From the Faculty of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama 226-8501, Japan
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ABSTRACT |
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Heterotrimeric G protein Mitogen-activated protein kinases
(MAPKs)1 are proline-directed
serine/threonine kinases and play important functions as mediators of
cellular responses to a variety of stimuli such as growth factors, cytokines, hormones, and environmental stresses (1-4). In mammalian cells, MAPKs have been classified into at least four subfamilies: ERK/MAPK, JNK/SAPK, p38 MAPK, and BMK1/ERK5. ERK is activated by many
growth factors and cytokines and phosphorylates transcription factors,
translational factors, cytoskeleton proteins, and other protein kinases
(1, 2). ERK is implicated in cell growth, differentiation, and
survival. Various stressors such as chemical agents and ultraviolet
irradiation, tumor necrosis factor- MAPK cascades consist of three conserved components: a family of
serine/threonine kinases called MAPKKK/MEKK, which activate MAPKK/MEK,
which in turn activates MAPK by a simultaneous phosphorylation on
threonine and tyrosine residues (1-4). Raf and Tpl-2 activate ERK
through MEK1 and MEK2. On the other hand, it has been shown that
overexpression of Tpl-2, MEKK1, MEKK2, MEKK3, MEKK4/MTK1, MAPKKK5/ASK1, TAK1, and MLK family protein kinases induce the activation of JNK and/or p38 MAPK cascade(s). JNK and p38 MAPK are
phosphorylated and activated by MKK4/SEK1/JNKK1/SKK1 (2-4). More
recently, MKK7/JNKK2/SKK4 has been cloned and shown to phosphorylate and activate only JNK (4). In contrast, MKK3/SKK2 and MKK6/SKK3 have
been known to phosphorylate and activate specifically p38 MAPK (2-4).
MKK5/MEK5 has been reported to phosphorylate BMK1 (3).
Heterotrimeric guanine nucleotide-binding proteins (G proteins) are
composed of Different G protein-coupled receptors stimulate the ERK pathway through
different G protein subunits in various types of cells (11). In the
case of the Gq/11-coupled m1 muscarinic acetylcholine and
On the other hand, it has been demonstrated that JNK is also activated
by an agonist stimulation of m1 and m2 muscarinic acetylcholine receptors expressed in NIH3T3, Rat-1, and COS-7 cells (15-17). JNK
activation by muscarinic acetylcholine receptors has been shown to be
mediated primarily by G During investigations on JNK activation by stimulation of the m1
muscarinic acetylcholine receptor, we found that its activation was
mediated by both G Antibodies and Inhibitors--
Mouse monoclonal antibody (B-14)
against Schistosoma japonicum GST was purchased from Santa
Cruz Biotechnology, Inc. Mouse monoclonal antibodies M2 and 12CA5
against FLAG epitope and HA epitope were obtained from Eastman Kodak
Co. and Boehringer Mannheim, Inc., respectively. Rabbit polyclonal
antibodies 06-238 and T-20 against G Construction of Mammalian and Bacterial Expression
Plasmids--
Complementary DNAs for human MKK4 (27) and human SKK4
(28), a human homolog of mouse MKK7, were amplified from a human fetal
brain cDNA library (CLONTECH) by polymerase
chain reaction using Ex Taq polymerase (TaKaRa). The DNAs
were inserted into the BamHI restriction site of mammalian
GST tag expression vector (pCMV-GST) which was generated by J. Suzuki,
and into the BglII/BamHI restriction sites of
mammalian FLAG tag expression vector (pCMV-FLAG). DNAs encoding
kinase-deficient mutants of MKK4 and MKK7, MKK4K95R (27) and MKK7K63R
(29), were produced by polymerase chain reaction-mediated mutagenesis
using Pfu polymerase (Stratagene) and inserted into
pCMV-FLAG. The SR Cell Culture and Transfection--
HEK 293 cells (ATCC CRL 1573)
were maintained in Dulbecco's modified Eagle's medium (Sigma)
containing 100 µg/ml kanamycin (Nacalai) with 10% heat-inactivated
fetal bovine serum (Life Technologies, Inc.). The cells were cultured
at 37 °C in humidified atmosphere containing 10% CO2.
Plasmid DNAs were transfected into HEK 293 cells by the calcium
phosphate precipitation method. The final amount of transfected DNA for
a 60-mm dish was adjusted to 30 µg by empty vector, pCMV. Five µg
of SR Purification of Recombinant Proteins from Escherichia
coli--
E. coli strain BL21 (DE3) cells were transformed
with pET15b-JNK, pET32a-c-Jun-(1-223), or pGEX2T-c-Jun-(1-223). An
isolated colony was inoculated in 10 ml of LB medium containing 100 µg/ml carbenicillin and shaken at 30 °C overnight. This culture
was diluted in 500 ml of medium and grown at 30 °C until
A600 reached 0.2 and added to 0.4 mM isopropyl-1-thio-
All purification steps were performed at 4 °C. For the purification
of hexahistidine-JNK or Trx-c-Jun-(1-223), the supernatants were
applied to NTA-agarose (Qiagen, Inc.) and washed with column buffer A
(20 mM HEPES-NaOH (pH 8.0), 1 mM
phenylmethanesulfonyl fluoride, 1 µg/ml aprotinin, 1 µg/ml
leupeptin) containing 20 mM imidazole. Hexahistidine-JNK
and Trx-c-Jun-(1-223) were eluted with column buffer A containing 200 mM imidazole. Hexahistidine-JNK was diluted to the
concentration of 0.3 mg of protein/ml with column buffer A containing
200 mM imidazole before dialysis. For the purification of
GST-c-Jun-(1-223), the supernatant was applied to
glutathione-Sepharose 4B (Amersham Pharmacia Biotech) and washed with
column buffer B (20 mM HEPES-NaOH (pH 7.5), 1 mM dithiothreitol, 1 mM phenylmethanesulfonyl
fluoride, 1 µg/ml aprotinin, 1 µg/ml leupeptin, 1 mM
EGTA), and eluted with column buffer B containing 10 mM
glutathione. The eluate was dialyzed against column buffer B and stored
at MAPK Assays--
After 24 h of serum starvation, the cells
transfected together with SR MAPKK Assays--
After 24 h of serum starvation, the
cells transfected together with pCMV-GST-MKK4 or pCMV-GST-MKK7 were
treated with or without 10 µM carbachol at 37 °C for
15 min and lysed in 600 µl of lysis buffer A on ice. Aliquots (500 µg) of the supernatants after centrifugation were mixed with
glutathione-Sepharose 4B for 2 h at 4 °C. GST-MKK4 or GST-MKK7
was precipitated by centrifugation and washed with lysis buffer A and
reaction buffer A. The precipitates were incubated in 30 µl of
reaction buffer A containing 2 µg of hexahistidine-JNK, 10 µg of
Trx-c-Jun-(1-223), 20 µM ATP, and 5 µCi of
[ Immunoblotting--
Aliquots of cell lysates were boiled in
Laemmli sample buffer. The boiled samples were separated by
SDS-polyacrylamide gel electrophoresis, and the proteins were
transferred to nitrocellulose membranes. After the membranes were
blocked, the separated proteins were immunoblotted with various
antibodies. The bound antibodies were visualized by an enhanced
chemiluminescence detection system, using anti-rabbit or mouse Ig
antibody conjugated with horseradish peroxidase as a secondary antibody.
Protein Assay--
Protein concentrations were determined using
Bradford reagent (Nacalai) with bovine serum albumin as the standard.
JNK Activation by the m1 Muscarinic Acetylcholine Receptor Is
Mediated by Both G
Next, we examined whether JNK activation by the m1 muscarinic
acetylcholine receptor is mediated by G Agonist Stimulation of the m1 Muscarinic Acetylcholine Receptor
Activates JNK through MKK4 and MKK7--
To explore whether two JNK
kinases MKK4 and MKK7 are involved in JNK activation by the m1
muscarinic acetylcholine receptor, the cells were transfected with
plasmids expressing the receptor and HA-JNK, and MKK4K95R or MKK7K63R.
MKK4K95R and MKK7K63R are the kinase-deficient mutants in which a
crucial lysine residue of ATP binding site is replaced by an arginine
and act as dominant-interfering mutants by sequestering upstream
components (27, 29). As shown in Fig. 2,
A and B, cotransfection of either MKK4K95R or
MKK7K63R partially attenuated the m1 muscarinic acetylcholine
receptor-mediated JNK activation, indicating that MKK4 and MKK7 are
involved in this cascade.
Next, the cells were transfected with plasmids expressing the m1
muscarinic acetylcholine receptor and GST-MKK4 or GST-MKK7, which was
tagged with GST at the NH2 terminus (27, 29). Using a
glutathione-Sepharose 4B, GST-MKK4 or GST-MKK7 was affinity precipitated from lysates of the transfected cells, and the kinase activity was measured by the reconstitution assay using recombinant hexahistidine-tagged JNK and Trx-tagged c-Jun (amino acids 1-223). In
each experiment, we examined the expression level of GST-MKK4 or
GST-MKK7 by immunoblotting to monitor the transfection efficiency (see
Figs. 2-7). Activation of the m1 muscarinic acetylcholine receptor with carbachol stimulated MKK4 and MKK7 activities (Fig. 2,
C and D). These results suggest that the m1
muscarinic acetylcholine receptor activates JNK through at least two
JNK kinases, MKK4 and MKK7.
MKK4 Is a Major Mediator for JNK Activation by
G
Next, we investigated whether G G MKK4 Activation by G PP2 and PP1, Specific Tyrosine Kinase Inhibitors,
Significantly Inhibit G G MKK4 and MKK7 have been shown to respond to environmental stresses
and inflammatory cytokines such as tumor necrosis factor- Cotransfection of kinase-deficient MKK4 completely inhibited
G The number of MAPKKK involved in JNK pathway is currently growing, and
the regulation of MAPKKK is very divergent and complicated (3, 4).
Several groups reported recently that MEKK1, MEKK4, MLK2, and MLK3
specifically associate with Cdc42 and Rac and may mediate JNK
activation through Cdc42 and Rac (39-41). Because Cdc42 and Rac were
involved in the activations of MKK4 and MKK7 by G We have shown previously that G We found differential involvement of Rho family GTPases in
G It is likely that tyrosine kinase is required for the MKK4 but not MKK7
activation by G Crespo et al. (43) reported recently that tyrosine
phosphorylation of Vav guanine nucleotide exchange factor promoted the exchange of GDP to GTP on Rac1 and to a lesser extent on Cdc42. It is
conceivable that G Pretreatment of specific phosphatidylinositol 3-kinase inhibitors
wortmannin and LY294002 failed to inhibit MKK4 and MKK7 activation by
G The present study presents some hints for elucidating the mechanism by
which G subunit (G
)
mediates signals to two types of stress-activated protein kinases,
c-Jun NH2-terminal kinase (JNK) and p38
mitogen-activated protein kinase, in mammalian cells. To investigate
the signaling mechanism whereby G
regulates the activity of JNK,
we transfected kinase-deficient mutants of two JNK kinases,
mitogen-activated protein kinase kinase 4 (MKK4) and 7 (MKK7), into
human embryonal kidney 293 cells. G
-induced JNK activation was
blocked by kinase-deficient MKK4 and to a lesser extent by
kinase-deficient MKK7. Moreover, G
increased MKK4 activity by
6-fold and MKK7 activity by 2-fold. MKK4 activation by G
was
blocked by dominant-negative Rho and Cdc42, whereas MKK7 activation was
blocked by dominant-negative Rac. In addition, G
-mediated MKK4
activation, but not MKK7 activation, was inhibited completely by
specific tyrosine kinase inhibitors PP2 and PP1. These results indicate
that G
induces JNK activation mainly through MKK4 activation
dependent on Rho, Cdc42, and tyrosine kinase, and to a lesser extent
through MKK7 activation dependent on Rac.
INTRODUCTION
Top
Abstract
Introduction
References
, interleukin-1, CD40 ligand, and
Fas/CD95 ligand stimulate the activities of JNK and p38 MAPK, which
appear to be involved in cell cycle arrest and apoptosis (2-4). JNK
phosphorylates transcription factors including c-Jun, Elk-1, and ATF2,
whereas p38 MAPK phosphorylates not only transcription factors such as
CHOP/GADD153, MEF2C, Elk-1, and ATF2, but also MAPKAP kinases
(2-4). BMK1 has been reported to be a redox-regulated kinase and
phosphorylates a transcription factor MEF2C, although its physiological
role has remained unclear (3).
and
subunits (G
and G
) and transduce signals from G protein-coupled receptors to intracellular effectors (5-7). A GDP-bound G
forms a complex with G
. Upon ligand
stimulation, the receptor stimulates the GDP-GTP exchange of G
,
which leads dissociation of G
·GTP from G
. Both the GTP-bound
G
and the free G
regulate downstream effectors including
adenylyl cyclases, phospholipase C
isozymes, ion channels,
phosphatidylinositol 3-kinase
(8, 9), and Tec family tyrosine
kinases (10).
1-adrenergic receptors, the activation of ERK is
mediated mainly by G
q/11. Gi-coupled m2
muscarinic acetylcholine and
2-adrenergic receptors, and
Gs-coupled
-adrenergic receptor induce the ERK activation primarily through G
. Many studies suggest that a signal transduction pathway from G
to ERK starts at the direct activation of phosphatidylinositol 3-kinase
, which increases the
activities of Src family tyrosine kinases, in turn leading to tyrosine
phosphorylation of Shc (11-14). Subsequent recruitment of the Grb2-Sos
complex to plasma membranes promotes the exchange of GDP to GTP on Ras
and activates a sequential kinase cascade that includes Raf, MEK, and
ERK (11-14).
in COS-7 cells (17). However, the
mechanism by which G
induces JNK activation has not been fully
understood, although it has been suggested that phosphatidylinositol 3-kinase
, Ras and Rac, and STE20-like kinase Pak1 are involved in
JNK activation by G
in COS-7 cells (17, 18). Furthermore, overexpression of constitutively activated G
q,
G
16, G
12, and G
13 has been
reported to induce the activation of JNK in some cells (19-25).
and G
q/11 in human embryonal
kidney (HEK) 293 cells. We have reported recently that JNK activation by the m1 muscarinic acetylcholine receptor and G
q/11
partially involves the activation of protein kinase C and Src family
tyrosine kinases (26). To clarify the signaling mechanism of JNK
activation by G
, we investigated whether G
stimulates the
activities of two JNK kinases MKK4 and MKK7. In this paper, we describe
that G
regulates MKK4 and MKK7 differentially through different
signaling pathways.
MATERIALS AND METHODS
were obtained from Upstate
Biotechnology, Inc. and Santa Cruz Biotechnology, Inc., respectively.
Rabbit polyclonal antibodies C-14 and GC/2 against G
11
and G
o were purchased from Santa Cruz Biotechnology,
Inc. and New England Biolabs, respectively. Goat anti-mouse (NA931) and
anti-rabbit (NA934) Ig antibodies conjugated with horseradish
peroxidase were from Amersham Pharmacia Biotech. Tyrosine kinase
inhibitors (PP2/AG1879 and PP1/AG1872) were kindly provided by A. Levitzki (Hebrew University). Phosphatidylinositol 3-kinase inhibitors
(wortmannin and LY294002) were purchased from Calbiochem-Novabiochem Co.
-HA-JNK1 and SR
-HA-ERK2 were kindly provided by
M. Karin (University of California, San Diego). pCMV-m1 muscarinic
acetylcholine receptor was kindly provided by E. M. Ross
(University of Texas Southwestern Medical Center). cDNAs of G
1
and G
2 were generously provided by M. I. Simon (California Institute of Technology) and T. Nukada (Tokyo Institute of Psychiatry), respectively, and were subcloned into pCMV as described previously (30,
31). pCMV-G
11Q209L and pCMV-G
o were
generated as described before (32, 33). cDNAs of H-RasS17N was
inserted into pCMV as described previously (30). cDNAs of RhoA and
Rac1 were kindly provided by K. Kaibuchi (Nara Institute of Science and
Technology). pCMV-FLAG-RhoAT19N and pCMV-FLAG-Rac1T17N were constructed
and kindly provided by Y. Yamazaki and H. Koide. Cdc42Hs cDNA was kindly provided by R. A. Cerione (Cornell University).
pCMV-FLAG-Cdc42HsT17N was constructed and kindly provided by K. Nishida. JNK1 cDNA was amplified by polymerase chain reaction using
SR
-HA-JNK1 as a template and subcloned into the EcoRI
restriction site of hexahistidine tag expression vector pET15b
(Novagen, Inc.). Expression plasmid pGEX2T-c-Jun (amino acids 1-223)
was kindly provided by M. Karin (University of California, San Diego)
and digested by BamHI and EcoRI. The DNA fragment
of c-Jun-(1-223) was subcloned into the BglII and
EcoRI sites of Trx-tag expression vector pET32a (Novagen, Inc.). DNA sequences were confirmed by DNA sequencer (LI-COR 4000L) using thermo sequenase (Amersham Pharmacia Biotech).
-HA-JNK, SR
-HA-ERK, pCMV-GST-MKK4, or pCMV-GST-MKK7 was
transfected with 0.3 µg of pCMV-m1 muscarinic acetylcholine receptor,
5 µg of pCMV-G
1, 5 µg of pCMV-G
2, 10 µg of pCMV-G
11Q209L, 10 µg of
pCMV-G
o, 10 µg of pCMV-FLAG-MKK4K95R, 10 µg of
pCMV-FLAG-MKK7K63R, 15 µg of pCMV-RasS17N, 15 µg of
pCMV-FLAG-RhoT19N, 15 µg of pCMV-FLAG-RacT17N, or 15 µg of
pCMV-FLAG-Cdc42T17N. The cells were starved with serum-free medium
containing 1 mg/ml bovine serum albumin (Nacalai) for 24 h
post-transfection.
-D-galactopyranoside and
further incubated for 3 h. The cells were harvested by
centrifugation, washed with phosphate-buffered saline, and stored at
80 °C until use. The frozen cells were suspended and sonicated
briefly in 10 ml of extraction buffer A (20 mM HEPES-NaOH
(pH 8.0), 1 mM phenylmethanesulfonyl fluoride, 1 µg/ml
aprotinin, 1 µg/ml leupeptin, 0.5% Nonidet P-40) for
hexahistidine-JNK and Trx-c-Jun-(1-223) or extraction buffer B (20 mM HEPES-NaOH (pH 7.5), 1 mM dithiothreitol, 1 mM phenylmethanesulfonyl fluoride, 1 µg/ml aprotinin, 1 µg/ml leupeptin, 1 mM EGTA, 0.5% Nonidet P-40) for
GST-c-Jun-(1-223) on ice, and shaken for 20 min at 4 °C. Cell
debris was removed by centrifugation at 150,000 × g
for 30 min at 4 °C.
80 °C until use.
-HA-JNK or SR
-HA-ERK were treated
with or without carbachol at 37 °C and lysed in 600 µl of lysis
buffer A (20 mM HEPES-NaOH (pH 7.5), 3 mM
MgCl2, 100 mM NaCl, 1 mM
dithiothreitol, 1 mM phenylmethanesulfonyl fluoride, 1 µg/ml leupeptin, 1 mM EGTA, 1 mM
Na3VO4, 10 mM NaF, 20 mM
-glycerophosphate, and 0.5% Nonidet P-40) on ice.
The lysates were centrifuged at 14,000 rpm for 10 min at 4 °C.
Aliquots (500 µg) of the supernatants were mixed with protein
A-Sepharose CL-4B preabsorbed with a mouse anti-HA antibody for 1 h at 4 °C. The immune complexes were precipitated and washed twice
with lysis buffer A and twice with reaction buffer A (20 mM
HEPES-NaOH (pH 7.5), 10 mM MgCl2, 0.1 mM phenylmethanesulfonyl fluoride, 0.1 µg/ml leupeptin,
0.1 mM EGTA, 0.1 mM
Na3VO4, and 0.2 mM
-glycerophosphate). The precipitates were incubated in 30 µl of
reaction buffer A containing 3 µg of GST-c-Jun-(1-223) for JNK assay
or 7.5 µg of myelin basic protein (Sigma) for ERK assay, 20 µM ATP, and 5 µCi of [
-32P]ATP (NEN
Life Science Products) at 30 °C for 10 min. The reaction was stopped
by adding 10 µl of 4 × Laemmli sample buffer (50 mM Tris-HCl (pH 6.8), 2% SDS, 30 mM dithiothreitol, and 10%
glycerol). The boiled samples were subjected to SDS-polyacrylamide gel
electrophoresis, and the radioactivity incorporated into
GST-c-Jun-(1-223) or myelin basic protein was measured by an imaging
analyzer (Fuji BAS 2000), and detected by autoradiography.
-32P]ATP at 30 °C for 20 min. The reaction was
stopped by adding 10 µl of 4 × Laemmli sample buffer and
boiling. Samples were subjected to SDS-polyacrylamide gel
electrophoresis, and the radioactivity incorporated into
Trx-c-Jun-(1-223) was measured by an imaging analyzer (Fuji BAS 2000)
and detected by autoradiography. When the reaction was performed
without hexahistidine-JNK, no obvious incorporation of radioactivity to
Trx-c-Jun-(1-223) was observed.
RESULTS
and G
q/11 in HEK 293 Cells--
To confirm that the m1 muscarinic acetylcholine receptor
stimulated JNK activity, we transfected plasmids encoding the receptor and the HA-tagged JNK into HEK 293 cells. Using an anti-HA antibody, HA-JNK was immunoprecipitated from lysates of the transfected cells,
and the kinase activity was assayed using recombinant GST-c-Jun (amino
acids 1-223) as a specific substrate. Fig.
1A shows the time course of
JNK activation after stimulation by carbachol, which is an agonist of
the receptor. Mock-transfected cells did not respond to carbachol (data
not shown). In each experiment, we examined the expression level of
HA-JNK by immunoblotting to monitor the transfection efficiency (see
Figs. 1-3). The persistent activation of JNK was observed from 10 min
to at least 30 min after the receptor stimulation. JNK activation by
the m1 muscarinic acetylcholine receptor was dependent on the
concentration of carbachol to maximum response at 10 µM
and decreased slightly at 100 µM (Fig.
1B).
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Fig. 1.
JNK activation by stimulation of the m1
muscarinic acetylcholine receptor is mediated by G and
G
q/11. HEK 293 cells were transfected with plasmids
carrying cDNAs for HA-JNK (pan els A-E), the m1 muscarinic
acetylcholine receptor (panels A-C),
G
o (
o, panels
C-E), G
1 (
1, panel
D), G
2 (
2, panel
D), and G
11Q209L (
11Q209L,
panel E). JNK activity was measured as described
under "Materials and Methods." JNK activity is shown at different
time points after the addition of 10 µM carbachol
(panel A), 15 min after the addition of
increasing concentrations of carbachol (panel B),
and 15 min after the addition of 10 µM carbachol
(panel C). Values shown represent the mean ± S.E. from three separate experiments. The phosphorylation of
GST-c-Jun and the expression of HA-JNK and G protein subunits in the
cell lysates are shown.
. It has been demonstrated previously that ERK phosphorylation (30) and p38 MAPK activation (33)
induced by G protein-coupled receptors and G
are blocked by
cotransfection of G
o, and G
o forms a
complex with G
in HEK 293 cells (32). It is likely that exogenous
G
o is able to sequester free endogenous G
dissociated from G
upon stimulation of the receptor, and free
exogenous G
(33). The expression of endogenous G
o
was below detectable level of immunoblotting in HEK 293 cells (Fig. 1,
C-E). As shown in Fig. 1C, activation of JNK by
the m1 muscarinic acetylcholine receptor was reduced to approximately
50% by cotransfection of G
o, indicating that both
G
and G
q/11 may mediate the signal from the m1
muscarinic acetylcholine receptor to JNK. To verify that inhibition of
the m1 muscarinic acetylcholine receptor-mediated JNK activation by G
o was due to the sequestration of G
,
G
o was cotransfected with G
or constitutively
activated G
11 (G
11Q209L) into the cells
(Fig. 1, D and E). As reported previously (26,
32), transfection of G
or G
11Q209L stimulated JNK
activity by more than 5-fold in HEK 293 cells. JNK activation by
G
, but not by G
11Q209L, was blocked almost
completely by cotransfection of G
o. These results
indicate that JNK activation by the m1 muscarinic acetylcholine
receptor is mediated by both G
and G
q/11 in HEK 293 cells.
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Fig. 2.
JNK activation by stimulation of the m1
muscarinic acetylcholine receptor is mediated by MKK4 and MKK7.
The cells were transfected with plasmids carrying cDNAs for HA-JNK
(panels A and B), GST-MKK4
(panel C), GST-MKK7 (panel
D), m1 muscarinic acetylcholine receptor (panels
A-D), FLAG-MKK4K95R (panel A), and
FLAG-MKK7K63R (panel B). The activities of JNK,
MKK4, and MKK7 were measured at 15 min after the addition of 10 µM carbachol as described under "Materials and
Methods." Values shown represent the mean ± S.E. from three or
four separate experiments. The phosphorylation of GST-c-Jun and
Trx-c-Jun and the expression of HA-JNK, FLAG-MKK4K95R, and
FLAG-MKK7K63R in the cell lysates are shown. GST-MKK4 and GST-MKK7 were
precipitated with glutathione-Sepharose 4B from the cell lysates and
immunoblotted with anti-GST antibody.
--
Because JNK activation by the m1 muscarinic acetylcholine
receptor appears to be mediated through MKK4 and MKK7, we investigated whether MKK4 and MKK7 may be involved in G
-mediated JNK
activation. As shown in Fig. 3,
A and B, JNK activation by G
was reduced by
about 90 and 50% by cotransfection of kinase-deficient MKK4 and MKK7,
respectively, raising the possibility that either MKK4 or MKK7 may
function as MAPKK in the pathway from G
to JNK, and MKK4 may play
a major role in this cascade.
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Fig. 3.
MKK4 is a major mediator of
G -induced JNK activation. The cells were transfected with
plasmids carrying cDNAs for HA-JNK (panels A
and B), GST-MKK4 (panel C), GST-MKK7
(panel D), G
1 (
1,
panels A-D), G
2 (
2,
panels A-D), FLAG-MKK4K95R (panel
A), and FLAG-MKK7K63R (panel B). The
activities of JNK, MKK4, and MKK7 were measured as described under
"Materials and Methods." Values shown represent the mean ± S.E. from three or four separate experiments. The phosphorylation of
GST-c-Jun and Trx-c-Jun and the expression of HA-JNK, FLAG-MKK4K95R,
FLAG-MKK7K63R, and G
1 in the cell lysates are shown.
GST-MKK4 and GST-MKK7 were precipitated with glutathione-Sepharose 4B
from the cell lysates and immunoblotted with anti-GST antibody.
activates MKK4 and MKK7. G
stimulated MKK4 activity by more than 5-fold (Fig. 3C). In contrast, G
activated MKK7 by about 2-fold (Fig. 3D).
On the other hand, G
11Q209L only weakly activated MKK4
and MKK7 (data not shown). Together with data using
dominant-interfering mutants, it is suggested that G
stimulates
JNK activity mainly through MKK4 and to a lesser extent through MKK7.
Activates MKK4 and MKK7 in a Ras-independent
Manner--
Ras is known to be essential for the ERK activation by
G
(11, 30). It was reported that G
-mediated JNK activation was blocked by dominant-negative Ras (17). To examine whether Ras is
involved in the pathway from G
to MKK4 and MKK7, we utilized dominant-negative mutant of Ras (RasS17N), which inhibits the activation of endogenous Ras by sequestering guanine nucleotide exchange factors. Cotransfection of RasS17N completely inhibited the
ERK activation by G
(Fig.
4C), whereas RasS17N had no
effect on the activations of MKK4 and MKK7 by G
(Fig. 4,
A and B).
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Fig. 4.
G activates MKK4 and MKK7 in a
Ras-independent manner. The cells were transfected with plasmids
carrying cDNAs for GST-MKK4 (panel A),
GST-MKK7 (panel B), HA-ERK (panel
C), G
1 (
1, panels
A-C), G
2 (
2,
panels A-C), and RasS17N (panels
A-C). The activities of MKK4, MKK7, and ERK were measured
as described under "Materials and Methods." Values shown represent
the mean ± S.E. from three separate experiments. The
phosphorylation of Trx-c-Jun and myelin basic protein (MBP)
and the expression of HA-ERK, G
1, and Ras in the cell
lysates are shown. GST-MKK4 and GST-MKK7 were precipitated with
glutathione-Sepharose 4B from the cell lysates and immunoblotted with
anti-GST antibody.
Is Dependent on Rho and Cdc42, whereas
MKK7 Activation Is Dependent on Rac--
It has been reported that Rho
family GTPases are also involved in JNK activation upon various stimuli
(34). Therefore, we investigated the role of these proteins on the
G
-mediated MKK4 and MKK7 activations by cotransfection of
RhoT19N, RacT17N, or Cdc42T17N, which act as dominant-negative mutants
analogous to RasS17N (35, 36). The MKK4 activation by G
was
blocked completely by RhoT19N and Cdc42T17N, but not RacT17N (Fig.
5). On the other hand, the MKK7
activation by G
was blocked by RacT17N, but not RhoT19N and
Cdc42T17N (Fig. 5). These results suggest that G
regulates MKK4
and MKK7 differentially through different Rho family GTPases. We also
examined the effect of dominant-negative mutants of Rho family GTPases
on JNK1 activation by G
. RhoT19N, Cdc42T17N, and RacT17N reduced
JNK activation by 80, 60, and 30%, respectively. Data using
dominant-negative mutants of Rho family GTPases also support that
G
stimulates JNK activity mainly through MKK4.
View larger version (36K):
[in a new window]
Fig. 5.
MKK4 activation by G is dependent on
Rho and Cdc42, whereas MKK7 activation by G
is dependent on
Rac. The cells were transfected with plasmids carrying cDNAs
for GST-MKK4 (panels A, C, and
E), GST-MKK7 (panels B, D,
and F), G
1 (
1,
panels A-F), G
2
(
2, panels A-F), FLAG-RhoT19N
(panels A and B), FLAG-RacT17N
(panels C and D), and FLAG-Cdc42T17N
(panels E and F). The activities of
MKK4 and MKK7 were measured as described under "Materials and
Methods." Values shown represent the mean ± S.E. from three
separate experiments. The phosphorylation of Trx-c-Jun and the
expression of G
1 and FLAG-tagged Rho family GTPases in
the cell lysates are shown. GST-MKK4 and GST-MKK7 were precipitated
with glutathione-Sepharose 4B from the cell lysates and immunoblotted
with anti-GST antibody.
-induced MKK4 but Not MKK7
Activation--
In a previous study (26), we demonstrated that
JNK activation by the m1 muscarinic acetylcholine receptor was
partially reduced by PP2 and PP1, which are known to be specific
tyrosine kinase inhibitors (37). Therefore, we explored a possible
involvement of tyrosine kinase in the signaling pathway from G
to
MKK4 and MKK7. The transfected cells were incubated with various
concentrations of PP2 or PP1. These inhibitors attenuated the MKK4
activation by G
in a dose-dependent manner (Fig.
6). The IC50 value of PP2 and
PP1 for the inhibition of G
-induced MKK4 activation is
approximately 5 and 10 µM, respectively. On the other
hand, the MKK7 activation by G
was not inhibited by these
inhibitors (Fig. 6). It is likely that G
regulates MKK4 in a
tyrosine kinase-dependent manner and MKK7 in an independent
manner.
View larger version (24K):
[in a new window]
Fig. 6.
Activation of MKK4 but not MKK7 by G is
dependent on tyrosine kinase. The cells were transfected with
plasmids carrying cDNAs for GST-MKK4 (panels
A and C), GST-MKK7 (panels
B and D), G
1 (
1,
panels A-D), and G
2
(
2, panels A-D) and treated with
or without increasing concentrations of PP2 (panels
A and B) and PP1 (panels C
and D) for 18 h after 24 h post-transfection. The
activities of MKK4 and MKK7 were measured as described under
"Materials and Methods." Values shown represent the mean ± S.E. from three separate experiments. The phosphorylation of Trx-c-Jun
and the expression of G
1 in the cell lysates are shown.
GST-MKK4 and GST-MKK7 were precipitated with glutathione-Sepharose 4B
from the cell lysates and immunoblotted with anti-GST antibody.
-induced MKK4 and MKK7 Activations Do Not Depend on
Phosphatidylinositol 3-Kinase--
To test the possibility that
phosphatidylinositol 3-kinase is involved in the MKK4 and MKK7
activations by G
, we investigated the effect of wortmannin and
LY294002, which are specific inhibitors of phosphatidylinositol
3-kinase. The transfected cells were treated with 100 nM
wortmannin or 100 µM LY294002 as described previously (14, 18). These inhibitors had no effect on the MKK4 and MKK7 activations (Fig. 7). In addition, we
also observed that JNK activation by the m1 muscarinic acetylcholine
receptor was not inhibited by the treatment of these inhibitors (data
not shown). Under the same experimental conditions, the ERK activation
by G
was effectively attenuated by these
inhibitors,2 being consistent
with reports that G
activate ERK pathway via phosphatidylinositol
3-kinase (13, 14). Therefore, phosphatidylinositol 3-kinase appears not
to be necessary for the JNK pathway mediated by G
in HEK 293 cells.
View larger version (21K):
[in a new window]
Fig. 7.
MKK4 and MKK7 activation by G does not
depend on phosphatidylinositol 3-kinase. The cells were
transfected with plasmids carrying cDNAs for GST-MKK4
(panels A and C), GST-MKK7
(panels B and D), G
1
(
1, panels A-D), and
G
2 (
2, panels A-D)
and treated with or without 100 nM wortmannin
(panels A and B) and 100 µM LY294002 (panels C and
D) for 20 min after 48 h post-transfection. The
activities of MKK4 and MKK7 were measured as described under
"Materials and Methods." Values shown represent the mean ± S.E. from three separate experiments. The phosphorylation of Trx-c-Jun
and the expression of G
1 in the cell lysates are shown.
GST-MKK4 and GST-MKK7 were precipitated with glutathione-Sepharose 4B
from the cell lysates and immunoblotted with anti-GST antibody.
DISCUSSION
and
interleukin-1 (27-29, 38). However, the regulation of MKK4 and MKK7 by
signaling through G protein-coupled receptors remained to be
elucidated. In this paper, we showed that in HEK 293 cells, JNK
activation by the m1 muscarinic acetylcholine receptor was mediated by
G
as well as G
q/11 (26). We next demonstrated that
the receptor-induced JNK activation required at least two JNK kinases,
MKK4 and MKK7. Thus, we attempted to determine which signal component,
including MKKs, small GTPases, tyrosine kinases, and
phosphatidylinositol 3-kinases, is involved in the signaling pathway
from G
to JNK. We found that JNK activation by G
was mediated mainly by MKK4 and partially by MKK7. Neither MKK4 nor MKK7
activation by G
was inhibited by dominant-negative Ras. However,
G
-induced MKK4 activation was blocked by dominant-negative Rho
and Cdc42, whereas G
-induced MKK7 activation was blocked by
dominant-negative Rac. Furthermore, the MKK4 but not MKK7 activation by
G
was inhibited by tyrosine kinase inhibitors. Finally,
G
-induced activations of MKK4 and MKK7 were independent on
phosphatidylinositol 3-kinase activity. These results indicate that
G
regulates MKK4 and MKK7 differentially through different
signaling molecules.
-mediated JNK activation, whereas that of kinase-deficient MKK7
partially inhibited JNK activation (Fig. 3). However, we could not rule
out the possibility that JNK kinase(s) other than MKK4 and MKK7 might
be involved in the pathway. In fact, Moriguchi et al. (38)
have reported that the activity of a JNK kinase rather than of MKK4 and
MKK7 is detected in unabsorbed fraction of anion-exchange
chromatography in the process of fractionating extracts from L5178Y and
KB cells exposed to hyperosmolarity. Further studies are necessary for
clarifying MAPKK respondent to the signal from G
in HEK 293 cells.
, respectively
(Fig. 5), MEKK1, MEKK4, MLK2, and/or MLK3 might act upstream of MKK4
and MKK7 in these pathways.
increases the level of the
GTP-bound form of Ras in HEK 293 cells (30). Furthermore, it has been
reported that oncogenic Ras is a potent activator of the JNK pathway
(35, 36). We thought that G
might activate MKK4 and MKK7 through
a Ras-dependent pathway. However, neither MKK4 nor MKK7
activation by G
was blocked by cotransfection of
dominant-negative Ras (Fig. 4). Collins et al. (22) reported that constitutively activated G
12 stimulates JNK
activity in a Ras-independent manner, although G
12 is
able to activate Ras in HEK 293 cells. It appears that Ras is not
essential for the JNK pathway mediated by G
12 and
G
in HEK 293 cells.
-induced MKK4 and MKK7 activation (Fig. 5). Because MKK4
activation by G
is blocked by both dominant-negative Rho and
Cdc42, it is possible that the MKK4 activation is mediated by a guanine nucleotide exchange factor specific for Rho and Cdc42, e.g.
Dbl and Ost (34). On the other hand, MKK7 activation by G
is
blocked only by dominant-negative Rac. Thus, G
may activate MKK7
through a guanine nucleotide exchange factor specific for Rac,
e.g. Tiam1 (34).
(Fig. 6). We observed that G
and the m1
muscarinic acetylcholine receptor induced tyrosine phosphorylation of
intracellular proteins, and tyrosine-phosphorylated proteins were
reduced by the treatment with
PP2.3 Many lines of evidence
suggest that Src family tyrosine kinases act downstream of G
in
various cells (11, 12). In addition, PP2 and PP1 preferentially inhibit
Src family tyrosine kinases, and the IC50 value of PP2 for
the inhibition of Src is 15 µM in intact
cells.4Thus, we considered
that Src family tyrosine kinases might contribute to G
-mediated
MKK4 activation, and we transfected plasmids encoding a negative
regulator of Src family tyrosine kinases (Csk) and kinase-negative Fyn
or Lyn into the cells. However, these plasmids did not affect the MKK4
pathway.5 It was also
reported that G
directly activated Tec family tyrosine kinases
Tsk and Btk in the presence of plasma membrane fractions in
vitro (10), and Btk regulated JNK activity in vivo
(42). This is unlikely to be a general mechanism for JNK regulation by
G
because Tsk and Btk appear to be expressed in very limited tissue distribution. However, another Tec family tyrosine kinase may be
involved in the MKK4 activation.
may induce the tyrosine phosphorylation of
guanine nucleotide exchange factors exerted on Rho family GTPases and
may increase the intrinsic exchange activity, leading to the MKK4 activation.
in HEK 293 cells (Fig. 7). Very recently, Lopez-Ilasaca et al. (18) demonstrated that in COS-7 cells, JNK
stimulation induced by G
was effectively suppressed by wortmannin
or LY294002 and partially blocked by coexpression of a kinase-deficient
mutant of phosphatidylinositol 3-kinase
. This discrepancy may be
caused by the difference of cell types.
induces MKK4 and MKK7 activations. In conclusion, G
activates JNK through at least two distinct pathways: one pathway is
dependent on Rho and Cdc42 and tyrosine kinase, and the other is
dependent on Rac. Further studies are needed to prove how G
differentially regulates the activities of MKK4 and MKK7.
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ACKNOWLEDGEMENTS |
---|
We thank Drs. E. M. Ross, M. I. Simon, T. Nukada, M. Karin, R. A. Cerione, and K. Kaibuchi for supplying the plasmids. We are grateful to Dr. A. Levitzki for providing PP2 and PP1. We also thank Dr. K. Tago for advice regarding recombinant protein expression and purification, and Dr. H. Koide, K. Nishida, J. Kato, M. Nagao, J. Suzuki, and Y. Yamazaki for helpful discussions and plasmid construction.
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FOOTNOTES |
---|
* This work was supported in part by grants from Core Research for Evolutional Science and Technology and from the Ministry of Education, Science, Sports, and Culture. Our laboratory at the Tokyo Institute of Technology is supported by funding from the Schering-Plogh Corporation.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 45 924 5746; Fax: 81 45 924 5822; E-mail:
hitoh{at}bio.titech.ac.jp.
The abbreviations used are:
MAPK(s), mitogen-activated protein kinase(s); ERK, extracellular
signal-regulated kinase; JNK/SAPK, c-Jun NH2-terminal
kinase/stress-activated protein kinase; MAPKK/MEK, MAPK kinase/MAPK or
ERK kinase; MAPKKK/MEKK, MAPKK kinase/MEK kinase; MKK, MAPK kinase; MLK, mixed lineage kinase; SKK1, SAPK kinase; G protein, heterotrimeric
guanine nucleotide-binding regulatory protein; G, G protein
subunit; G
, G protein
subunit; HEK, human embryonal
kidney; GST, glutathione S-transferase; HA, hemagglutinin; CMV, cytomegalovirus; Trx, thioredoxin.
2 A. Ito, Y. Kaziro, and H. Itoh, unpublished results.
3 M. Nagao, Y. Kaziro, and H. Itoh, unpublished results.
4 A. Levitzki, personal communication.
5 J. Yamauchi, Y. Kaziro, and H. Itoh, unpublished results.
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REFERENCES |
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