(Received for publication, September 4, 1996, and in revised form, October 28, 1996)
From the Université de Nice, Centre de Biochimie, CNRS UMR 134, Parc Valrose, 06108 Nice Cedex 02, France
Mitogen-activated protein (MAP) kinase phosphatase-1 (MKP-1) and MKP-2 are two members of a recently described family of dual specificity phosphatases that are capable of dephosphorylating p42/p44MAPK. Overexpression of MKP-1 or MKP-2 inhibits MAP kinase-dependent intracellular signaling events and fibroblast proliferation. By using specific antibodies that recognize endogenous MKP-1 and MKP-2 in CCL39 cells, we show that MKP-1 and MKP-2 are not expressed in quiescent cells, but are rapidly induced following serum addition, with protein detectable as early as 30 min (MKP-1) or 60 min (MKP-2). Serum induction of MKP-1 and MKP-2 is sustained, with protein detectable up to 14 h after serum addition. Induction of MKP-1 and, to a lesser extent, MKP-2 temporally correlates with p42/p44MAPK inactivation.
To analyze the contribution of the MAP kinase cascade to MKP-1 and
MKP-2 induction, we examined CCL39 cells transformed with either
v-ras or a constitutively active direct upstream activator of MAP kinase, mitogen-activated protein kinase kinase-1 (MKK-1; MKK-1(SD/SD) mutant). In both cell models, MKP-1 and MKP-2 are constitutively expressed, with MKP-2 being prevalent. In addition, in
CCL39 cells expressing an estradiol-inducible Raf-1::ER
chimera, activation of Raf alone is sufficient to induce MKP-1 and
MKP-2. The role of the MAP kinase cascade in MKP induction was
highlighted by the MKK-1 inhibitor PD 098059, which blunted both the
activation of p42/p44MAPK and the induction of MKP-1 and
MKP-2. However, the MAP kinase cascade is not absolutely required for
the induction of MKP-1, as this phosphatase, but not MKP-2, was induced
to detectable levels by agents that stimulate protein kinases A and C. Thus, activation of the p42/p44MAPK cascade promotes the
induction of MKP-1 and MKP-2, which may then attenuate
p42/p44MAPK-dependent events in an inhibitory
feedback loop.
A major characteristic of protein phosphorylation is its reversibility. In the cell, a dynamic balance exists between phosphorylation and dephosphorylation, resulting from interplay between protein phosphatases and protein kinases. As a consequence, a modification of either component is likely to have an equally important impact on signal transduction (1, 2, 3).
Protein phosphatases are generally classified into either serine/threonine or tyrosine phosphatases, depending on phosphoamino acid specificity. Dual specificity phosphatases capable of removing both phosphotyrosine and phosphothreonine from protein targets are a relatively recent discovery. However, an ever increasing number of such phosphatases exist, including Cdc25, which dephosphorylates Thr14 and Tyr15 of Cdc2 (4), and Cdi1 (or KAP) (5, 6), which dephosphorylates Thr160 of Cdk2. A novel family of dual specificity enzymes harboring the canonical motif (I/V)HCXAGXXR(S/T)G, originally identified in the active site of the VH-1 phosphatase (7), and able to dephosphorylate, at least in vitro, an archetypal substrate, p42/p44MAPK (8, 9), has recently been identified. These phosphatases have been named MAP1 kinase phosphatases (MKPs), with at least eight known members currently identified: MKP-1 (CL100, XCL100, HVH-1, 3CH134, or erp) (10, 11, 12, 13, 14); MKP-2 (hVH-2, TYP1) (15, 16); hVH-3 (B23) (17, 18); MKP-3 (rVH-6, Pyst1) (19, 20, 23); hVH-5 (21), PAC1 (22); and two incomplete sequences, MKP-X (19) and Pyst2 (23).
p42/p44MAPK are highly homologous ternary members of a group of ubiquitously expressed serine/threonine kinases that, in fibroblasts, are activated in response to all mitogenic stimuli (24). Sustained activation of the MAP kinase cascade is an absolute requirement for fibroblasts to exit from the quiescent G0 state and to pass the restriction point in G1. Thus, blockade of MAP kinase signaling with antisense MAP kinase constructs or dominant-negative MAP kinase molecules (25) or overexpression of MKP-1 (26) all prevent cell cycle reentry. The MAP kinase family is exemplified by three distinct subtypes: p42/p44MAPK (27), p38MAPK (28), and p46/p54JNK (29), which are predominately cytoplasmic proteins in quiescent cells. Upon stimulation, p42/p44MAPK and p46/p54JNK translocate to the nucleus, where they may phosphorylate nuclear transcription factors and thus regulate gene transcription (30, 31, 32). Full enzymatic activity of MAP kinase requires a dual phosphorylation on a Thr-X-Tyr motif, which is performed in vivo by specific upstream activators termed MAP kinase kinase (MKK-1/2, MKK-3/6, and MKK-4 for the p42/p44MAPK, p38MAPK, and p46/p54JNK enzymes, respectively) (33, 34, 35). Consequently, each MAP kinase family member is a candidate for dual dephosphorylation by MKPs. This raises the question of specificity between cognate kinase-phosphatase partners. However, previous studies have shown that MKP-1, MKP-2, MKP-3, and PAC1 (restricted to hematopoietic cell lines) are all able to inactivate p42/p44MAPK and that specificity is correspondingly low (8, 15, 19, 36).
Several MKP family members were identified by virtue of their being encoded by immediate-early genes (10, 37). With the exception of Pyst1 (23), MKPs are not expressed in quiescent cells and may be induced upon stimulation with agonists that include mitogens, oxidative stress, heat shock, and UV irradiation (9). Specificity of MKPs for the various MAP kinase members may therefore depend on their presence during a particular cellular event. An understanding of the mechanisms controlling MKP induction may explain how MAP kinase signaling pathways are regulated.
We report that two dual specificity phosphatases (MKP-1 and MKP-2) may be induced in resting CCL39 fibroblasts following serum exposure. We have attempted to correlate their induction with the activity status of signal transduction pathways involving different MAP kinase family members. To this end, we investigated the level of MKP-1 and MKP-2 in cells expressing either constitutively active or inducible forms of each element of the Ras/Raf-1/MKK module. Our results suggest that activation of the p42/p44MAPK cascade is sufficient to promote the expression of MKP-1 and MKP-2. We propose that p42/p44MAPK down-regulation when cells progress through G1 is dependent on a feedback loop that involves MKP family members.
Materials
[-32P]ATP and the enhanced chemiluminescence
(ECL) immunodetection system were obtained from Amersham Corp.;
antiserum E1B, which specifically recognizes p42 and
p44MAPK on Western blots, and antiserum Kelly, which
specifically immunoprecipitates p42/p44MAPK activity, were
as previously detailed (38). Antisera specifically immunoprecipitating
p44MAPK were a kind gift of Dr. Sylvain Meloche (39).
Anti-cyclin D1 antisera were a kind gift of Dr. Véronique Baldin.
The GST-Jun-(1-79) and GST-ATF-2-(1-149) expression vectors were kind
gifts of Dr. M. Karin and Dr. R. Davis, respectively, and were as
described (40). The Flag-p46JNK expression vector was a
kind gift of Dr. B. Dérijard (35). The specific
p38MAPK inhibitor SB 203580 was supplied by SmithKline
Beecham. The specific MKK-1/2 inhibitor PD 098059 was purchased from
New England Biolabs Inc. Bovine myelin basic protein and bovine serum
albumin were purchased from Sigma. Triton X-100 and
Nonidet P-40 were from Pierce.
Cells and Culture Conditions
The established Chinese hamster lung fibroblast line CCL39
(American Type Culture Collection) and its derivatives (clones Ras5C
and MKK-1(SD/SD)) were cultivated in Dulbecco's modified Eagle's
medium (DMEM; Life Technologies, Inc.) containing 7.5% fetal calf
serum, 50 units/ml penicillin, and 50 µg/ml streptomycin sulfate.
CCL39-derived Raf-1::ER-expressing cells were cultivated in DMEM without phenol red in the presence of 7.5% fetal calf serum
and G418 (400 µg/ml) (42). HEK 293 cells were cultivated in DMEM
containing 7.5% decomplemented fetal calf serum. Cells were
growth-arrested by serum starvation for 16-24 h.
DNA Constructs and Expression in HEK 293 Cells
The EcoRI fragment of the full-length cDNA of CL100 (human MKP-1) previously introduced into the pECE vector (Stratagene) was digested by SmaI/XbaI and cloned into the pcDNAneoI mammalian expression vector (EcoRV and XbaI sites). The EcoRI/EcoRI fragment of the full-length cDNA of hVH-2 (human MKP-2) in the pBS vector was digested by BamHI/XhoI and cloned into the pcDNAneoI expression vector (BamHI and XhoI sites) (Invitrogen). HEK 293 cells were seeded at a density of 0.5 × 106 cells/well (six-well plate) and transfected the following day by the classical calcium phosphate coprecipitation technique with 10 µg of the corresponding construct (43). 48 h after transfection, cells were lysed for Western blot analysis with Laemmli sample buffer (45).
Preparation of Polyclonal Antiserum against MKP-1
A synthetic peptide corresponding to the last 12 C-terminal amino acids of 3CH134 protein (mouse MKP-1) conjugated to keyhole limpet hemocyanin was used for rabbit immunization. This antibody is referred to here as Alb-1. The specificity of Alb-1 was tested by Western blotting using cell lysates from HEK 293 cells transfected with the pcDNAneo/MKP-1 construct, the pcDNAneo/MKP-2 construct, or an empty vector.
Expression of MAP Kinase and MAP Kinase Phosphatase Family Members in CCL39 Cells
CCL39 cells were seeded at a density of 0.8 × 106 cells/dish (60-cm plate) (day 0) and transfected the following day (day 1) by the classical calcium phosphate coprecipitation technique with 2 µg of the relevant HA or Flag epitope-tagged kinase construct associated either with 25 µg of empty vector or with 25 µg of phosphatase-expressing vector. The cells were then serum-starved overnight (day 2), and on day 3, cells were stimulated by serum or anisomycin for 30 min. Kinase assays were performed as described below. For HA-p44MAPK and HA-p38MAPK, immunoprecipitation was performed with monoclonal anti-HA antibodies. For Flag-p46JNK, immunoprecipitation was performed with monoclonal anti-Flag antibodies. In experiments to determine the effect of MKP-1 and MKP-2 on CCL39 cell growth, CCL39 cells were seeded at a density of 3 × 105 cells/well (six-well plate) and transfected with plasmid encoding the Na+/H+ antiporter NHE-3 (2 µg) either with pcDNAneo alone or with pcDNAneo encoding MKP-1 or MKP-2 (20 µg). Cells were selected by the established proton-killing technique (25) performed over a 2-week period, after which colonies were stained with Giemsa blue (10% in PBS) and counted.
Northern Blot Analysis
CCL39 cells were seeded in 10-cm plates and, when confluent, serum-starved for 24 h. Cells were then stimulated with the appropriate agonist and, following a suitable time period, washed twice with cold PBS and lysed with Bioprobe RNA preparation (acid phenol and guadininium chloride). RNA was prepared by phenol/chloroform extraction and sodium acetate precipitation. Following RNA separation (30 µg of total RNA) on formaldehyde-agarose gels, RNA was transferred to nitrocellulose and probed by the technique of Church and Gilbert (44). The MKP-1 probe was obtained following purification from a StuI digestion (780 base pairs) of pcDNAneo/CL100. The MKP-2 probe was purified as a StuI/SacII digest (1055 base pairs) of pcDNAneo/hVH-2.
Western Blot Analysis
Cells were washed twice with cold PBS and lysed in Triton X-100
lysis buffer (50 mM Tris-HCl, pH 7.5, 100 mM
NaCl, 50 mM NaF, 5 mM EDTA, 40 mM
-glycerophosphate, 200 µM sodium orthovanadate, 10
4 M phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, 1 µM pepstatin A, and 1% Triton
X-100) for 15 min at 4 °C. Insoluble material was removed by
centrifugation at 12,000 × g for 2 min at 4 °C. Proteins from cell lysates (100 µg) were separated by 10% SDS-PAGE and electrophoretically transferred to Hybond-C Extra membranes (Amersham Corp.) in 25 mM Tris-HCl and 0.19 M
glycine. Membranes were blocked in Tris-buffered saline (25 mM Tris-HCl, pH 7.5, and 137 mM NaCl)
containing 5% nonfat dry milk. The blots were then incubated with
antiserum Alb-1 (1:300) in blocking solution for 2 h at room
temperature. After washing in Tris-buffered saline and 0.1% Triton
X-100, blots were incubated with horseradish peroxidase-conjugated goat
anti-rabbit IgG (1:3000) in blocking solution for 1 h and revealed
with ECL. Where indicated, the activity status of
p42/p44MAPK was determined by a mobility shift assay in
which, following cell lysis, proteins were separated by SDS-PAGE
(12.5% acrylamide, 0.0625% bisacrylamide) and Western blotting was
performed with antiserum E1B, which preferentially recognizes
p42MAPK.
Immune Complex Kinase Assays
p42/p44MAPKCCL39 cells or their derivatives
were seeded in 12-well plates and rendered quiescent at confluence by
serum starvation for 24 h. Cells were stimulated in DMEM with 10%
serum (CCL39) or in phenol red-free DMEM with either estradiol (1 µM) or serum (5%) (Raf-1::ER) at 37 °C
for the times indicated, prior to being washed with ice-cold PBS and
lysed with Triton X-100 lysis buffer as described for Western blot
analysis. Proteins from lysates (200 µg) were incubated with specific
anti-p44MAPK antibodies preadsorbed to protein
A-Sepharose-coated beads for 2 h at 4 °C. Immune complexes were
washed three times with Triton X-100 lysis buffer and twice with kinase
buffer (20 mM HEPES, pH 7.4, 20 mM
MgCl2, 1 mM dithiothreitol, and 10 mM p-nitrophenyl phosphate).
p42/p44MAPK activity was assayed by resuspending the final
pellet in 40 µl of kinase buffer containing 50 µM
[
-32P]ATP (5000 cpm/pmol) and 0.25 mg/ml myelin basic
protein. The reaction was incubated for 10 min at 30 °C and stopped
by addition of Laemmli sample buffer (45).
Cells were deprived of serum for
18 h in DMEM and stimulated with FCS (10%), IL-1 (10 ng/ml),
sorbitol (300 mM), or sodium arsenite (200 µM). Cells were washed and lysed with the same protocol as that described for p42/p44MAPK. Cleared lysates (500 µg of protein) were incubated with glutathione-Sepharose coupled with
6 µg of GST-Jun-(1-79) for 3 h. Complexes were washed three
times with 150 mM Tris, pH 7.5, and 1% Nonidet P-40; once with PBS and 0.5 M LiCl; and twice with kinase buffer prior
to the kinase reaction.
Following immunoprecipitation of p38MAPK from cell lysates (400 µg of protein), immune complexes were treated exactly as described for p42/p44MAPK, with myelin basic protein being substituted in the kinase assay for GST-ATF-2 (5 µg).
In each case, samples were heated at 95 °C for 2 min and separated by 10% SDS-PAGE. Gels were exposed, and the relevant bands were quantified using a Fuji phosphorimager.
Protein Determinations
Protein determinations were evaluated using the bicinchoninic acid protein assay kit (Pierce) with bovine serum albumin as standard.
Data Reproducibility
All experiments were performed at least three times. Where indicated, data shown are representative of at least two additional experiments that gave qualitatively identical results.
Previous studies that have examined the
expression of MKP family members have principally employed Northern
blot analysis. In serum-stimulated (2 h), but not quiescent, CCL39
cells, two RNA species that migrate with the size expected of MKP-1 and
MKP-2 (~2.4 kilobase pairs) were detected using probes that were
designed to specifically identify either MKP-1 or MKP-2 mRNA
species, respectively (Fig. 1A). A more
detailed analysis revealed that the two mRNA species that were
identified with MKP-1 and MKP-2 probes were induced by serum with a
time course similar to that previously noted for MKP-1 and MKP-2 (data
not shown).
To specifically identify MKP-1 and MKP-2 proteins, we raised rabbit polyclonal antiserum directed against a synthetic peptide corresponding to the last 12 C-terminal amino acids of mouse MKP-1 (3CH134 protein) (37). This sequence has only one amino acid change from human MKP-1 (Lys to Gln) (10) and one amino acid change in the same position in MKP-2 (Lys to His in both rat and human sequences) (15, 46). In contrast, the C-terminal regions of the other MKPs bear little or no primary structure similarity. Thus, we expected that our anti-MKP antisera would be capable of recognizing both MKP-1 and MKP-2, but not other MKP family members. In Western blots of HEK 293 cells transiently transfected with expression vectors encoding MKP-1 or MKP-2, but not with empty vector, two single major bands were identified by anti-MKP-1/2 antiserum Alb-1 with apparent molecular masses of 40 and 42 kDa, in close agreement with the calculated molecular mass from the primary sequence of MKP-1 and MKP-2, respectively (Fig. 1B).
We next performed Western blotting on cell lysates derived from serum-deprived or serum-stimulated CCL39 cells. Following serum stimulation (3.5 h) of quiescent CCL39 cells, Alb-1 antisera recognized two major bands with the expected molecular mass of MKP-1 and MKP-2 (Fig. 1B). In addition, these two bands comigrated with MKP-1 and MKP-2 transiently expressed in HEK 293 cells. When immunoblotting was performed in the presence of the specific peptide used for rabbit immunization, but not with an unrelated peptide sequence, the two bands with apparent molecular masses of 40 and 42 kDa were no longer detectable. Additionally, serum-mediated induction of MKP-1 and MKP-2 was dose-dependent in nature (Fig. 1C). These data strongly suggest that our anti-MKP-1/2 antiserum is capable of specifically detecting the hamster homologues of MKP-1 and MKP-2 in CCL39 cells.
MKP-1 and MKP-2 Are Rapidly but Differentially Induced in Response to Serum: Correlation with p42/p44MAPK Inactivation Time CourseTo analyze the induction of MKP-1 and MKP-2 in CCL39
cells, we performed Western blotting on cell lysates from
serum-stimulated cells (Fig. 2A). In
quiescent CCL39 cells, both MKP-1 and MKP-2 are undetectable. Upon
addition of serum, MKP-1 and MKP-2 are induced, with MKP-1 present as
early as 1 h after addition of serum. Comparison of a range of
experiments shows that MKP-1 is often detectable as early as 30 min
after addition of serum (data not shown). In contrast, MKP-2 is induced
later, with detectable protein present at 2-3 h after serum addition.
Each phosphatase is present for at least 14 h after serum
addition. After 10-12 h of serum addition, the CCL39 cell populations
had started to enter S phase, correlating with the expression of cyclin
D1. Thus, MKP-1 and MKP-2 are induced and present during
G0-G1 transition and S phase entry in CCL39
cells. As we have previously reported (38), serum stimulation of
p44MAPK is rapid, being maximal after 5 min.
p44MAPK then declines slowly, until 1-2 h following serum
addition, where there is a significant loss of activity (Fig.
2B, lower panel) (47). This loss of activity
correlates temporally with the induction and presence of MKP-1 protein
and, to a lesser extent, with the production of MKP-2 (Fig. 2,
upper panel). As most members of the MKP family are encoded
by immediate-early genes, addition of protein synthesis inhibitors to
quiescent CCL39 cells should block their induction. To examine the
contribution of MKP-1 and MKP-2 to the inactivation of
p42/p44MAPK, we examined the time course of MAP kinase
activity in the presence or absence of cycloheximide (Fig.
2C). As detailed in Fig. 2B, serum-stimulated
p42/p44MAPK activity is maximal at 5 min and then declines
such that ~30% of the maximal activity elicited by serum (5-min time
point) is present 4 h after addition of serum. However, in the
presence of cycloheximide, the long-term inactivation of
p42/p44MAPK is reduced such that 54% of the maximal
activity elicited by serum (5-min time point) is present 4 h after
addition of serum. Additional experiments revealed that cycloheximide
inhibited the serum-mediated induction of both MKP-1 and MKP-2 in a
dose-dependent manner that correlated with the extent of
protein synthesis inhibition (data not shown). Thus, although a
considerable fraction of the p42/p44MAPK inhibitory
activity is independent of cycloheximide treatment, immediate-early
genes and probably MKP-1 and MKP-2 play a role in setting the level of
p42/p44MAPK activity in CCL39 cells.
MKP-1 and MKP-2 Block the Activation of p44MAPK, p46JNK, and p38MAPK and Inhibit CCL39 Cell Proliferation
To explore the substrate specificity of two MKP
family members, MKP-1 and MKP-2 expressed by CCL39 cells, we performed
cotransfection assays with each phosphatase together with
epitope-tagged p44MAPK, p38MAPK, or
p46JNK (Fig. 3A). In response to
serum, HA-p44MAPK may be activated and can phosphorylate
its substrate, myelin basic protein. However, following cotransfection
with either MKP-1 or MKP-2, p44MAPK is no longer activable.
In a similar manner, in response to specific stress stimulus, such as
anisomycin, both HA-p38MAPK and Flag-p46JNK are
able to phosphorylate their respective substrates in the absence, but
not in the presence, of either MKP-1 or MKP-2. Thus, the transient
expression of both MKP-1 and MKP-2 attenuates the activation in
vivo of each of the three kinases examined: p44MAPK,
p38MAPK, and p46JNK.
We have previously shown that deregulated expression of MKP-1 in CCL39 fibroblasts prevents cell cycle reentry (26). As MKP-2 is also able to inhibit p42/p44MAPK in vivo (Fig. 3A), we expected that MKP-2 would also exert a strong antiproliferative effect. To examine this hypothesis, we transfected CCL39 cells with either MKP-1 or MKP-2 and determined the colony-forming ability of transfected cells (Fig. 3B). In the presence of both MKP-1 and MKP-2, colony formation and hence cell division are blocked by up to 80%, demonstrating that both MKP-1 and MKP-2 inhibit the proliferation of CCL39 cells.
Inactivation of Stress Kinases Does Not Require MKP-1 or MKP-2The transient expression studies described above (Fig. 3)
show that overexpression of MKP-1 or MKP-2 can inhibit
p38MAPK and p46JNK activity. However, this does
not unequivocally demonstrate that endogenous MKP-1 and MKP-2 are
required for inactivation of either kinase. Hence, we examined the time
course of activation of p46/p54JNK in response to a range
of agonists, together with the possible induction of MKP-1 or MKP-2
(Fig. 4). Activation of p46/p54JNK by
osmotic shock (sorbitol), sodium arsenite (equivalent to heat shock
(48)), or IL-1 was slow in comparison with agonist-mediated activation of p42/p44MAPK (Fig. 2), peaking at ~30 min
after addition of either sorbitol or IL-1
. In the case of sodium
arsenite and sorbitol, this activity was sustained, lasting for at
least 3 h in the continual presence of agonist. In contrast,
stimulation of p46/p54JNK by IL-1
was transient, having
returned to basal values within 2-3 h after addition of agonist. None
of the agonists tested were able to induce MKP-1 or MKP-2 to detectable
levels, in contrast to control cells, which were stimulated with serum.
Thus, inactivation of p46/p54JNK proceeds in the absence of
detectable MKP-1 and MKP-2.
Serum Induction of MKP-1 and MKP-2 Does Not Require p38MAPK Activity
p38MAPK is weakly,
although significantly activated by serum in CCL39 cells (data not
shown) and could therefore play a role in MKP induction. Pretreatment
of CCL39 cells with the specific p38MAPK inhibitor SB
203580 (49, 50) completely blocks the activation of p38MAPK
(51). However, following inhibitor pretreatment, the ability of FCS
both to stimulate p42/p44MAPK and to induce MKP-1 and MKP-2
was unimpaired (Fig. 5). Hence, activation of the
p38MAPK signaling pathway is not a requirement for the
serum-mediated induction of MKP-1 and MKP-2.
MKP-1 and MKP-2 Are Constitutively Expressed in CCL39 Cells Expressing v-ras or MKK-1(SD/SD)
As both MKP-1 and MKP-2 are able
to inactivate p42/p44MAPK and agents that activate MAP
kinase also provoke the induction of MKP-1 and MKP-2, we hypothesized
that p42/p44MAPK might regulate the induction of its own
inhibitor. Activation of p42/p44MAPK requires the
sequential activation of Ras, Raf-1, and MKK-1/2, with
p42/p44MAPK being the last step in the kinase cascade. To
examine the contribution of the MAP kinase cascade to the induction of
MKP-1 and MKP-2, we analyzed CCL39 cells expressing constitutively
active members of this pathway. In CCL39 cells expressing
v-ras, p42/p44MAPK is constitutively active
(Ref. 52 and data not shown), and MKP-2 is constitutively expressed,
with MKP-1 present at a much lower level, but still detectable.
Addition of serum results in the induction of MKP-1, with no
appreciable change in MKP-2 (Fig. 6). In addition to the
p42/p44MAPK cascade, Ras is also known to control signaling
pathways such as those linked to phosphatidylinositol 3-kinase or
Rac/Rho proteins (53). Hence, to specifically activate only
p42/p44MAPK, we analyzed CCL39 cells transformed by a
mutated, constitutively active MKK (MKK-1(SD/SD)) (clone SS3 (54)). As
with CCL39 cells transformed by v-ras,
p42/p44MAPK is constitutively active (54), MKP-2 is
constitutively expressed, and MKP-1 is barely detectable. Addition of
serum provokes the induction of MKP-1 in SS3 cells, with MKP-2 levels
not modified to a significant degree (Fig. 6). Therefore, it appears
that sustained p42/p44MAPK activity is sufficient to
promote the expression of both MKP-1 and MKP-2, with MKP-2 being
preferentially induced.
Activation of Raf-1 Is Sufficient to Induce MKP-1 and MKP-2
The transformation of fibroblasts by the ectopic expression
of oncogenic proteins may result in the secretion of growth factors or
hormones, which may then stimulate the cell population in an autocrine/paracrine manner. Indeed, conditioned serum-free medium from
v-ras-transformed, but not wild-type, CCL39 cells can modify signal transduction pathways when added back to wild-type CCL39 cells
(data not shown). Hence, the constitutive expression of MKP-1/2 in
v-ras- or MKK-1(SD/SD)-transformed CCL39 cells may not be
due solely to the constitutive activation of the
p42/p44MAPK cascade, but may involve additional signaling
pathways. To rigorously assess the role of the p42/p44MAPK
cascade alone in the regulation of MKP induction, we used CCL39 cells
expressing an inducible member of the MAP kinase signaling cascade,
Raf-1::ER (42, 55). This chimeric protein has been shown
to be conditionally activable by exposure to the estrogen analogue
estradiol and to be able to specifically activate p45MKK-1
and p42/p44MAPK with no interference with stress MAP kinase
pathways (55). In
Raf-1::ER cells, estradiol addition
potently activates p42/p44MAPK (Ref. 42 and data not shown)
and promotes MKP-1 and MKP-2 expression (Fig. 7),
whereas in parental cells, estradiol alone has no effect on
p42/p44MAPK activities and MKP-1/2 expression (data not
shown). The ability of the
Raf-1::ER construct to induce
MKP-1 and more markedly MKP-2 is augmented in the presence of serum.
Hence, activation of Raf-1, MKK-1, and, by consequence,
p42/p44MAPK is sufficient to induce expression of MKP-1 and
MKP-2.
Serum-mediated Induction of MKP-1 and MKP-2 Requires MKK-1/2 Activity
The experiments above (Fig. 7) show that activation of
Raf-1 is sufficient to induce MKP-1 and MKP-2. To determine whether activation of p42/p44MAPK is necessary for the
serum-mediated induction of MKP-1 and MKP-2, we employed a specific
inhibitor of MKK-1/2, PD 098059 (56, 57). In the presence of PD 098059 (10 µM), the ability of serum to stimulate
p42MAPK is inhibited by 70%, and the induction of MKP-1
and MKP-2 is significantly, although not completely attenuated (Fig.
8). Higher concentrations of PD 098059 are able to
completely block the activation of p42/p44MAPK and the
induction of MKP-1 and MKP-2, but also inhibit protein synthesis.2 However, this inhibitor, when
used at a concentration of 10 µM, allows us to conclude
that p42/p44MAPK are required for full serum-mediated
induction of MKP proteins in CCL39 cells.
MKP-1 and MKP-2 Are Differentially Induced by Protein Kinase C- and cAMP-elevating Agents
Addition of the tumor-promoting agent PMA
to fibroblasts leads to the stimulation of protein kinase C and
p42/p44MAPK, but not p38MAPK and
p46/p54JNK. When CCL39 cells are treated with PMA, MKP-1,
but not MKP-2, is transiently induced, with maximal induction evident
1 h after addition of agonist (Fig. 9A,
upper panel). To determine whether protein kinase C pathways
are also associated with the serum-dependent induction of
MKP-1/2, we pretreated quiescent cells with a specific inhibitor of
protein kinase C enzymes, GF 109203X (58), and followed the
serum-mediated induction of MKP-1 and MKP-2. In addition to attenuating
the ability of PMA to induce MKP-1 (Fig. 9B), the serum-mediated induction of MKP-1 and MKP-2 is also reduced by pretreatment with GF 109203X. In addition to an AP-1 element present in
the MKP-1 promoter, two cyclic AMP-responsive elements (CRE) are
evident. Sustained activation of protein kinase A in CCL39 cells leads
to the induction of MKP-1, with MKP-2 undetectable over a 3-h time
course. Maximal induction of MKP-1 is evident after 1 h of agonist
addition. The level of induction of MKP-1 is significantly lower than
that elicited by serum (Fig. 9A, lower panel). As
activation of protein kinase A does not stimulate the p42/p44MAPK pathway in CCL39 cells, this suggests that
although full induction of MKP-1 and MKP-2 requires
p42/p44MAPK activation, p42/p44MAPK activity is
not an absolute requirement.
Dual phosphorylated p42/p44MAPK is an excellent substrate for the MKP family of dual specificity phosphatases in vitro (9), and all MKP family members tested have been shown to inactivate p42/p44MAPK in vivo. At least eight MKPs are known to exist in mammalian systems (see the Introduction), which, together with the identification of additional MAP kinase family members, reflects signaling complexity (59).
MKPs, with the exception of Pyst1 (23), are the product of immediate-early genes and, based on Northern analysis, share overlapping tissue distributions (17, 46). Purified MKPs are constitutively active (15, 60, 61). Although this does not rule out the possibility that post-translational regulation of MKP activity occurs, it suggests that the principal point of MKP regulation is at the level of transcription. Hence, the specificity of interaction between MAP kinase and MKP family members may depend on the specific induction of one or more MKP. A detailed analysis of the factors required for induction of MKPs would increase our understanding of their physiological role.
We show that MKP-1 and MKP-2 are expressed in the well established CCL39 fibroblast cell line (Fig. 1). Both phosphatases are induced by serum, albeit with a different time course of induction, suggesting that the mechanisms involved in their induction may not be identical (Fig. 2). Induction of MKP-1 and, to a lesser extent, MKP-2 correlates with an attenuation of p44MAPK activity. It is therefore possible that p44MAPK is specifically targeted by MKP-1 in CCL39 cells. However, it should be noted that in contrast to NIH3T3 fibroblasts (8) and Rat-1 cells (data not shown), where there is very little inactivation of p42/p44MAPK in the presence of cycloheximide, in CCL39 cells, ~50% of the p42/p44MAPK inactivating activity is insensitive to cycloheximide and hence does not involve the majority of MKP family members. Overexpression of either MKP-1 or MKP-2 completely blocks the activity of p44MAPK and two members of the stress kinase family, p38MAPK and p46JNK. Thus, both MKP-1 and MKP-2 are potent inhibitors of CCL39 cell cycle reentry (Fig. 3). We cannot conclude that this block is entirely due to p42/p44MAPK inhibition or is a result of inhibition of p42/p44MAPK, stress kinases, and some of the more recently identified MAP kinase family members. However, it is important to note that overexpression of MKP-1 does not result in a complete loss of substrate specificity as we have previously shown that activation of p70S6K occurs normally in cells that overexpress MKP-1 (42). Our results are not in total agreement with those reported by Chu et al. (62), who have demonstrated by a similar transient transfection technique of several cell types that while MKP-1 dephosphorylates p42MAPK, p38MAPK, and p54JNK, MKP-2 is more discriminating and will dephosphorylate p42MAPK and p54JNK, but not p38MAPK. This discrepancy may arise from differences in the expression levels of each phosphatase.
To define specificity between endogenous MAP kinases and MKPs, we
examined the ability of a range of agonists known to stimulate either
the mitogenic p42/p44MAPK pathway or the stress kinase
pathways to induce MKP-1 or MKP-2. In apparent contradiction to
previous reports based on Northern blot analysis (63), we found that
none of the stress agents tested, IL-1, osmotic shock, or sodium
arsenite, were capable of inducing MKP-1 or MKP-2 to detectable levels.
However, with the exception of IL-1
, all of these agents are potent
inhibitors of protein synthesis in CCL39 cells. Hence, it is not
surprising that we were unable to detect protein expression of two
immediate-early genes. In addition, protein synthesis inhibition has
been shown to up-regulate immediate-early gene mRNA induction, most
probably through an increase in mRNA stability (64). Thus, an
analysis of MKP mRNA induction is response to stress agents may be
difficult to interpret. Stimulation of CCL39 stress pathways with the
above agonists, in particular IL-1
, resulted in only a transient
activation of both p46/p54JNK and p38MAPK (data
not shown). As we were unable to detect induction of MKP-1 or MKP-2 in
response to any stress agonists tested, we conclude that neither
phosphatase is involved in the inactivation of stress kinases following
addition of these agonists alone (Fig. 4). Unfortunately, it is not
possible to block production of MKP family members with protein
synthesis inhibitors and then follow a time course of stress kinase
activity in response to agonist stimulation, as an inhibition of
protein synthesis by itself activates stress kinases (Ref. 65 and data
not shown). The inhibition of p38MAPK failed to modify
serum-stimulated induction of MKP-1 and MKP-2, demonstrating that
activation of this stress kinase is not a requirement for MKP-1 and
MKP-2 induction. Whether stress kinase inactivation in response to
IL-1
stimulation requires the expression of other members of the MKP
phosphatase family remains to be determined.
Stimulation of CCL39 cells results in the activation of a range of
signaling pathways, one or more of which lead to the induction of MKP-1
and MKP-2. To fully analyze the contribution of the MAP kinase cascade
to the induction of MKP-1 and MKP-2, we employed three different
approaches: (i) CCL39 cells transformed by constitutively active
components of the MAP kinase pathway; (ii) CCL39 cells expressing an
inducible Raf-1 construct, Raf-1::ER; and finally, (iii) a
chemical inhibitor of MKK-1, the direct upstream activator of
p42/p44MAPK. We thus identified the activation of MKK-1 as
being sufficient to strongly induce MKP-2, with MKP-1 induced more
weakly. A similar pattern of induction was obtained in cells
transformed by v-ras (Fig. 6). These experiments suggest
that activation of the MAP kinase pathway is more tightly linked to the
induction of MKP-2 than of MKP-1. However, one should be careful in
analyzing signal transduction pathways in transformed cell lines.
Hence, in untransformed CCL39 cells expressing
Raf-1::ER
(Fig. 7), we show that activation of the Raf/MKK/MAP kinase module is
sufficient for the induction of MKP-1 and MKP-2. These results are
similar to those of Krautwald et al. (66), who have shown
that expression of v-raf in macrophages can lead to the
induction of mRNA encoding MKP-1.
Although activation of the p42/p44MAPK pathway induces MKP-1 and MKP-2, it seems that additional pathways may be implicated in their regulation. MKP-1, but not MKP-2, is detectably induced in CCL39 cells in response to protein kinase C- and protein kinase A-activating agents (Fig. 9). In addition, in cultured glomerular mesangial cells, atrial natriuretic peptide, which does not activate the p42/p44MAPK cascade, is able to induce MKP-1 expression (67).
The promoter sequence of the genes encoding both MKP-1 and pac1 have recently been described (14, 68, 69), providing information regarding the intracellular events controlling their transcription. An analysis of the PAC1 gene in murine B- and T-lymphocytes demonstrates that up-regulation of PAC1 transcription is mediated via an AP-2- and an E-box-binding protein. Furthermore, induction of PAC1 in response to both v-ras and v-raf is attenuated following transfection of "dominant-negative" p42MAPK, thus highlighting a role for the MAP kinase cascade (70). In addition to an AP-2 element and an E-box, the MKP-1 promoter contains an AP-1-binding site, which may respond to phorbol esters (71), and two CRE elements, which may bind and have their activity modified by CRE-binding protein family members including c-Jun, CREM, and ATF-2 (72). We have recently shown that activation of the Raf-1::ER chimera in CCL39 cells may lead to a p42/p44MAPK-independent activation of p70S6K (42). As activation of p70S6K can lead to the activation of CREM- and CRE-dependent gene expression (73), it is possible that the induction of MKP-1 and MKP-2 by Raf-1::ER in CCL39 cells is a consequence of both p42/p44MAPK and p70S6K activation. However, this is unlikely as insulin, a potent stimulator of both p70S6K and protein synthesis in CCL39 cells, fails to induce either MKP-1 or MKP-2 (data not shown).
In the yeast Schizosaccharomyces pombe, the MAP kinase homologue Spc1 is activated in response to osmotic stress and is inactivated by its cognate phosphatase, Pyp2 (74). Interestingly, Pyp2 is also transcriptionally induced by osmotic stress, indicating that a Spc1-Pyp2 negative feedback loop exists (41). In agreement with this model, we suggest that p42/p44MAPK down-regulation may depend on a similar negative feedback loop involving MKP family members.
Finally, we have shown that MKP-1 and MKP-2 are principally induced in CCL39 cells by the MAP kinase cascade, but maximal induction involves the interplay of at least one additional signaling pathway. However, we have been unable to address whether the endogenous level of expression of MKP-1 or MKP-2 is sufficient to dephosphorylate p42/p44MAPK and whether p42/p44MAPK is preferentially dephosphorylated by endogenous MKP-1 or MKP-2. In an attempt to address the latter question, we are adopting an antisense strategy to specifically target and prevent the induction of endogenous MKP-1 and MKP-2. Current research in this laboratory is aimed at developing cell models in which MKPs are maximally inducible in the absence of activation of the MAP kinase pathway, which may allow us to answer the former question.
We thank Drs. S. Keyse and K. Guan for CL100 cDNA and hVH-2 cDNA, respectively; Dr. V. Dulic for critical discussions and comments on the manuscript; Dr. M. Bouaboula for providing protein kinase C inhibitors; D. Grall for technical assistance; and M. Valetti for manuscript preparation.