Stimulation of Jun N-Terminal Kinase (JNK) by Gonadotropin-Releasing Hormone in Pituitary
T31 Cell Line Is Mediated by Protein Kinase C, c-Src, and CDC42
Nurel L. Levi,
Tamar Hanoch,
Outhiriaradjou Benard,
Meirav Rozenblat,
Dagan Harris,
Nachum Reiss,
Zvi Naor and
Rony Seger
Department of Biological Regulation (N.L.L., T.H., O.B., M.R.,
R.S.) The Weizmann Institute of Science Rehovot, 76100
Israel
Department of Biochemistry (M.R., D.H., N.R.,
Z.N.) George S. Wise Faculty of Life Sciences Tel Aviv
University Ramat Aviv 69978, Israel
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ABSTRACT
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The signaling of ligands operating via
heterotrimeric G proteins is mediated by a complex network that
involves sequential phosphorylation events. Signaling by the G
protein-coupled receptor GnRH was shown to include elevation of
Ca2+ and activation of phospholipases, protein
kinase C (PKC) and extracellular signal-regulated kinase (ERK). In this
study, GnRH was shown to activate Jun N-Terminal Kinase (JNK)/SAPK in
T31 cells in a PKC- and tyrosine kinase-dependent manner. GnRH as
well as tumor-promoting agent (TPA) also increased c-Src activity,
which peaked at 2 min after GnRH stimulation and was sensitive both to
PKC and to tyrosine kinase inhibitors. Coexpression of Csk, which
serves as a Src-dominant interfering kinase, and constitutively active
forms of Src, together with JNK, confirmed the involvement of c-Src
downstream of PKC in the GnRH-JNK pathway. Coexpression of dominant
negative and constitutively active forms of CDC42, Rac1, Ras, MEKK1,
and MEK1 with JNK indicated that JNK activation by GnRH and TPA is
mediated by CDC42 and MEKK1. Ras and MEK1, which are involved in a
related mitogen-activated protein kinase (MAPK) pathway, did not affect
JNK activation in
T31 cells. Taken together, our results suggest
that GnRH stimulation of JNK activity is mediated by a unique pathway
that includes sequential activation of PKC, c-Src, CDC42, and probably
also MEKK1.
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INTRODUCTION
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Intracellular transmission of extracellular signals is mediated in
large by several groups of sequentially activated protein kinases,
which are collectively known as the mitogen-activated protein kinase
(MAPK) cascades. In growth factor signaling, the key elucidated MAPK
cascade is the extracellular signal-regulated kinase (ERK, also known
as p42/44 MAPK) cascade (reviewed in Ref.1), which is initiated by the
small GTP-binding protein (sGP) Ras. Upon stimulation, Ras assumes its
active, GTP-bound form, and recruits the protein kinase Raf-1 to the
plasma membrane where the kinase can be activated (2, 3). At this
stage, the signal dissociates from the plasma membrane via a sequential
activation of protein kinases, which are MEK, ERK, p90RSK,
and under some conditions, also glycogen-synthase kinase 3. However,
the ERK cascade is not the only link between membranal receptors and
their intracellular targets, and in the past years several other
ERK-like cascades have been identified (1). One of the best studied of
these cascades is the Jun N-terminal kinase [JNK; also known as
stress-activated protein kinase ( SAPK) (4, 5)] cascade that utilizes
a sequential activation of PAK1/MLK, MEKK1, SEK1/MKK7, and JNK1/2 to
activate transcription factors such as c-Jun, ATF2, and Elk1 (reviewed
in Refs. 6 and 7). Additional MAPK cascades are the p38RK [HOG, CSBP
(8, 9, 10)] and ERK5 [BMK (11)]. The extent of activation of these
different cascades varies according to stimuli, and the ratio between
the signals in each cascade may determine signaling specificity. For
example, JNK appears to be highly activated by stress, whereas the ERKs
are mainly activated by mitogens. Interestingly, MKK4 [SEK1 (12, 13)]
activates both JNK and p38RK, thus indicating the existence of some
cross-talk between the cascades in mammalian cells.
GnRH, which is a hypothalamic decapeptide, serves as a key
regulator of the reproductive system. It induces the synthesis and
release of the pituitary gonadotropin LH and FSH. When GnRH binds to
its seven-transmembrane receptor, it induces interaction of the
receptor with the heterotrimeric Gq protein that leads to activation of
phospholipase C and formation of inositol 1,4,5- trisphosphate and
diacylglycerol, leading to elevation of Ca2+ and activation
of protein kinase C (PKC). Phospholipase D, phospholipase
A2, and the formation of bioactive lipoxygenase products
are also activated by GnRH, although the mechanisms involved are not
yet known (reviewed in Refs. 14 and 15). Recently, the stimulation of
the ERK-signaling cascade by GnRH has been demonstrated by several
laboratories (16, 17, 18, 19). ERK activation in response to GnRH is unique as
it was shown to be fully mediated by PKC and Ca2+, which
seem to act in a Ras-independent manner (19). Furthermore, the protein
tyrosine kinase (PTK) inhibitor genistein partially inhibited this
pathway, which indicates the involvement of a PTK in the GnRH-induced
ERK activation.
In this study, stimulation of the JNK pathway by GnRH was examined. Our
results show that GnRH-induced JNK activation was greater but occurred
more slowly than that of ERK. The signaling pathway that mediates the
GnRH stimulation of JNK appears to involve PKC, c-Src, CDC42/Rac1, and
probably also MEKK1, and to act independently of the ERK cascade. Thus,
the
T31 cells appear to utilize a unique pathway that links a G
protein-coupled receptor to the activation of the JNK-signaling cascade
through PKC, c-Src, and CDC42.
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RESULTS
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Stimulation of JNK Activity and Jun Expression by GnRH
Recently, we have shown that the activity of ERK1 and ERK2 is
stimulated by GnRH in pituitary
T31 cells (19). To examine whether
GnRH can also activate the JNK pathway, serum-starved
T31 cells
were stimulated with a GnRH analog (GnRH-a, 10-7
M), and JNK activity was monitored by a solid phase assay
(20). As shown in Fig. 1
, JNK activity
was significantly (25- to 55-fold) stimulated by GnRH-a, to an extent
similar to that caused by the potent stimulating agents, peroxovanadate
(VOOH, 15 min) and the PKC activator tumor-promoting agent (TPA, 30
min). This magnitude of stimulation was higher than that observed for
ERK1/2 (5- to 15-fold) in the same cells. However, the time course of
JNK activation by GnRH-a was slower than the rapid activation of the
ERKs. JNK activation was detected 5 to 10 min after the initiation of
the GnRH-a treatment, it peaked by 30 min, and it decreased over the
next 90 min (Fig. 1B
). Thus, although both ERK and JNK might be key
mediators of GnRH signaling, they may regulate different cellular
processes.
Since activation of the JNK cascade is known to induce c-Jun expression
in several cell systems (7), we examined whether c-Jun expression is
also induced by GnRH in
T31 cells. Northern blot analysis, using
c-Jun as a probe, revealed that GnRH-a induced the expression of c-Jun
mRNA in these pituitary cells (Fig. 1C
), thus confirming the findings
of Cesnjaj et al. (21). Taken together, these results
indicate that the JNK cascade does indeed play a role in GnRH signaling
in
T31 cells.
Effects of PKC and PTK Inhibitors on JNK Activation
Activation of ERKs by GnRH-a requires PKC and to some extent also
protein tyrosine phosphorylation (16, 17, 18, 19). Therefore, the role of PKC
and PTKs in GnRH-induced JNK activation was examined. Exposure of
T31 cells to TPA or to VOOH (general activator of tyrosine
kinases) caused a pronounced stimulation of JNK activity (Fig. 1A
),
which suggested that PKC and PTK might stimulate JNK activity in this
cell line. Whether PKC is indeed involved in GnRH signaling was
determined using PKC depletion and inhibition. Thus,
T31 cells
were pretreated with TPA (1 µM, 16 h) which depletes
most PKC isoforms, or with the selective PKC inhibitor GF109203X. In
both cases there was a significant (8085%) inhibition of the
GnRH-induced JNK activity (Fig. 2A
).
These results suggest that, as in the case of ERK (19), activation of
JNK by GnRH-a in these cells is mainly mediated by PKC.

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Figure 2. Effect of PKC and PTK Inhibitors on GnRH-a and TPA
Stimulation of JNK Activity
A, Inhibition of PKC: T31 cells were either pretreated with 1
µM TPA (TPA dep.) for 16 h or with 3
µM GF109203X for 15 min before stimulation or left
untreated. GnRH-a (10-7 M) was added for 0,
30, 60, or 120 min, and JNK activity was measured by a solid phase
assay using GST-Jun as a substrate as described in Materials and
Methods. B, Inhibition of PTKs: T31 cells were pretreated
with 200 µM genistein for 15 min or left untreated as
control. GnRH-a (10-7 M) was then added for 0,
15, 30, and 60 min, and JNK activity was measured as above. C,
Inhibition of TPA-stimulated JNK activity by genistein: T31 cells
were pretreated with 200 µM genistein for 15 min or left
untreated as control. TPA (200 nM) was then added for 0,
15, 30, and 60 min, and JNK activity was measured as above. D,
Quantification of the experiments in A, B, and C by an imaging
densitometer. , GnRH-a; , GnRH-a+ TPA depletion; ,
GnRH-a+GF109203X; x, GnRH-a+genistein; , TPA; , TPA +
genistein. All results are from a representative experiment that was
reproduced three times.
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To determine whether PTKs are involved in the GnRH-induced activation
of JNK, we pretreated the
T31 cells with the PTK inhibitor,
genistein. Genistein completely abolished the GnRH-stimulated JNK
activity for up to 1 h after stimulation (Fig. 2B
) but only inhibited
ERK activation by
30% (19), indicating the existence of a
genistein-sensitive PTK that mediates the activation of JNK by GnRH-a.
To determine whether this PTK is located upstream or downstream to PKC,
genistein-treated
T31 cells were stimulated with TPA. As shown in
Fig. 2
, C and D (that quantitate the results of panels AC),
stimulation of JNK activity by TPA was most prominent after 60 min
(
30-fold), and this stimulation was completely abolished by
pretreatment with genistein. TPA bypasses the GnRH receptor-Gq complex
and activates PKC directly; these results indicate that PKC is located
upstream to the PTK(s) in the pathway that mediates the activation of
JNK by GnRH. Sequential activation of PKC and PTKs that leads to JNK
activation in G-protein coupled receptors signaling seems to be unique
to GnRH-stimulated
T31 cells, as will be discussed below.
c-Src Is Involved in the GnRH-JNK Signaling Pathway
The involvement of a putative PTK that was implicated by the use
of genistein initiated an effort to identify the PTK that is involved
in JNK activation by GnRH. c-Src or other member(s) of the Src family
of PTKs are genistein-sensitive protein kinases that have been
previously reported to participate in G-protein signaling (22, 23, 24). To
examine whether a c-Src family member is the PTK that mediates the
effect of GnRH on JNK, we first asked whether these kinases are
expressed in the
T31 cells. Western blot analysis revealed that
the
T31 cells contain a substantial amount of c-Src, a small
amount of Fyn, and, as expected, no Lyn (Fig. 3A
). Activation of c-Src by GnRH-a was
probed by immunoprecipitating c-Src from GnRH-a-stimulated cells using
specific antibodies and measuring its activity using enolase as a
substrate (25). GnRH-a caused an
3.2-fold increase in c-Src
activity, which peaked at 5 min after stimulation and was maintained
for an additional 15 min (Fig. 3
, B and C). Although the fold
activation seems to be small, it is comparable to the activation
reported in other cellular systems (22) and might be due to a high
basal activity of this PTK. c-Src was activated also by TPA (2.6-fold;
Fig. 3
, B and C), and the activation of c-Src by GnRH was sensitive
both to PKC and to PTK inhibitors, thus indicating that the activation
of c-Src in response to GnRH-a is downstream of PKC.

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Figure 3. Content and Activation of Src by GnRH-a and TPA in
T31 Cells
A, Western blot analysis of Src family members in T31 cells. Total
cell extracts were separated by SDS-PAGE and subjected to Western
blotting using anti-Src, anti-Fyn, and Anti-Lyn anti-peptide antibodies
(Santa Cruz, according to the manufacturer instruction and using
alkaline phosphatase-coupled secondary antibody). To assure the
specificity of the antibodies, the antigenic peptide was used (100
µg/ml) for competition. B, c-Src activation by GnRH-a and TPA:
T31 cells were treated with GnRH-a (10-7
M) for the indicated times, or pretreated with GF109203X (3
µM, 15 min) and with genistein (200 µM, 15
min) and then treated with GnRH-a (top panel).
Alternatively, the cells were stimulated with 200 nM TPA
(lower panel) Stimulation was terminated by washing with
ice-cold PBS followed by c-Src immunoprecipitation. c-Src activity
toward acid-denatured enolase was determined as described in
Materials and Methods. C, Quantification of the results
in panel B. The reactions were analyzed by an SDS-PAGE, and the amount
of phosphate incorporated into enolase was determined by imaging
densitometer. These results are an average of two experiments. ,
GnRH-a activation of Src; , GnRH-a + GF109203X; , GnRH-a +
genistein. Inset, TPA stimulation.
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The involvement of c-Src in GnRH-induced JNK activation was then
examined by cotransfecting Csk, which serves as a Src-dominant
interfering kinase (22), together with hemagglutinin (HA)-JNK2 into the
T31 cells. GnRH-stimulated JNK activity was markedly inhibited by
the overexpression of Csk, whereas the basal activity of JNK was not
affected (Fig. 4A
), indicating that most,
if not all, the GnRH signal toward JNK is indeed mediated by c-Src. To
further verify this point we also cotransfected the constitutively
active form of c-Src, Y527F-Src, together with HA-JNK2 into the same
cells. Y527F-Src by itself activated JNK up to 4-fold above basal
activity, and this activity was not enhanced by the addition of GnRH-a
to the cells (Fig. 4
, B and C), further indicating that most of the
GnRH signal is funneled through c-Src. Moreover, the activation of JNK
by Y527F-Src was sensitive to genistein (Fig. 4
, B and C), indicating
that c-Src by itself is a target for genistein and therefore might be
the genistein- sensitive PTK in the GnRH-JNK pathway. As expected,
GF109203X did not influence the Y527F-Src activation of JNK (up to 60
min treatment with the inhibitor). Therefore, GnRH signal toward JNK
appears to be mediated mainly via the genistein-sensitive c-Src (or a
family member), operating downstream of PKC.

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Figure 4. The Effect of Y527F-Src and Csk on GnRH-a
Stimulation of JNK Activity
A, T31 cells were cotransfected with either the Src-dominant
interfering kinase Csk or with vector control together with HA-JNK2.
Two days after transfection the cells were serum starved for 16 h
and then either treated with GnRH-a (10-7 M)
or left untreated. JNK activity toward GST-Jun was determined as
described above (GST-Jun). The amount of immunoprecipitated HA-JNK was
determined by Western blot analysis with anti-JNK antibody (Sigma). B,
T31 cells were cotransfected with either the constitutively active
Y527F-Src (Src) or vector control (left lane) together with HA-JNK2.
Two days after transfection the cells were serum starved for 16 h
and then either treated with GnRH-a (right lane),
treated with GF109203X (GF, 3 µM, 15 min), genistein
(Gen, 200 µM, 15 min), or left untreated (two left
lanes). JNK activity toward GST-Jun was determined as described
above. Similar results were obtained when the GF109203X was added for
1 h. The amount of immunoprecipitated HA-JNK was determined by
Western blot analysis with anti-JNK antibody (HA-JNK). C, The amount of
phosphate in GST-Jun was determined by an imaging densitometer. These
results are an average of three experiments.
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JNK Stimulation by GnRH Is Mediated by CDC42/Rac1 but Not Ras
The JNK signaling cascade is known to act downstream of several
sGPs, such as Rac1, CDC42, and Ras (26, 27). Therefore, we examined
which, if any, of the sGPs is involved in the GnRH-stimulated,
genistein-sensitive, JNK activation. Thus, the dominant interfering
mutants of three sGPs (T17N-Rac2, T17N-CDC42, and S17N-H-Ras) were
cotransfected into
T31 cells together with HA-JNK2. After cell
treatment with the desired stimuli and inhibitors, lysis of the cells
and immunoprecipitation with anti-HA antibodies, JNK2 activity was
measured using GST-Jun as a substrate. All three inactive forms of the
sGP had an inhibitory effect on the GnRH-stimulated JNK2 activity (Fig. 5
, A and B). T17N-CDC42
inhibited GnRH-stimulated JNK activity by as much as 62 ± 5%,
the T17N-Rac1 by 36 ± 4%, and the S17N-H-Ras by only 17 ±
4%.

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Figure 5. The Involvement of CDC42 and Rac1, But Not Ras, in
GnRH-a and TPA Stimulation of JNK Activity
A, T31 cells were cotransfected with HA-JNK together with the
dominant negative (DN-sGP) and constitutively active (CA-sGP) forms of
CDC42, Rac1 and H-Ras, or with a vector control (Control and GnRH
lanes). Two days after transfection the cells were starved for 16
h followed by a treatment with GnRH-a (10-7 M,
30 min, two left lanes), with GF109203X (GF, 3
µM, 15 min), with genistein (Gen, 200 µM,
15 min) or left untreated (two central lanes). The cells
were then harvested and JNK activity was determined as described in
Materials and Methods. It should be noted that the
results of the different sGPs are from separate experiments and therefore the intensity of the
32p incorporated GST-Jun should only be compared within each gel strip.
B, The amount of phosphate incorporated into GST-Jun in cells
transfected with DN-sGP was quantified by an imaging densitometer. The
average of three experiments is presented. C, The influence of
GF109203X (3 µM, 0.25, 1, and 2 h) and genistein
(200 µM, 0.25, 1, and 2 h) over prolonged time on
JNK activation by the constitutively active CDC42, Rac1, and Ras was
determined as described above. , GF109203X; , genistein; , JNK
activation by GnRH-a (10-7 M, 30 min) under
these conditions. The fold activation in time 0 represents fold
activation by the constitutively activated sGPs, and the broken
line represents a time course of GF109203X and genistein
influence on these activations. The results in this figure are from one
representative experiment that was reproduced four times. D, T31
cells were cotransfected with HA-JNK together with the dominant
negative forms of CDC42 (right lanes) or vector control
(left lanes). Two days after transfection the cells were
starved (16 h) and then treated with TPA (200 nM, 30 min)
or left untreated. The cells were then harvested, and JNK activity was
determined as above. Comparable amounts of JNK were detected in the
immunoprecipitants of all the above experiments (data not shown).
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Since the inhibition of GnRH stimulation of JNK activity by the
dominant interfering forms of the sGPs was incomplete, some of the
GnRH-induced, genistein-sensitive signaling may have been mediated by
an sGP-independent pathway. To test this possibility, constitutively
active forms of the same sGPs (Q61L-CDC42, Q61L-Rac1, and Q61L-H-Ras)
were cotransfected into
T31 cells together with HA-JNK2, and the
stimulation of JNK activity was determined as above. The constitutively
active CDC42 stimulated JNK activity greatly [8.8 ± 1.3 fold
(n = 3), which is 65% of GnRH stimulated JNK activity under these
conditions]. The constitutively active form of Rac1 had a moderate
effect (3.8 ± 0.5 fold), and the constitutively active H-Ras had
only a marginal effect (1.9 ± 0.7 fold; Fig. 5C
). The stimulation
of HA-JNK2 by Q61L-CDC42 or by Q61L-Rac1 was not affected by the PKC
inhibitor GF109203X nor by the PTK inhibitor genistein (Fig. 5C
). In
addition, TPA stimulation of JNK activity was significantly inhibited
by the T17N-CDC42 (Fig. 5D
), which supports CDC42 and also, to a lesser
extent, Rac1 acting downstream of PKC and c-Src. The effects of the PKC
and PTK inhibitors on cells cotransfected with the constitutively
active form of H-Ras could not be determined because of the low
stimulation with this construct.
The above results indicate that most of the genistein-sensitive,
GnRH-induced JNK activation is mediated by CDC42. Although in many
cases CDC42 and Rac1 seem to use similar guanine nucleotide exchange
factors, the effect of Rac1 on the GnRH-JNK pathway seems to be
limited. Similar phenomenon, where CDC42 transmits downstream signals
without the involvement of Rac1, was recently reported for
serum-induced proliferation (28). Ha-Ras does not seem to be
significantly involved in GnRH-induced signaling that contributes to
the JNK cascade.
GnRH Stimulation of JNK Is Mediated by MEKK1 But Not by MEK1
Several reports have recently suggested that the stress-activated
JNK cascade consists of JNK, JNKK (SEK1, MKK7), MEKK1 (6), and either
PAK1 (29) or MLK (30). To determine whether the genistein-sensitive
GnRH signal toward JNK is mediated via this cascade,
T31 cells
were cotransfected with HA tagged-JNK2 and the truncated,
constitutively active form of MEKK1 (31) and processed as described for
the sGPs above. The constitutively active MEKK1 elicited a strong
activation (
25 fold) of JNK2, which was somewhat lower than the
activation caused by GnRH-a (data not shown) but could not be further
activated by treatment of the transfected cells with GnRH-a (Fig. 6
). The lack of synergistic activation
with MEKK1 and GnRH indicates (although does not prove) that MEKK1
might be involved in the GnRH-JNK signaling pathway. Indeed, the MEKK1
effect was not blocked by GF109203X or genistein when added for 15
(Fig. 6
), 60, or 120 min (data not shown), thus indicating that PKC and
the genistein-sensitive PTK(s) are probably situated upstream of MEKK1,
(although they may lie on a distinct pathway in case MEKK1 is not the
activatory component as suggested above). On the other hand,
coexpression of HA-JNK2 with the constitutively active form of MEK1
[
N-EE-MEK (32, 33)] which is involved in the distinct ERK
signaling cascade, did not affect JNK activity under conditions in
which MEKK1 caused its strong activations (data not shown). This later
observation is in agreement with our previous findings on the effects
of
N-EE-MEK in COS7 cells (32). Thus, in
T31 and COS7 cells,
but unlike in U937 cells (34), the JNK signaling cascade appears to be
completely distinct from the MEK-ERK cascade.

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Figure 6. Effect of Constitutively Active MEKK on JNK
Activity
T31 cells were cotransfected with either N-terminally deleted,
constitutively active MEKK1 (MEKK), or vector control together with
HA-JNK2 as described in Materials and Methods. Two days
after transfections the cells were serum starved for 16 h followed
by a treatment with GF109203X (GF, 3 µM, 15 min),
genistein (Gen, 200 µM, 15 min), GnRH-a
(10-7 M, 30 min), or control PBS as indicated.
GF109203X and genistein had no effect even when added for up to 2
h. Comparable amounts of JNK were detected in the immunoprecipitants of
all lanes (data not shown).
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Taken together, our results support a model wherein GnRH-stimulated JNK
activation is mediated by a pathway involving PKC, c-Src, CDC42/Rac1,
and possibly MEKK1, which might lead to the expression of Jun detected
upon GnRH stimulation of the
T31 cells. A divergence of the
GnRH-stimulated ERK and JNK pathways at the level of PKC stimulation of
PTK activity was suggested by the differential sensitivity of the two
MAPKs to genistein (Fig. 7
).

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Figure 7. A Schematic Representation of GnRH-Induced ERK and
JNK Signaling Pathways.
For details see text.
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DISCUSSION
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Binding of GnRH to its membranal receptor elicits a series of
signaling pathways (14), including activation of the ERK cascade
(16, 17, 18, 19). However, the ERK cascade is not the only route by which GnRH
communicates with the nucleus. Here the JNK pathway is shown to be
significantly activated in response to GnRH-a, to a much greater extent
than the ERK cascade. The time course of this GnRH-induced JNK
activation in response to GnRH-a is slower than that of ERK (Fig. 1B
)
and is similar to time courses observed for the induction of JNK by
other heterotrimeric G protein-coupled receptors (35) and tyrosine
kinase receptors (36). However, this slower time course does not occur
in all cells. For example, in nerve growth factor-treated PC12 cells,
the short-term JNK activation is rapid (1030 min) and transient (37).
Thus, like the ERK pathway (38), the duration of JNK activity may
determine the outcome and specificity of the signal. The delayed
response of JNK observed here (Fig. 1
) may indicate that JNK is
involved in a later stage of transcription regulation and suggests that
the signaling machinery that is involved in the activation of JNK is
different from that leading to ERK activation. This upstream machinery
was elucidated in this study, and our results suggest that GnRH binding
to its receptors triggers the activation of PKC, which occurs most
probably via the heterotrimeric Gq protein and PLC. The signal is then
transmitted to a Src-family PTK and further to the sGPs CDC42 and
possibly Rac1. The sGPs activate, in turn, a sGP-activated kinase whose
identity is still controversial and which might be PAK1 (29), MLK (30),
or another unknown protein kinase. Finally, the JNK cascade, including
JNKKK (probably MEKK1), JNKK, and JNK (reviewed in Ref.6), is
activated, causing the stimulation of several transcription factors
including, most likely, c-Jun (6) and the expression of c-Jun protein
(Fig. 7
).
It is well established by now that PKC mediates many of the downstream
effects of GnRH, including the activation of the ERK cascade (14). In
T31 cells, JNK activation, like that of ERK (19), is activated by
TPA and markedly inhibited (8085%) by the PKC-specific inhibitor
GF109203X or by TPA depletion of PKC (Fig. 2
). This degree of
activation would imply that most of the GnRH signal toward JNK is
mediated by PKC, although the small amount of residual activity upon
PKC inhibition could suggest an additional signaling machinery. This
involvement of PKC in JNK activation by GnRH seems to be unique to our
system. In other cell lines TPA does not stimulate JNK activity (35, 39, 40) or may even cause a decrease in the basal activity of JNK (32).
A clue regarding the identity of the putative components of GnRH
signaling was first achieved by the effect of PTK inhibitors on JNK
activity. Thus, the PTK inhibitor, genistein, completely blocked
GnRH-stimulated JNK activation (Fig. 2
) whereas it inhibited only
30% of ERK activation by GnRH (19). Moreover, TPA-induced JNK
activation was also blocked by genistein (Fig. 2
), indicating that the
PTK is located downstream to PKC. Since TPA bypasses the GnRH
receptor-Gq complex and activates PKC directly, our results indicate
that the PKC-PTK step is located in the pathway leading to GnRH
activation of JNK. This is reminiscent of the involvement of PTKs in
PKC signaling toward ERK in other systems (19, 41, 42). The difference
in the extent of inhibition by genistein suggests that two (or more)
distinct PTKs may be involved in the activation of ERK and JNK by GnRH.
Therefore, the activation of PTKs by PKC might be the point of
divergence between the JNK and ERK cascades in
T31 cells (Fig. 7
).
Recently, Src family protein kinases have been implicated in some G
protein-coupled receptor signaling pathways. Lyn is involved in the
activation of MEK in DT40 cells (23), and c-Src is involved in
Angiotensin II signaling in COS cells (43). Our results showing that
c-Src is involved in the stimulation of JNK activity (Figs. 3
and 4
) in
a GF109203X-independent, but genistein-dependent, manner implies that
c-Src (or a member of its family) is the PTK that mediates
PKC-dependent signaling toward JNK. Since JNK activation by GnRH-a was
completely blocked by a Src-dominant interfering kinase (Csk), and JNK
could not be further activated by GnRH in cells transfected with the
constitutively active form of c-Src, this PTK seems to act as a central
component that funnels all the PKC-mediated GnRH signals toward JNK.
The mechanism by which PKC induces c-Src activation in the GnRH-JNK
system is not clear. PKC has already been shown to directly activate
c-Src by phosphorylation of its serines 12 and 48 (44), which are
required for the enhanced response to ß-adrenergic agonists in cells
overexpressing c-Src. The importance of the c-Src phosphorylation on
serine 12 was supported also by Liebenhoff et al. (45), who
suggested that this phosphorylation by PKC induces cytoskeletal
association necessary for c-Src activation. Furthermore, G
protein-coupled receptors may mediate their signal through a
cascade of PTK that involves activation of c-Src by Pyk2 (22) or c-Src
and FAK (46). Therefore, in the GnRH-JNK pathway studied here, PKC may
activate an upstream PTK, which in turn activates c-Src. However, since
some reports have implied that PKC may be located downstream of c-Src
in the signaling pathway leading to proliferation (47, 48), the
relationship between PKC and c-Src is not yet fully understood, and
some other signaling components may be involved in the activation of
c-Src by PKC.
The activation of JNK involves in most cases the sGPs, CDC42 and Rac1,
that might operate downstream to Ras (26, 27). Both dominant-negative
and constitutively active forms of these G proteins were used to
determine whether they participate in the genistein-sensitive
GnRH-stimulated pathway leading to JNK activation. Here we show that
CDC42 and, to some extent, also Rac1 are involved in this activation,
whereas the involvement of Ras is unlikely (Fig. 5
). The activation of
Rac1 and CDC42 by stress-activated signals was previously shown to be
Ras-dependent (27). Therefore, the activation of JNK by GnRH in
T31 cells seems to differ and involves a Ras-independent
activation of Rac1 and CDC42 by c-Src. Although the mechanism of
activation is not yet known, it might involve a distinct set of adaptor
molecules and nucleotide exchange factors.
In summary, we demonstrated here the activation of the JNK cascade by a
unique signaling pathway that involves PKC, c-Src family PTK,
CDC42/Rac1, and probably MEKK1, which leads to JNK activation and most
likely to the c-Jun induction in response to GnRH. Similar to the ERK
cascade, the activation of JNK by GnRH is PKC dependent, and the point
of divergence between the two cascades seems to be the activation of
PTK downstream of PKC. Furthermore, the JNK pathway in these cells
seems to be different from most other signaling pathways that link G
protein-coupled receptors to the activation of JNK in that it includes
the activation of CDC42/Rac1 by PKC and c-Src in a Ras-independent
mechanism.
 |
MATERIALS AND METHODS
|
---|
Buffers
Homogenization buffer (buffer H) consisted of 50 mM
ß-glycerophosphate (pH 7.3), 1.5 mM EGTA, 1
mM EDTA, 1 mM dithiothreitol, 0.1
mM sodium orthovanadate, 1 mM benzamidine,
aprotinin (10 µg/ml), leupeptin (10 µg/ml), and pepstatin (2
µg/ml). HEPES lysis buffer consisted of 20 mM HEPES (pH
7.6), 10 mM EGTA, 40 mM ß-glycerophosphate,
1% Nonidet P-40, 2.5 mM MgCl2, 1
mM dithiothreitol, 2 mM sodium orthovanadate,
aprotinin (20 µg/ml), leupeptin (20 µg/ml), and 1 mM
phenylmethylsulfonyl fluoride. The JNK buffer consisted of 20
mM HEPES (pH 7.6), 20 mM MgCl2, 40
mM ß-glycerophosphate, 0.1 mM sodium
orthovanadate, and 2 mM dithiotritol. The PTK buffer
consisted of 50 mM HEPES (pH 7.6), 150 mM NaCl,
5 mM MnCl2, 5 mM MgCl2,
0.1 mM sodium orthovanadate, and 1 mM
dithiothreitol.
Stimulants, Inhibitors, Antibodies, and Miscellaneous
Reagents
[D-Trp6]-GnRH, a stable GnRH analog,
genistein (PTK inhibitor), enolase, and protein A-Sepharose were
obtained from Sigma Chemical Co. (St. Louis, Mo). GF109203X (a PKC
inhibitor) and TPA were purchased from Calbiochem (La Jolla, CA).
GST-Jun (197) was prepared as previously described (20). Mouse
monoclonal antihemagglutinin (HA)-antibodies were produced by the
Antibody Unit of The Weizmann Institute of Science. Rabbit polyclonal
anti-c-Src antibodies, N-16 and SRC 2, and anti-Fyn and anti-Lyn
antibodies and their control peptides were purchased from Santa Cruz
(San Diego, CA). Mouse monoclonal anti-active MAPK antibodies were from
Sigma (Rehovot, Israel).
Plasmids
The CDC42 constructs (Q61L and T17N-CDC42) were a gift from Dr. G.
Bokoch (The Scripps Research Institute, La Jolla CA). The Csk construct
was a gift from Dr. S. Courtneidge (Sugen, Inc. Redwood City, CA).
MEKK1, GST-c-Jun JNK2, and Rac constructs were provided by Dr. Y.
Ben-Neriah (Hebrew University, Jerusalem, Israel).
N-EE-MEK was
cloned in pCDNA1 (Invitrogen, San Diego, CA) as previously described
(32).
Solid Phase Assay for JNK Activity
Pituitary
T31 cells were grown to 80% confluency serum
starved for 16 h, and the examined stimulants were added for
various time intervals. The cells were then washed (twice with PBS and
once with buffer H), scraped into 250 µl of buffer H and sonicated
(50 W, 2 x7 sec), all at 4 C. After centrifugation (20,000 x
g, 15 min, 4 C), aliquots of the resulting supernatant were
assayed by the Coomassie protein assay (Pierce, Rockford, IL) for
protein. JNK activity was detected according to Hibi et al.
(20). Briefly, aliquots (100150 µg protein) of the cell extracts
were incubated (2 h, 4 C) with GST-Jun to allow the JNKs to bind to the
substrate. After extensive washing, the JNK activity was measured by
phosphorylation of the GST-Jun, which was mediated by the bound kinase
in the presence of 20 mM MgCl2, 20
µM [
-32P]-ATP (300 cpm/pmol) for 20 min
at 30 C. The reactions were terminated by the addition of sample
buffer, and the samples were subjected to SDS-PAGE analysis. The gels
were dried, autoradiographed on Kodak X-100 films, and the
phosphorylation of GST-Jun was quantitated by densitometry (Bio-Rad 690
densitometer, Bio-Rad, Richmond, CA).
Transfection of
T31 Cells
Subconfluent
T31 cells were cotransfected with 5 µg
each of HA-JNK2-SR
and one of the following plasmids
(MEKK-pCMV5, Q61L-Rac1-pCDNA3, T17N-Rac1-pCDNA3,
Q61L-CDC42-pCDNA3, T17N-CDC42-pCDNA3, Q61L-H-Ras-pCMV5,
S17N-H-Ras-pCMV5, Y527F-Src-RK5, and Csk-pRK5) using the calcium
phosphate technique (49). The total amount of plasmid was adjusted to
10 µg with vector DNA in the control experiments. The transfection
efficiency was 1030%, as determined by transfection with a plasmid
that contained ß-Gal and appropriate staining. Two days after
transfection, the cells were serum starved for 16 h and incubated
for the desired time intervals with GnRH-a in the presence or absence
of various inhibitors. The cells were then lysed with 250 µl of HEPES
lysis buffer at 4 C, vortexed, and kept on ice for 5 min. After
centrifugation (20,000 x g, 20 min), the supernatant
was assayed for protein content as above.
Immunoprecipitation with Anti-HA Antibodies
Lysates from transfected cells (400500 µg protein in HEPES
lysis buffer) were incubated (1 h, 4 C) with anti HA-antibodies (3 µg
Ab/reaction). The immunocomplexes were precipitated with Protein
A-Sepharose, and the resulting precipitates were washed twice with PBS
containing 1% Nonidet P-40 and 2 mM sodium vanadate, once
with 100 mM Tris (pH 7.5) containing 0.5 M LiCl
and once with JNK buffer. The immunoprecipitates were then resuspended
in 30 µl of JNK buffer, and the JNK activity was measured (30 min, 30
C with constant mixing) using GST-c-Jun as a substrate. Phosphorylation
of GST-c-Jun was monitored by autoradiography.
Immunoprecipitation with Anti c-Src Antibodies
Cell lysates (400500 µg protein in HEPES lysis buffer) were
incubated (1 h at 4 C) with anti-c-Src-antibodies (5 µg/reaction) and
then precipitated with Protein A-Sepharose. The immunocomplexes were
washed twice with PBS containing 1% Nonidet P-40 and 2 mM
sodium vanadate, once with 100 mM Tris (pH 7.5) containing
0.5 M LiCl, and once with PTK buffer. The washed
immunoprecipitates were resuspended in PTK buffer and the c-Src
activity was determined by using acid-denatured enolase (3
mM) as substrate (final volume of 40 µl) in the presence
of 20 µM [
32P]-ATP (8,000 cpm/pmol). The
enzymatic reactions were terminated by the addition of sample buffer.
The samples were then subjected to SDS-PAGE, autoradiography, and
densitometry analysis.
RNA Extraction and Northern Blot Analysis
Total RNA was isolated from cells by extraction in guanidium
thiocyanate containing 8% ß-mercaptoethanol by the LiCl method. For
Northern blot analysis, total RNA (15 µg) was fractionated on 1.2%
denaturing agarose gel and transferred to GeneScreen membrane (Dupont,
NEN, Boston, MA). After baking and prehybridization, the membranes were
hybridized (16 h) with the specifc cDNA probes labeled to high specific
activity using a random primer labeling kit (Boehringer Mannheim,
Indianapolis, IN). The filters were washed at high stringency and
autoradiographed.
 |
ACKNOWLEDGMENTS
|
---|
We would like to thank Dr. B. Schick for helping in the
preparation of the manuscript.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Dr. Rony Seger, Department of Biological Regulation, Weizmann Institute of Science, Rehovot 76100, Israel.
This work was supported by grants from the Israeli Ministry of Health,
the Forschheimer Fund, and Keren Naftali (to R.S.) and a postdoctoral
fellowship award from the Israel Cancer Research Fund (to N.L.L). R.S.
is an incumbent of the Samuel and Isabela Friedman Career Development
Chair.
Received for publication December 15, 1997.
Revision received February 4, 1998.
Accepted for publication February 19, 1998.
 |
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