(Received for publication, September 27, 1995; and in revised form, November 8, 1995)
From the
The intracellular mechanisms involved in the activation of extracellular signal-regulated kinase (ERK) are relatively well understood. However, the intracellular signaling pathways which regulate the termination of ERK activity remain to be elucidated. Mitogen-activated protein kinase phosphatase 1 (MKP-1) has been shown to dephosphorylate and inactivate ERK in vitro and in vivo. In the present study, we show in NIH3T3 fibroblasts that activation of the stress-activated protein kinase (SAPK) pathway by either specific extracellular stress stimuli or via induction of MEKK, an upstream kinase of SAPK, results in MKP-1 gene expression. In contrast, selective stimulation of the ERK pathway by 12-O-tetradecanoylphorbol-13-acetate or following expression of constitutively active MEK, the upstream dual specificity kinase of ERK did not induce the transcription of MKP-1. Hence, these findings demonstrate the existence of cross-talk between the ERK and SAPK signaling cascades since activation of SAPK induced the expression of MKP-1 that can inactivate ERK. This mechanism may modulate the cellular response to stimuli which employ the SAPK signal transduction pathway.
Mitogen-activated protein (MAP) ()kinases are
important components in the intracellular regulatory network that
transduce extracellular cues to intracellular responses. The complete
reconstitution of two distinct Ras-dependent MAP kinase cascades has
been described in mammalian cells. One is the intensively investigated
Raf-MEK-ERK cascade and the other recently described kinase cascade is
initiated by MEKK (MEK kinase) leading to activation of SEK1, the
upstream dual specificity kinase of stress-activated protein kinase
(SAPK), that in turn phosphorylates and activates SAPK. Extracellular
signal-regulated kinase (ERK) mediates cellular proliferation and
differentiation (1) whereas the SAPK, also referred to as
Jun-N-terminal kinase (JNK), pathway plays a pivotal role in the
response to extra- and intracellular stress stimuli and generally
promotes inhibition of cell growth(2, 3, 4) .
Activation of both ERK and SAPK is mediated by dual phosphorylation on tyrosine and threonine residues(4) . Therefore, recently cloned dual specificity protein-tyrosine phosphatases, like MKP-1 (MAP kinase phosphatase 1, encoded by the murine gene 3CH134), which exhibit dual catalytic activity toward phosphotyrosine and phosphothreonine are of special interest in the regulation of intracellular MAP kinase signaling pathways(5) . MKP-1 dephosphorylates and inactivates ERK in vivo and in vitro(5, 6, 7) . Little is known whether ERK itself induces MKP-1 expression, thereby terminating its own activation, or whether other signaling cascades distinct from ERK induce MKP-1, thereby providing a mechanism to maintain signaling specificity.
Since MKP-1 is principally regulated at the transcriptional level(8, 9) , we have studied the MKP-1 gene expression in response to induction of both ERK or SAPK in order to determine the signaling cascades involved in the regulation of MKP-1. We demonstrate the induction of MKP-1 in response to activation of the SAPK signaling pathway but not after stimulation of ERK. Since MKP-1 is capable of inactivating ERK, its induction may play a pivotal role in the cellular stress response.
As described
previously(3) , the EE epitope-tagged C-terminal 320 amino
acids of MEKK1 (MEKK) was expressed in NIH3T3 cells using the
lacSwitch promoter (Stratagene). Parental cells and stably transfected
cells that express catalytically active
MEKK in response to
isopropyl-1-thio-
-D-galactosidase (IPTG) were incubated
with 1 mM IPTG for the indicated times.
After stimulation of quiescent, parental NIH3T3 cells with
serum, MKP-1 expression was induced rapidly (Fig. 1A).
To investigate the regulation of MKP-1 gene expression, we used
extracellular stimuli that activate either the ERK or the SAPK pathway.
TPA induced a strong activation of ERK, detected by electrophoretic
retardation indicating phosphorylated protein forms (Fig. 1B), with no effect on SAPK activity (Fig. 1C). In contrast, UV-light and anisomycin
potently activated p54 and p46
, but
induced only a weak and transient stimulation of ERK (Fig. 1, B and C). These data were confirmed by blotting with
anti-phosphotyrosine antibodies for ERK phosphorylation and by
measurements of the kinase activity of SAPK toward GST-Jun (data not
shown).
Figure 1:
Regulation
of MKP-1 mRNA expression in response to specific extracellular stimuli
and effect of TPA, anisomycin, and short wavelength UV-light (UV-C) on the activation of ERK and SAPK in NIH3T3 cells. A, quiescent NIH3T3 cells were stimulated with 10% fetal
bovine serum (FBS), TPA (100 nM), UV-C (40
J/m), or anisomycin (500 nM) for different time
periods as indicated. Total cellular RNA was isolated as described
above. MKP-1 mRNA (2.2 kilobases in length) was detected by Northern
blot analysis. Blots were reprobed with glyceraldehyde-3-phosphate
dehydrogenase gene (GAPDH) as a loading control. B,
quiescent NIH3T3 cells were stimulated with TPA (100 nM),
anisomycin (500 nM), or UV-C (40 J/m
) for 5 and 30
min. Both ERK isoforms, p42 and p44, are detected in whole cell lysate
by Western blot. Activation is identifiable by the appearance of bands
with delayed mobility indicating phosphorylated protein forms
(indicated by stars). C, quiescent NIH3T3 cells were
stimulated with TPA (100 nM), anisomycin (500 nM), or
UV-C (40 J/m
) for 5 and 30 min. Both SAPK isoforms, p46 and
p54, are detected in whole cell lysate by Western blot as described
above. Activation is identifiable by the appearance of bands with
delayed mobility indicating phosphorylated protein forms (indicated by stars). D, time curve of ERK activation after
stimulation of quiescent NIH3T3 cells with 10% FBS alone or together
with 20 µg/ml cycloheximide (Cx). The upper two panels show Western blot analysis detecting ERK as described above (stars indicate phosphorylated protein forms). The lower
two panels show ERK activity assayed by the ability of
immunoprecipitated ERK to phosphorylate myelin basic
protein.
While TPA activated ERK, it did not induce MKP-1 gene expression. Other groups (15, 16) have reported TPA to be a weak stimulator of MKP-1 mRNA. In contrast, UV-light and anisomycin were potent stimuli of MKP-1 mRNA expression (Fig. 1A). These data suggest that activation of the SAPK pathway by cellular stress induces MKP-1.
In the presence of the protein synthesis inhibitor cycloheximide, serum induced a sustained activation of ERK (Fig. 1D). These data were confirmed using actinomycin D, an inhibitor of transcriptional activity (data not shown). Thus, in accordance with findings by Sun et al.(6) , the synthesis of new protein, presumably of a transcriptionally regulated dual specificity protein-tyrosine phosphatase like MKP-1, is required for the inactivation of ERK in NIH3T3 cells.
We used NIH3T3 cells stably transfected with vectors encoding mutants of MEK, the upstream kinase of ERK, in order to investigate the role of the MEK-ERK module in the regulation of MKP-1(10) . As shown previously (10) , the constitutively active mutants of MEK exhibit a 55-fold increase in activity compared to wild type, resulting in the morphological transformation of transfected cells (10) and the stimulation of ERK (data not shown). However, cells expressing constitutively active MEK did not show increased gene expression of MKP-1 compared to unstimulated wild-type or neo-transfected cells (Fig. 2A, lanes 10, 1, and 4). In fact, transcription of MKP-1 after stimulation with serum was diminished in wild-type compared to neo-transfected cells and was inhibited in cells expressing constitutively active MEK (Fig. 2A). Moreover, MKP-1 gene expression following stimulation with serum (Fig. 2A) or anisomycin (Fig. 2B) was enhanced in cells expressing catalytically inactive MEK. In accordance with this finding, prestimulation of the ERK cascade with TPA potently inhibited the MKP-1 gene expression in response to serum in parental NIH3T3 cells (Fig. 2C). Thus, in these cells in which ERK is selectively regulated by mutants of MEK as well as in the experiments using selective extracellular agonists, MKP-1 gene expression is not induced by activation of ERK. Moreover, from these results, it appears that activation of the MEK-ERK module correlates with an inhibition of MKP-1 gene expression.
Figure 2:
Effect of mutants of MEK on the gene
expression of MKP-1. MKP-1 mRNA was detected by Northern blot analysis
as described above. Blots were reprobed with glyceraldehyde-3-phosphate
dehydrogenase gene (GAPDH) as a loading control. A,
quiescent NIH3T3 cells were stimulated with 10% FBS for different time
periods as indicated. NIH3T3 cells were stably transfected with vector
alone (vector) or together with vectors encoding wild-type (WT) MEK,
catalytically inactive MEK (K97M), or constitutively active MEK
(N3-S222D). B, stimulation of quiescent NIH3T3 cells with
anisomycin (500 nM) for time periods as indicated. NIH3T3
cells were stably transfected with vector alone (vector) or together
with vectors encoding wild-type (WT) MEK or inactive MEK (K97M). C, quiescent parental NIH3T3 cells were stimulated with 10%
serum (FBS) for the indicated time periods alone or after
prestimulation with TPA (100 nM) for 15
min.
Previously, we (3) and others (17, 18, 19) showed that MEK kinase (MEKK)
selectively phosphorylates and activates SEK1, which subsequently
phosphorylates and activates SAPK. In vivo MEKK is suggested
to stimulate SAPK rather than HOG1 kinase(17, 19) , a
recently identified MAP kinase-like kinase. Stably transfected NIH3T3
cells expressing active MEKK (MEKK) in response to IPTG (3) were used to investigate the effect of the SAPK pathway on
MKP-1 expression. As shown previously(3) , SAPK activity
increased 6- to 8-fold after induction of
MEKK for 12 to 23 h in
comparison to parental NIH3T3 cells. IPTG did not affect MKP-1 gene
expression in parental NIH3T3 cells but induced MKP-1 mRNA in
MEKK-inducible cell lines (Fig. 3). These findings, as well
as the positive effect of UV-light and anisomycin on MKP-1 gene
expression, reinforce the conclusion that activation of the SAPK
pathway induces MKP-1.
Figure 3: MEKK expression in NIH3T3 cells induces MKP-1. Parental cells and stably transfected cells that express active MEKK in response to IPTG were incubated with 1 mM IPTG for indicated times. MKP-1 mRNA was detected by Northern blot analysis as described above. MKP-1 hybridization signals were quantified by scanning densitometry, normalized to the glyceraldehyde-3-phosphate dehydrogenase signal, and expressed as -fold increases over unstimulated levels.
Previously we have shown that induction of
MEKK in NIH3T3 cells inhibits ERK activation in response to
TPA(3) . In accordance with this finding, we show in Fig. 4A that prestimulation with UV-light, a potent stimulus
of the SAPK cascade and hence MKP-1 expression (Fig. 1),
inhibits the TPA-stimulated ERK activation as detected by the reduced
appearance of phosphorylated ERK protein forms and reduced ERK
activity. Thus, our data provide in vivo evidence for the
regulation of ERK activity by SAPK-induced MKP-1 in response to
cellular stress.
Figure 4:
Cross-talk between the MAP kinase pathways
Raf-MEK-ERK and MEKK-SEK-SAPK. A, quiescent parental NIH3T3
cells were prestimulated with UV-light (40 J/m) for 75 min
(+) or untreated(-) prior to stimulation with TPA (100
nM) for the indicated periods of time. The upper panels shows a Western blot analysis detecting p42
as
described above (stars indicate phosphorylated protein forms),
and the lower panel shows ERK activity assayed by the ability
of immunoprecipitated ERK to phosphorylate myelin basic protein (MBP). B, we describe the induction of MKP-1, that is
capable of inactivating ERK, by the MEKK-SEK-SAPK pathway. Thereby SAPK
may control the activity of the functionally antagonistic ERK pathway.
Furthermore, we show that activation of the MEK-ERK module correlates
with an inhibition of the MKP-1 gene expression (dotted
line).
Our results demonstrate cross-talk between the Raf-MEK-ERK signaling pathway and the recently identified MEKK-SEK-SAPK pathway(2, 3, 17) (Fig. 4B). We show that the expression of MKP-1 is induced by activation of the SAPK pathway but not by stimulation of ERK. These data support the existence of an important mechanism involved in the maintenance of signaling specificity, whereby activation of the MEKK-SEK-SAPK pathway induces a phosphatase that is capable of inactivating the Raf-MEK-ERK pathway. Since activation of ERK is known to mediate cell growth, this cross-talk of MAP kinase cascades could contribute to the growth inhibition observed following increased MEKK activity and SAPK stimulation(3) . In addition, our data suggest that activation of ERK may inhibit the induction of MKP-1. This mechanism may contribute to our previously described finding that the expression of constitutively active MEK induces loss of cell growth control in NIH3T3 cells(10) . Recently, it was shown that MKP-1 may play a role in the down-regulation of the cellular stress response since transfection of HeLa cells with MKP-1 inhibited the activation of SAPK(20) . Therefore, the induction of MKP-1 by SAPK may also be important for the deactivation of SAPK in the way of a classical feedback mechanism. In vitro and in vivo data in rat fibroblasts, however, support a relative selectivity of MKP-1 for ERK over SAPK (7) .
Complex factors like serum and growth
factors like platelet-derived growth factor or epidermal growth factor
induce MKP-1 gene transcription(16) . Based on our data, it is
likely that growth factors induce MKP-1 expression due to their
stimulation of signaling pathways distinct from the ERK cascade such as
the SAPK cascade. Growth factors stimulate ERK (4) and SAPK (21, 22) in a Ras-dependent way (18) , whereas
SAPK is also inducible in a Ras-independent way by tumor necrosis
factor (18) . Therefore, it is likely that the mitogenic
and Ras-dependent activation of ERK by growth factors is controlled by
a simultaneous stimulation of the SAPK pathway that induces MKP-1
expression (Fig. 4B).