complex formation to enhance transcription (Fang, 2013).
Although Axin was previously recognized as a negative regulator
of canonical Wnt signaling, suppressing cell division by
recruiting GSK-3β and β-catenin into the β-catenin destruction
complex for β-catenin degradation, the present
results show that cytoplasmic Axin and nuclear Axin act
distinctly from canonical Wnt signaling through specific binding
to GSK-3β and β-catenin, respectively. Therefore, the current findings
corroborate the notion that Wnt signaling components play
multifaceted roles in NPCs during neurogenesis, independent
of canonical Wnt signaling as demonstrated in previous studies (Fang, 2013).
In conclusion, the present study identified distinct roles of
Axin in IP amplification and neuron production. The results
demonstrate that the modulation of Axin levels, subcellular localization,
phosphorylation, and its interaction with key signaling
regulators (e.g., GSK-3β and β-catenin) in NPCs ultimately control
neuron production and expansion of the cerebral cortex.
Given that Axin is a key regulator of the switch from IP amplification
to differentiation, the characterization of the signals that
control this switch will not only advance current understanding
of how the cerebral cortex expands during evolution but also
provide important insights into neurodevelopmental disorders
such as microcephaly (Fang, 2013).
Balanced control of neural progenitor maintenance and neuron production is crucial in establishing functional neural circuits during brain development, and abnormalities in this process are implicated in many neurological diseases. However, the regulatory mechanisms of neural progenitor homeostasis remain poorly understood. This study shows that mammalian target of rapamycin (mTOR see Drosophila Tor) is required for maintaining neural progenitor pools and plays a key role in mediating glycogen synthase kinase 3 signaling during brain development. First, conditional mutant mice exhibiting deletion of mTOR were generated and characterized in neural progenitors and neurons in the developing brain using Nestin-cre and Nex-cre lines, respectively. The elimination of mTOR resulted in abnormal cell cycle progression of neural progenitors in the developing brain and thereby disruption of progenitor self-renewal. Accordingly, production of intermediate progenitors and postmitotic neurons were markedly suppressed. Next, it was discovered that GSK3, a master regulator of neural progenitors, interacts with mTOR and controls its activity in cortical progenitors. Finally, it was found that inactivation of mTOR activity suppresses the abnormal proliferation of neural progenitors induced by GSK3 deletion. These findings reveal that the interaction between mTOR and GSK3 signaling plays an essential role in dynamic homeostasis of neural progenitors during brain development (Ka, 2014).
Congenital hypertrophy/hyperplasia of the retinal pigmented epithelium is an ocular lesion found in patients harboring mutations in the APC tumor suppressor gene. Apc-deficient zebrafish display developmental abnormalities of both the lens and retina. Injection of dominant-negative Lef reduced Wnt signaling in the lens but did not rescue retinal differentiation defects. In contrast, treatment of apc mutants with all-trans retinoic acid rescued retinal differentiation defects but had no apparent effect on the lens. Rdh5 was identified as a retina-specific retinol dehydrogenase controlled by APC. Morpholino knockdown of Rdh5 phenocopied the apc mutant retinal differentiation defects and was rescued by treatment with exogenous all-trans retinoic acid. Microarray analyses of apc mutants and Rdh5 morphants revealed a profound overlap in the transcriptional profile of these embryos. These findings support a model wherein Apc serves a dual role in regulating Wnt and retinoic acid signaling within the eye and suggest retinoic acid deficiency as an explanation for APC mutation-associated retinal defects such as congenital hypertrophy/hyperplasia of the retinal pigmented epithelium (Nadauld, 2006).
The retinal pigment epithelium (RPE) is indispensable for vertebrate eye development and vision. In the classical model of optic vesicle patterning, the surface ectoderm produces fibroblast growth factors (FGFs) that specify the neural retina (NR) distally, whereas TGFbeta family members released from the proximal mesenchyme are involved in RPE specification. However, it was previously proposed that bone morphogenetic proteins (BMPs) released from the surface ectoderm are essential for RPE specification in chick. This study now shows that the BMP- and Wnt-expressing surface ectoderm is required for RPE specification. Wnt signalling from the overlying surface ectoderm is involved in restricting BMP-mediated RPE specification to the dorsal optic vesicle. Wnt2b is expressed in the dorsal surface ectoderm and subsequently in dorsal optic vesicle cells. Activation of Wnt signalling by implanting Wnt3a-soaked beads or inhibiting GSK3beta at optic vesicle stages inhibits NR development and converts the entire optic vesicle into RPE. Surface ectoderm removal at early optic vesicle stages or inhibition of Wnt, but not Wnt/beta-catenin, signalling prevents pigmentation and downregulates the RPE regulatory gene Mitf. Activation of BMP or Wnt signalling can replace the surface ectoderm to rescue MITF expression and optic cup formation. Evidence is provided that BMPs and Wnts cooperate via a GSK3beta-dependent but beta-catenin-independent pathway at the level of pSmad to ensure RPE specification in dorsal optic vesicle cells. A new dorsoventral model of optic vesicle patterning is proposed, whereby initially surface ectoderm-derived Wnt signalling directs dorsal optic vesicle cells to develop into RPE through a stabilising effect of BMP signalling (Steinfeld, 2013).
Chondrocytes and osteoblasts are two primary cell types in the skeletal system
that are differentiated from common mesenchymal progenitors. It is believed that
osteoblast differentiation is controlled by distinct mechanisms in
intramembranous and endochondral ossification. Ectopic
canonical Wnt signaling leads to enhanced ossification and suppression of
chondrocyte formation. Conversely, genetic inactivation of β-catenin, an
essential component transducing the canonical Wnt signaling, causes ectopic
formation of chondrocytes at the expense of osteoblast differentiation during
both intramembranous and endochondral ossification. Moreover, inactivation of
β-catenin in mesenchymal progenitor cells in vitro causes chondrocyte
differentiation under conditions allowing only osteoblasts to form. These results
demonstrate that β-catenin is essential in determining whether mesenchymal
progenitors will become osteoblasts or chondrocytes regardless of regional
locations or ossification mechanisms. Controlling Wnt/β-catenin signaling is a common molecular mechanism underlying chondrocyte and osteoblast differentiation
and specification of intramembranous and endochondral ossification (Day, 2005).
Inactivation of β-catenin in mesenchymal progenitors prevents
osteoblast differentiation; inactivation of Lrp5, a gene encoding a
likely Wnt coreceptor, results in low bone mass (osteopenia) by decreasing bone
formation. These observations indicate that Wnt signaling controls osteoblast
differentiation and suggest that it may regulate bone formation in
differentiated osteoblasts. This study deals with later events and it was found that stabilization of β-catenin in differentiated osteoblasts results in high
bone mass, while its deletion from differentiated osteoblasts leads to
osteopenia. Surprisingly, histological analysis showed that these mutations
primarily affect bone resorption rather than bone formation. Cellular and
molecular studies have shown that β-catenin, together with TCF proteins,
regulates osteoblast expression of Osteoprotegerin, a major inhibitor of
osteoclast differentiation. These findings demonstrate that β-catenin, and
presumably Wnt signaling, promote the ability of differentiated osteoblasts to
inhibit osteoclast differentiation; thus, they broaden knowledge of the
functions Wnt proteins have at various stages of skeletogenesis (Glass, 2005).
Using K14ΔNβ-cateninER transgenic mice, it has been shown that short-term,
low-level β-catenin activation stimulates de novo hair follicle formation
from sebaceous glands and interfollicular epidermis, while only sustained,
high-level activation induces new follicles from preexisting follicles. The
Hedgehog pathway is upregulated by β-catenin activation, and inhibition of
Hedgehog signaling converts the low β-catenin phenotype to wild-type
epidermis and the high phenotype to low. β-catenin-induced follicles
contain clonogenic keratinocytes that express bulge markers; the follicles
induce dermal papillae and provide a niche for melanocytes, and they undergo
4OHT-dependent cycles of growth and regression. New follicles induced in
interfollicular epidermis are derived from that cellular compartment and not
through bulge stem cell migration or division. These results demonstrate the
remarkable capacity of adult epidermis to be reprogrammed by titrating
β-catenin and Hedgehog signal strength and establish that cells from
interfollicular epidermis can acquire certain characteristics of bulge stem
cells (Silva-Vargas, 2005).
Shh signaling induces proliferation of many cell types during development and
disease, but how Gli transcription factors regulate these mitogenic responses
remains unclear. By genetically altering levels of Gli activator and repressor
functions in mice, it has been demonstrated that both Gli functions are involved in
the transcriptional control of N-myc and Cyclin D2 during
embryonic hair follicle development. The results also indicate that additional
Gli-activator-dependent functions are required for robust mitogenic responses in
regions of high Shh signaling. Through posttranscriptional mechanisms, including
inhibition of GSK3-β activity, Shh signaling leads to spatially restricted
accumulation of N-myc and coordinated cell cycle progression. Furthermore, a
temporal shift in the regulation of GSK3-β activity occurs during
embryonic hair follicle development, resulting in a synergy with β-catenin
signaling to promote coordinated proliferation. These findings demonstrate that
Shh signaling controls the rapid and patterned expansion of epithelial
progenitors through convergent Gli-mediated regulation (Mill, 2005).
The regulation of cell adhesion in epithelia is a fundamental process governing morphogenesis in embryos and a key step in the progression of invasive cancers. The molecular pathways controlling the epithelial organisation of somites have been examined. Somites are mesodermal epithelial structures of vertebrate embryos that undergo several changes in cell adhesion during early embryonic life. Wnt6 in the ectoderm overlaying the somites, but not Wnt1 in the neighbouring neural tube, is the most likely candidate molecule responsible for the maintenance of the epithelial structure of the dorsal compartment of the somite: the dermomyotome. The signalling pathway that mediates Wnt6 activity have been analyzed. The experiments suggest that the Wnt receptor molecule Frizzled7 probably transduces the Wnt6 signal. Intracellularly, this leads to the activation of the ß-catenin/LEF1-dependent pathway. Finally, it is demonstrated that the bHLH transcription factor paraxis, which has been shown to be a major player in the epithelial organisation of somites, is a target of the ß-catenin signal. It is concluded that ß-catenin activity, initiated by Wnt6 and mediated by paraxis, is required for the maintenance of the epithelial structure of somites (Linker, 2005).
Glycogen synthase kinase-3 (GSK3) has been implicated in major neurological disorders, but its role in normal neuronal function is largely unknown. GSK3β mediates an interaction between two major forms of synaptic plasticity in the brain, NMDA receptor-dependent long-term potentiation (LTP) and NMDA receptor-dependent long-term depression (LTD). In rat hippocampal slices, GSK3β inhibitors block the induction of LTD. Furthermore, the activity of GSK3β is enhanced during LTD via activation of PP1. Conversely, following the induction of LTP, there is inhibition of GSK3β activity. This regulation of GSK3β during LTP involves activation of NMDA receptors and the PI3K-Akt pathway and disrupts the ability of synapses to undergo LTD for up to 1 hr. It is concluded that the regulation of GSK3β activity provides a powerful mechanism to preserve information encoded during LTP from erasure by subsequent LTD, perhaps thereby permitting the initial consolidation of learnt information (Peineau, 2007).
NMDA receptor-dependent LTD is due to the internalization of AMPA receptors and involves protein interactions directly associated with the AMPA receptor subunits, particularly GluR2. It was reasoned that GSK3β might form a complex with AMPA receptors, and thus attempts were made to investigate this by probing for an association of native GSK3β with AMPA receptors in the CA1 area of hippocampal slices. A specific antibody against GSK3β was able to coimmunoprecipitate the GluR1 and GluR2 AMPA receptor subunits, and conversely, immunoprecipitation of AMPA receptors produced coimmunoprecipitation of GSK3β. To determine the functional status of AMPA receptor-associated GSK3β, AMPA receptors were immunoprecipitated, using antibodies against either GluR1 or GluR2, and then assayed for kinase activity. GSK3β activity was readily detected in both GluR1 and GluR2 immunoprecipitates relative to the background IgG control, demonstrating that endogenous GSK3β associates with native AMPA receptors in the brain, and that the bound GSK3β is functionally active. This association of GSK3β with AMPA receptors suggests a compartmentalization of this enzyme for the efficient regulation of AMPA receptors during LTD (Peineau, 2007).
It was asked whether the GSK3β activity that is associated with AMPA receptors could be regulated. Previous work has shown that transient exposure of cultured neurons to a solution containing sucrose plus glycine leads to an NMDA receptor-dependent insertion of AMPA receptors into the plasma membrane. Interestingly, this effect is associated with an increase in AMPA receptor-associated PI3K activity. Since PI3K is an upstream regulator of GSK3β, whether this treatment also affected the AMPA receptor-associated GSK3β enzyme activity was investigated. Neurons were treated with sucrose (200 mM) plus glycine (100 µM) for 2 min, and this led to the insertion of AMPA receptors into the plasma membrane as determined approximately 15 min later using surface biotinylation assays. This chemically induced AMPA receptor insertion was associated with a decrease in AMPA receptor-associated GSK3β activity (Peineau, 2007).
This study identified a form of regulation of synaptic plasticity in which the transient synaptic activation of NMDA receptors, as occurs during LTP, leads to inhibition of LTD. This regulation is very powerful since LTD is fully inhibited immediately following the conditioning stimulus and the effect lasts for approximately 1 hr. Also some of the signaling pathways responsible for this potent regulation of synaptic plasticity have been identified. GSK3β activity is an absolute requirement for the induction of LTD and the conditioning stimulus inhibits its activity via activation of the PI3K-Akt pathway. Finally, there is a correlation between the phosphorylation state of GSK3β ser9 and whether NMDA receptor activation leads to the induction or inhibition of LTD (Peineau, 2007).
GSK3β is an unusual kinase that has been implicated in many diseases. However, very little is known about its normal function in the nervous system. It is important during early development and it has been shown to play a key role in cell polarity and in the growth of neuromuscular junctions. Recently, it has been shown that GSK3β is important for determining neuronal polarity during the development of hippocampal neurons. However, though GSK3β is also highly expressed in the mature brain, its function in the nervous system has, hitherto, been largely unexplored. In the nucleus of hippocampal neurons, GSK3β is involved in the regulation of gene transcription by promoting the nuclear export of the transcription factor NF-ATc4. In addition, it has been shown that overexpression of GSK3β impairs spatial learning, though the mechanism underlying this effect is unknown. This study shows that in 2-week-old rats, an age at which the expression of GSK3β is near its peak, GSK3β activity is essential for NMDA receptor-dependent LTD in the hippocampus. This form of LTD is widespread throughout the brain and has been strongly implicated in development and learning and memory. Therefore, this novel GSK3β-dependent mechanism may be of general significance in regulating the interaction between LTP and LTD throughout the brain (Peineau, 2007).
GSK3β, unlike most enzymes, possesses high basal level constitutive activity and can be bidirectionally regulated to either further increase or decrease its activity. During LTD there is additional activation of GSK3β, probably via dephosphorylation of ser9. This effect is prevented by an inhibitor of PP1/PP2A. This suggests that the activation of PP1, which is known to occur during LTD, is responsible for the activation of GSK3β, via its dephosphorylation of ser9. LTD is associated with inhibition of Akt, probably also via the activation of PP1. These data suggest that GSK3β activity is increased during LTD because the phosphatase concomitantly inhibits Akt and directly dephosphorylates ser9 of GSK3β (Peineau, 2007).
Interestingly, the alteration in the phosphorylation status of GSK3β persists beyond the delivery of low-frequency stimulation (LFS), and lithium completely blocks LTD when applied after the delivery of LFS. These data suggest that GSK3β is required for the LTD process beyond the initial induction phase. Further studies are required to determine the full time course of the involvement of GSK3β in LTD (Peineau, 2007).
GSK3β has several upstream regulators and numerous downstream targets. In the present study, two of its upstream regulators have been identified. During LTD, GSK3β is activated via an okadaic acid-sensitive protein phosphatase, which is probably PP1. During LTP, GSK3β is inhibited via the PI3K-Akt pathway. Since GSK3β is such a ubiquitous kinase, it needs mechanisms to localize its access to its substrates. This is achieved in part via direct interactions with other proteins to form complexes. For example, in the canonical Wnt pathway, GSK3β binding proteins control access of β-catenin. It seems likely that the association between GSK3β and AMPA receptors serves to localize the kinase close to substrates that are involved in the trafficking of these receptors during synaptic plasticity. Further studies are required to establish the mechanism of this interaction as well as the downstream pathways mediated by GSK3β in the regulation of LTD (Peineau, 2007).
The finding that the synaptic activation of NMDA receptors during LTP inhibits NMDA receptor-dependent LTD raises an intriguing issue: what determines whether the synaptic activation of NMDA receptors leads to the induction or inhibition of LTD? Evidence is presented that the phosphorylation state of ser9 of GSK3β is a critical determinant. Thus, during LTP, activation of the PI3K-Akt pathway results in phosphorylation of GSK3β, and hence inhibition of its activity. In contrast, during LTD, activation of PP1 results in inhibition of Akt and the dephosphorylation of GSK3β at ser9, and this leads to an increase in the enzyme's activity. The activation of PI3K-Akt and inhibition of PP1 during LTP, but inhibition of Akt during LTD as well as the selective activation of PP1 during LTD, can be explained by the differences in the magnitude and spatiotemporal properties of the Ca2+ rise associated with the synaptic activation of NMDA receptors during these two forms of synaptic plasticity (Peineau, 2007).
Previous work has described other ways in which synaptic plasticity can be powerfully influenced by the prior history of synaptic activity. However, the mechanisms involved in these forms of metaplasticity are not known. Why synapses need such regulatory mechanisms is a matter of conjecture. One intriguing possible role for the regulation described in this study is to stabilize a synaptic modification over the short term by protecting synapses from the effects of additional NMDA receptor-dependent plasticity until the information can be either consolidated or erased by NMDA receptor-independent mechanisms (Peineau, 2007).
The regulation of synaptic plasticity is further complicated by the involvement of mGluRs, which are involved in depotentiation, LTD of baseline transmission, heterosynaptic LTD, and metaplasticity. So that focus could be placed on interactions between the NMDA receptor-dependent forms of synaptic plasticity, the additional complication of mGluR-dependent synaptic plasticity were eliminated by using the broad spectrum mGluR antagonist LY341495 and by employing stimulus protocols optimized for NMDA receptor-dependent synaptic plasticity. However, given that PI3K has been implicated in a chemically induced form of mGluR-dependent LTD and heterosynaptic LTD, it will be interesting to determine whether GSK3β is also involved in these forms of synaptic plasticity. One possibility is that the PI3K-Akt-GSK3β pathway serves to inhibit NMDA receptor-dependent LTD both homosynaptically following the induction of LTP and heterosynaptically following the induction of LTD (Peineau, 2007).
The finding that in the normal brain activation of GSK3β is essential for NMDA receptor-dependent LTD, and that its activity can be regulated by LTP, may offer clues to the pathological role of this enzyme in neurological disorders. For example, the primary therapeutic action of lithium in bipolar disorders may be via inhibition of GSK3β. Indeed, specific inhibition of GSK3β has recently been shown to produce antidepressive-like activity in vivo. Overactivity of GSK3β may, therefore, lead to this mood disorder by affecting the balance and interplay between NMDA receptor-dependent LTP and LTD (Peineau, 2007).
Glycogen synthase kinase 3 (GSK3) is a multifunctional serine/threonine kinase that participates in numerous signalling pathways involved in diverse physiological processes. Several of these pathways are implicated in disease pathogenesis, which has prompted efforts to develop GSK3-specific inhibitors for therapeutic applications. However, before now, there has been no strong rationale for targeting GSK3 in malignancies. This study reports pharmacological, physiological and genetic studies that demonstrate an oncogenic requirement for GSK3 in the maintenance of a specific subtype of poor prognosis human leukaemia, genetically defined by mutations of the MLL proto-oncogene. In contrast to its previously characterized roles in suppression of neoplasia-associated signalling pathways, GSK3 paradoxically supports MLL leukaemia cell proliferation and transformation by a mechanism that ultimately involves destabilization of the cyclin-dependent kinase inhibitor p27Kip1. Inhibition of GSK3 in a preclinical murine model of MLL leukaemia provides promising evidence of efficacy and earmarks GSK3 as a candidate cancer drug target (Wang, 2008).
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