From the Department of Chemistry and Biochemistry,
¶ Howard Hughes Medical Institute, University of Colorado,
Boulder, Colorado 80309
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ABSTRACT |
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Inactivation of growth factor-regulated mitogen-activated protein (MAP) kinases (ERK1 and ERK2) has been proposed to occur in part through dephosphorylation by the dual specificity MAP kinase phosphatase-1 (MKP-1), an immediate early gene that is induced by mitogenic signaling. In this study, we examined the effect of MKP-1 on signaling components upstream of ERK1 and ERK2. Coexpression of MKK1 or MKK2 with MKP-1 resulted in 7-10-fold activation of mitogen-activated protein kinase kinase (MKK), which required the presence of regulatory serine phosphorylation sites. Endogenous MKK1 and MKK2 were also activated upon MKP-1 expression. Raf-1, a direct regulator of MKK1 and MKK2, was activated under these conditions, and a synergistic activation of MKK was observed upon coexpression of Raf-1 and MKP-1. This effect did not appear to involve synthesis of autocrine growth factors or the inhibition of basal extracellular signal-regulated kinase (ERK) activity but was inhibited by a dominant negative Ras mutant, indicating that MKP-1 enhances Ras-dependent activation of Raf-1 in a cell autonomous manner. This study demonstrates positive feedback regulation of Raf-1 and MKK by the MKP-1 immediate early gene and a potential mechanism for activating Raf-1/MKK signaling pathways alternative to those involving ERK.
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INTRODUCTION |
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The mitogen-activated protein (MAP)1 kinase cascade has emerged as a key signaling pathway regulating factor-dependent cell growth and differentiation through intracellular phosphorylation (1, 2). Growth factor regulation of this pathway involves the phosphorylation and activation of MAP kinases, ERK1 and ERK2, by MAP kinase kinases, MKK1 and MKK2 (3-6). ERKs 1 and 2 phosphorylate various targets including upstream and downstream protein kinases, cell surface receptors, and nuclear transcription factors (1, 7). MKKs 1 and 2, in turn, can be activated through phosphorylation by members of the Raf protein kinase family, including Raf-1, which is ubiquitously expressed (8, 9). Receptor-dependent activation of Raf-1 involves its interaction with p21 Ras, through a mechanism that is not completely defined, but appears to involve Raf-1 dimerization and phosphorylation by heterologous protein kinases (10-13).
The product of the immediate early gene, MAP kinase phosphatase (MKP-1), is able to dephosphorylate phosphoserine/threonine as well as phosphotyrosine residues, and shows selectivity for ERKs 1 and 2 in vitro, with lower activity toward other MAP kinases such as JNK and p38 MAP kinase (14, 15). MKP-1 inactivates ERK following growth factor stimulation in intact cells and also suppresses signaling downstream of ERK at the level of gene transcription and proliferation (15-17), most likely through its inhibitory effects on MAP kinase. Interestingly, MKP-1 is transcriptionally induced by stress-regulated pathways such as those occurring in response to UV treatment or activation of stress-regulated protein kinases (18, 19), suggesting that MKP-1 may function as a mediator of cross-regulatory pathways between mammalian protein kinase cascades. A similar cross-regulation is observed between osmotic stress- and pheromone-activated protein kinase cascades in yeast involving transcriptional induction of protein-tyrosine phosphatases (20-23).
To obtain a more complete understanding of how MKP-1 regulates cell signaling, we examined the effects of this enzyme on other components in the MAP kinase pathway in NIH 3T3 cells. Our studies demonstrate the novel finding that MKP-1 positively regulates Raf-1 and MKK components upstream of ERK in an ERK-independent manner, providing a means by which different pathways downstream of Raf-1 might be differentially controlled.
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MATERIALS AND METHODS |
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Cell Culture-- NIH 3T3 cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with penicillin (100 units/ml), streptomycin (100 µg/ml), and 10% fetal bovine serum (FBS, Life Technologies, Inc.).
Transfections-- Cells were seeded at 2 × 105 cells per well into six-well plates (35 mm diameter) and transfected at 50-80% confluence using 5 µl of LipofectAMINE (Life Technologies, Inc.) according to manufacturer's instructions and 1 µg of cDNA unless otherwise noted. Transfection efficiency was estimated as 20-30%, based on fluorescence of cells transfected in parallel with a construct (pK7-GFP) expressing green fluorescent protein (a gift of Dr. Ian Macara). The cDNA constructs for expression of wild type and mutant hemagglutinin (HA)-tagged MKKs 1 and 2 in pMCL have been described previously (24-26). Constructs for expression of Myc-tagged wild type MKP-1 and MKP-1 (C258S) in a pCEP4 vector were a gift of Drs. Hong Sun and Nicholas Tonks (15). A construct for expression of Flag-tagged Raf-1 was a gift of Dr. Roger Davis (27). Dominant negative Raf-1 (S621A) construct was a gift of Dr. Deborah Morrison (28). Dominant negative H-Ras (S17N) construct was a gift of Dr. Melanie Cobb.
Immunoprecipitations and Phosphorylation Assays--
Cells were
harvested 40-48 h following transfection by washing twice in cold
phosphate-buffered saline and extraction in 350 µl of tissue lysis
buffer (20 mM Tris, pH 7.4, 137 mM NaCl, 2 mM EDTA, 1% Triton X-100, 25 mM
-glycerophosphate, 2 mM sodium pyrophosphate, 10%
glycerol, 1 mM sodium orthovanadate, 1.0 mM phenylmethylsulfonyl fluoride, and 1 mM benzamidine)
followed by centrifugation at 15,000 rpm for 10 min to clarify lysates. Antibodies (0.3 µg) recognizing ERK2 (C-14, Santa Cruz
Biotechnology), MKK1 (17020, Transduction Laboratories), MKK2 (N-20,
Santa Cruz), hemagglutinin tag (12CA5, BabCo), or Raf-1 (C-12, Santa
Cruz) were added to separate aliquots of lysates, which were allowed to
incubate on ice for 1 h. Immune complexes were then incubated end-over-end at 4 °C with 20 µl of protein A- or protein
G-Sepharose (Pharmacia Biotech Inc.) for 1 h. The immune complexes
were washed twice with kinase buffer (25 mM Hepes, pH 7.4, 25 mM
-glycerophosphate, 25 mM
MgCl2, 2 mM dithiothreitol, and 1.0 mM sodium orthovanadate) and incubated with 30 µl of
kinase buffer containing 10 µCi of [
-32P]ATP, 30 µM cold ATP, and substrate for 10 min at 30 °C.
Substrates used were 5 µg of myelin basic protein for measurement of
ERK activity, 1 µg of catalytically inactive ERK2 (K52R) for MKK
activity, or 1 µg of catalytically inactive MKK1 (K97 M)
for Raf-1 activity. Reactions were quenched with Laemmli sample buffer
and resolved by SDS-PAGE. 32P incorporation was quantified
by PhosphorImager analysis.
[35S]Methionine Labeling of Cells-- To confirm MKP-1 expression, cells were transfected with MKP-1 cDNA and incubated for 40 h in DMEM containing 10% FBS. Cells were removed to methionine-free DMEM containing 4% FBS for 1 h, then incubated with the same medium containing 75 µCi/ml [35S]methionine for 4 h. Extracts were prepared, and MKP-1 was immunoprecipitated using monoclonal antibody 9E10 and resolved by SDS-PAGE followed by autoradiography.
Transcription Assays--
AP-1 promoter-dependent
transcription was assayed by transfecting cells with a TRE-CAT reporter
construct (pTE3S-N) (a gift of Drs. Ami Aronhein and Michael Karin)
and v-Raf-3611-MSV (a gift of Dr. Peter Shaw), constitutively active
"MKK1-G1C" (
N4/S218E/S222D) (26), or constitutively active
"MKK2-KW+71" (
N4/S222D/S226D) (25) in the presence or absence of
MKP-1 cDNA. Forty-eight hours after transfection, cells were
harvested and chloramphenicol acetyltransferase (CAT) activity was
measured in 20 µg of protein extract by incorporation of tritium from
[3H]acetyl coenzyme A into chloramphenicol in 30 min.
Western Blotting-- ERK, MKK, Raf-1, or Ras guanine nucleotide exchange factor (SOS) were detected by resolving proteins using SDS-PAGE followed by semidry transfer (Ellard Instruments) onto Immobilon P (Millipore). Blots were blocked for 1 h with Tris-buffered saline (TBS, 50 mM Tris, pH 8, 0.15 M NaCl), Tween 20 (0.1%), nonfat dry milk (5%), followed by incubation with antibodies (listed above, IM2 for the Flag epitope on Raf-1 (Kodak), C-23 for SOS (Santa Cruz)) in TBS/Tween 20 (1:1000 dilution in all cases). The blots were washed four times with TBS/Tween 20, then incubated with horseradish peroxidase or alkaline phosphatase secondary antibodies (1:5000). Protein detection was performed using enhanced chemiluminescence (ECL, Amersham) with horseradish peroxidase secondary antibodies or nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate with alkaline phosphatase secondary antibodies. In some cases, anti-HA and anti-ERK primary antibodies were incubated together to detect HA-MKK and ERK proteins simultaneously.
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RESULTS |
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MKP-1 Expression Activates MKK1 and MKK2-- Cells were transiently transfected with wild type MKK1 or MKK2 and MKP-1, and the activity of expressed MKK was measured. As shown in Fig. 1A, coexpression with wild type MKP-1 resulted in 7-10-fold enhancement of the activity of expressed MKK1 or MKK2. Controls from one experiment showed comparable MKK protein levels present in immunoprecipitates (Fig. 1B), demonstrating enhanced specific activity of wild type MKK. This effect was not observed with MKP-1 (C258S), indicating that the effect requires MKP-1 activity. Under conditions where MKK was activated by MKP-1, endogenous ERK2 activity was low (Fig. 1, C and D), as was coexpressed wild type ERK2 (data not shown).
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MKK1 Activation Requires Regulatory Serine Phosphorylation
Sites--
MKK1 is activated by phosphorylation at Ser-218 and Ser-222
(29, 30). The dependence of MKK1 activation by MKP-1 on these phosphorylation sites was examined by coexpressing MKP-1 with a series
of MKK1 mutants with varying specific activities. MKK1(S218E/S222D), which substitutes regulatory phosphorylation sites with acidic amino
acids, MKK1(N4), which deletes amino acid residues 44-51 but leaves
Ser-218 and Ser-222 intact, and MKK1(
N4/S218A/S222A), which
substitutes regulatory phosphorylation sites with Ala in the
N4
background, are constitutively active mutants with 65-fold, 80-fold,
and 40-fold greater activity than basal wild type MKK1, respectively
(26). MKK1(S218A/S222A) has activity comparable to basal wild type MKK1
(24). MKP-1 expression resulted in activation of wild type MKK1 as well
as MKK1(
N4) (Fig. 4A). In
contrast, MKP-1 had no effect on any of the MKK1 mutants containing
Ser-218 or Ser-222 substitutions. Controls demonstrated comparable
protein levels of each MKK1 in the immune complex assays (Fig.
4B). These data suggest that an upstream activator that
targets Ser-218 and Ser-222 is required for MKP-1-dependent
activation of MKK.
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Involvement of Raf-1 in MKK1 Activation in Cells Expressing MKP-1-- Raf-1 has been shown to be a major upstream activator of MKK1 and MKK2 (8, 9). The dependence of Raf-1 activity on MKP-1 was explored by transfecting cells with HA-tagged MKK1 and varying amounts of Raf-1 cDNA in the presence or absence of MKP-1. A small amount of Raf-1 expression significantly enhanced the MKK activation by MKP-1 (Fig. 5A). Increasing the amount of overexpressed Raf-1 did not further augment MKK1 activation by MKP-1 (Fig. 5A). Western blots probing the Flag epitope showed increased expressed Raf-1 levels in transfected cells, but no variation due to MKP-1 (data not shown). Endogenous MKK activity was also elevated with increasing Raf-1 levels in the presence of MKP-1 compared with MKK activity observed in the absence of MKP-1 (data not shown).
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Involvement of Ras in MKK and Raf-1 Activation by MKP-1-- Involvement of Ras was further investigated using a dominant negative H-Ras mutant (S17N), which inhibits growth factor-dependent activation of ERK. Coexpression of Ras (S17N) with MKP-1 and wild type MKK1 showed partial inhibition of MKK activation by MKP-1 (Fig. 6A) under conditions of comparable loading of MKK immunoprecipitates. Parallel controls confirmed the dominant interfering effect of Ras (S17N) on Ras signaling by showing a complete inhibition of PDGF-stimulated ERK activity (Fig. 6B). The data strongly suggest that Raf-1 activation by MKP-1 occurs through a Ras-dependent mechanism.
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DISCUSSION |
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The growth factor-stimulated MAP kinase cascade has been shown to be inactivated at the level of ERK1 and ERK2 by the dual specificity phosphatase, MKP1 (14-17, 36-38). Previous studies proposed that MKP-1 might function as a feedback regulator of ERK signaling. Originally, MKP-1 was identified through its transcriptional induction following growth factor stimulation of cells and was described as an immediate early gene whose message is rapidly induced in a manner independent of protein synthesis (39). The kinetics of MKP-1 induction are thus consistent with its potential role in catalyzing ERK dephosphorylation, which occurs during a phase of inactivation observed in response to many types of stimuli. However, the induction of MKP-1 and dephosphorylation of ERK are uncorrelated in some studies, suggesting that this may not occur in all cells. For example, the inactivation of ERK in PC12 cells that occurs several minutes following epidermal growth factor-dependent activation is unaffected by cycloheximide, conditions that suppress MKP-1 induction (40, 41). These findings suggest that alternative functions of MKP-1 exist that have yet to be described.
In this study, we show for the first time that MKP-1 positively regulates enzymes upstream of ERK, namely MKK1, MKK2, and Raf-1, thus providing an additional pathway for feedback regulation of Raf-1 by immediate early gene expression. This effect appears to occur through a cell autonomous mechanism and is partially blocked by dominant negative Ras, indicating that Ras-mediated activation of Raf-1 is most likely involved.
Several mechanisms for negative feedback regulation of upstream signaling targets by ERK, including cell surface receptors (epidermal growth factor receptor), Ras guanine nucleotide exchange factor (SOS), or Raf-1 have been proposed (33-35, 42, 43). In particular, SOS phosphorylation by ERK has been shown to down-regulate signaling through dissociation of Grb2-SOS (33-35). Furthermore, others have reported Raf-1 activation in unstimulated cells treated with the MKK1 inhibitor, PD98059 (60). It is possible that part of the stimulatory effect we observe with MKP-1 can be accounted for by relief of signal repression by ERK. However, it is unlikely that ERK inhibition accounts for all of the effects because our data were taken in cells treated under basal conditions and reproduced under serum-starved conditions where ERK activity is low or undetectable. Consistent with this, under basal or serum-starved conditions, we found no evidence for retardation in SOS gel mobility, which has been attributed to feedback phosphorylation by ERK- or MKK-dependent kinases (33, 34, 44). In addition, PDGF-induced SOS phosphorylation and gel mobility retardation were unaffected by MKP-1 expression, suggesting that altered SOS activity due to phosphorylation is not involved. The available data suggest the possibility that MKP-1 activates Raf-1 through Ras, involving a mechanism that may not involve inactivation of ERK.
The incomplete effect of Ras (S17N) on MKP-1 activation of MKK suggests that Ras-independent mechanisms for Raf-1 activation might also be regulated by MKP-1. Ras-independent means of Raf-1 activation might occur through Raf dimerization or through direct phosphorylation by tyrosine kinases, protein kinase C, ceramide-activated protein kinase, or kinase suppressor of ras, KSR (11-13, 45-53). Raf-1 inhibition may involve phosphorylation of Ser-43 or Ser-621 by protein kinase A (54-56). Alternatively, Raf-1 may be regulated by interactions with other signaling components such as 14-3-3 proteins (57-59). It is conceivable that alternative mechanisms for Raf-1 activation may be regulated indirectly through dephosphorylation of cellular targets by MKP-1. A significant implication of our study is that MKP-1 may alter the flux through different Raf-1-dependent pathways, thus providing a means of uncoupling different regulatory pathways downstream of Ras. For example, Raf-1 has been shown to phosphorylate other proteins, including IkB and the proapoptotic Bad protein (61-63), suggesting that it targets other substrates and downstream pathways in addition to MKK1 and MKK2 and the ERK pathway. Under some conditions, signaling through Raf-1 may also activate p70 S6 kinase through an ERK-independent mechanism (64). Our results would predict that induction of MKP-1 (e.g. through immediate early gene expression or activation of stress-activated protein kinase pathways) might enhance signaling through alternative Raf-1 targets under conditions where ERK signaling is suppressed. Another possibility that has yet to be demonstrated is that MKK targets other cellular substrates in addition to ERK, in which case phosphorylation of these substrates might also be enhanced by MKP-1.
In summary, this study reveals the ability of MKP-1 to serve as a positive regulator of Raf-1 signaling and presents an important consideration for studies utilizing MKP-1 as an inhibitor of ERK signaling. The results suggest a novel approach to address Raf-1 regulation by both Ras-independent and dependent mechanisms, and provide the groundwork for future studies examining the control of multiple signaling pathways downstream of Raf-1.
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ACKNOWLEDGEMENTS |
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We thank Hong Sun, Nicholas Tonks, Melanie Cobb, Peter Shaw, Ian Macara, Roger Davis, Ami Aronhein, and Michael Karin for providing several cDNA constructs used in these studies. We also thank Anne M. Whalen and Tim Lewis for critical reading of the manuscript.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants R01 GM48521 (to N. G. A.) and F32 GM18151 (to P. S. S.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed: Dept. of Chemistry and Biochemistry, Campus Box 215, University of Colorado, Boulder, CO 80309. Tel.: 303-492-7794; Fax: 303-492-2439.
1 The abbreviations used are: MAP, mitogen-activated protein; MKP-1, MAP kinase phosphatase-1; MKK, mitogen-activated protein kinase kinase; ERK, extracellular signal-regulated kinase; HA, hemagglutinin; SOS, Son of Sevenless Ras-guanine nucleotide exchange factor; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; PAGE, polyacrylamide gel electrophoresis; CAT, chloramphenicol acetyltransferase; TBS, Tris-buffered saline; PDGF, platelet-derived growth factor.
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REFERENCES |
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