From the Max Planck Research Unit "Molecular Cell Biology,"
Medical Faculty, University of Jena, 07747 Jena, Germany and the
Molecular Signaling Unit, Laboratory of Cellular
Development and Oncology, NIDR, National Institutes of Health,
Bethesda, Maryland 20892
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ABSTRACT |
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Jun kinases (JNK) are involved in the stress
response of mammalian cells. Stimulation of JNK can be induced by
stress factors and by agonists of tyrosine kinase and G protein-coupled
receptors. G protein-dependent receptors stimulate JNK via
G subunits of heterotrimeric G proteins, but the subsequent
signaling reaction has been undefined. Here we demonstrate JNK
activation in COS-7 cells by G
-stimulated phosphoinositide
3-kinase
(PI3K
). Signal transduction from PI3K
to JNK can be
suppressed by dominant negative mutants of Ras, Rac, and the protein
kinase PAK. These results identify PI3K
as a mediator of
G
-dependent regulation of JNK activity.
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INTRODUCTION |
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Interaction of cells with a wide variety of agonists results in a stimulation of intracellular mitogen-activated protein kinase (MAPK)1 cascades. MAPKs control the expression of genes that are important for the regulation of many cell functions including proliferation and differentiation. In mammalian cells three parallel MAPK pathways have been characterized so far (1). The canonical MAPK cascade composed of Raf, MEK, and ERK is regulated by the Ras GTPase in response to agonists of tyrosine kinase and G protein-coupled receptors. ERK species catalyze the phosphorylation of transcription factors including Elk-1, thus controlling the expression of several genes (2).
A second MAPK cascade that is stimulated by osmotic stress regulates the activity of the protein kinase p38. Signal transduction to p38 and the function of this pathway are still unclear. Like p38 the elements of the Jun kinase (JNK) cascade as a third MAPK pathway are involved in the stress response of mammalian cells. JNK can be stimulated by stress factors like interleukin 1 and tumor necrosis factor but also by agonists of tyrosine kinase and G protein-coupled receptors (3, 4). Available evidences point to an involvement of the small GTPase Rac and several protein kinases in the regulation of JNK activity (5). In some cellular systems the STE 20 homologue PAK was found to act as a JNK kinase kinase kinase (6). PAK is able to activate JNK via sequential stimulation of the protein kinases MEKK and SEK.
The mechanism of signal transduction from G protein-coupled receptors
to the JNK cascade is only partially understood. Using COS-7 cells as a
transient expression system a recent report showed the involvement of
G subunits of heterotrimeric G proteins in the stimulation of JNK
by agonists of the G protein-coupled muscarinergic receptor m2 (7).
Additionally the small GTPases Ras and Rac have been demonstrated to
mediate signal transduction from G protein-coupled receptor to JNK, but
the topology of the signaling path from G
to Ras and Rac remains
unknown (7-9).
One candidate for the link of G protein-coupled receptor and JNK
cascade is phosphoinositide 3-kinase (PI3K
). This subspecies of
the PI3K family is stimulated in vitro by G
and
recently has been shown to be involved in signal transduction from G
protein-coupled receptor to the ERK path of MAPK cascades (10). We now
present evidences that JNK stimulation by an agonist of the m2
muscarinergic receptor and by G
also implies a PI3K.
Overexpression of PI3K
in COS-7 cells induces a significant increase
of JNK activity. Stimulation of JNK by G
can be suppressed by the
PI3K inhibitor wortmannin and a lipid kinase negative mutant of PI3K.
Dominant negative mutants of Ras, Rac, and PAK significantly reduce the stimulatory effect of PI3K
on JNK. Thus PI3K
seems to act as an
intermediate connecting G protein-coupled receptors to the JNK
cascade.
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EXPERIMENTAL PROCEDURES |
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Cell Culture and Transfection-- COS-7 cells (ATCC) were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. Cells were split the day before transfection. Subconfluent cells were transfected with pcDNAIII-HA-JNK or pcDNAIII-HA-MAPK and additional DNAs, following the DEAE-dextran technique, adjusting the total amount of DNA to 5 µg per plate with vector DNA when necessary. Assays were performed 48 h after transfection (9). For JNK assays the cells were serum-starved for 2 h, whereas for MAPK the cells were starved overnight in serum-free medium.
DNA Constructs--
Expression plasmids for an epitope-tagged
JNK and MAPK, pcDNA3 HA-JNK and pcDNA3 MAPK, respectively, as
well as expression plasmids for the dominant negative mutants of the
small GTP-binding proteins Ras, RhoA, Rac1, and Cdc42 have been
described (9, 11). PI3K and dominant negative PI3K
were used as
described previously (10). pcDNA3-p101 was kindly provided by Dr.
Len Stephens and prepared as published recently (12). An
expression plasmid for the N-terminal 150-amino acid noncatalytic
domain of Pak1, which contains the Rac/Cdc42 binding region, was cloned into pcDNA3 vector coding the NH2-terminal
myristoylation membrane localization signal from c-Src (13, 14). The
final construct was designated pcDNA3-myr-PAK(N). Efficacy of
dominant negative mutants of PI3K
, Ras, RhoA, Rac1, Cdc42, and PAK
to suppress muscarinergic response in COS-7 cells has been established
recently (7, 10, 14).
Kinase Assays--
JNK assays in cells transfected with an
epitope-tagged JNK construct (HA-JNK) was determined as described
previously (9) using purified, bacterially expressed GST-ATF2(96)
fusion protein as a substrate. Briefly, serum-starved cells left
untreated or stimulated with various agents were lysed, and after
centrifugation, clarified supernatants were immunoprecipitated with an
anti-hemagglutinin monoclonal antibody 12CA5 (Babco, Richmond, CA) for
1-2 h at 4 °C, and immunocomplexes were recovered with the aid of
GammaBind (Pharmacia, Uppsala). Pellets were then washed three times
with phosphate-buffered saline solution, supplemented with 1% Nonidet
P-40, and 2 mM sodium vanadate, once with 0.5 M
LiCl in 100 mM Tris, pH 7.5, and once with kinase reaction
buffer (12.5 mM MOPS, pH 7.5, 12.5 mM
-glycerophosphate, 7.5 mM MgCl2, 0.5 mM EGTA, 0.5 mM sodium fluoride, 0.5 mM vanadate). Reactions were performed in a 30-µl volume
of kinase reaction buffer containing 1 µCi of
[
-32P]ATP/reaction, 20 µM unlabeled ATP,
and 1.5 mg/ml substrate at 30 °C for 30 min. Reactions were
terminated by addition of 5× Laemmli buffer, boiled, and
electrophoresed in 12% polyacrylamide gel. Phosphorylated GST-ATF2(96)
was visualized by autoradiography and quantified with a PhosphorImager.
MAPK activity in cells transfected with an epitope-tagged MAPK
(HA-MAPK) was determined as described previously (11). In each case,
parallel samples were immunoprecipitated with anti-HA antibody and
processed by Western blot analysis using the appropriate antibody
(Santa Cruz Technology, Santa Cruz, CA). Immunocomplexes were
visualized by enhanced chemiluminescence detection (Amersham) with the
use of goat antiserum to rabbit or mouse immunoglobulin G coupled
to horseradish peroxidase (Santa Cruz Technology).
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RESULTS AND DISCUSSION |
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To investigate signaling from G protein-coupled receptor to JNK
the muscarinergic m2 receptor has been transiently expressed in COS-7
cells. Confirming previous results (7) we observed an increase of JNK
activity after treatment with the receptor agonist carbachol (Fig.
1A). Overexpression of G
mimics the effect of carbachol on JNK. As shown in Fig. 1A
both m2- and G
-mediated stimulation could be effectively
suppressed by wortmannin, a specific inhibitor of PI3K. In contrast JNK
activation by the stress-inducing agent anisomycin was unaffected by
wortmannin, demonstrating the specificity of this approach. Another
PI3K inhibitor LY 294002 decreased JNK stimulation induced by G
in a dose-dependent manner (Fig. 1B). Together
these data point to an involvement of PI3K in the stimulation of JNK by
G protein-coupled receptor.
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Trying to outline the PI3K species that induces
G-dependent JNK activation we expressed PI3K
in
COS-7 cells. As shown in Fig.
2A, PI3K
moderately
stimulated JNK activity. This effect could be significantly enhanced by
coexpression of p101, a PI3K
-binding protein recently discovered
(12). p101 alone was unable to induce JNK stimulation. For a comparison
Fig. 2A expresses the individual effects of p101, PI3K
,
and PI3K
+ p101 on MAP kinase (ERK) activity. Interestingly p101
exhibited significantly lower potency to affect PI3K
-dependent MAPK activity than JNK induced by
PI3K
. To examine possible autocrine reactions of the transfectants
we analyzed the effects of conditioned media from COS-7 cells
expressing PI3K
and/or p101 on naive cells. Supernatants of the
transfected cells did not induce any measurable stimulation of JNK
activity in untreated cells, thus providing evidence for direct
signaling from PI3K
and p101 to JNK (data not shown). Fig.
2B demonstrates the dependence of JNK activation on the
expression levels of p101 and PI3K
. Thus optimal stimulation of JNK
seems to depend on PI3K
and p101.
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The activatory effect of PI3K on JNK could be completely inhibited
by wortmannin suggesting an important role of the kinase activities of
the PI3K (Fig. 3A). PI3K
recently has been shown to exhibit a significant ability to catalyze
autophosphorylation in addition to its lipid kinase activity (15). Both
kinase activities depend on magnesium and can be inhibited by nanomolar
concentrations of wortmannin. A recently described mutant of PI3K
K799R, which does not express significant lipid kinase and protein
kinase activities, was able to suppress the JNK stimulation induced by
G
(Fig. 3B). Together with our previous report (10)
these findings provide clear evidence that PI3K
links both JNK and
MAPK to G protein-coupled receptors. Involvement of PI3K
, which
recently has been shown to mediate signaling of platelet-derived growth
factor receptor tyrosine kinase to JNK (16) seems improbable.
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The recently described involvement of Ras and Rac in signal
transduction from G protein-coupled receptors to JNK prompted us to
investigate a possible role of these small GTPases in JNK stimulation induced by PI3K. As shown in Fig.
4A coexpression of dominant
negative forms of Ras and Rac prevented activation of JNK by PI3K
.
In contrast coexpression of the negative mutants of two other small
GTPases RhoA and Cdc42 did not affect the stimulation of JNK by
PI3K
. Therefore Ras and Rac1 seem to represent essential elements of
the signaling path from PI3K
to JNK. Expression of
ARK as a
binding protein for G
(9, 10) also suppressed JNK stimulation
induced by PI3K
(data not shown). This effect points to an essential
role of G
as a link of G protein-coupled receptors to
PI3K
.
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We next explored a possible involvement of protein kinase PAK in
downstream signaling from PI3K to JNK. The coexpression of a
dominant negative mutant of this protein kinase also induced a partial
suppression of JNK stimulation by G
, PI3K
, and a constitutively active mutant of Rac (Fig. 4B). Thus
signaling from G protein-coupled receptor to JNK appears to require
G
, PI3K
, Ras, Rac, and PAK.
Nevertheless the sequence of events connecting PI3K and JNK remains
unclear. A PI3K
effect on Ras has been proposed in our previous
report on ERK activation by PI3K
(10). In this paper we provide
evidences that Ras could be activated by PI3K
via a Src-type
tyrosine kinase, the adapter proteins Shc and Grb2, and the guanine
nucleotide exchange factor Sos. Stimulated in this way Ras could act as
a dissociation point for signals from PI3K
to ERK and JNK. Such a
bifurcating function of Ras as a regulator of the Raf-ERK path and
signal transduction to Rac, PAK, and JNK has been proposed (6).
Despite the attractiveness of this model some experimental data point
to a more complex relation of the signaling proteins involved. Thus the
differential effects of p101 on the PI3K-dependent stimulation of ERK and JNK cannot be explained by simple sequential signal transduction from PI3K
via the lipid kinase product
PIP3 to Ras and subsequent stimulation of JNK and ERK
paths. One possible explanation for the distinct effects of PI3K
and
its ligands on the MAPK cascades could be the involvement of signaling
activities of the enzyme in addition to PIP3 production.
Wortmannin-sensitive protein kinase activity of PI3K
has been
reported (15). If G
and p101 produce divergent effects on the
lipid kinase and protein kinase activities of PI3K
and if both
activities are important for signal transduction different downstream
events could be expected. The individual signaling functions of PI3K
lipid kinase and protein kinase activities are currently under investigation. The effects of PI3K
on different MAPK cascades clearly represent another example for the complex interrelations of
these intracellular signaling proteins.
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ACKNOWLEDGEMENTS |
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We are very grateful to Drs. Len Stephens and Phill Hawkins for providing us with the pcDNA3-p101 construct.
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FOOTNOTES |
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* This work was supported by Deutsche Forschungsgemeinschaft Grants SFB 197.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: Max-Planck Research Unit "Molecular Cell Biology," Drackendorfer Straße 1, D 07747 Jena, Germany. Tel.: 49-3641-304-460; Fax: 49-3641-304-462; E-mail: i5rewe{at}rz.uni-jena.de.
1 The abbreviations used are: MAPK, mitogen-activated protein kinase; PI3K, phosphoinositide 3-kinase; ERK, extracellular signal-regulated kinase; JNK (or SAPK), c-Jun NH2-terminal kinase (or stress-activated protein kinase); PIP3, phosphatidylinositol 3,4,5-trisphosphate; HA, hemagglutinin; MOPS, 4-morpholinepropanesulfonic acid.
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REFERENCES |
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