(Received for publication, June 5, 1995; and in revised form, December 20, 1995)
From the
In airway smooth muscle cells ligand binding to the
seven-transmembrane endothelin and thrombin receptors stimulates cell
growth. Rapid activation of the extracellular regulated kinase 2 and
c-Jun NH-terminal kinase groups of mitogen-activated
protein kinases was also observed. The results demonstrate a novel
mechanism of seven-transmembrane receptor signaling involving
activation of the Jun kinase pathway. Receptor coupling to Jun kinase
activation may involve heterotrimeric G proteins since the kinase was
enzymatically activated in cells treated with aluminum fluoride. The
activity of Raf-1, measured by immune complex kinase assay, revealed
that platelet-derived growth factor and phorbol 12-myristate 13-acetate
both stimulated Raf-1 activity, while thrombin and endothelin did not
appreciably stimulate Raf-1. The data suggest that endothelin and
thrombin stimulate Raf-1-independent mechanisms of mitogen-activated
protein kinase activation. Endothelin- or thrombin-induced activation
of mitogen-activated protein kinases was significantly inhibited by
activation of cyclic AMP-dependent protein kinase by forskolin.
Proliferation of airway smooth muscle cells, measured by incorporation
of [
H]thymidine into DNA, was also greatly
attenuated by forskolin.
The endothelin and thrombin receptors are members of the
seven-transmembrane receptor superfamily that are thought to transduce
mitogenic information by coupling to G proteins. Airway smooth muscle
(ASM) ()cells treated with endothelin and thrombin are known
to activate second messenger systems, including increases in cytosolic
calcium, phospholipase C stimulation, and activation of protein kinase
C (PKC)(1, 2, 3, 4) . However, the
intracellular molecular circuitry that transduces the mitogenic
information from these receptors is not completely defined.
The MAP kinases, or ERKs, are 42- and 44-kilodalton serine/threonine kinases encoded by the ERK2 and ERK1 genes, respectively, that are enzymatically activated by tyrosine and threonine phosphorylation in response to numerous different stimuli(5, 6, 7, 8) . The ERKs are phosphorylated and activated by a dual specificity tyrosine/threonine kinase, MKK1, also called MAP or ERK Kinase (MEK)(9, 10, 11, 12) . In addition to MKK1 a second MEK isoform, MKK2, has been isolated that shares a high degree of amino acid identity with MKK1(13) . MEK is catalytically activated by phosphorylation on serines 218 and 222 by the serine kinase Raf-1 (14, 15, 16) . Raf-1 is currently thought to be the primary physiological activator of MEK, although in adipocytes treated with insulin or PMA, a MEK kinase distinct from Raf-1 appears to be activated within 20 s and inactivated in 30 s(17) .
The activation of the MAP kinases appears to be a common requirement for agents that induce cell growth and differentiation. For example, PC12 cell differentiation is blocked by a dominant negative MEK mutant in which both serine phosphorylation sites have been substituted with alanine(18) . Furthermore, activating mutants of MEK in which the serine phosphorylation sites have been substituted with acidic residues results in constitutive kinase activation and cell transformation(19) . Inhibition of MAP kinase synthesis by antisense oligonucleotides depletes MAP kinase protein and blocks the ability of insulin or serum to stimulate DNA synthesis(20) .
Agents that induce cellular stress, such as
UV light and tumor necrosis factor (TNF-
), activate the JNK
protein kinases. The JNKs are distant relatives of the MAP kinases and
were initially identified as c-Jun NH
-terminal kinases
(JNK) based on their phosphorylation and activation of
c-Jun(21, 22, 23) . More recently the JNKs
have also been found to phosphorylate and activate the ATF2
transcription factor(24) . The human JNK1 and JNK2 genes encode
46- and 55-kDa kinases, respectively, that are distantly related to the
MAP kinases and are similarly activated by phosphorylation on a single
threonine and single tyrosine residue. The regulatory phosphorylation
sequence of the ERKs and JNKs resides within subdomain VIII of the
catalytic domain, a region thought to be involved in the regulation of
many protein kinases(25, 26) . The JNK regulatory
sequence is Thr-Pro-Tyr, while the ERK phosphorylation sequence is
Thr-Glu-Tyr(21) . MKK4 is a recently identified human protein
kinase with homology to MKK1 and MKK2 that activates JNK1 by
phosphorylation of the Thr-Pro-Tyr sequence(27) . SEK1 is the
murine homolog of MKK4 that also activates JNK1(28) . Kyriakis et al.(29) have identified the rat homologs of the
JNKs, referred to as stress-activated protein kinases. The JNKs may
also be activated by tyrosine kinase growth factors such as epidermal
growth factor in a Ras-dependent mechanism(30) .
While JNK
activation is known to follow stimulation of the TNF- receptor, as
well as tyrosine kinase receptors such as the epidermal growth factor
receptor, this report demonstrates that seven-transmembrane receptors
also activate the JNK subgroup of MAP kinases. Activation of these
kinases is inhibited by forskolin, which increases the enzymatic
activity of cyclic AMP-dependent protein kinase (PKA). Furthermore, the
ability of endothelin and thrombin to induce the proliferation of ASM
cells is inhibited by activating PKA, suggesting that activation of the
ERK and JNK kinase cascades is an essential component of the
proliferative effects of endothelin and thrombin.
For Raf-1 immunoprecipitation 100-mm plates
were serum-starved overnight and scraped into 0.6 ml of radioimmune
precipitation (RIPA) buffer (50 mM Hepes, pH 7.5, 150 mM NaCl, 2 mM EDTA, 1% Nonidet P-40, 0.1% SDS, 1% aprotinin,
2 mM sodium orthovanadate, and 0.5 µM microcystin). Rabbit anti-Raf-1 polyclonal antibody (C-12, Santa
Cruz Biotechnology) was added to the clarified lysate at a 1:100
dilution and incubated on ice for 2 h. The washed immune complexes were
incubated in 25 µl of KB, 5 µCi of
[P]ATP, and 0.5 µg of recombinant
kinase-inactive MKK1 (32) at 30 °C for 20 min.
The
activity of JNK1 was assayed by immunoprecipitation with JNK1 antiserum
or by affinity purification with a GST-Jun-(1-79) fusion protein.
To immunoprecipitate JNK1, cell lysates were prepared in radioimmune
precipitation buffer. A rabbit polyclonal antibody raised against JNK1 (24) was added to the clarified lysates at a 1:1000 dilution.
The washed immune complexes were incubated with 25 µl of KB, 5
µCi of [P]ATP, and 2 µg
GST-Jun-(1-79) for 20 min at 30 °C. To affinity purify JNK1
with GST-Jun, cell lysates were prepared in EB (25 mM Hepes,
pH 7.5, 0.3 M NaCl, 5 mM MgCl
, 0.2 mM EDTA, 0.1% Triton X-100, 1 mM phenylmethylsulfonyl
fluoride, 1 mM benzamidine, 0.5 µM microcystin,
and 0.2 mM sodium orthovanadate). The cleared lysates were
diluted 1:2 in BB (20 mM Hepes, pH 7.5, 50 mM NaCl,
2.5 mM MgCl
, 0.1 mM EDTA, 0.05% Triton
X-100, 1 mM phenylmethylsulfonyl fluoride, 1 mM benzamidine, 0.5 µM microcystin, and 0.2 mM vanadate), 20 µg of GST-Jun-(1-79) was added, and the
complexes were incubated on ice for 2 h. The washed complexes were
resuspended in 30 µl of KB and 5 µCi of
[
P]ATP for 20 min at 30 °C. The
phosphorylation of each substrate protein was quantitated using
densitometry. The data are expressed relative to the unstimulated
control sample in each experiment.
Figure 1: Activation of ERK2 in airway smooth muscle cells. Quiescent serum-starved ASM cells were treated with or without 10 µM forskolin for 15 min prior to treatment with PDGF (15 ng/ml), thrombin (40 nM), endothelin (200 nM), or PMA (1 µM). ERK2 activity was determined by Western analysis and immune complex kinase assays. Western blots of ERK2 immunoprecipitates (panel A) reveal the phosphorylated form of ERK2 (upper band) was present in the samples with ERK2 kinase activity. Immune complex kinase assays on the immunoprecipitates shown in panel A, using GST-Myc as an in vitro substrate, demonstrate increased catalytic activity of ERK2 following agonist stimulation (panel B). Forskolin pretreatment significantly decreased ERK2 catalytic activity in PMA-, endothelin-, or thrombin-treated cells. The relative intensities of the phosphorylation of the GST-Myc substrate were compared to the unstimulated control. Lane 1, 1; lane 2, 4.6; lane 3, 3.8; lane 4, 2.8; lane 5, 4.8; lane 6, 1.2; lane 7, 5.2; lane 8, 1.8; lane 9, 1.4; lane 10, 1.4.
Pretreatment of the cells with forskolin, an agent that increases the catalytic activity of PKA, inhibited the ability of endothelin, thrombin, and PMA to activate the ERK2 MAP kinase (Fig. 1). The ability of PDGF to activate the MAP kinase pathway appears to be independent of the activity of PKA.
Figure 2:
Activation of the JNKs in airway smooth
muscle cells. A, dose response of JNK activation in
endothelin- or thrombin-treated ASM cells. Serum-starved ASM cells were
treated with increasing doses of endothelin or thrombin. JNK activity
was measured in GST-JunJNK complexes by measuring the
incorporation of [
P]ATP into the GST-Jun
substrate as described under ``Materials and Methods.'' Each
point is the mean of two replicates. B, time course of JNK
activation in endothelin-treated ASM cells. JNK activity was measured
as described for A. The kinetics of thrombin activation of JNK
were similar to endothelin. In both cases maximal activation was at 15
min. C, activation of JNK by seven-transmembrane receptors and
inhibition by forskolin. Serum-starved quiescent ASM cells were treated
with or without 10 µM forskolin for 15 min prior to
stimulation with TNF-
(10 ng/ml), endothelin (200 nM), or
thrombin (40 nM) for 15 min. JNK enzymatic activity was
measured by phosphorylation of the GST-Jun fusion protein as described
under ``Materials and Methods.'' An autoradiogram of the
phosphorylated Jun fusion protein is shown. TNF-
, endothelin, and
thrombin activate the JNKs; pretreatment with forskolin blocks
agonist-induced activation of the JNKs. The intensities of the GST-Jun
bands are expressed relative to the unstimulated control. Lane
1, 1; lane 2, 5; lane 3, 5; lane 4,
3.6; lane 5, 0.6; lane 6, 1.6; lane 7, 0.8; lane 8, 0.8.
Figure 3:
Immunoprecipitation of JNK1 from
stimulated ASM cells. ASM cells were pretreated with forskolin and
subsequently stimulated with the indicated agonists as described in the
legend of Fig. 2. The JNK1 kinase was immunoprecipitated with a
JNK1 antibody. Kinase activity was measured by immune complex kinase
assay using GST-Jun as a substrate. Increased phosphorylation of
GST-Jun was observed with TNF-, endothelin, or thrombin treatment.
Forskolin pretreatment inhibited agonist-induced activation of JNK1 and
phosphorylation of GST-Jun. The intensities of the GST-Jun bands are
expressed relative to the unstimulated control. Lane 1, 1; lane 2, 2.9; lane 3, 2.4; lane 4, 3.7; lane 5, 1.2; lane 6, 1.4; lane 7, 1.4; lane 8, 1.5.
Seven-transmembrane receptors bind to and activate heterotrimeric
GTP-binding proteins, or G proteins, which subsequently interact with
downstream effector molecules. Activation of G proteins can be mimicked
pharmacologically using aluminum fluoride
(AlF). Fluoroaluminate is thought to
interact with GDP in the nucleotide-binding site of the G protein and
mimic the
phosphate of GTP, thereby activating the G protein.
AlF
has been frequently used in
characterizing signaling pathways that employ G proteins(33) .
To determine if JNK activation may occur as a result of G protein
activation, ASM cells were treated with 0.1, 1, and 5 mM AlF
for 15 min prior to assaying the
catalytic activity of the JNKs. Treatment of ASM cells with
AlF
activated the JNK pathway as
indicated by increased JNK catalytic activity (Fig. 4).
Figure 4:
JNK activation by aluminum fluoride. ASM
cells were treated with TNF- (10 ng/ml), endothelin (200
nM), thrombin (40 nM) or 0.1, 1, or 5 mM aluminum fluoride for 15 min prior to assaying JNK catalytic
activity as in Fig. 2. AlF
resulted in rapid activation of JNK catalytic activity in a
dose-dependent manner. The intensities of the GST-Jun bands are
expressed relative to the unstimulated control. Lane 1, 1; lane 2, 5.4; lane 3, 5.4; lane 4, 4.6; lane 5, 2.1; lane 6, 2.1; lane 7,
4.9.
Figure 5: Raf-1 activity in agonist-stimulated ASM cells. ASM cells were pretreated with 10 µM forskolin and subsequently stimulated with PDGF (10 ng/ml), thrombin (40 nM), endothelin (200 nM), and PMA (1 µM). The Raf-1 kinase was immunoprecipitated, and MEK kinase activity was determined by immune complex kinase assay using kinase-inactive recombinant (KR) MEK as an in vitro substrate. PDGF-, PMA-, and thrombin-stimulated cells showed increased Raf-1 activity as measured by MEK phosphorylation. Endothelin resulted in little appreciable Raf-1 activation. The intensities of the GST-Jun bands are expressed relative to the unstimulated control. Lane 1, 1; lane 2, 16.8; lane 3, 10.5; lane 4, 8.0; lane 5, 17.1; lane 6, 3.0; lane 7, 11.1; lane 8, 0.8; lane 9, 0.3; lane 10, 17.3.
Activation of PKA by increasing cAMP has been reported to inhibit the ability of epidermal growth factor to activate Raf-1 and the ERKs(34) . The effects of PKA activation in ASM cells were examined by activation of PKA prior to agonist treatment. Activation of PKA resulted in a decrease in the ability of PDGF and PMA to activate Raf-1, although the ability of PDGF to activate ERK2 was unaffected by PKA activation (Fig. 5).
Figure 6:
Inhibition of endothelin- or
thrombin-induced DNA synthesis by forskolin. DNA synthesis in
agonist-induced ASM cells was measured by
[H]thymidine incorporation. Pretreatment with
forskolin inhibited the ability of endothelin or thrombin to stimulate
DNA synthesis. Each condition is an average of four replicates. The
data are representative of three experiments.**, indicates statistical
significance, p < 0.01.
In the present studies, we have determined that stimulation of the endothelin or thrombin receptors in airway smooth muscle cells results in activation of both the MAP kinase and Jun kinase pathways, as measured by catalytic activity of the ERK2 and JNK1 kinases. The catalytic activity of JNK1 was determined by affinity purification using a GST-Jun-(1-79) fusion protein that contains the c-Jun transactivation phosphorylation sites Ser-63 and Ser-73 and is an excellent substrate for JNK1(35) . To confirm our findings regarding the activation of the JNKs by endothelin and thrombin, the JNK1 kinase was immunoprecipitated with a JNK1-specific antibody. The immune complex kinase assay revealed a similar pattern of activation of JNK1 by endothelin and thrombin. Activation of the JNK kinase cascade by seven-transmembrane receptors is a novel mechanism of intracellular signaling by these receptors. The mechanism of coupling of the receptors to the ERK and JNK pathways is unclear at this point. Activation of G proteins with aluminum fluoride is known to activate the MAP kinase pathway (36) and activated ERK2 in these ASM cells (not shown). The present study demonstrates that JNK kinase activity can also be stimulated by activation of G proteins, suggesting that the seven-transmembrane receptors may be activating the ERKs and JNKs by a G protein-mediated mechanism.
The upstream kinases that couple the endothelin or thrombin receptors to activation of the ERK and JNK pathways are not defined at this point. Endothelin and thrombin receptor activation stimulates phospholipase activity and results in increased diacylglycerol levels (37, 38) as well as activation of PKC(4, 39) . PKC is reported to phosphorylate and activate Raf-1(40) , suggesting a possible PKC-dependent mechanism of ERK activation. However, the ability of PKC to activate Raf-1 is controversial since PKC phosphorylation of Raf-1 appears to preferentially stimulate its autokinase activity rather than its MEK kinase activity(41) . Our results in airway smooth muscle cells indicate that endothelin is a very poor activator of Raf-1 catalytic activity (but a strong activator of the ERKs and JNKs), suggesting that these agonists employ Raf-1-independent mechanisms of activating the ERKs. Furthermore, our experiments indicate that in ASM cells the ERK and JNK activator(s) stimulated by endothelin or thrombin appears to be inhibited by PKA.
Dissecting the effects of cellular cAMP on cell growth has been enigmatic since cAMP appears to be growth stimulatory in some cells, such as 3T3, and growth inhibitory in other cell types, such as T cells and Src-transformed cell lines(42, 43) . Several reports indicate that the PKA-induced inhibition of the ERKs is mediated at least in part by phosphorylation of Raf-1. For instance, agonists that activate PKA, such as forskolin and dibutyryl cAMP, inhibit growth factor-induced activation of Raf-1 and the MAP kinases(34, 44) . PKA phosphorylates Raf-1 on Ser-43, and this phosphorylation inhibits the in vitro interaction between Ras and Raf-1(45) , suggesting a mechanism for the inhibition of the MAP kinase pathway by forskolin. However, this may not be the sole mechanism of PKA inhibition of Raf-1 since Ser-43 is not conserved in B-Raf, which is similarly inhibited by PKA(46) . Interestingly, activation of ERK2 in response to PDGF does not appear to be inhibited by activation of PKA (Fig. 2), suggesting that seven-transmembrane receptors and tyrosine kinase growth factor receptors couple to the MAP kinase pathway via distinct mechanisms in airway smooth muscle cells. The inhibition of the ERKs and JNKs by forskolin correlates with an inhibition in endothelin- or thrombin-induced mitogenesis, suggesting that these kinases may regulate the mitogenic effects of these agonists. Although forskolin has striking effects on MAP kinase activation, PKA activation has pleiotropic effects on cell growth that are poorly understood, making it difficult to establish causality between inhibition of the ERKs or JNKs and inhibition of mitogenesis.
While the mechanism of inhibition of the ERKs and JNKs by PKA in ASM
cells is currently unknown, there are interesting parallels with
another seven-transmembrane receptor, the -adrenergic receptor,
which is desensitized by phosphorylation(47) . Perhaps
PKA-induced receptor phosphorylation inhibits seven-transmembrane
coupling to the MAP kinase pathway. The mechanism of
seven-transmembrane receptor activation of these MAP kinases is an area
of intense study.