(Received for publication, October 20, 1995; and in revised form, December 28, 1995)
From the
Stimulation of a variety of cell surface receptors enhances the
enzymatic activity of mitogen-activated protein kinases (MAPKs). MAPKs
have been classified in three subfamilies: extracellular
signal-regulated kinases (ERKs), stress-activated protein kinases or
c-Jun NH-terminal kinases (SAPKs/JNKs), and p38 kinase.
Whereas the pathway linking cell surface receptors to ERKs has been
partially elucidated, the mechanism of activation of JNKs is still
poorly understood. Recently, we have shown that stimulation of G
protein-coupled receptors can effectively induce JNK in NIH 3T3 cells
(Coso, O. A., Chiariello, M., Kalinec, G., Kyriakis, J. M., Woodgett,
J., and Gutkind, J. S.(1995) J. Biol. Chem. 270,
5620-5624). In the present study, we have used the transient
expression in COS-7 cells of m1 and m2 muscarinic receptors (mAChRs) as
a model system to study the signaling pathway linking G protein-coupled
receptors to JNK. We show that stimulation of either muscarinic
receptor subtype leads to JNK activation; however, this effect was not
mimicked by expression of activated forms of
,
,
, or
G protein
subunits. In contrast, overexpression of G
subunits
potently induced JNK activity. Furthermore, we show that signaling from
m1 and m2 mAChRs to JNK involves
subunits of heterotrimeric
G proteins, acting on a Ras and Rac1-dependent pathway.
Stimulation of a variety of cell surface receptors causes a
rapid elevation of the enzymatic activity of a family of closely
related serine-threonine kinases, known as mitogen-activated protein
kinases (MAPKs)()(2) . The function of MAPKs is to
convert extracellular stimuli to intracellular signals which, in turn,
control the expression of genes that are essential for many cellular
processes, including cell growth and differentiation(3) . MAPKs
have been classified in three subfamilies: extracellular signal
regulated kinases (ERKs), including ERK1 and ERK2, also known as
p44
and p42
,
respectively; stress-activated protein kinases (SAPKs), also termed
c-Jun NH
-terminal kinases (JNKs); and p38
kinase(2) . ERKs phosphorylate and regulate the activity of
certain enzymes, including phospholipase A
and
p90
, and nuclear proteins, such as the ternary
complex factor p62
or Elk-1(4) . The latter
represents a critical event in controlling the expression of several
genes, including c-fos(5) . JNKs phosphorylate the
amino-terminal transactivating domain of c-Jun and ATF2(6) ,
thereby increasing their transcriptional activity. p38 is the homologue
of the Saccharomyces cerevisiae HOG1 gene, and its function is
still unknown, although recently available information suggests that
p38 might play a critical role in the inflammatory
response(7) .
Recent findings have helped to unveil the
pathway linking cell surface receptors to ERKs(3) . In
contrast, the mechanism of activation of JNKs is still poorly
understood. JNKs were shown to be activated by a variety of stimuli
distinct from those that elevate the enzymatic activity of ERKs,
including protein synthesis inhibitors, heat shock, changes in
osmolarity, and ultraviolet irradiation(6, 8) . JNKs
can be also activated by agents acting on cell surface receptors, such
as tumor necrosis factor-, interleukin-1, or epidermal growth
factor (8) . Furthermore, available evidence suggest that
whereas Ras controls the activation of ERKs, members of the Rho family
of small GTP-binding proteins, Rac1 and Cdc42, regulate the activity of
JNKs(9) .
Recently, we have shown that stimulation of
certain G protein-coupled receptors expressed in NIH 3T3 cells can
effectively induce JNK activity, however, following a time course
clearly distinct from that of ERK activation(1) . In the
present study, we have used the transient expression in COS-7 cells of
m1 and m2 muscarinic receptors (mAChRs) as a model system to study the
biochemical route connecting G protein-coupled receptors to an
epitope-tagged JNK. We present evidence that signaling from m1 and m2
mAChRs to JNK involves subunits of heterotrimeric G
proteins, acting on a Ras- and Rac1-dependent pathway.
In order to study the mechanism controlling the activation of
JNK by m1 and m2 G protein-coupled receptors, we coexpressed these
receptors together with an epitope-tagged JNK (HA-JNK) in COS-7 cells.
We observed that when cotransfected with the HA-JNK cDNA, both m1 and
m2 were expressed at similar high levels (data not shown), and the
epitope-tagged JNK was efficiently expressed and readily detectable
upon immunoprecipitation with the anti-HA monoclonal
antibody(9) . In cells expressing either muscarinic receptor,
the cholinergic agonist carbachol induced an increase in JNK activity,
as judged by its in vitro phosphorylating activity using
GST-ATF2(96) as a substrate (Fig. 1). Whether mediated by m1 or
m2 receptors, induction of GST-ATF2(96) phosphorylation showed a peak
at approximately 15 min after stimulation, being of 2-4-fold
higher than the control with a slow decrease at later times (Fig. 1). Thus, JNK is activated by either m1 or m2 G
protein-coupled receptors when expressed in COS-7 cells. Whereas m1
receptors are typical of those coupled through G proteins of the
G family to phospholipase C activation, m2 is known to
couple through G
to a number of effector pathways,
including to the inhibition of adenylyl cyclases (12) .
Interestingly, both m1 and m2 mAChRs appear to activate JNK
irrespective of their G protein-coupling specificity.
Figure 1: The cholinergic agonist carbachol induces JNK (SAPK) activity in COS-7 cells transfected with expression plasmids for m1 or m2 muscarinic receptors. COS-7 cells were transfected with expression plasmids for m1 or m2 mAChRs (1 µg/plate) together with a plasmid expressing an epitope-tagged JNK (pcDNA3-HA-JNK, 1 µg/plate), as indicated. Cultures were stimulated by addition of carbachol (10 µM) for the indicated time, cells were lysed, and JNK activity was determined in the HA immunoprecipitates as described under ``Experimental Procedures.'' Data represent the mean ± S.E. of three independent experiments, expressed as -fold increase in JNK activity with respect to nonstimulated cells.
As an approach
to investigate which G proteins mediate the activation of JNK, we took
advantage of the observation that the expression of GTPase-deficient,
mutationally activated forms of G protein subunits can activate
effector pathways by obviating the need for receptor
stimulation(13) . Thus, we coexpressed the epitope-tagged JNK
together with GTPase-deficient mutants for G
,
G
, G
, and G
, which
are representative members for each of the four G
subunit families (14) . Expression of each G protein
subunit, when
transfected into COS-7 cells, could be demonstrated by immunoblotting
with subtype-specific antibodies (data not shown; (11) and (12) ). However, these activated mutants enhanced JNK activity
only to a very limited extent, either when each G
subunit was
expressed alone (Fig. 2) (15) or in all possible
combinations (data not shown). Thus,
subunits of heterotrimeric G
proteins might not mediate JNK activation by G protein-coupled
receptors.
Figure 2:
Overexpression of subunits of
heterotrimeric G proteins results in stimulation of JNK activity. COS-7
cells were transfected with pcDNA3-HA-JNK (1 µg/plate) together
with pcDNA3 vector (control) or with an expression vector carrying
cDNAs for the activated (QL) forms of the
subunits of
G
, G
, G
, and G
, or
expressing
,
, or
*
G protein subunits, alone or in combination, as indicated (2
µg/plate in each case). Kinase reactions and Western blot (WB) analysis were performed in anti-HA immunoprecipitates
from the corresponding lysates, as described under ``Experimental
Procedures.'' Autoradiograms correspond to representative
experiments.
P-Labeled products as well as specific bands
detected by the anti-HA antibody are indicated with an arrow.
Data represent the mean ± S.E. of four to five independent
experiments, expressed as -fold increase with respect to
vector-transfected cells (control).
When activated, receptors linked to G proteins catalyze
the replacement of GDP by GTP-bound to the subunit and induce the
dissociation of
-GTP from
dimers. Although the
subunits were thought to be solely responsible for coupling receptors
to second-messenger-generating systems, recent work has established a
critical role for
dimers in signal
transduction(10, 14) . Thus, the failure of
mutationally activated G protein
subunits to elevate JNK activity
prompted us to explore whether
dimers participate in
signaling to JNK. We observed that, when cotransfected,
subunits induce a remarkable
increase in the phosphorylating activity of the epitope-tagged JNK,
although expression of the HA-JNK was similar for each transfected cell
population (Fig. 2). Similar results were obtained when
subunits were expressed (data not
shown). In contrast, JNK was poorly activated when coexpressed with
or
alone or when cotransfected with
and an altered form of the
subunit,
designated
*, that lacks an isoprenylation signal and
fails to associate to the plasma membrane (10) (Fig. 2).
We conclude that membrane-bound
subunits of heterotrimeric G
proteins, but not G
subunits, can effectively stimulate JNK
activity in COS-7 cells.
We next explored a role for
complexes in JNK stimulation by mAChRs. To approach this question, we
employed a chimeric molecule combining the extracellular and
transmembrane domain of CD8 fused to the carboxyl-terminal domain of
ARK, which includes the
-binding region(11) .
This chimeric molecule expresses the CD8 antigen at the cell surface,
localizing the
ARK carboxyl-terminal domain to the inner face of
the plasma membrane. The CD8-
ARK chimera is expected to bind and
sequester free
complexes when dissociated from G
subunits upon receptor stimulation, thus blocking
-dependent
pathways(11) . As shown in Fig. 3, coexpression of
CD8-
ARK with the m1 or m2 mAChRs nearly abolished the activation
of JNK in response to carbachol, while CD8 alone had no demonstrable
effect. In contrast, JNK activation by stress-inducing agents such as
anisomycin (9) was unaffected by CD8-
ARK, demonstrating
the specificity of this approach (Fig. 3). Taken together, these
findings strongly suggest that signaling from m1 and m2 mAChRs to JNK
is mediated by
subunits of heterotrimeric G proteins.
Figure 3:
Effect of a scavenging protein
in JNK activation. pcDNA3-HA-JNK was cotransfected with expression
plasmids for m1 or m2 mAChRs (1 µg/plate), and
and
subunits (2 µg per plate), together
with vector alone (vector) or plasmids expressing the CD8 receptor or a
chimeric molecule CD8-
ARK, as indicated. Cells were stimulated
with carbachol (10 µM) or anisomycin (10 µg/ml) for 15
min or left untreated. Quantitation of JNK activity present in anti-HA
immunoprecipitates was performed as in Fig. 1. Data represent
the mean ± S.E. of three independent experiments, expressed as
-fold increase with respect to unstimulated
cells.
We
have recently shown that the Rho-related small GTP-binding proteins
Rac1 and Cdc42 are integral components of signaling pathways linking
certain cell surface receptors to JNK(9) . Thus, we set out to
investigate whether these small GTPases mediate JNK activation by G
protein-coupled receptors, using as a tool the expression of
dominant-negative mutants for Ras, RhoA, Rac1, and Cdc42. None of these
dominant-inhibitory small GTP-binding proteins affected the JNK
activation in response to anisomycin (Fig. 4). In contrast, as
shown in Fig. 4, coexpression of N17Ras and N17Rac1 prevented
JNK activation by either mAChRs, or when induced by
complexes, N17Cdc42 diminished JNK stimulation by m1 mAChRs, and the
dominant-negative form of RhoA did not display any significant effect.
Similarly, we have recently shown that JNK activation by EGF in COS-7
cells is also blocked by N17Ras, N17Rac1, and N17Cdc42(9) .
Thus, these findings support a role for Ras and Rac1 or Cdc42 in
linking both tyrosine kinase and G protein-coupled receptors to the JNK
pathway.
Figure 4:
Effect of dominant-negative mutants for
Ras, RhoA, Rac1, and Cdc42 on JNK activation. COS-7 cells were
transfected with pcDNA3-HA-JNK and expression plasmids for m1 and m2
receptors (1 µg/plate) or for and
G protein subunits (2 µg/plate), together with either pcDNA3
(vector) without insert or carrying cDNAs for N17Ras, N19RhoA, N17Rac1,
or N17Cdc42 (1 µg/plate), and cells were left untreated or
stimulated with carbachol (10 µM) or anisomycin (10
µg/ml) for 15 min, as indicated. Cell lysates were processed as in Fig. 1. Data represent the mean ± S.E. of four to five
independent experiments, expressed as percent of activation with
respect to the corresponding vector cotransfected control
(100%).
Although Ras controls the activity of ERKs, recent
available evidence suggests that Rac1 and/or Cdc42 regulate JNK
activation(9) . Thus, distinct small GTP-binding proteins
appear to link cell surface receptors with independent signaling
pathways leading to the activation of each member of the MAPK
superfamily. In this study, we present evidence supporting a role for
subunits of heterotrimeric G proteins in communicating G
protein-coupled receptors with the JNK pathway, acting on a Ras- and
Rac1-dependent biochemical route. Furthermore, we and others have
previously shown that
complexes link this class of cell
surface receptors to ERKs, in this case acting through
Ras(10, 16) . Thus, taking these findings together we
can postulate that
heterodimers provide a link between
heterotrimeric G proteins and small GTP-binding proteins.
The
molecular basis for this interaction is still poorly
defined(16) . However, it is strikingly similar to that of the
pathway linking the G protein-coupled pheromone receptors to
MAPK-related enzymes in the budding yeast S. cerevisiae. In
this case, extracellular ligands ( or a factors) activate
pheromone receptors which, in turn, induce the dissociation of a
heterotrimeric G protein into
(GPA1) and
(Ste4, Ste18)
subunits (see (17) for review). Free
dimers then
activate a serine-threonine kinase (Ste20), initiating activity from a
linear cascade of kinases, including sequentially Ste11, Ste7, and the
yeast MAPK homologues Fus3 and Kss1. Extensive search for molecules
linking yeast
complexes to Ste20 has led to the recent
discovery that Cdc42 participates in Ste20 activation(18) , and
that Ste4 might directly bind and activate Cdc24, a nucleotide exchange
factor for Cdc42(19) . Available data suggest that additional
proteins might also be involved, including a protein designated Ste5,
which plays a role as a platform or scaffold recruiting Ste11, Ste7,
Kss1/Fus3(20) , and another small GTP-binding protein, the Ras
homologue Rsr1(21) . The latter also appears to play an
important role by binding Cdc24 and, probably, by positioning this
guanine nucleotide exchange factor within the cell, thus allowing its
interaction with Cdc42 and its targets(21) . These observations
represent an interesting example of convergence between Ras-like (Rsr1)
and Rho-like (Cdc42) biochemical routes and might also provide a clue
regarding the pathway connecting mammalian
to JNK. Based
upon these observations in yeast and our present findings, we can
hypothesize that upon receptor stimulation
dimers might
recruit a yet to be identified guanine nucleotide exchange factor for
Rac1 and/or Cdc42 and that Ras functioning may be necessary for the
effective activation of these GTP-binding proteins and the consequent
stimulation of JNK. We conclude that our present study might represent
a biologically relevant example of a signal transduction pathway
extraordinarily conserved from yeast to mammals and might provide an
attractive model to elucidate the nature of those molecules linking
heterotrimeric G proteins to small GTP-binding proteins.