(Received for publication, December 11, 1996, and in revised form, February 18, 1997)
From ONYX Pharmaceuticals, Inc., Richmond, California 94806 and ¶ DNAX Research Institute, Palo Alto, California 94304
Stimulation of Rat-1 cells with
lysophosphatidic acid (LPA) or epidermal growth factor (EGF) results in
a biphasic, sustained activation of extracellular signal-regulated
kinase 1 (ERK1). Pretreatment of Rat-1 cells with either cycloheximide
or sodium orthovanadate had little effect on the early peak of ERK1
activity but potentiated the sustained phase. Cycloheximide also
potentiated ERK1 activation in Rat-1 cells expressing Raf-1:ER, an
estradiol-regulated form of the oncogenic, human Raf-1. Since
cycloheximide did not potentiate MEK activity but abrogated the
expression of mitogen-activated protein kinase phosphatase (MKP-1)
normally seen in response to EGF and LPA, we speculated that the level
of MKP-1 expression may be an important regulator of ERK1 activity in
Rat-1 cells. Inhibition of LPA-stimulated MEK and ERK activation with
PD98059 and pertussis toxin, a selective inhibitor of
Gi-protein-coupled signaling pathways, reduced
LPA-stimulated MKP-1 expression by only 50%, suggesting the presence
of additional MEK- and ERK-independent pathways for MKP-1 expression.
Specific activation of the MEK/ERK pathway by
Raf-1:ER had little or
no effect on MKP-1 expression, suggesting that activation of the
Raf/MEK/ERK pathway is necessary but not sufficient for MKP-1
expression in Rat-1 cells. Activation of PKC played little part in
growth factor-stimulated MKP-1 expression, but LPA- and EGF-induced
MKP-1 expression was blocked by buffering [Ca2+]i, leading to a potentiation of the
sustained phase of ERK1 activation without potentiating MEK activity.
In Rat-1
Raf-1:ER cells, we observed a strong synergy of MKP-1
expression when cells were stimulated with estradiol in the presence of
ionomycin, phorbol 12-myristate 13-acetate, or okadaic acid under
conditions where these agents did not synergize for ERK activation.
These results suggest that activation of the Raf/MEK/ERK pathway is
insufficient to induce expression of MKP-1 but instead requires other
signals, such as Ca2+, to fully reconstitute the response
seen with growth factors. In this way, ERK-dependent and
-independent signals may regulate MKP-1 expression, the magnitude of
sustained ERK1 activity, and therefore gene expression.
One of the major signal pathways responsible for regulating reentry into the cell cycle leads to activation of the extracellular signal-regulated kinases (ERKs)1 p44ERK1 and p42ERK2 (also called mitogen-activated protein kinases or MAP kinases) (1-4). Growth factor-induced activation of p74Raf-1 leads to the phosphorylation and activation of the dual specificity protein kinase MEK, which in turn activates the ERK/MAP kinases by phosphorylation of threonine and tyrosine residues in the motif TEY (5-9). p74Raf-1 is recruited to the plasma membrane and regulated by the Ras GTPase proteins (10-12). This cascade is activated by receptor tyrosine kinases such as the EGF receptor (reviewed in Ref. 13), cytokine receptors such as the GMCSF-receptor (14), and G-protein-coupled receptors such as the lysophosphatidic acid (LPA) or thrombin receptors (15, 16, 20).
Several observations demonstrate the importance of this signaling pathway in cell growth. First, activating mutations in Ras (17), Raf (9), or MEK (18) are sufficient to activate ERK, induce gene expression, or cause oncogenic transformation. Second, inhibition in this signaling pathway is associated with inhibition of ERK activation and cell growth (19). Third, among the substrates for ERK1 and ERK2 are transcription factors of the Ets family, Elk-1 and Sap1a (4, 21, 22), which regulate transcription of c-Fos (4). Finally, many growth factors stimulate the sustained activation of ERKs (20, 23, 24), which is temporally associated with their accumulation in the nucleus (25-27). In this way, the ERK cascade provides a link between receptor signaling events at the plasma membrane and regulated gene expression in the nucleus.
Whereas much attention has focused on the mechanisms by which this cascade is activated by growth factors and oncoproteins, several studies have turned to the question of which protein phosphatases are responsible for dephosphorylating and thereby inactivating each kinase in the cascade. In the case of ERK, dephosphorylation and inactivation is of considerable interest because the activating phosphorylations are on both threonine and tyrosine (6) and because sustained ERK activation, which seems to be important for proliferative signaling (20, 23, 24), is compartmentalized within the nucleus (25-27).
Recent work has identified a family of dual specificity protein phosphatases that dephosphorylate both the tyrosine (Tyr183) and threonine (Thr185) residues on ERK (28-32). The prototype of this family is MAP kinase phosphatase-1 (MKP-1) (which is encoded by human CL100 and mouse 3CH134 or erp genes, which are immediate early genes induced by oxidative stress and mitogenic stimulation (28, 29, 32)). Overexpression of MKP-1 blocks activation of ERK (31) and cell cycle reentry (33, 34). Furthermore, pretreatment of cells with cycloheximide, to block de novo expression of MKP-1, is able to potentiate sustained ERK activation by serum in NIH3T3 cells (31). Since MKP-1 appears to be expressed exclusively in the nucleus, a simple model envisages growth factor-stimulated activation and nuclear accumulation of ERK leading to increased MKP-1 expression, which in turn accumulates in the nucleus and inactivates ERK, affording exquisite fine tuning of the sustained phase of ERK activation (35). This model has been challenged by the observation that in PC12 cells and endothelial cells MKP-1 is induced but may not be the phosphatase that regulates ERK activity; it was suggested that PP2A and an unidentified PTPase were responsible for dephosphorylation of ERK (36).
In Rat-1 fibroblasts, there is a strong correlation between sustained ERK activation and DNA synthesis in response to mitogenic stimulation with LPA (24); factors that regulate this sustained ERK activation are therefore of great interest. In this report, we demonstrate a role for MKP-1 or a closely related molecule in regulating sustained ERK activity in Rat-1 cells and characterize the pathways regulating expression of MKP-1 in response to growth factor stimulation. Induction of MKP-1 clearly reflects activation of the MEK/ERK cascade, but this pathway alone is insufficient; maximal expression of MKP-1 requires synergistic activation of the ERK pathway and additional ERK-independent signals including Ca2+. In addition, we show that preventing Ca2+-dependent MKP-1 expression potentiates sustained ERK activation, suggesting that MKP-1 is a focus for regulatory cross-talk to the ERK pathway from other signal pathways.
Cell culture reagents were from Irvine
Scientific. Prepoured SDS-PAGE reagents were from Novex Gel Systems.
LPA was obtained from Avanti Polar Lipids. EGF was from Boehringer
Mannheim. Okadaic acid was from LC Laboratories.
[-32P]ATP was from DuPont NEN. Goat-anti-rabbit
horseradish peroxidase-conjugated secondary antibodies were from
Bio-Rad. All other reagents including myelin basic protein,
cycloheximide, and sodium orthovanadate were from Sigma. Antibodies to
ERK1 (E1.2) have been described previously (15). Antibodies to MKP-1
and MKP-2 (Alb-1), generated using a peptide derived from the
C-terminal 12 amino acids of mouse CL100 (YLKSPITTSPSC) were the very
generous gift of Dr. Fergus McKenzie and Prof. Jacques Pouysségur
(Center de Biochimie, Université de Nice, Nice, France).
Monoclonal antibodies to ERK and MEK were from Pharmingen and Zymed,
respectively. Phosphospecific antibodies for ERK and MEK were from New
England Biolabs. The MEK inhibitor PD98059 was prepared by Cheri Blume
and Dr. Dan Rogers in the Chemistry Group at ONYX Pharmaceuticals and
confirmed by NMR analysis.
The Rat-1 cells used in this and previous studies (15, 24, 37) were originally provided by Dr. J. L. Bos (Department of Physiological Chemistry, University of Utrecht, The Netherlands). Rat-1 cells were cultured in Dulbecco's modified Eagle's medium containing penicillin/streptomycin, glutamine, and 10% fetal bovine serum. Cells were washed once in serum-free medium and then placed in fresh serum-free medium for at least 24 h prior to the experiments described herein. Pretreatments with various agents were as follows: 50 µg/ml cycloheximide for 45 min prior to growth factor addition, 100-1000 µM sodium orthovanadate for 30 min prior to growth factor addition, 40 µM PD98059 for 30 min prior to growth factor addition, and 100 µg/ml pertussis toxin for 18 h prior to growth factor addition.
The derivation and characterization of R1Raf-1:ER-4 cells will be
described elsewhere.2 These are a clone of
Rat-1 cells expressing the conditional form of oncogenic human Raf-1 in
which the catalytic domain of Raf-1 is fused to the hormone-binding
domain of the human estrogen receptor (38), allowing
estrogen-dependent activation of MEK and ERK independently
of Ras. R1
Raf-1:ER-4 cells were maintained in the same medium as
Rat-1 cells but supplemented with 400 µg/ml G418. G418 selection was
maintained throughout and only removed during serum deprivation for the
last 24 h.
For ERK1 assays, experiments were performed upon six-well plates of confluent, quiescent cells that had been serum-starved for 24-36 h. Following the addition of the indicated drug or inhibitor, cells were stimulated by the addition of 10 × solutions of growth factors, and stimulations proceeded at 37 °C for the time indicated. Incubations were terminated by aspiration and the addition of ice-cold TG lysis buffer (20 mM Tris/HCl (pH 8), 1% Triton X-100, 10% glycerol, 137 mM NaCl, 1.5 mM MgCl2, 1 mM EGTA, 50 mM NaF, 1 mM Na3VO4, 1 mM Pefabloc, 20 µM leupeptin, 10 µg/ml aprotinin). Clarified cell lysates were prepared as described previously (15, 24, 39).
Immune Complex Kinase Assays for ERK1Anti-peptide antibodies directed to the extreme C termini of ERK1 (E1.2) and the assay of immunoprecipitated ERK1 were described previously (15, 24, 37, 39). We have been unable to derive immunoprecipitating antiserum for ERK2. However, we have derived a Rat-1 cell line that stably expresses physiological levels of a Myc epitope-tagged version of ERK2. In all experiments performed, we have noted that ERK1 and MycERK2 are regulated identically in response to the agents used in this study.3
Assay of ERK and MEK Activation by Western Blot Analysis with Phosphospecific AntibodiesConfluent 10-cm dishes of Rat-1 cells were serum-starved in 5 ml of serum-free Dulbecco's modified Eagle's medium for 24 h before being treated and stimulated as indicated. Cells were then washed briefly in ice-cold PBS before the addition of ice-cold TG lysis buffer. Following removal of detergent-insoluble material, the clarified supernatant was boiled in sample buffer, and equal amounts of cell lysate were resolved on 10 or 12% SDS-PAGE gels until the 30-kDa marker approached the bottom. Gels were transferred to PVDF using a Bio-Rad transblot apparatus, and the filter was stained with Coomassie Brilliant Blue to confirm equal loading of lanes. Filters were washed thoroughly in 0.1% (v/v) Tween 20 in PBS (TPBS) and then "blocked" overnight in TPBS, 5% (w/v) Carnation powdered milk (TPBS/milk). Filters were then probed at room temperature for 1 h in TPBS/milk with anti-peptide antiserum that specifically recognizes the phosphorylated, activated versions of ERK1 and ERK2 or MEK1 and MEK2. Following five 5-min washes with TPBS, the second antibody, goat anti-rabbit horseradish peroxidase, was used at a 1:5000 dilution in TPBS/milk for 1 h at room temperature. Following five washes in TPBS, the filter was dried and incubated with Amersham ECL reagents according to the manufacturer's instructions; exposures to hyperfilm were typically 1-2 min. In addition, duplicate blots were probed with conventional, nonphosphospecific monoclonal antibodies to ERK1/2 and MEK1/2 to confirm that equal amounts of these proteins were present in each sample.
Western Blot Analysis of Endogenous MKP-1 ExpressionCell samples were fractionated, transferred to PVDF and "blocked" in TPBS/milk as described above. Filters were then probed at room temperature for 1 h in TPBS/milk with a 1:500 dilution of crude antiserum Alb-1 raised against the C-terminal peptide of mouse 3CH134 (40). Following five 5-min washes with TPBS, the second antibody, goat anti-rabbit horseradish peroxidase, was used at a 1:4000 dilution in TPBS/milk for 1 h at room temperature. Following five washes in TPBS, the filter was dried and incubated with Amersham ECL reagents according to the manufacturer's instructions; exposures to hyperfilm were typically 10-30 s.
Reproducibility of ResultsResults are from single experiments representative of between three and six experiments giving similar results. For ERK1 MBP kinase assays, results are expressed as raw cpm of 32P incorporated into MBP from single point assays. This is a sensitive and highly reproducible assay (15, 20, 23). In addition, some cells were stimulated and assayed in duplicate and gave identical results with errors generally less than 10%. Data were pooled for statistical analysis by student's t test. Experiments involving Western blotting were performed at least three or four times with identical results; a representative experiment is shown in each case.
Potentiation of Sustained ERK Activity in Rat-1 Cells by Cycloheximide
Stimulation of Rat-1 cells with 100 µM LPA resulted
in a biphasic increase in ERK1 activity, which peaked at 5-10 min
before declining rapidly until 30 min, after which a smaller second
phase persisted above basal level for up to 3 h (Fig.
1A). Pretreatment of cells with 50 µg/ml
cycloheximide for 45 min followed by challenge with LPA had no effect
on the magnitude of peak ERK1 activity or the rapid decline after 10 min of stimulation but did potentiate the sustained phase of ERK1
activity from 60 min onward (Fig. 1A). For example, the
magnitude of LPA-stimulated ERK1 activity, measured as a percentage of
the maximum response at 10 min, was 27 ± 15% after 120 min in
control cells but 135 ± 28% after 120 min in
cycloheximide-treated cells; the difference between the control and
cycloheximide-treated values was statistically significant (p < 0.01). Similar results were obtained when EGF was
the stimulus (Fig. 1B). Prolonged treatment with
cycloheximide alone had a small, poorly reproducible effect on ERK1
activity (see Fig. 1C).
We also examined the effect of cycloheximide on ERK1 activation by
Raf-1:ER (38) in the R1Raf-1:ER-4 cell line. Activation of
Raf-1:ER by
-estradiol (
-E2) results in the rapid activation of MEK and ERK in these cells, thereby circumventing the other pathways
activated by receptor signaling. The ability of
Raf-1:ER to activate
ERK1 was greatly potentiated by pretreatment with cycloheximide (Fig.
1C). These results suggest that a labile protein acts as a
negative regulator of sustained ERK1 activity, whether stimulated by a
receptor tyrosine kinase, a G-protein-coupled receptor, or the
conditional form of the human c-Raf-1 protooncogene.
This effect was not confined to ERK1. Western blotting with a phosphospecific antibody that only recognizes the activated versions of ERK1 and ERK2 (phosphorylated at Tyr185 in the TEY motif) confirmed that both ERK1 and ERK2 isoforms were activated in a strongly sustained manner in the presence of cycloheximide (Fig. 1D). Furthermore, duplicate blots probed with a phosphospecific antibody that only recognizes activated MEK1 and MEK2 confirmed that cycloheximide did not amplify or prolong LPA-stimulated MEK activation under these conditions (Fig. 1D). These results demonstrate that cycloheximide exerts its effect at the level of ERK, not MEK, by preserving the activating phosphorylation sites of ERK1 and ERK2. Since these sites are substrates for MKP family phosphatases, we were interested in seeing if MKPs were expressed in response to growth factors in these cells.
Induction of MKP-1 Expression by LPA, EGF, and Serum in Rat-1 Cells
We investigated the expression of MKP-1 in response to LPA, EGF,
and serum in Rat-1 cells by Western blotting whole cell lysates with an
antibody raised to the C terminus of human 3CH134 (Alb-1) (40). This antibody recognizes MKP-1 (CL100/3CH134) and MKP-2 (hVH2)
(41, 42) but not MKP-3 (rVH6, Pyst1) (43-45). As a control for the
MKP-1 antiserum, we immunoblotted samples from COS cells transfected
with empty vector (EXV) or EXV-CL100. Transfected cells were
lysed and Western blotted as described under "Experimental Procedures." We detected a strong immunoreactive band of 39-40 kDa
in lysates from CL100-transfected cells that was absent in the cells transfected with empty vector (Fig.
2A). A band of similar molecular weight was
strongly induced in quiescent Rat-1 cells stimulated with LPA (Fig.
2A). This protein is consistent with the molecular weight of
MKP-1 and indicates that antiserum Alb-1 recognizes both recombinant
MKP-1 and endogenous MKP-1 induced by growth factor stimulation.
The ability of LPA, EGF, or FBS to induce MKP-1 protein expression was completely blocked by pretreatment of cells with cycloheximide (Fig. 2B), indicating that MKP-1 expression is a growth factor-stimulated event that requires de novo protein synthesis.
To examine the kinetics of MKP-1 expression, confluent, quiescent Rat-1 cells were stimulated with LPA or FBS for various times from 8 min to 8 h, and lysates were analyzed by Western blot with the MKP-1 antiserum. MKP-1 was barely detectable in quiescent Rat-1 cells but was strongly induced after 30-60 min of LPA stimulation (Fig. 2C, upper panel). This correlated well with the earliest time points at which we could observe a potentiation of ERK1 activation by cycloheximide (Fig. 1A). In response to continuous stimulation with LPA and FBS MKP-1, expression peaked at 2-4 h before declining, but MKP-1 levels were clearly elevated even after 8 h (Fig. 2C). This correlates with the sustained ERK1 activation seen for up to 8 h in response to stimulation with LPA, EGF, or FBS (24).
We were unable to detect MKP-2 expression in quiescent, growth factor-stimulated, or serum-stimulated Rat-1 cells although the antibody used readily detects this enzyme and it is expressed in serum-stimulated CCL39 cells (40). MKP-2 exhibits a slightly reduced mobility on SDS-PAGE, resolving just above MKP-1 with an apparent molecular mass of 42-43 kDa. MKP-2 is expressed in Rat-1 cells, since we have recently shown that it is induced in response to stimulation with the kinase inhibitor Ro-31-8220, which activates the JUN N-terminal kinase (JNK) stress kinase pathway (39). MKP-3/Pyst1 is constitutively expressed and is therefore not likely to account for the effects of cycloheximide in this system (45), whereas PAC1 is restricted hematopoietic cells (46). This antibody is not able to detect hVH3/B23 (47, 48) or hVH5 (49), other members of the family of MKPs, so we cannot rule out the possibility that they may also contribute to the regulation of ERK activity in these cells.
Sodium Orthovanadate Potentiates Sustained ERK1 Activation in Rat-1 Cells
To obtain additional evidence for MKP-1 or a related phosphatase
playing a role in the sustained phase of ERK1 activation, we examined
the effect of sodium orthovanadate, an inhibitor of protein-tyrosine
phosphatases including MKP-1 (30). Treatment of Rat-1 cells with sodium
orthovanadate alone resulted in activation of ERK1 in a time- and
dose-dependent fashion. ERK1 activation by sodium
orthovanadate exhibited an EC50 value of 200-300
µM3 and was slightly slower than that
observed with LPA, peaking after 15-30 min before gradually declining
(Fig. 3A). In several experiments, maximal
doses of sodium orthovanadate resulted in ERK1 activation to a similar
degree as that seen with LPA. Therefore, to investigate the effect of
sodium orthovanadate on growth factor-stimulated ERK1 activation, we
used suboptimal doses to pretreat Rat-1 cells prior to the addition of
LPA. In the presence of sodium orthovanadate, we were able to observe a
clear potentiation of the sustained phase of ERK1 activation in
response to LPA as previously seen with cycloheximide (Fig.
3A). For example, the magnitude of LPA-stimulated ERK
activity, measured as a percentage of the maximum response at 10 min,
was 27 ± 15% after 120 min in control cells but 107 ± 25%
after 120 min in sodium orthovanadate-treated cells; the difference
between the control and orthovanadate-treated values was statistically
significant (p < 0.01). Activation of ERK1 by this low
dose of orthovanadate alone was weak and transient, and it was
therefore clear that the potentiation seen with LPA following sodium
orthovanadate treatment was synergistic rather than additive. Similar
results were obtained when we studied the effect of sodium orthovanadate on the EGF response.3,4
Importantly, the doses of sodium orthovanadate used did not prevent LPA-stimulated expression of MKP-1 (Fig. 3B), so the
potentiation we observed was not due to inhibition of protein
synthesis. The simplest conclusion from these results is that the
magnitude of sustained ERK1 activity in Rat-1 cells is regulated by the
de novo expression of a growth factor-induced,
orthovanadate-sensitive phosphotyrosine phosphatase.
Characterization of the Pathways Regulating Growth Factor-stimulated MKP-1 Expression
We sought to define which signaling pathways are responsible for MKP-1 expression in response to LPA. Whereas the ERK cascade itself is a likely candidate, both LPA and EGF activate multiple signaling pathways that may be regulate gene expression.
Activation of MEK/ERK Is Necessary for Maximal MKP-1 Expression in Rat-1 CellsPretreatment of Rat-1 cells with the MEK inhibitor
PD98059 (50) resulted in a dose-dependent inhibition of
LPA- or EGF-stimulated ERK activation such that ERK activity was
completely abolished by 25 or 30 µM PD98059 (Fig.
4A). Pretreatment of Rat-1 cells with 40 µM PD98059 resulted in inhibition of approximately 50% of LPA- and EGF-stimulated MKP-1 induction (Fig. 4B),
suggesting that MKP-1 induction is partially dependent upon activation
of the Raf-MEK-ERK cascade. However, we could not observe complete inhibition of LPA- or EGF-stimulated MKP-1 expression by PD98059 even
at doses at which ERK1 was completely inhibited, suggesting that
additional pathways are also used by LPA and EGF to regulate MKP-1
expression. This point is further emphasized by the fact that in the
same experiments PMA-stimulated MKP-1 expression was completely
inhibited by PD98059 (Figs. 4B and 6B).
In Rat-1 cells, LPA activates ERK1 by at least two pathways:
(a) a major and sustained pathway, which is mediated by a
pertussis toxin-sensitive Gi protein and involves Ras, and
(b) a quantitatively minor and transient pathway, which may
involve PKC and Ca2+ (15, 16, 24, 51, 52). Pretreatment of
Rat-1 cells with pertussis toxin inhibited approximately 50% of
LPA-stimulated MKP-1 induction (Fig. 4C). Under the same
conditions, pertussis toxin inhibits 70% of peak ERK1 activation and
all of the sustained response (24). These results together with the
PD98059 data are consistent with a Gi-mediated pathway of
ERK1 activation playing a role in MKP-1 expression by LPA. However, we
could not rule out the possibility that the "Gi
pathway" for MKP-1 expression was totally independent of that for ERK
activation. To address this issue, we utilized Rat-1 cells expressing
Raf-1:ER.
Activation of R1Raf-1:ER in Rat-1 cells results in
a sustained increase in ERK1 activity of similar magnitude to that
observed with LPA although the peak of the response is delayed by about 20 min (Fig. 5A); similar results are
observed in 3T3 cells expressing
Raf-1:ER.5 Surprisingly, we observed
that, despite activation of ERK1 to the same extent as that with LPA,
stimulation of
Raf-1:ER resulted in a barely detectable increase in
MKP-1 expression compared with that observed with serum (Fig.
5B) or LPA (Fig. 5C), even when stimulated for up
to 4 h. This result suggested that sustained activation of the ERK
cascade may be necessary but is not sufficient to
induce MKP-1.
Treatment of R1Raf-1:ER-4 cells with pertussis toxin inhibited
LPA-stimulated MKP-1 expression (Fig. 5C, lanes 2 and 3). However, activation of
Raf-1:ER together with LPA
bypassed the effect of pertussis toxin on LPA-induced MKP-1 expression
so that the maximal response to LPA was fully reconstituted (Fig.
5C, lane 4). Under these conditions,
Raf-1:ER
activation induced little MKP-1 alone (Fig. 5C, lane
5) but could fully complement for the loss of Gi or
Go-mediated signal pathways, whereas LPA was presumably
providing non-Gi or -Go signals. These results suggest that of all the pathways possibly regulated by Gi
or Go in Rat-1 cells, the ERK cascade is sufficient to
complement for their loss and synergize with LPA-stimulated
non-Gi signals to reconstitute MKP-1 expression.
Stimulation of Rat-1 cells with the phorbol ester PMA resulted in a strong induction of MKP-1 protein (Figs. 4B and 6) despite the fact that this agonist elicits only a transient and small ERK activation in these cells (24, 53). The induction of MKP-1 protein by PMA was completely inhibited by PD98059 in experiments in which the responses to LPA and EGF exhibited a significant MEK-independent component (Figs. 4B and 6B), suggesting that induction of MKP-1 by PKC absolutely requires the MEK/ERK pathway, whereas LPA and EGF can use other signal pathways.
To assess the role of PKC in LPA-stimulated ERK activation, we
down-regulated PKC expression by pretreating cells with 1 µM PMA for 48 h (39). Under such conditions we
observe depletion of essentially all PKC-, -
, and -
but not
PKC-
(39). Such treatment resulted in complete inhibition of
PMA-stimulated MKP-1 expression (Fig. 6A) but had only a
small effect upon the LPA response and no effect on the EGF response.
We previously observed that the selective PKC inhibitor GF109203X had
little effect on LPA-stimulated MKP-1 expression although it completely
inhibited the PMA response (39). This suggests a small role for PKC in regulating MKP-1 expression by these growth factors.
Ca2+ plays a major role in regulating gene expression in a variety of systems (54, 55). We used ionomycin to investigate the potential role of Ca2+ in the regulation of MKP-1 expression. Ionomycin treatment led to induction of MKP-1 expression (Fig. 6, A and B) although it or A23187 stimulated only a transient activation of ERK1 in Rat-1 cells (24, 53). The induction of MKP-1 expression by ionomycin was not affected by chronic PMA treatment, suggesting that the effect of Ca2+ is not mediated via activation of PKC (Fig. 6A). Ionomycin-stimulated MKP-1 expression was strongly inhibited by PD98059, suggesting that ionomycin, like PMA, absolutely requires activation of the MEK/ERK cascade to induce MKP-1 (Fig. 6B).
To assess the requirement for Ca2+ in the induction of
MKP-1 by growth factors in Rat-1 cells, we used a combination of 3 mM EGTA and 20 µM BAPTA-AM, to buffer
agonist-stimulated Ca2+ mobilization and Ca2+
entry. This treatment completely blocked MKP-1 induction in response to
LPA, ionomycin, or EGF (Fig. 7A). This
complete abolition of response was not due to toxicity, since, for
example, the same treatment did not block growth factor-stimulated ERK1
activation (see below; Fig. 7, B and C). It
appears that there is a strong requirement for Ca2+ in the
induction of MKP-1.
Calcium mobilization is necessary for growth
factor-stimulated MKP-1 expression in Rat-1 cells. A,
serum-starved Rat-1 cells were pretreated with 20 µM
BAPTA-AM plus 3 mM EGTA (+) or vehicle controls () for 20 min before stimulating with LPA (50 µM), EGF (10 nM), PMA (100 nM), or ionomycin (1.5 µM) for 1 h. MKP-1 expression was assayed by Western
blot as described under "Experimental Procedures." B and C,
serum-starved Rat-1 cells were pretreated with vehicle (
) or 20 µM BAPTA-AM and 3 mM EGTA (
) for 20 min
prior to stimulating with LPA (B) or EGF (C).
Some cells received vehicle controls (
) or were incubated with
BAPTA-AM/EGTA alone (
). Cells were then lysed, and ERK1 was
immune-precipitated and assayed in the immune complex as described
under "Experimental Procedures." Results are from individual
experiments typical of three (LPA) or four (EGF) others giving similar
results. D, serum-starved Rat-1 cells were pretreated with
vehicle (control) or 20 µM BAPTA-AM plus 3 mM
EGTA (B + E) for 20 min prior to stimulating with LPA for
the times indicated. Cell lysates were then resolved by SDS-PAGE, transferred to PVDF membranes, and immunoblotted with antibodies specific for phospho-ERK1 and -ERK2, ERK1 and ERK2, phospho-MEK1 and
-MEK2, or MEK1 and MEK2. Results are from a representative experiment.
The requirement for Ca2+ in regulating MKP-1 expression raised the question of whether Ca2+, by increasing MKP-1 levels, might serve to regulate ERK activity by way of a cross-talk pathway. To address this possibility, we pretreated Rat-1 cells with BAPTA-AM and EGTA and then stimulated them with LPA or EGF for various time points up to 2 or 3 h. We found that buffering Ca2+ had only a small effect on the early peak of ERK1 activity, but we observed a strong potentiation of the sustained phase of the response to both growth factors (Fig. 7, B and C), as we had previously seen with cycloheximide or sodium orthovanadate.
This effect was not confined to ERK1, since immunoblotting with the phosphospecific ERK1/2 antibody confirmed that both ERK1 and ERK2 were activated in a strongly sustained manner in the BAPTA-AM/EGTA-treated cells compared with untreated cells (Fig. 7D). Furthermore, calcium chelation did not prolong or amplify LPA-stimulated MEK activation, monitored with a phosphospecific MEK antibody (Fig. 7D), suggesting that BAPTA-AM/EGTA exerts its effects by preserving ERK phosphorylation and not by amplifying the upstream signal pathway. Thus, Ca2+-regulated MKP-1 expression seems to be functionally relevant to regulation of sustained ERK activity.
Synergy betweenAlthough activation of Raf-1:ER in Rat-1 cells
elicited only a small increase in MKP-1, when we pretreated
R1
Raf-1:ER-4 cells with okadaic acid the combination of
Raf-1:ER
and okadaic acid resulted in a strong, synergistic induction of MKP-1
(Fig. 8A).
We also observed synergy between PMA or ionomycin and
Raf-1:ER. In R1
Raf-1:ER-4 cells, the induction of MKP-1
expression by PMA and ionomycin was reduced compared with normal Rat-1
cells, and this allowed us to look at combinatorial stimulations. In these cells, the combination of PMA and
Raf-1:ER clearly gave a
synergistic induction of MKP-1 (Fig. 8A). Maximal doses of
ionomycin in combination with
Raf-1:ER seemed to be synergistic, but
to be sure we titrated ionomycin doses from 1 µM down to
125 nM with or without
Raf-1:ER activation. Under these
conditions, we observed synergy between ionomycin and
Raf-1:ER at
each dose of ionomycin examined (Fig. 8B). Under the same
conditions, PMA and ionomycin did not synergize with
Raf-1:ER to
activate ERK, and only rarely did we observe an additive response (Fig.
8C).5 These results strongly suggest that the
synergy observed between
Raf-1:ER and PMA and ionomycin is not due
to enhancement of ERK activation but instead is more probably due to
the synergistic combination of different signaling pathways.
These results again suggested a role for Ca2+ and PKC in
regulating MKP-1 levels in Rat-1 cells. To address the question of which is the major signal cooperating with the ERK pathway in the
response to LPA, we returned to the synergy between Raf-1:ER and LPA
in pertussis toxin-treated R1
Raf-1:ER-4 cells. Under these
conditions, most of the LPA-stimulated ERK activation is lost,
suggesting that some other signal is being provided by LPA to synergize
with
Raf-1:ER-stimulated ERK activation. This synergy was abolished
by BAPTA-AM/EGTA treatment and substantially reduced by BAPTA alone but
only weakly inhibited by the selective PKC inhibitor
GF109203X.5 These results are consistent with the relative
efficacy of inhibiting PKC or Ca2+ on MKP-1 expression
(Figs. 6 and 7) (39) and suggest that the major signal generated by LPA
that regulates MKP-1 levels (albeit in concert with
Raf-1:ER) is
Ca2+ with little role for PKC.
The hormone binding domain of the estrogen receptor contains an
estrogen-inducible transactivation domain, which could have contributed
directly to the regulation of MKP-1 expression, independently of ERK
activation. However, we observed identical results when 4-hydroxytamoxifen was substituted for -E2.5 Since
4-hydroxytamoxifen is able to inactivate the "protein repressor" function of the hormone binding domain without inducing the
transactivation function, these results indicate that the synergy
between
Raf-1:ER and PMA, ionomycin, or okadaic acid is specifically
due to derepression of the Raf kinase moiety and concomitant activation
of the ERK cascade.
The magnitude and duration of ERK activation appears to be a key determinant in cell fate signaling (3, 20, 23, 24, 27), providing the connection between receptors and nucleus (4, 52). Inactivation of ERKs within the nucleus is catalyzed by MKPs, such as MKP-1, which are induced de novo in response to proliferative and stress signals. These observations lead to a simple model for attenuation of ERK activity in which activation and nuclear accumulation of ERK results in increased expression of MKP-1, which in turn inactivates ERK (35). However, some cells clearly use different mechanisms of ERK dephosphorylation regardless of MKP-1 expression (36).
The sustained phase of ERK1 activation correlates well with proliferation in LPA-stimulated Rat-1 cells (24), and our results suggest that an inducible MKP serves to regulate sustained ERK activity in these cells. The ability of cycloheximide to potentiate the second, sustained phase of ERK activation (Fig. 1) is generally considered to be a hallmark of an MKP-like phosphatase inactivating ERK (31, 36). The effect of cycloheximide on ERK activity is correlated well with the kinetics and cycloheximide-sensitive expression of MKP-1 in response to LPA, EGF, and FBS (Fig. 2). These results, together with the ability of Ca2+-chelating agents to block MKP-1 expression and potentiate sustained ERK1 activity (Fig. 7), support a role for MKP-1 acting as an ERK phosphatase in Rat-1 cells. In addition, we have recently shown that the kinase inhibitor Ro-31-8220 is able to block MKP-1 expression and potentiate ERK activation by growth factors in these cells (39). Finally, sodium orthovanadate, an inhibitor of protein-tyrosine phosphatases including MKP-1 (30), can potentiate the sustained phase of ERK1 activation without inhibiting MKP-1 expression, providing biochemical evidence that a PTPase negatively regulates ERK activity in Rat-1 cells.
Significantly, the agents that blocked MKP-1 expression and potentiated sustained ERK activity did not amplify MEK activity, indicating that they exert their effects at the level of ERK by preserving the phosphorylation state and therefore the activity of the enzyme. In fact, both cycloheximide and BAPTA-AM/EGTA treatment caused a reduction in long term MEK activation (Figs. 1D and 7D); this may be due to the amplification of ERK activity under these conditions, since ERK can retrophosphorylate and inhibit MEK1 (56).
In contrast to studies in CCL39 cells (40), we have been unable to demonstrate growth factor-stimulated expression of MKP-2 in Rat-1 cells although we have shown that this molecule is expressed in response to Ro-31-8220, which activates the JNK stress kinase pathway (39). Since many of the MKP family members are able to inactivate ERK in vitro and in cells (40-48), it seems that selectivity is quite low and may be achieved rather by distinct temporal or spatial expression patterns in response to different agonists. Indeed, our preliminary experiments indicate that MKP-2 expression in Rat-1 cells correlates with activation of the JNK and p38 stress kinase pathways rather than with growth factor stimulation.5 The antiserum used in this study will not recognize the other members of the MKP family (MKP-3, hVH3, and hVH5), some of which are inducible immediate early genes, so we cannot presently rule out the possibility that other MKP family members may also contribute to the regulation of sustained ERK activity by cycloheximide in this system.
One significant difference between the effects of cycloheximide and orthovanadate should be pointed out. ERK activity in LPA-stimulated cells peaks at 5-10 min and quickly declines at 15-30 min before persisting in a second phase. In many experiments, this early sharp decline was prevented or delayed by orthovanadate but not by cycloheximide at a time point at which MKP-1 is only barely detectable. This may suggest that the rapid decline in activity after 10 min of LPA treatment may be due to a nonlabile, cytosolic PTPase distinct from MKP-1. Indeed, Keyse and co-workers (45) have recently reported the cloning of an MKP-1-related dual specificity phosphatase called Pyst-1 or MKP-3 that is highly selective for the ERK family, is constitutively expressed, and is located in the cytosol. Perhaps a similar molecule regulates the early peak of ERK activity in Rat-1 cells.
With these caveats in mind, our results suggest that in Rat-1 cells, orthovanadate and cycloheximide potentiate sustained ERK1 activity because they are able to inhibit the activity or the expression of MKP-1 and perhaps other MKPs, which act as ERK phosphatases.
MKP-1 Expression Requires the Integration of Coincident ERK- and Ca2+-dependent Signaling Pathways in Rat-1 CellsThese results are consistent with a model in which activation and nuclear translocation of ERK results in expression of the MKP-1 gene, which in turn serves to negatively regulate ERK activity. However, although ERK activation is required for maximal induction of MKP-1 by LPA and EGF in Rat-1 cells (Fig. 4), it is not sufficient (Fig. 5); maximal MKP-1 expression also requires other signal pathways that are coincident with ERK activation.
Two observations lead us to this conclusion. Blocking activation of the
ERK cascade with PD98059 results in complete inhibition of
LPA-stimulated ERK activity but can only reproducibly inhibit 50% of
LPA-stimulated MKP-1 expression; clearly, ERK-independent signals
contribute to this response also. Reexamination of the role of the ERK
pathway using Raf-1:ER showed that specific and persistent
activation of the ERK cascade alone is not sufficient to induce
expression of MKP-1. In this respect, our results differ significantly
from those in CCL39 hamster fibroblasts (40), where PD98059 strongly
inhibited MKP-1 expression and activation of
Raf-1:ER was sufficient
to induce MKP-1 expression to the same degree as that with serum. Since
we have used the identical antiserum to detect MKP-1, we can only
speculate that this disparity represents a cell type-specific
difference in the regulation of expression of MKP-1.
A major question arising from our results is why the sustained
activation of ERK seen with Raf-1:ER cannot result in MKP-1 expression. Two explanations seemed plausible. First, there is a
threshold of ERK activation required to initiate MKP-1 expression, and
the sustained response seen with
Raf-1:ER is insufficient; perhaps
the early burst of ERK activity seen with growth factors at 5-10 min
is an important signal. A second explanation is that MKP-1 expression,
while requiring ERK activation, also requires other additional signals
provided by growth factor receptor signaling, and it is the detection
and integration of these coincident signals that serves to induce
MKP-1. The former argument seems unlikely, since we observed that
whereas ERK1 activation by
Raf-1:ER is slightly delayed compared
with that for LPA, the magnitude of the response is the same as that
for LPA. We might therefore expect to see MKP-1 expression in response
to
Raf-1:ER but with delayed kinetics. In fact, even after 4 h
the expression of MKP-1 is vanishingly low compared with that seen with
LPA or FBS.
For a number of reasons, we favor a model in which MKP-1 expression is
the result of synergy between coincident signals including Ca2+ and the ERK cascade and in which any one signal is not
sufficient for this response. For example, the inhibition of either
signal (with PD98059 or BAPTA-AM/EGTA) results in the inhibition of
MKP-1 expression (Figs. 4 and 7), whereas modest activation of either with Raf-1:ER and ionomycin will give synergistic expression of
MKP-1 (Fig. 8). In the case of PMA, PKC can presumably activate the ERK
pathway by virtue of activating Raf (57, 58) as well as activating
another PKC-dependent pathway; again, synergistic integration of these signals would explain why the PMA response is also
dependent upon MEK/ERK and why we see a pronounced synergy between
Raf-1:ER and PMA. It is important to stress that under these
conditions we do not observe synergy for ERK activation, suggesting
that the different signals are not simply cooperating to boost the ERK
signal.
The relative importance of Ca2+ and PKC signals is
demonstrated by the fact that chelation of Ca2+ (Fig. 7)
but not inhibition of PKC (Fig. 6) completely inhibits MKP-1 expression
by growth factors and blocks the synergy between Raf-1:ER and
non-Gi-mediated LPA signals in pertussis toxin-treated R1
Raf-1:ER-4 cells. Thus, it would appear that Ca2+ is
the major "non-Gi" signal generated by LPA for
regulating MKP-1 expression. Again, this highlights significant
differences in the regulation of expression of MKP-1 between Rat-1
cells and CCL39 cells (40). Although the role of Ca2+ was
not examined in CCL39 cells, it was clear that inhibition of PKC
significantly inhibited serum-stimulated MKP-1 expression, whereas our
results using GF109203X and PKC "down-regulation" suggest little
role for PKC in the responses to growth factors (Ref. 39 and this
work).
The requirement for both the MEK-ERK pathway and other signals generated by classical second messenger pathways for MKP-1 expression raises the possibility that MKP-1 may be the focus for regulatory cross-talk from parallel, ERK-independent pathways. One such example is the observation that buffering [Ca2+]i to block MKP-1 expression potentiates sustained ERK activity. This may partly explain why agonists that elicit strong Ca2+ mobilization by the Gq-phospholipase C pathway activate ERK in a transient manner (23); the difference in kinetics of ERK activity may reflect a Ca2+-stimulated expression of MKP-1, leading to inhibition of sustained ERK activity.
Analysis of the c-fos promoter in transgenic mice suggests
that multiple promoter elements are required for maximal c-fos induction by growth factors in whole cells (59). Lacking a
suitable MKP-1 promoter to analyze, we have dissected the pathways by
which MKP-1 protein is induced by growth factors using classical and novel biochemical inhibitors and the conditional Raf-1:ER allele to
specifically reconstitute the Raf-MEK-ERK cascade. The picture that
emerges, of multiple pathways converging on multiple promoter elements,
is consistent with what is known about the structure of the MKP-1
promoters. Both the CL100 (60) and 3CH134/erp
(32) promoters possess AP-1 and CRE sites, which can respond to PKC and
Ca2+/cAMP signals. There is no obvious serum response
element, but a recent study of the PAC-1 promoter, a
hematopoietic-specific MKP, suggests that an E-box and AP-2 motifs may
mediate the effects of the ERK cascade (46); these motifs are conserved
in the CL100 and 3CH134 promoters. Finally, the
synergy between the ERK and Ca2+ or PKC pathways is
reminiscent of that seen with different promoter elements in the
c-fos gene (59). It seems that synergistic integration of
signaling pathways at various 5
promoter and enhancer elements may
turn out to be a common means of transducing often low level extracellular signals into significant changes in gene expression. In
the case of MKP-1, this may be relevant to the regulation of the ERK
cascade by various signals, both growth-promoting and growth-inhibitory, thereby allowing fine tuning of ERK activity in
response to different environmental conditions.
S. J. C. particularly thanks Dr. Fergus Mckenzie and Prof. Jacques Pouysségur for the generous gift of Alb-1 anti-MKP-1 antiserum and Fergus Mckenzie for many stimulating discussions. We thank Drs. Gideon Bollag and David Stokoe for suggestions and comments and David Stokoe for providing samples from COS cells transfected with CL100 for testing the anti-MKP-1 antiserum.