From the Department of Bio-Signal Analysis, Yamaguchi
University Graduate School of Medicine, Yamaguchi, 755-8505 Japan,
the ¶ Division of Cellular Therapy, Advanced Clinical Research
Center, Institute of Medical Science, University of Tokyo, Tokyo
108-8639 Japan, and the
Division of Molecular Metabolism and
Diabetes, Department of Internal Medicine, Tohoku University Graduate
School of Medicine, Sendai, 980-8575 Japan
Received for publication, June 19, 2002, and in revised form, November 18, 2002
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ABSTRACT |
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MEK kinase 1 (MEKK1) has been shown to contribute
to the regulation of cell migration, whereas focal adhesion kinase
(FAK) is a major player involved in both cell migration and integrin signaling. Here we show that MEKK1 and FAK are co-immunoprecipitated from mouse fibroblasts. Moreover, the association between MEKK1 and FAK
appears to be physiologically relevant, as it is enhanced by treatment
with epidermal growth factor (EGF). Targeting FAK to the membrane also
enhanced its association with MEKK1, indicating that MEKK1 is localized
to a membrane-related subcellular domain, perhaps focal adhesions.
Interestingly, the expression of insulin receptor substrate-1 (IRS-1)
was diminished in MEKK1-deficient fibroblasts, which is similar to an
earlier finding in FAK-deficient fibroblasts. Insulin-like growth
factor 1 (IGF-1)-induced ERK activation was diminished in
MEKK1-deficient cells, but phosphatidylinositol 3-kinase/Akt activation
was not. Although integrin reportedly regulates the transcription of
the IRS-1 gene via FAK-mediated JNK activation, no impairment of
fibronectin-stimulated activation of FAK, ERK, or JNK was observed in
MEKK1-deficient cells. Reconstitution of MEKK1 expression restored
IRS-1 expression as well as IGF-1-induced ERK activation. Taken
together, these findings indicate that MEKK1 interacts with FAK in
focal adhesions and regulates IRS-1 expression.
MEKK11 is a 196-kDa
serine-threonine kinase activated in response to a variety of stimuli,
including EGF, lysophosphatidic acid, osmotic stress, and microtubule
toxins (1, 2). Upon activation, MEKK1 participates in the regulation of
the JNK and ERK pathways and is involved in the activation of NF- FAK is protein tyrosine kinase found at sites of adhesion (7). It is
activated by integrin-mediated adhesion and serves as a signaling
protein within cytoskeleton-associated networks (7). The Src-family
protein tyrosine kinases p130 Cas, Shc, and Grb2 act in concert with
FAK to transduce integrin-generated signals to the ERK/JNK MAP kinase
cascades (7). Experiments using FAK-deficient cells have established
that FAK is essential for integrin-stimulated cell migration and
important for linking activation of the PDGF and EGF receptors to the
cellular machinery that promotes directed cell migration (8). The fact
that MEKK1 is enriched in membranes and colocalizes with Insulin and IGF-1 exert diverse biological effects by binding to and
activating their cognate tyrosine kinase receptors. IRS-1 is a major
substrate for the insulin and IGF-1 receptors, which rapidly
phosphorylate it on multiple tyrosine residues upon ligand binding.
Recently, it was reported that targeted disruption of FAK eliminates
IRS-1 expression in MEFs and that interactions between cells and the
extracellular matrix regulate the transcription of the IRS-1 gene via
FAK-mediated JNK activation (11). Here, we show the following. 1) MEKK1
interacts with FAK in vivo. 2) Like FAK, MEKK1 is required
for IRS-1 expression. 3) Targeted disruption of MEKK1 alters
IGF-1-induced ERK activation but not PI3K/Akt activation.
Cell Culture--
MEFs were harvested from wild-type and
MEKK1 Materials--
Mouse recombinant EGF and IGF-1 were purchased
from Sigma. Anti-FAK and anti-IRS-1 Abs were from Santa Cruz
Biotechnology (Santa Cruz, CA). The anti-IRS-2 polyclonal Ab and the
4G10 anti-phosphotyrosine mAb are described elsewhere (13).
Xpress-tagged MEKK1 expression vector (pcDNA3.1/MEKK1) was
constructed by subcloning full-length MEKK1 into pcDNA3.1 HisB
(Invitrogen, Carlsbad, CA). HA-tagged, wild-type FAK expression vector
(pRcCMV/FAK) was a gift from Dr. S. Hanks (Vanderbilt University) (14).
A myristoylated FAK expression vector (pCEFL Myr FAK) was a gift from
Dr. S. Gutkind (National Institutes of Health). Myristoylated FAK was
constructed by fusing the amino-terminal myristoylation signal from
c-Src with FAK, resulting in a constitutively active, heavily
tyrosine-phosphorylated form of FAK that is targeted to the membrane
(15). Transient transfections were performed using FuGENE-6 (Roche
Diagnostics). Recombinant retrovirus with pMY
vector2 was prepared as
described previously (16). MEKK1 Immunoprecipitation and Immunoblotting--
Cells were washed
twice with PBS and lysed in 20 mM Tris-HCl (pH 7.6), 0.5%
Nonidet P-40, 250 mM sodium chloride, 3 mM
EDTA, 3 mM EGTA, 1 mM phenylmethylsulfonyl
fluoride, 2 mM sodium orthovanadate, 20 µg/ml aprotinin,
1 mM dithiothreitol, and 5 µg/ml leupeptin. The lysates
were centrifuged at 14,000 × g for 10 min at 4 °C, after which the supernatant was incubated first with the appropriate Abs for 16 h at 4 °C and then with protein G-Sepharose
(Amersham Biosciences) for an additional 1 h. The Sepharose beads
were then washed three times in lysis buffer and resuspended in Laemmli sample buffer. The immunoprecipitates were resolved by electrophoresis. For IGF-1 stimulation, cells were serum starved overnight in medium containing 0.1% fetal calf serum and then exposed to IGF-1 (100 ng/ml)
for the indicated times.
Northern Blotting--
Total RNA was extracted from cells
using ISOGEN (Nippongene, Japan), after which 15-µg samples were
denatured with formaldehyde/formamide, resolved by electrophoresis, and
transferred to Hybond membranes (Amersham Biosciences). The
probes used were an IRS-1 cDNA fragment and the full-length
GAPDH cDNA.
Cell Adhesion Assay--
Culture dishes were coated with 10 µg/ml bovine plasma fibronectin (Sigma) or 10 µg/ml mouse
sarcoma-derived laminin (Sigma) in PBS at 4 °C overnight. The dishes
were then rinsed twice with PBS and warmed for 1 h at 37 °C
before use. MEFs were serum starved in IMDM with 0.1% fetal calf serum
overnight and then detached using 0.05% trypsin and 2 mM
EDTA. After the addition of trypsin inhibitor (Sigma), the cells were
pelleted by centrifugation and then resuspended in IMDM containing
0.1% fetal calf serum. The cells were then kept in suspension for
2 h, after which they were then plated on the coated dishes for
the indicated times, harvested, and lysed.
Analysis of Kinase Activity--
To determine ERK and Akt
activities, cell lysates were immunoblotted with either an
anti-phospho-MAPK or anti-phospho-Akt Ab (Cell Signaling Technology,
Inc.). NIH Image 1.62 was used for quantitative analysis of kinase
activity. ERK activity was also measured by in vitro kinase
assay with myelin basic protein as substrate (5). PI3K and JNK
activities were measured as described previously (1, 13).
MEKK1 Interacts with FAK--
After transiently co-transfecting
HEK293T cells with expression vectors encoding Xpress-tagged MEKK1 and
HA-tagged FAK, we were able to co-immunoprecipitate the expressed MEKK1
and FAK using anti-Xpress and anti-HA Abs (Fig.
1A). We then co-expressed MEKK1 and wild-type or myristoylated FAK in HEK293T cells and found
that the latter more readily interacted with MEKK1 than the former
(Fig. 1B). Moreover, endogenous FAK and MEKK1 were co-immunoprecipitated from MEFs, and this interaction was enhanced by
treatment with EGF (Fig. 1C). EGF induced the activation of both ERK and JNK in MEFs (Fig. 1D). These data suggests that
MEKK1 interacts with FAK in vivo and that the interaction is
physiologically regulated.
To identify the MEKK1 domain that interacts with FAK, HEK293T cells
were transfected with plasmids encoding various Xpress-tagged MEKK1
mutants and HA-tagged, wild-type FAK. The MEKK1 mutants were
immunoprecipitated using anti-Xpress Ab and then blotted with anti-HA
Ab (Fig. 2). Co-immunoprecipitation of
MEKK1 with FAK was still observed when the MEKK1 kinase domain (amino
acids 1171-1493) was deleted; conversely, the MEKK1 kinase domain
expressed in HEK293 cells was not co-immunoprecipitated with FAK (data
not shown). When we examined the N-terminal regulatory region of MEKK1, we found that the region encompassing amino acids 1-144 (Fig. 2,
d1) did not interact with FAK, but regions encompassing
amino acids 1-443 (Fig. 2, d2) or more (Fig. 2,
d3-d5) were co-immunoprecipitated with FAK.
Thus, a MEKK1 domain encompassing amino acids 145-443 is essential for
binding to FAK.
MEKK1 Regulates IRS-1 Expression--
It was recently reported
that IRS-1 is not expressed in FAK-null fibroblasts (11). To further
investigate the relationship between MEKK1 and FAK, we evaluated IRS-1
expression in MEKK1
We found that expression of the IRS-1 protein was increased when MEF
cultures reached confluence (Fig. 3A). We therefore tested whether IRS-1 expression was also cell density dependent in HEK293T cells. In one set of experiments, the same numbers of the cells were
seeded on day 0 and then harvested on day 1, 2, or 4. To minimize the
effect of humoral factors, the culture medium was changed every day. In
another set of experiments, different numbers of the cells were seeded
on day 0 and then harvested after 48 h. In both experiments,
expression of the IRS-1 mRNA and protein was increased as a
function of cell density (Fig. 3C). Notably, changes in cell
density affected IRS-1 expression similarly in MEKK1
Cell adhesion to fibronectin and vitronectin reportedly induces the
expression of IRS-1 (11). In that regard, the activation of integrin in
turn activates FAK, which is required for the fibronectin-stimulated activation of JNK, suggesting that IRS-1 expression is regulated at
least in part by a FAK-JNK pathway (11). We therefore investigated whether adhesion to fibronectin would induce activation of FAK, JNK,
ERK, and NF- MEKK1 Is Involved in IGF-1 MEKK1 reportedly binds to a number of signaling proteins including
Ras, Raf-1, Rac1, cdc42Hs, Nck-interacting kinase, SEK1, and JNK (1,
17, 18, 19-21); consequently, MEKK1 has been proposed to function,
like yeast Pbs2p, as a scaffold protein (22) . In the present study, we
showed that MEKK1 also interacts with FAK in vivo,
suggesting that among the subcellular regions to which MEKK1 is
localized are focal adhesions. The finding that a membrane-targeted
form of FAK (myristoylated FAK) associated with MEKK1 more readily than
did wild-type FAK supports that idea. In addition, the finding that EGF
enhanced the interaction between endogenous MEKK1 and FAK suggests that
the interaction is physiologically relevant. In that regard, FAK is
known to associate with the activated EGF receptor signaling complex
and to be required for EGF-stimulated cell motility (8). MEKK1 has been
shown to be activated in the EGF receptor signaling pathway (1), and
EGF indeed stimulated activation of ERK and JNK in MEFs. EGF might
recruit MEKK1 to focal adhesion complexes containing FAK and EGF
receptor. The function of MEKK1 within focal adhesions is unclear,
although it is known to regulate JNK activation and survival in cells
challenged with mild hyperosmolarity and microtubule toxins (2, 5). The
fact that these stimuli elicit changes in cell shape suggests that
MEKK1 localized in focal adhesions might be involved in sensing cytoskeletal dynamics.
Our data also show that the N terminus of MEKK1 (amino acids 145-443)
is important for binding FAK. This region has a proline-rich segment
containing putative binding sites for proteins having SH3 domains (23).
FAK does not contain an SH2 or SH3 domain, but it does contain SH2
domain-interacting phosphotyrosines and SH3 domain-interacting
proline-rich regions. It may be that one or more other adaptor proteins
(e.g. Grb2) mediates the interaction of MEKK1 with FAK.
Additional studies will be necessary to precisely define the nature of
the interaction between MEKK1 and FAK as well as the physiological
significance of that interaction.
Integrins are transmembrane proteins that mediate cell adhesion with an
extracellular matrix (7). Using FAK-deficient cells, Lebrun et
al. (11) showed that integrins regulate transcription of the
IRS-1 gene, in part via FAK-mediated JNK activation. That report prompted us to examine IRS-1 expression, which led to the finding that levels of IRS-1 protein and mRNA are diminished in MEKK1 In the above mentioned study, Lebrun et al. (11) also
reported that cell adhesion to fibronectin increases IRS-1 expression and that JNK in not activated in FAK The results of this and our earlier study (6) show that, like FAK,
MEKK1 positively regulates IRS-1 expression and cell motility. On the
other hand, the ectopic expression of IRS-1 in prostate cancer cells
that do not express IRS-1 endogenously increased cell adhesion and
decreased cell motility (24). Although this finding is apparently
opposite to ours, it is nevertheless consistent with the idea that
IRS-1 plays a key role in regulating cell motility. IRS-1 also mediates
various metabolic and growth-promoting actions of insulin and IGF-1.
For instance, mice lacking IRS-1 display retardation of somatic growth
and enhanced Upon insulin stimulation, IRS-1 interacts with
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B
(3-5). In addition, MEKK1 senses microtubule integrity, protects cells from committing to apoptosis, and contributes to the migration of
fibroblasts and epithelial cells. The phenotype of the MEKK1-null mouse
includes an eyelid closure defect that is also seen in mice lacking the
EGF receptor and TGF-
(6). This suggests the possibility that MEKK1
is required for EGF receptor control of cell migration. Consistent with
that idea, overexpression of MEKK1 induces the formation of a large
lamellipodia-like structure in epithelial cells (6). Still, the
mechanism by which MEKK1 influences cell motility remains unclear.
-actinin
along actin stress fibers at focal adhesions (9, 10) prompted us to
investigate the possibility that MEKK1 is associated with FAK.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
14.5 day embryos (6), after which they were immortalized
using the 3T3 protocol (12). MEFs and HEK293T cells were cultured in
IMDM (Invitrogen) supplemented with 100 units/ml penicillin, 100 mg/ml streptomycin (Invitrogen), 10% fetal calf serum (Gemini
Bio-Products, Woodland, CA) and 15 × 10
5
M monothioglycerol (Sigma).
/
MEFs were stably
transfected using pMY-HA-tagged, full-length, wild-type MEKK1 or
pMY-full-length IRS-1.
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Expression of MEKK1 and FAK.
A, co-immunoprecipitation of MEKK1 and FAK in 293T cells.
293T cells were transiently transfected with empty vector (pCDNA
3.1 or pRcCMV), Xpress-tagged, full-length recombinant MEKK1
(Xpress-MEKK1), or HA-tagged, full-length recombinant FAK
(HA-FAK). Immunoprecipitation (IP) and
immunoblotting (IB) were performed with anti-Xpress and
anti-HA Abs (12CA5) as indicated. B, enhanced interaction of
membrane-targeted FAK with MEKK1. MEKK1 was coexpressed with FAK
containing the N-terminal myristoylation signal from c-Src (Myr.
FAK), wild-type FAK (WT-FAK), or empty vector
(Mock) in HEK293T cells. Immunoprecipitation and
immunoblotting were performed with the Abs against endogenous MEKK1 or
FAK, as indicated. C, co-immunoprecipitation of endogenous
MEKK1 and FAK in mouse embryonic fibroblasts. MEFs were treated with
100 ng/ml EGF or without EGF (control) for 10 min.
Immunoprecipitation and immunoblotting were performed with Abs against
endogenous FAK and MEKK1 as indicated. D, MEFs were treated
with 100 ng/ml EGF for the indicated periods. ERK and JNK activations
were measured by in vitro kinase assay using myelin basic
protein and GST-Jun as substrates, respectively. The autoradiogram
shown is representative of three independent experiments yielding
similar results.
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Fig. 2.
The N-terminal region of MEKK1 is essential
for its interaction with FAK. Xpress-tagged MEKK1 deletion mutants
and wild-type FAK were co-transfected into HEK293T cells. Deletion
mutants of MEKK1 were as follows: d1 (amino acids 1-144),
d2 (1-443), d3 (1-785), d4 (1-975)
and d5 (1-1230). Cell lysates were prepared,
immunoprecipitated (IP) with anti-Xpress mAb, and
immunoblotted (IB) with anti-HA mAb. To detect exogenous
expression of MEKK1 mutants and FAK, immunoblotting with anti-Xpress
and anti-HA Ab was performed.
/
MEFs. Although there was no significant
difference in the growth rates of MEKK1
/
and wild-type MEFs (data
not shown), expression of the IRS-1 protein was diminished in two
independent MEKK1
/
MEF clones (Fig.
3A). By contrast, no changes
in the expression of IRS-2, another IRS family protein, or FAK were
observed in MEKK1-deficient cells. This indicates that the decreased
expression of IRS-1 is not due to altered expression of FAK and that
the expression of IRS-1, but not IRS-2, is regulated by MEKK1. As expected, Northern blot analysis showed levels of IRS-1 mRNA to be
diminished in the MEKK1
/
MEF clones, whereas levels of GAPDH mRNA were similar in MEKK1
/
and wild-type cells (Fig.
3B).
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Fig. 3.
MEKK1-dependent IRS-1
expression. A, decreased IRS-1 expression in MEKK1 /
MEFs. Wild-type MEF (+/+) and two independent
MEKK1
/
MEF clones (
/
) were seeded on day 0 at a density of 2 ×105 cells/10-cm dish. The cells were harvested on day 2, 3, or 4, and the cell lysates were immunoblotted for IRS-1, IRS-2, and
FAK. Equivalent proteins were loaded on each lane. B,
decreased expression of IRS-1 mRNA in MEKK1
/
MEFs. MEFs were
seeded at a density of 2 × 105 cells/10-cm dish on
day 0 and harvested on day 2. Total RNA was extracted, resolved on
formaldehyde gels, and subjected to Northern blotting. IRS-1 and GAPDH
mRNA were detected using IRS-1 and GAPDH cDNAs as probes.
C, cell density-dependent expression of IRS-1 in
293T cells. Cells were seeded at a density of 1 × 106/10-cm dish and harvested on day 1, 2, or 4 (left panel). Alternatively, cells were seeded at
1 × 106, 5 × 106, or 1 × 107 cells/10-cm dish and harvested after 48 h
(right panel). Immunoblotting was performed using
an IRS-1 Ab. Northern blotting was performed using IRS-1 cDNA as a
probe. Ethidium bromide-stained 28 S rRNA levels demonstrate similar
loading in each lane.
/
and wild-type
cells, suggesting that MEKK1 plays a role in regulating basal IRS-1
expression but not cell density-dependent expression.
Finally, stably transfecting MEKK1
/
MEFs with full-length MEKK1
cDNA using a retroviral vector showed that expression of the IRS-1
protein was restored by re-expression of MEKK1, confirming that MEKK1
is required for IRS-1 expression in MEFs (Fig.
4).
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Fig. 4.
Restoration of IRS-1 expression by
reconstitution of MEKK1 in MEKK1 /
MEFs. MEKK1
/
MEFs were
stably transfected with recombinant retrovirus harboring full-length
MEKK1 cDNA. MEKK1
/
MEFs re-expressing MEKK1 (AB) and
those that were not (
/
) were seeded at 2 × 105
cells/10-cm dish on day 0. The cells were harvested and lysed on day 2, and the cell lysates were immunoblotted for IRS-1 and MEKK1.
B in MEKK1
/
cells. We found that fibronectin stimulated FAK, JNK and ERK activation to a similar degree in wild-type
and MEKK1
/
cells (Fig. 5) and that
NF-
B was not activated in either cell type (electrophoresis mobility
shift assay; data not shown). Apparently, MEKK1 is not required for
fibronectin-induced FAK, JNK, and ERK activation. We also tested
whether laminin, another extracellular matrix protein, would induce
FAK, JNK, or ERK activation and found a slight activation of FAK that
was not different in MEKK1
/
and wild-type cells. Neither JNK nor
ERK was activated by laminin in either cell type (data not shown).
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Fig. 5.
MEKK1 is not required for
fibronectin-stimulated activation of FAK, JNK, and ERK. MEKK1 /
(
/
) or wild-type (+/+) MEFs were serum
starved overnight, detached by limited trypsin/EDTA treatment, and
either suspended for 2 h or replated on fibronectin-coated (10 µg/ml) dishes for the indicated times in the absence of serum. FAK
was then immunoprecipitated (IP) from the lysates and
analyzed by immunoblotting (IB) with anti-phosphotyrosine Ab
(4G10). JNK activation was analyzed using an in
vitro kinase assay with GST-Jun as a substrate. ERK activation was
analyzed using an anti-phospho-MAPK Ab. The autoradiogram shown is
representative of three independent experiments yielding similar
results.
induced ERK Activation but Not
PI3K/Akt Activation--
IGF-1 exerts a wide variety of
effects including cell proliferation, differentiation, survival, and
migration, in part via IRS-1, a substrate and downstream signaling
molecule for the IGF-1 receptor. Because IRS-1 expression was
diminished in MEKK1
/
MEFs, we examined IGF-1 signal transduction in
MEKK1
/
MEFs. We found that IGF-1 strongly activated ERK and
PI3K/Akt and weakly activated JNK in wild-type MEFs. The ERK activation
was significantly diminished in the MEKK1
/
MEFs, whereas the PI3K
and Akt activation were similar in both cell types (Fig.
6, and data not shown), as was the JNK
activation (data not shown). Reconstitution of MEKK1 expression
restored IGF-1-induced ERK activation in MEKK1
/
cells (Fig.
7A). MEKK1 thus appears to
contribute to IGF-1-induced ERK activation but not to PI3K/Akt
activation. It is possible that a decreased IRS-1 protein reduces ERK
activation in MEKK1
/
MEFs. It is also possible, however, that there
exists both IRS-1-independent and MEKK1-dependent pathways
leading to ERK activation. To investigate these possibilities,
IRS-1 was stably transfected to MEKK1
/
cells. Although IRS-1 was
abundantly expressed in MEKK1
/
cells, IGF-1-induced ERK
activation was not restored (Fig. 7B). This indicates
that the decreased activation of ERK by IGF-1 in MEKK1
/
cells is
not due to the altered expression of IRS-1.
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Fig. 6.
IGF-1-induced ERK and Akt activation on
wild-type and MEKK1 /
MEFs. A, Decreased
IGF-1-induced ERK activation in MEKK1
/
MEFs. Wild-type
(+/+) and MEKK1
/
(
/
) MEFs were stimulated
with 100 ng/ml IGF-1 for the indicated periods. Cell lysates were then
analyzed by immunoblotting with phospho-ERK Ab. Reprobing the membranes
with Abs against total ERK showed that the same amount of protein was
loaded on each lane. The intensity of phosphorylated p44 ERK1 was
quantified using NIH Image 1.62. The autoradiogram shown is
representative of three independent experiments yielding similar
results; *, p < 0.05 (Student's t test).
B, IGF-1-induced Akt activation was the same in MEKK1
/
and wild type MEFs. Cells were stimulated with 100 ng/ml of IGF-1 for
the indicated times. Immunoblot analysis was performed with
anti-phospho-Akt (Ser-473) and anti-Akt1 Abs. The autoradiogram shown
in the bottom panel is representative of three
independent experiments yielding similar results.
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Fig. 7.
Restoration of ERK activation by
reconstitution of MEKK1 but not by overexpression of IRS-1 in
MEKK1 /
MEFs. A, MEKK1
/
MEFs (
/
) and a MEKK1
re-expression clone (AB) were challenged with 100 ng/ml
IGF-1 for the indicated periods. Then, the cell lysates were
immunoblotted with anti-phospho-ERK and anti-ERK2 Abs. B,
wild-type (+/+), MEKK1
/
MEFs (
/
), and
stably transfecting MEKK1
/
MEFs with IRS-1 (IRS-1) were
stimulated with 100 ng/ml IGF-1 for the indicated periods. The cell
lysates were then immunoblotted with anti-IRS-1, anti-phospho-ERK, and
anti-ERK2 Abs.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
cells and that the ectopic expression of MEKK1 in MEKK1
/
cells is sufficient to restore expression of the protein. These results
demonstrate unequivocally that MEKK1 regulates expression of the IRS-1
message and protein. Interestingly, IRS-1 protein levels were increased
as a function of cell density in both MEFs and HEK293T cells. In this
case, IRS-1 expression may be up-regulated as the number of
cell-to-cell interactions via adhesion molecules increases. Such a
situation would be consistent with an earlier report that cell adhesion
to the extracellular matrix up-regulates IRS-1 expression (11). The
fact that cell density-dependent IRS-1 expression was also
observed in MEKK1
/
MEFs indicates that a MEKK1-independent pathway
to IRS-1 expression also exists.
/
MEFs when integrin engages fibronectin. They therefore concluded that FAK is essential for fibronectin-stimulated JNK activation. In the present study, by contrast, adhesion to fibronectin induced a similar activation of JNK,
ERK, and FAK in wild-type and MEKK1
/
MEFs, suggesting that integrin
induces IRS-1 expression via an integrin-FAK-JNK pathway that is
independent of MEKK1. The signaling molecules involved in the
regulation of IRS-1 expression thus remain to be determined. It is
possible that, like FAK, MEKK1 is involved in organizing the cortical
cytoskeleton and that disorganization due to the loss of MEKK1
participates in the down-regulation of IRS-1 expression.
-cell mass (25, 26); effects on cell migration have not
been reported, however.
v
3 integrins (27). FAK binds to both
IRS-1 and integrins (7, 28), suggesting that IGF-1/insulin and cell
adhesion induce the formation of focal adhesion complexes that include
IRS-1, FAK, and MEKK1. We found that IGF-1-induced ERK activation was
significantly diminished in MEKK1
/
MEFs. The decreased activation
of ERK by IGF-1 in MEKK1
/
MEFs was independent of IRS-1 expression.
Although MEKK1 contributes significantly to IGF-1-stimulated ERK
activation, its effect is only partial. The activation of other MAPK
kinase kinases (e.g. Raf-1 and B-Raf) that also activate ERK
might partially compensate for the loss of MEKK1. In contrast to ERK
activation, IGF-1-induced PI3K/Akt activation was not diminished in
MEKK1
/
MEFs. Although IRS-1 is a key mediator of
IGF-1/insulin-induced PI3K signaling, in this case other IRS proteins
(e.g. IRS-2 which is abundant in MEFs) might have
compensated for the diminished levels of IRS-1. In summary, MEKK1
interacts physiologically with FAK and regulates IRS-1 expression,
which in turn contributes to the regulation of IGF-1/insulin-induced
signaling and cell migration.
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ACKNOWLEDGEMENTS |
---|
We thank Drs. S. Hanks and S. Gutkind for the generous gift of plasmids. We also thank Dr. G. L. Johnson for valuable discussions and Yukari Kora and Atsuko Tanimura for technical assistance.
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FOOTNOTES |
---|
* This work was supported by research fellowships of the Japan Society for the Promotion of Science for Young Scientists, The Naito Foundation, The Kanae Foundation, and Yamanouchi Foundation for Research on Metabolic Disorders (to T. Y.).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.
§ To whom correspondence should be addressed. Tel.: 81-836-22-2251; Fax: 81-836-22-2342; E-mail: yujirit@yamaguchi-u.ac.jp.
Published, JBC Papers in Press, November 27, 2002, DOI 10.1074/jbc.M206087200
2 T. Kitamura, unpublished data.
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ABBREVIATIONS |
---|
The abbreviations used are:
MEKK1, mitogen-activated protein kinase/extracellular signal regulated kinase
kinase 1;
MAP, mitogen-activated protein;
MAPK, MAP kinase;
ERK, extracellular signal-regulated kinase;
MEK, MAPK/ERK kinase;
EGF, epidermal growth factor;
JNK, c-Jun N-terminal kinase;
TGF-, transforming growth factor-
;
FAK, focal adhesion kinase;
PDGF, platelet-derived growth factor;
IGF-1, insulin-like growth factor 1;
IRS-1, insulin receptor substrate 1;
PI3K, phosphatidyl inositol
3-kinase;
MEF, mouse embryonic fibroblast;
HEK293, human embryonic
kidney 293 (cell line);
IMDM, Iscove's modified Eagle's medium;
Ab, antibody;
mAb, monoclonal Ab;
HA, hemagglutinin;
PBS, phosphate-buffered saline;
GAPDH, glyceraldehyde-3-phosphate
dehydrogenase;
SH, Src homology;
GST, glutathione
S-transferase.
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