From the National Creative Research Initiative Center
for Cell Death, Graduate School of Life Science and Biotechnology,
Korea University, Seoul 136-701, Korea and ¶ Department of Animal
Science, Konkuk University, Seoul 143-702, Korea
Received for publication, January 9, 2003, and in revised form, February 10, 2003
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ABSTRACT |
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Glycogen synthase kinase 3 Glycogen synthase kinase 3 Mitogen-activated protein kinase (MAPK) signaling cascades are
evolutionarily conserved from yeast to humans and are involved in a
variety of cellular functions, including cell growth, differentiation, and cell death (16-19). Each MAPK signaling pathway includes three components, a MAPK, a MAPK kinase, and a MAPK kinase kinase. An activated MAPK kinase kinase phosphorylates serine or threonine residues of a MAPK kinase, which in turn phosphorylates and thereby activates a MAPK (16-18). The mammalian family of MAPKs includes at
least three subgroups, extracellular signal-regulated kinases, c-Jun
NH2-terminal kinases (JNKs), also known as stress-activated protein kinases (SAPKs), and p38 MAPK (17, 18). The extracellular signal-regulated kinase pathway is activated by various mitogens (19),
whereas the JNK/SAPK and p38 pathways are activated by proinflammatory
cytokines such as tumor necrosis factor- To provide further insight into the regulatory role of GSK3 Antibodies--
Mouse monoclonal antibodies to GSK3 Plasmid Constructs--
MEKK1 constructs used were prepared as
described previously (28). GSK3 Cell Culture and Transfection--
Human embryonic kidney 293 (HEK293) and rat neuroblastoma B103 cells were maintained under a
humidified atmosphere of 5% CO2 at 37 °C in Dulbecco's
modified Eagle's medium supplemented with 10% heat-inactivated fetal
bovine serum and 100 units/ml penicillin/streptomycin (Invitrogen).
Cells were transiently transfected with the use of GenePORTER II (Gene
Therapy Systems), by the calcium phosphate method, or by electroporation.
Apoptotic Cell Death--
B103 cells were transfected for
48 h with pEGFP (Clontech) and the appropriate
vector constructs, fixed with 4% paraformaldehyde, and then stained
with 4',6-diamidino-2-phenylindole (10 µg/ml) for 10 min. The
4',6-diamidino-2-phenylindole-stained nuclei of cells expressing green
fluorescence protein (GFP) were examined for apoptotic morphology with
a Zeiss Axiovert fluorescence microscope. The percentage of apoptotic
cell death was determined as the number of GFP-expressing cells with
apoptotic nuclei divided by the total number of GFP-expressing cells.
More than 200 cells were counted in each experiment.
Co-immunoprecipitation--
Cells were lysed in buffer A
consisting of 20 mM Tris-HCl (pH 7.4), 150 mM
sodium chloride, 1% Triton X-100, 1% deoxycholate, 12 mM
Immunocomplex Kinase Assays--
Cells were lysed in buffer A,
and the cell lysates were subjected to immunoprecipitation as described
above. The resulting immunoprecipitates were assayed for enzymatic
activities of the indicated protein kinases as described previously
(29, 30). Phosphorylated proteins were separated by SDS-PAGE, and the
extent of phosphorylation was quantified with a Fuji BAS2500 imager. Bacterially expressed glutathione S-transferase (GST) fusion
proteins of c-Jun-(1-79), SAPK In Vitro Binding Analysis--
GSK3 GSK3
We next examined whether GSK3
We then examined the possible effect of GBP, an intracellular inhibitor
of GSK3 GSK3 GSK3
Next, we examined whether GSK3 Akt Inhibits GSK3
Insulin initiates the activation of PI3K that in turn induces Akt
activation (38-40). We thus investigated whether insulin is also able
to block the GSK3 The Kinase-inactive Mutant MEKK1(K1253M) Blocks GSK3 Reduced MEKK1 Activity in MEFs Derived from GSK3 We have shown that GSK3 Axin, a GSK3 In this study we demonstrate that insulin inhibited the activation of
MEKK1 by GSK3 GSK3 (GSK3
) is
implicated in many biological events, including embryonic
development, cell differentiation, apoptosis, and insulin response.
GSK3
has now been shown to induce activation of the
mitogen-activated protein kinase kinase kinase MEKK1 and thereby to
promote signaling by the stress-activated protein kinase pathway.
GSK3
-binding protein blocked the activation of MEKK1 by
GSK3
in human embryonic kidney 293 (HEK293) cells. Furthermore,
co-immunoprecipitation analysis revealed a physical association between
endogenous GSK3
and MEKK1 in HEK293 cells. Overexpression of axin1,
a GSK3
-regulated scaffolding protein, did not affect the physical
interaction between GSK3
and MEKK1 in transfected HEK293 cells.
Exposure of cells to insulin inhibited the activation of MEKK1 by
GSK3
, and this inhibitory effect of insulin was abolished by the
phosphatidylinositol 3-kinase inhibitor wortmannin. Furthermore, MEKK1
activity under either basal or UV- or tumor necrosis factor
-stimulated conditions was reduced in embryonic fibroblasts derived
from GSK3
knockout mice compared with that in such cells from
wild-type mice. Ectopic expression of GSK3
increased both basal and
tumor necrosis factor
-stimulated activities of MEKK1 in
GSK3
/
cells. Together, these observations
suggest that GSK3
functions as a natural activator of MEKK1.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(GSK3
)1 is a
serine/threonine kinase that is thought to contribute to a variety of
biological events such as embryonic development, metabolism,
tumorigenesis, and cell death (1-4). GSK3
is a constitutively
active kinase and regulates many intracellular signaling pathways by
phosphorylating substrates such as
-catenin. The phosphorylation of
-catenin by GSK3
is facilitated by the scaffold protein axin and
is inhibited either by GSK3
-binding protein (GBP), also known as
Frat (Frequently rearranged in advanced T-cell lymphomas), or by
Dishevelled (5-7). The GSK3
-catalyzed phosphorylation of
-catenin results in its ubiquitin-mediated proteolysis (8, 9). The
function of GSK3
is also regulated through phosphorylation by other
protein kinases including Akt (10-13). Akt is a serine/threonine
kinase that is activated by phosphatidylinositol 3-kinase (PI3K)
signaling and phosphorylates GSK3
on Ser9, thereby
inactivating it (10, 14, 15).
(TNF-
) as well as
cellular stresses (16-18). The JNK/SAPK pathway, which is associated
with stress-induced cellular events including cell death (20-27), is
composed of JNK/SAPK, a MAPK kinase such as MKK4 (also known as SEK1 or
JNKK1) and MKK7, and a MAPK kinase kinase such as MEKK1 (16-18).
in
intracellular signaling cascades, we have now investigated the effect
of this enzyme on the JNK/SAPK pathway. We show that GSK3
physically
associates with and activates MEKK1, thereby stimulating the JNK/SAPK
pathway. The PI3K-Akt signaling inhibited the GSK3
-induced
activation of MEKK1. Furthermore, the activity of endogenous MEKK1 was
reduced in mouse embryonic fibroblasts (MEFs) derived from GSK3
knockout (GSK3
/
) mice compared with that in MEFs
from GSK3
+/+ mice. These findings suggest that GSK3
functions as an endogenous activator of MEKK1.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and to
axin as well as rabbit polyclonal antibodies against MEKK1 and
apoptosis signal-regulating kinase 1 (ASK1) were purchased from Santa
Cruz Biotechnology. Mouse monoclonal antibody to JNK1 was from BD
Pharmingen. Mouse monoclonal antibodies to the hemagglutinin epitope
(HA) and to the FLAG epitope were obtained from Roche Molecular
Biochemicals and Stratagene, respectively.
, GSK3
(K85A), and
GSK3
(S9A) cDNAs were isolated by the polymerase chain reaction using
primers (5'-GCGGAATTCATGTCAGGGCGGCCCAGA-3' and
5'-CGCCTCGAGGGTGGAGTTGGAAGCTGATGC-3') and cloned into pCMV5-FLAG (Eastman Kodak Co.). cDNAs for GSK3
deletion mutants (NT, CEN,
N,
and
C) were made by the polymerase chain reaction and cloned into
the EcoRI/XhoI sites of pcDNA3-HA (Invitrogen).
-glycerophosphate, 10 mM sodium fluoride, 5 mM EGTA, and 1 mM phenylmethylsulfonyl
fluoride. Cell lysates were subjected to centrifugation at 12,000 × g for 15 min at 4 °C, and the resulting supernatants
were subjected to immunoprecipitation with the appropriate antibodies.
The resulting precipitates were washed four times with buffer A and
then examined by SDS-PAGE and immunoblot analysis.
(K55R), SEK1(K129R), and
-catenin were used as substrates for JNK/SAPK, SEK1, MEKK1, and
GSK3
, respectively.
and its variants were
translated in vitro in the presence of
[35S]methionine with the use of a rabbit reticulocyte
lysate system (Promega). The resulting 35S-labeled GSK3
proteins were then incubated for 3 h at 4 °C with bacterially
expressed GST fusion proteins of MEKK1 variants in a solution
containing 50 mM Tris, pH 7.5, 150 mM NaCl, 2 mM EDTA, 1 mM dithiothreitol, 0.1% Nonidet
P-40, and 5 mg/ml bovine serum albumin. The GST fusion proteins were
recovered with the use of glutathione-agarose beads. The beads were
then washed three times with washing buffer (50 mM Hepes,
pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol, 0.1% Tween 20). The associated
35S-labeled proteins were eluted from the beads and
analyzed by SDS-PAGE and autoradiography.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Induces the Activation of MEKK1--
To investigate
the possible effect of GSK3
on the SAPK/JNK signaling pathway, we
first transfected HEK293 cells with plasmid encoding HA-tagged SAPK
either alone or together with a vector for FLAG epitope-tagged GSK3
or GSK3
(K85A). Exposure of the transfected cells to UV resulted in
activation of the recombinant SAPK
(Fig.
1A). This effect of UV
irradiation was enhanced by ectopic expression of GSK3
but not by
that of the mutant GSK3
(K85A), which is catalytically inactive (31).
Rather, GSK3
(K85A) inhibited the UV-induced activation of SAPK
.
Whereas overexpressed GSK3
also increased the basal activity of
SAPK
, it did not enhance either the basal activity or the phorbol
12-myristate 13-acetate-stimulated activity of extracellular
signal-regulated kinase 2 (Fig. 1A).
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Fig. 1.
GSK3 induces the
stimulation of MEKK1, SEK1, and JNK. A, HEK293 cells
were transfected for 48 h with expression vectors encoding
HA-SAPK
(left panel) or HA-extracellular signal-regulated
kinase (HA-ERK2) (right panel) either alone or
together with vectors for FLAG-GSK3
or FLAG-GSK3
(K85A) as
indicated. The cells were then either unexposed or exposed to UV light
(80 J/m2) (left panel) or 200 nM
phorbol 12-myristate 13-acetate (right panel). After 30 min,
cell lysates were subjected to immunoprecipitation with mouse
monoclonal anti-HA antibody. The resulting immunoprecipitates were
examined for SAPK
activity (left panel) or extracellular
signal-regulated kinase 2 activity (right panel) by
immunocomplex kinase assays with GST-c-Jun-(1-79) or myelin basic
protein (MBP), respectively, as substrates. The fold
increase in activity relative to that of control cells is indicated.
Cell lysates were also subjected to immunoblot analysis (IB)
with antibodies to HA or to FLAG. Graphs in the bottom show
the means ± average deviation of three independent experiments.
B and C, HEK293 cells were transfected for
48 h with a vector encoding GST-SEK1 (B) or HA-MEKK1
(C) either alone or together with a vector for GSK3
-FLAG
or GSK3
(K85A)-FLAG, as indicated. The cells were then untreated or
treated with UV light (80 J/m2) and further incubated for
30 min. In B, GST-SEK1 expressed in the transfected cells
was isolated from cell lysates with the use of glutathione-agarose
beads and then assayed for kinase activity with GST-SAPK
(K55R) as
substrate. In C, cell lysates were subjected to
immunoprecipitation with antibody to HA, and the resulting precipitates
were assayed for MEKK1 activity with GST-SEK1(K129R) as substrate.
Graphs in the bottom show the means ± average
deviation for SEK1 and MEKK1 activity, respectively, of three
independent experiments. D, HEK293 cells were transfected
for 48 h with an expression vector encoding GST-JNK1 either alone
or together with the indicated combinations of vectors for
GSK3
-FLAG, HA-MEKK1, HA-MEKK1(K1253M), HA-ASK1, or HA-ASK1(K709R).
The cells were then unexposed or exposed to UV light (80 J/m2) and incubated for an additional 30 min. GST-JNK1 was
precipitated from cell lysates with glutathione-agarose beads and
assayed for kinase activity with GST-c-Jun-(1-79) as substrate.
affects the activities of SEK1 and
MEKK1, which function as MAPK kinase and MAPK kinase kinase of
JNK/SAPK, respectively. Overexpressed GSK3
increased in both the
basal and UV-stimulated activities of SEK1 and MEKK1 (Fig. 1, B and C). In contrast, GSK3
did not
increase the activity of ASK1 (data not shown), which also functions as
a MAPK kinase kinase of JNK/SAPK (32). Furthermore, the GSK3
-induced
activation of JNK1/SAPK
was inhibited by expression of
MEKK1(K1253M) (Fig. 1D), a catalytically inactive
mutant of MEKK1, but not by ASK1(K709R), a kinase-inactive mutant of
ASK1. Collectively, these results suggest that GSK3
induces the
activation of MEKK1, thereby triggering JNK/SAPK activation.
(7), on the activation of MEKK1 by GSK3
. Overexpressed
GBP inhibited the GSK3
-induced increase in MEKK1 activity (Fig.
2A). Furthermore, exposure of
HEK293 cells to LiCl, which functions as an inhibitor of GSK3
(33,
34), resulted in a concentration-dependent reduction in the
activities of endogenous JNK1 and MEKK1 (Fig. 2B).
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Fig. 2.
Effects of GBP and LiCl on the JNK/SAPK
signaling pathway. A, HEK293 cells were transfected for
48 h with plasmid encoding HA-MEKK1 either alone or together with
plasmids for GSK3 -FLAG and GBP as indicated. Cell lysates were then
subjected to immunoprecipitation with anti-HA antibody, and the
resulting precipitates were assayed for MEKK1 activity with
GST-SEK1(K129R) as substrate. Cells were also subjected to immunoblot
analysis with antibodies to HA or to FLAG. B, HEK293 cells
were treated for 30 min with LiCl at the indicated concentrations. Cell
lysates were then subjected to immunoprecipitation with antibodies to
JNK1 or to MEKK1. The resulting immunoprecipitates were assayed for
JNK1 or MEKK1 activity with GST-c-Jun-(1-79) or GST-SEK1(K129R),
respectively, as substrates. IB, immunoblot.
Induces MEKK1 Activation in an Axin-independent
Manner--
Axin, a GSK3
-regulated scaffolding protein (4), has
been shown to activate MEKK1 through a direct protein-protein
interaction (35, 36). Furthermore, axin interacts with both GSK3
and MEKK1, implying that it might function as a molecular linker for MEKK1
and GSK3
(36). We therefore examined whether axin mediates the
GSK3
-induced activation of MEKK1. HEK293 cells were cotransfected with an HA-MEKK1 vector and various combinations of GSK3
-FLAG, axin1, and axin1
-(217-353) constructs, and then cell lysates were
examined for MEKK1 activity. Axin1
-(217-353), which lacks the
MEKK1-interacting domain, exerts a dominant-negative effect on
axin-induced MEKK1 activation (36). Overexpression of either GSK3
or
axin1 induced MEKK1 activation in HEK293 cells, and these effects of
GSK3
and axin1 were additive (Fig. 3).
Whereas axin1
-(217-353) inhibited the activation of MEKK1 by axin1,
it did not affect that induced by GSK3
. These data suggest that
GSK3
induces MEKK1 activation in an axin-independent manner.
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Fig. 3.
Axin1 -(217-353)
does not inhibit GSK3
-induced activation of
MEKK1. HEK293 cells were transfected for 48 h with an
expression vector encoding HA-MEKK1 and the indicated combinations of
vectors for GSK3
-FLAG, axin1, and axin1
-(217-353). Cell lysates
were then subjected to immunoprecipitation with anti-HA antibody, and
the resulting immunoprecipitates were assayed for MEKK1 activity with
GST-SEK1(K129R) as substrate. Cell lysates were also subjected to
immunoblot (IB) analysis with anti-HA or anti-FLAG antibody
to show expression of HA-MEKK1, HA-axin1, HA-axin1
-(217-353), or
GSK3
-FLAG.
Physically Associates with MEKK1--
We next investigated
whether GSK3
interacts with MEKK1 in intact cells. HEK293 cells were
transfected with a GSK3
-FLAG vector either alone or together with
vectors for HA-MEKK1 or HA-axin1. Co-immunoprecipitation analysis
revealed the presence of HA-MEKK1 in GSK3
-FLAG immunoprecipitates
(Fig. 4A). HA-axin1 also
associated with GSK3
-FLAG, but it did not affect the extent of the
physical interaction between GSK3
-FLAG and HA-MEKK1. Similar
analysis of non-transfected cells with antibodies to GSK3
and to
MEKK1 also revealed an interaction of the two endogenous proteins (Fig. 4B). This interaction was not markedly affected by exposure
of the cells to UV radiation. In comparison, endogenous GSK3
did not
interact with endogenous ASK1.
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Fig. 4.
GSK3 physically
associates with MEKK1 in intact cells. A, interaction
of ectopic GSK3
and MEKK1 in transfected HEK293 cells. Cells were
transfected for 48 h with an expression vector encoding
GSK3
-FLAG either alone or together with vectors for HA-MEKK1 or
HA-axin1, as indicated. Cell lysates were then subjected to
immunoprecipitation (IP) with anti-FLAG antibody, and the
resulting precipitates were examined by immunoblot analysis with
anti-HA antibody. Cell lysates were also subjected to immunoblot
analysis with anti-HA or anti-FLAG antibody. B, physical
interaction between endogenous GSK3
and MEKK1 in HEK293 cells. Cells
were unexposed or exposed to UV (80 J/m2) and then
incubated for 15 min before immunoprecipitation with either mouse
preimmune IgG or anti-GSK3
antibody. The resulting
immunoprecipitates were subjected to immunoblot analysis with
anti-MEKK1 or anti-ASK1 antibody. IgGH, the heavy
chain of immunoglobulin G.
interacts directly with MEKK1
in vitro. In vitro translated
35S-labeled GSK3
was incubated with GST-MEKK1 fusion
proteins, and an interaction between the two proteins was examined by
GST pull-down analysis. 35S-Labeled GSK3
bound to
MEKK1-N1 (amino acids 1-401 of MEKK1) and MEKK1-N2 (amino acids
402-821) but not to MEKK1-
NC (amino acids 822-1172) or
MEKK1
(amino acids 1173-1493) (Fig.
5A). The
MEKK1 mutant is a
constitutively active form of MEKK1. In a separate in vitro
binding analysis with GST-MEKK1-N1 and 35S-labeled GSK3
variants, MEKK1-N1 bound to full-length GSK3
, GSK3
-NT (amino
acids 1-150), and GSK3
-
C (amino acids 1-300), but not to
GSK3
-CEN (amino acids 151-300) or GSK3
-
N (amino acids
151-420) (Fig. 5B). These results thus suggest that the NH2-terminal region that contains amino acids 1-150 of
GSK3
is required for binding to MEKK1. Neither GST-MEKK1-
NC nor
GST-
MEKK1 interacted with the various GSK3
deletion mutants (data
not shown). GSK3
phosphorylated both MEKK1 and the kinase-inactive
mutant MEKK1(K1253M) in vitro, and these phosphorylation
reactions were inhibited by LiCl (Fig. 5C). LiCl also
inhibited the autophosphorylation of GSK3b.
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Fig. 5.
Direct interaction between
GSK3 and MEKK1 in vitro.
A, 35S-labeled GSK3
was produced by in
vitro translation and incubated for 3 h at 4 °C with GST
alone or the indicated GST-fused deletion mutants of MEKK1. The GST
fusion proteins were recovered with the use of glutathione-agarose
beads, and the bead-bound proteins were eluted and analyzed by SDS-PAGE
and autoradiography. B, in vitro translated
35S-labeled GSK3
variants were incubated for 3 h at
4 °C with GST or GST-MEKK1-N1, after which the
35S-labeled GSK3
variants associated with GST-MEKK1-N1
were detected as in A. C, phosphorylation of
MEKK1 by GSK3
in vitro. HEK293 cells were transfected for
48 h with plasmids encoding GSK3
-FLAG, HA-MEKK1, or
HA-MEKK1(K1253M). Cell lysates were then subjected to
immunoprecipitation with anti-FLAG or anti-HA antibody. In
vitro phosphorylation assays were performed for 30 min at 30 °C
with the indicated combinations of HA-MEKK1, HA-MEKK1(K1253M), and
GSK3
-FLAG immunoprecipitates and 10 mM LiCl in the
presence of [
-32P]ATP (100 µCi/ml). Phosphorylated
proteins were analyzed by SDS-PAGE and autoradiography.
-induced MEKK1 Activation--
Akt
phosphorylates GSK3
on Ser9 and thereby inactivates this
enzyme (4, 10, 15). We therefore examined whether Akt negatively
regulates the activation of MEKK1 by GSK3
. HEK293 cells were
transfected with an HA-MEKK1 vector either alone or together with
various combinations of vectors for GSK3
-FLAG, GSK3
(S9A)-FLAG,
Akt-CA, and Akt(K179A). Akt-CA is a myristoylated form of Akt that is
constitutively active (37), and Akt(K179A) is a kinase-inactive mutant
of Akt. Ectopic GSK3
induced MEKK1 activation in the transfected
cells, and this activation was inhibited by Akt-CA but not by
Akt(K179A) (Fig. 6A). In
contrast, Akt-CA failed to inhibit MEKK1 activation induced by
GSK3
(S9A), which is resistant to the Akt-mediated phosphorylation as
a result of the substitution of Ser9 with Ala.
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Fig. 6.
PI3K and Akt inhibit
GSK3 -induced activation of MEKK1.
A, HEK293 cells were transfected for 48 h with a vector
encoding HA-MEKK1 and the indicated combinations of vectors for
GSK3
-FLAG, GSK3
(S9A)-FLAG, Akt-CA, and Akt(K129R). Cell lysates
were then subjected to immunoprecipitation with anti-HA antibody, and
the resulting immunoprecipitates were assayed for MEKK1 activity with
GST-SEK1(K129R) as substrate. Cell lysates were also subjected to
immunoblot (IB) analysis with antibodies to HA and to FLAG.
B, HEK293 cells were transfected for 48 h with a vector
encoding HA-MEKK1 either alone or together with a vector for
GSK3
-FLAG. The cells were incubated in the absence or presence of
200 nM wortmannin for 30 min and then without or with 1 µM insulin for 30 min. Cell lysates were then assayed for
MEKK1 activity as in A.
-induced activation of MEKK1. The activation of
MEKK1 by GSK3
in transfected HEK293 cells was inhibited by an
exposure of the cells to insulin (Fig. 6B). Furthermore, the
inhibitory effect of insulin was abolished by the PI3K inhibitor wortmannin. These results suggest that the insulin-activated PI3K negatively regulates the activation of MEKK1 by GSK3
.
-induced
Apoptosis--
GSK3
has been shown to induce cell death in several
studies (31, 41). Persistent activation of MEKK1 also induces apoptosis (42, 43). We therefore investigated the possibility that MEKK1 activation mediates apoptosis induced by GSK3
. Neuroblastoma B103 cells were transiently transfected with vectors encoding GSK3
or GSK3
(S9A) in the absence or presence of a vector for the
dominant-negative mutant MEKK1(K1253M), after which the cells were
examined for apoptosis (Fig. 7).
Overexpressed GSK3
or GSK3
(S9A) increased apoptosis in the
transfected cells, and co-expression of MEKK1(K1253M) inhibited this
effect. These results thus suggest that activation of MEKK1 contributes
to the induction of apoptosis by GSK3
. Insulin blocked the induction
of apoptosis by GSK3
but not that by the Akt-resistant mutant
GSK3
(S9A).
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Fig. 7.
Role of MEKK1 activation in
GSK3 -induced apoptosis in neuroblastoma B103
cells. Cells were transfected for 24 h with pEGFP and the
indicated combinations of expression vectors encoding GSK3
,
GSK3
(S9A), and MEKK1(K1253M). The cells were then untreated or
treated with 1 µM insulin for 16 h, fixed, and
stained with 4',6-diamidino-2-phenylindole. GFP-expressing cells were
examined for the apoptotic nuclei by fluorescence microscopy. The
percentage of apoptotic cells was determined, and data are the
means ± S.E. of values from the representative from two
independent experiments.
Null
Mice--
To confirm the biological relevance of GSK3
-induced
MEKK1 activation, we examined the basal, UV-stimulated, and
TNF
-stimulated activities of MEKK1 in MEFs derived from
GSK3
+/+ and GSK3
/
mice (Fig.
8A). The MEKK1 activities
under all three conditions were higher in MEFGSK3
(+/+)
than MEFGSK3
(
/
). Furthermore, ectopic expression of
GSK3
increased both the basal and TNF
-stimulated activities of
MEKK1 in MEFGSK3
(
/
) (Fig. 8B). In
comparison, expression of the kinase-inactive mutant GSK3
(K85A) did
not affect the basal or TNF
-stimulated activities of MEKK1 in
MEFGSK3
(
/
). These data thus suggest that GSK3
functions as a natural activator of MEKK1.
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Fig. 8.
MEKK1 activity is higher in MEF cells from
GSK3 (+/+) mice than in MEF cells
from GSK3
-null mice. A, MEF
cells from either GSK3
+/+ or GSK3
/
mice were left untreated, exposed to UV (80 J/m2), and then
incubated for 30 min or treated with TNF-
(20 ng/ml) for 15 min.
Cell lysates were then subjected to immunoprecipitation with anti-MEKK1
or anti- GSK3
antibody. The resulting immunoprecipitates were
examined for MEKK1 or GSK3
activity by immunocomplex kinase assay.
B, MEFGSK3
(
/
) cells were transfected for
48 h with a vector encoding HA-MEKK1 either alone or together with
a vector for GSK3
-FLAG or GSK3
(K85A)-FLAG. The cells were then
incubated for 15 min in the absence or presence of TNF-
(20 ng/ml).
Cell lysates were subjected to immunoprecipitation with anti-HA
antibody, and the resulting immunoprecipitates were assayed for MEKK1
activity as in A. IB, immunoblot.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
physically interacts with and activates
MEKK1, thereby triggering the JNK/SAPK signaling pathway. Insulin-activated PI3K and Akt inhibited the GSK3
-induced activation of MEKK1. Furthermore, MEKK1 activity was reduced in MEFs from GSK3
/
mice compared with that in MEFs from wild-type
animals, and forced expression of GSK3
increased MEKK1 activity in
the cells from GSK3
/
mice. Our data thus indicate
that GSK3
is an activator of MEKK1.
-interacting protein, was also recently shown to
interact directly with and to activate MEKK1 (36). It implies that axin
might mediate the activation of MEKK1 by GSK3
. Our results, however,
indicate that GSK3
physically associates with and activates MEKK1 in
a manner independent of axin. MEKK1 is activated as a result of
phosphorylation by upstream kinases or through binding to oligomeric
forms of upstream regulators such as TNF receptor associated factor 2 (44, 45). GSK3
might thus induce the activation of MEKK1 by
phosphorylation given that MEKK1 contains several potential sites for
GSK3
-catalyzed phosphorylation. Indeed, the kinase-inactive mutant
GSK3
(K85A) failed to activate MEKK1. Moreover, GSK3
phosphorylated MEKK1 in vitro. Further studies are needed to
identify a phosphorylation site(s) in MEKK1 targeted by GSK3
. We
cannot also exclude the possibility that the physical interaction
between GSK3
and MEKK1 induces the activation of MEKK1 through a
mechanism independent of protein phosphorylation.
and that this effect of insulin was abolished by the
PI3K inhibitor wortmannin. Furthermore, Akt inhibited GSK3
-induced MEKK1 activation. These observations thus suggest that PI3K and Akt
modulate the JNK/SAPK signaling pathway through inhibition of
GSK3
-induced MEKK1 activation. Many lines of evidence have previously demonstrated PI3K and Akt as negative regulators of the
JNK/SAPK cascade (46-50). Akt phosphorylates and inhibits both ASK1
(47) and SEK1 (50), both of which actions result in inhibition of
JNK/SAPK. Thus, PI3K and Akt appear to tightly regulate the JNK/SAPK
signaling pathway by means of multiple mechanisms.
is implicated in many biological events, including signaling
activated by Wnt and glycogen metabolism (4). GSK3
-deficient mice,
however, do not exhibit any marked alterations in the Wnt-induced embryonic development or glycogen metabolism (51). Interestingly, the
activation of the transcription factor NF-
B by TNF-
is impaired in MEFs from GSK3
-deficient mice (51). We now show that MEKK1 activity under either basal or UV- or TNF-
-stimulated condition is
reduced in MEFs from GSK3
-deficient mice compared with that in MEFs
from wild-type mice, consistent with the notion that GSK3
potentiates the function of MEKK1. Thus, our results demonstrate that
GSK3
is an endogenous activator of MEKK1. This function of GSK3
may be an important aspect of the mechanism by which this enzyme
modulates intracellular signaling, including that triggered by cellular stress.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. J. W. Woodgett for
providing wild-type and GSK3/
MEFs and SAPK
cDNA. We also thank Dr. R. J. Davis, Dr. G. Johnson, Dr. H. Ichijo, Dr. L. I. Zon, Dr. J. Chung, Dr. S. C. Lin, and Dr. D. Kimelman for providing JNK1, MEKK1, ASK1, SEK1, Akt, axin1, and
GBP cDNA vector constructs and Dr. W. A. Toscano, Jr. for critical reading of the manuscript.
![]() |
FOOTNOTES |
---|
* This work was supported by the Creative Research Initiatives Program of the Korean Ministry of Science and Technology and in part by a Korea University Grant (to E.-J. C.), and in part by the faculty research fund of Konkuk University (to S.-G. C.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Supported by a postdoctoral fellowship from the BK21 program of the Korean Ministry of Education.
To whom correspondence should be addressed. Tel.:
82-2-3290-3446; Fax: 82-2-3290-4741; E-mail: ejchoi@korea.ac.kr.
Published, JBC Papers in Press, February 12, 2003, DOI 10.1074/jbc.M300253200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
GSK3, glycogen
synthase kinase 3
;
GBP, GSK3
-binding protein;
PI3K, phosphatidylinositol 3-kinase;
MAPK, mitogen-activated protein kinase;
JNK, c-Jun N-terminal kinase;
SAPK, stress-activated protein kinase;
ASK1, apoptosis signal-regulating kinase 1;
HA, hemagglutinin;
MEF, mouse embryonic fibroblast;
GFP, green fluorescence protein;
TNF-
, tumor necrosis factor
;
HEK293, human embryonic kidney 293;
GST, glutathione S- transferase.
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