From the Institut de Recherches Cliniques de
Montréal and the Department of Pharmacology, Université de
Montréal, Montreal, Quebec H2W 1R7, Canada, the
§ Biotechnology Research Institute, National Research
Council of Canada, Montreal, Quebec H4P 2R2, Canada, and the
Biology Department, McGill University, Montreal,
Quebec H3A 1B1, Canada
Received for publication, November 21, 2002, and in revised form, December 20, 2002
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ABSTRACT |
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The MAPKKs MEK1 and MEK2 are activated by
phosphorylation, but little is known about how these enzymes are
inactivated. Here, we show that MEK1 is phosphorylated in
vivo at Ser212, a residue conserved among all MAPKK
family members. Mutation of Ser212 to alanine enhanced the
basal activity of MEK1, whereas the phosphomimetic aspartate mutation
completely suppressed the activation of both wild-type MEK1 and the
constitutively activated MEK1(S218D/S222D) mutant. Phosphorylation of
Ser212 did not interfere with activating phosphorylation of
MEK1 at Ser218/Ser222 or with binding to ERK2
substrate. Importantly, mimicking phosphorylation of the equivalent
Ser212 residue of the yeast MAPKKs Pbs2p and Ste7p
similarly abrogated their biological function. Our findings suggest
that Ser212 phosphorylation represents an evolutionarily
conserved mechanism involved in the negative regulation of MAPKKs.
Mitogen-activated protein kinase
(MAPK)1 pathways are
evolutionarily conserved signaling modules by which cells transduce
extracellular chemical and physical signals into intracellular
responses (reviewed in Refs. 1-3). These modules are organized into an
architecture of three sequentially acting protein kinases comprising a
MAPK kinase kinase (MAPKKK or MEK kinase), a MAPK kinase (MAPKK or MEK), and the MAPK itself. The propagation of the signal through MAPK
pathways is facilitated by specific protein-protein interactions between individual components of the pathway and scaffolding
proteins (3, 4).
The prototypical and most studied MAPK pathway is the ERK1/2 pathway,
which controls cell proliferation, differentiation, and development
(1). Stimulation of cells with growth and differentiation factors leads
to the activation of the MAPKKK Raf by a complicated mechanism
involving cellular relocalization and multiple phosphorylation events
(5, 6). Activated Raf isoforms bind to and activate the MAPKKs MEK1 and
MEK2 by phosphorylation of two serine residues (corresponding to
Ser218 and Ser222 in MEK1) in their activation
loop (7, 8). Substitution of the two regulatory serines with acidic
residues is sufficient to enhance the basal activity of MEK1/2 (7-12).
The dual-specificity kinases MEK1 and MEK2 then catalyze the
phosphorylation of the MAPKs ERK1 and ERK2 at threonine and tyrosine
residues within the activation loop motif Thr-Glu-Tyr (13), causing a
reorientation of the loop and activation of the enzyme (14). Both MEK1
and MEK2 stably associate with ERK1/2, and this association is required for efficient activation of the latter in cells (15, 16). The binding
site for ERK1/2 is located at the N terminus of MEK1/2 and consists of
a short basic region known as the D domain (16). MEK1 and MEK2 also
contain a unique proline-rich insert between subdomains IX and X, which
is required for full activation of ERK1/2 in intact cells (17, 18).
The magnitude and duration of MAPK activation are important
determinants of the cellular response to extracellular signals (19,
20). Therefore, a tightly regulated balance between activation and
inactivation mechanisms must exist to control the cellular activity of
ERK1/2. Inactivation of the ERK1/2 enzymes is mainly achieved by
dephosphorylation of the activating threonine and tyrosine residues.
Biochemical and genetic studies have implicated both tyrosine-specific
phosphatases and dual-specificity MAPK phosphatases in the negative
regulation of ERK1/2 and other MAPKs (21, 22). Much less is known about
the mechanisms that negatively regulate the pathway at the MAPKK level.
The serine/threonine phosphatase protein phosphatase 2A was identified
as the major phosphatase inactivating MEK1 in lysates of PC12 cells
(23). Furthermore, overexpression of SV40 small t antigen, which binds to the A subunit of protein phosphatase 2A and inactivates the enzyme,
was found to stimulate MEK and ERK activity in CV-1 cells (24). It is
not known whether protein phosphatase 2A activity for MEK1/2 is
regulated. Feedback inhibition of MEK1/2 activity may also occur by
direct phosphorylation. Several protein kinases, including Cdc2 (25),
ERK1/2 (9, 26-29), and Pak1 (30), have been shown to phosphorylate
MEK1 at sites that are phosphorylated in intact cells. However, the
impact of these phosphorylation events on the regulation of the ERK1/2
pathway remains uncertain. Here, we show that MEK1 is phosphorylated at
Ser212 in intact cells. Substitution of Ser212
with Ala enhanced the basal activity of MEK1 and MEK2, whereas phosphomimetic mutants completely inactivated the enzymes in
vivo. We further show that mutations of the analogous
Ser212 residue in the yeast MAPKKs Pbs2p and Ste7p
similarly regulate their biological activity.
Cell Culture and Transfections--
Rat1 fibroblasts were
cultured and synchronized by serum starvation as previously described
(31). Rat1 cells were transfected with MEK1 expression plasmids using
Lipofectin (Invitrogen). After 48 h, populations of stably
transfected cells were selected by their ability to grow in complete
minimum Eagle's medium containing 0.5 mg/ml Geneticin
(Invitrogen). Human embryonic kidney (HEK) 293 cells were cultured in
Dulbecco's modified Eagle's medium supplemented with 10% fetal
bovine serum and were growth-arrested by serum starvation for 24 h. The cells were transiently transfected by the calcium phosphate
precipitation method.
Plasmid Constructs and Mutagenesis--
The sources of the
plasmids used in this study were as follows: pGEX-2T/MEK2 (K.-L. Guan,
University of Michigan, Ann Arbor, MI), pMT3-HA-SEK1 (J. Woodgett,
Ontario Cancer Institute, Toronto, Canada), and pEF-Myc-MKK6 (A. Nebreda, European Molecular Biology Laboratory, Heidelberg,
Germany). The plasmid pFA-Elk-1, which encodes a Gal4-Elk-1
fusion protein, and the Gal4-dependent luciferase reporter
plasmid pFR-Luc were obtained from Stratagene.
The XbaI/HindIII fragment of pGEX-MEK1,
containing the entire human MEK1 coding sequence (32), and the
EcoRI/PvuII fragment of pGEX-MEK2 (33),
containing the human MEK2 coding sequence, were subcloned into pALTER-1
(Promega). To generate HA-tagged constructs of MEK1 and MEK2, a
synthetic oligonucleotide encoding the amino acid sequence YDVPDYASL
was inserted at the N terminus of the respective cDNAs (after the
initiator methionine) using the Altered Sites in vitro
mutagenesis system (Promega). HA-MEK1 and HA-MEK2 cDNA constructs
were then used as templates for in vitro mutagenesis to
generate the various mutants described in this study. All mutations
were confirmed by DNA sequencing. The HA-MEK1 and HA-MEK2 constructs
were subcloned into the expression vector pRc/CMV (Invitrogen).
Immunoblot Analysis and Protein Kinase Assays--
Cell lysis,
immunoprecipitation, and immunoblot analysis were performed as
described previously (34). Commercial antibodies were obtained from the
following suppliers: anti-phospho-Ser218/Ser222
MEK1/2 (Cell Signaling Technology) and anti-MEK1 (Transduction Laboratories). Monoclonal antibody 12CA5 raised against influenza was a
gift from M. Dennis (SignalGene). Immunoblot analysis of MEK1/2
activating loop phosphorylation was carried out according to the
manufacturer's specifications. The phosphotransferase
activities of endogenous or ectopically expressed MEK1 and MEK2 were
assayed by measuring their ability to increase the myelin basic protein kinase activity of recombinant ERK2 in vitro as previously
described (35).
Luciferase Reporter Gene Assays--
For reporter gene assays,
293 cells seeded in 24-well plates were cotransfected with 1 µg of
pFR-Luc reporter construct, 50 ng of pFA-Elk-1, 300 ng of pCMV- HPLC Purification and N-terminal Sequencing of
Phosphopeptides--
For analysis of phosphorylated peptides, 10 Petri
dishes (100 mm) of HEK 293 cells were transfected with HA-MEK1, and two of the dishes were metabolically labeled for 6 h with 2 mCi/ml [32P]phosphoric acid. Cell lysates were prepared, and
HA-MEK1 was immunoprecipitated as described above. The
immunoprecipitated proteins were resolved by SDS-gel electrophoresis,
and the gel was stained with Coomassie Brilliant Blue R-250 and exposed
to x-ray film. The protein band corresponding to
32P-labeled HA-MEK1 was excised from the gel, subjected to
dithiothreitol reduction and iodoacetamide alkylation, and then
digested overnight at 37 °C with 0.2 µg of sequencing-grade
trypsin (Promega) (36). The tryptic peptides were extracted with 1%
trifluoroacetic acid and 60% acetonitrile at 60 °C and separated by
reverse-phase HPLC on a Vydac microbore C18 column
using an Applied Biosystems 130A separation system. The column was
developed at a flow rate of 150 µl/min using the following gradient
program: 3 min in solvent A (0.1% trifluoroacetic acid in water),
0-50% solvent B (0.08% trifluoroacetic acid in 70% acetonitrile)
during the next 60 min, and 50-100% solvent B during the remaining 7 min. The peptides were detected by absorbance at 220 nm, and the peaks
were collected manually and subjected to Cerenkov counting to identify
the radioactive phosphopeptides. Where necessary, HPLC-purified tryptic
peptides were subjected to a second digestion with sequencing-grade
endoproteinase Asp-N (Roche Molecular Biochemicals). The HPLC fractions
were incubated for a total time of 5 h at 37 °C with two
additions of 0.1 µg of Asp-N protease. The labeled peptides were
applied to a Prosorb disc (Applied Biosystems) and subjected to
automatic Edman degradation on a Procise Model 494 cLC sequencer using
the general protocol of Hewick et al. (37). The
phenylthiohydantoin-derivatives were analyzed on-line using an Applied
Biosystems Model 140D capillary separation system and ultraviolet detection.
Yeast Strains and Standard Methods--
The yeast strains used
in this study were W303-1A In Vivo Recombination and Construction of Mutant
Plasmids--
The construction of both PBS2 and
STE7 plasmids and their mutant alleles was performed using
the in vivo recombination procedure in the yeast
Saccharomyces cerevisiae according to Jansen et
al.2 Two backbone
plasmids (low copy number) with the promoter region and the N-terminal
part of either PBS2 or STE7 were first
constructed: 1) pGREG506-PBS2-N, containing 701 bp of the
PBS2 promoter region and the first 507 amino acids of
PBS2 coding sequence followed by an added unique
NotI site and 2) pGREG506-STE7-N, containing 550 bp of the
STE7 promoter region and the first 352 amino acids of
STE7 coding sequence followed by a NotI site. To
generate the mutant plasmid constructs by the in vivo
recombination procedure, the backbone plasmids were first digested with
NotI and XhoI and co-transformed into the
appropriate yeast strain with the respective C-terminal parts of the
genes carrying the desired mutations generated by PCR with mutant
primers. The resulting mutants were sequenced to confirm the desired
mutation and subcloned into the Gal1-GST yeast expression vector
pGREG546 to verify the expression of the mutant proteins by anti-GST
immunoblot analysis.
Transient Activation of MEK1 Contrasts with Sustained Regulatory
Loop Phosphorylation in Mitogen-stimulated Cells--
MEK1 is
activated by phosphorylation at Ser218 and
Ser222 in the regulatory loop between kinase subdomains VII
and VIII. To better understand the regulation of MEK1 activity, we
monitored the enzymatic activation and
Ser218/Ser222 phosphorylation of MEK1 after
serum stimulation of Rat1 fibroblasts. Detailed kinetic analysis
revealed that MEK1 activation was very transient, reaching a peak at 5 min and returning to near basal levels by 15-30 min (Fig.
1A). A similar transient
activation of endogenous MEK1/2 has been observed in other cell types
(Ref. 23 and data not shown). In contrast, the phosphorylation of activating Ser218/Ser222 residues, which was
maximally induced at 3 min, was sustained for at least 3 h after
serum addition (Fig. 1B). These results indicate that
mechanisms other than dephosphorylation of regulatory Ser218/Ser222 residues must contribute to
inactivation of MEK1.
MEK1 Is Phosphorylated at Ser212 in
Vivo--
Phosphopeptide mapping analysis has revealed that MEK1 is
phosphorylated on multiple peptides in both quiescent and
serum-stimulated cells (Refs. 17 and 26 and data not shown), suggesting
that phosphorylation of residues other than the
Ser218/Ser222 activation loop may also be
involved in the regulation of the kinase. We initiated a series of
experiments to identify new regulatory phosphorylation sites of MEK1.
HEK 293 cells were transfected with HA-MEK1 and deprived of serum for
24 h. The cells were then metabolically labeled with
[32P]orthophosphate for 5 h, and ectopically
expressed MEK1 was immunoprecipitated with anti-HA antibody. After
resolution by SDS-gel electrophoresis, the 32P-labeled MEK1
protein band was cut from the gel, alkylated, and subjected to complete
in-gel trypsin digestion. The resulting tryptic peptides were separated
by reverse-phase HPLC, and the fractions recovered were counted for
radioactivity (Fig. 2A). The
radioactive fractions were subjected to automated Edman degradation, and the phenylthiohydantoin-derivatives were analyzed using a sensitive
capillary separation system. The fraction eluting at 49 min was found
to contain the peptide LCDFGVSGQLIDXMAN(S)FV, which corresponds to the
tryptic fragment Leu206-Arg227 of the human
MEK1 sequence (Fig. 2A). This peptide contains four potential phosphorylation sites: Ser212,
Ser218, Ser222, and Thr226. To
refine our analysis, the HPLC fractions containing the
Leu206-Arg227 fragment were pooled and
subjected to a second digestion with endoproteinase Asp-N, which
cleaves before aspartate residues. Analysis of Asp-N digestion product
by HPLC revealed the presence of a major radioactive peak (Fig.
2B). N-terminal sequencing of this peak yielded the sequence
DFG, which corresponds to the double-digested peptide
Asp208-Ile216. The only phosphorylatable
residue within this peptide is Ser212. These results
unambiguously demonstrate that MEK1 is phosphorylated at
Ser212 in vivo.
Ser212 Regulates the Activity of MEK1 and
MEK2--
Alignment of MAPKK sequences from different species revealed
that Ser212, which lies in the activation loop between
kinase subdomains VII and VIII, is conserved in all members of the
MAPKK family from yeast to mammals (Fig.
3). However, this residue is not found in
Raf MAPKKKs, MAPKs, cyclin-dependent kinases, or
cAMP-dependent protein kinase. Notably, replacement of
Ser212 with aspartic acid was shown to completely abolish
the basal kinase activity of MEK1 in vitro (40). To evaluate
the impact of Ser212 on the regulation of MEK1 activity in
intact cells, we generated a series of MEK1 mutants by site-directed
mutagenesis. The various HA-MEK1 constructs were transiently expressed
in HEK 293 cells, and their phosphotransferase activity was measured
using a specific ERK2 reactivation assay. Immunoblotting of total cell
extracts with anti-HA antibody confirmed that all mutants were
expressed to similar levels (Fig.
4A). Replacement of
Ser212 with alanine significantly enhanced the enzymatic
activity of MEK1 (from 3- to 5-fold) in exponentially growing HEK 293 cells, whereas mutation to the phosphomimetic acidic residue aspartate completely abolished it (Fig. 4A). As previously reported,
substitution of the activating phosphorylation sites Ser218
and Ser222 with acidic residues (S218D/S222D)
strongly potentiated the activity of MEK1, whereas substitution with
alanine residues (S218A/S222A) impaired activation. Replacement of
Ser212 with alanine did not further enhance the activity of
the MEK1(S218D/S222D) mutant, nor did it rescue the compromised
activation of the S218A/S222A mutant. However, substitution of
Ser212 with aspartate completely abrogated the constitutive
activation of the MEK1(S218D/S222D) mutant. We also tested whether the
equivalent Ser216 residue of the related MAPKK MEK2 had
similar regulatory effects. As shown in Fig. 4B, replacement
of Ser216 with alanine increased the basal activity of
MEK2, whereas the aspartate mutation completely suppressed the
activation of wild-type MEK2 and the constitutively activated
MEK2(S222D/S226D) mutant.
To examine the functional consequences of MEK1 regulation by
Ser212, we tested the ability of MEK1 mutants to potentiate
the transcriptional activation of the ERK1/2 target Elk-1 in
exponentially growing HEK 293 cells. Under these experimental
conditions, Elk-1-dependent reporter activity was not
significantly enhanced by expression of the wild-type MEK1 protein
(Fig. 4C). However, expression of the MEK1(S212A) mutant
caused a small but reproducible 2-fold stimulation of Elk-1
transcriptional activity. In agreement with the results of enzymatic
assays, transfection of activated MEK1(S218D/S222D) strongly
potentiated Elk-1-dependent transcription, and this effect was completely prevented by substitution of Ser212 with a
phosphomimetic Asp residue.
To further investigate the role of Ser212 in the regulation
of MEK1 activity, we generated populations of Rat1 fibroblasts stably expressing HA-MEK1 Ser212 mutants. The cells were made
quiescent by serum starvation and restimulated for different period of
times with serum, and the activity of ectopically expressed MEK1 was
measured. Similar to the endogenous protein, activation of ectopic MEK1
was transient, reaching a peak at 5 min and returning to basal levels
by 30 min (Fig. 5A). The
MEK1(S212A) mutant displayed constitutive activity in serum-deprived
cells. Stimulation with serum induced a further increase in MEK1(S212A)
activity at 5 min, which declined thereafter, but remained elevated for
at least 24 h. Mutation of Ser212 to Asp lowered the
basal activity of MEK1 and abrogated activation of the enzyme by serum
growth factors. Immunoblot analysis confirmed that the mutants were
expressed at levels comparable to the wild-type protein (Fig.
5B). These results are consistent with the idea that
phosphorylation of Ser212 plays a role in the regulation of
MEK1/2 activity and of downstream signaling events.
Mutations of Ser212 Do Not Modulate Activating Loop
Phosphorylation of MEK1 or Binding to ERK2--
MEK1 is activated by
phosphorylation of Ser218/Ser222 in the
activation loop. We therefore tested whether the effects of
Ser212 mutations on MEK1 activity could be related to
differences in activating phosphorylation of the kinase. For these
studies, HA-MEK1 constructs were transiently expressed in HEK 293 cells. The cells were serum-starved and restimulated with serum for 5 min, and the phosphorylation of MEK1 at
Ser218/Ser222 was analyzed by immunoblotting
using a phospho-specific antibody. Mutation of Ser212 to
Ala or Asp did not affect the phosphorylation of MEK1 at activating Ser218/Ser222 residues in serum-stimulated
cells (Fig. 6A).
We also investigated whether Ser212 mutations interfere
with the ability of MEK1 to bind its substrates ERK1 and ERK2. Cell
extracts prepared from HEK 293 cells transiently transfected with
HA-MEK1 constructs were incubated with His6-ERK2 beads, and
the resulting complexes were analyzed by anti-HA immunoblotting. No
differences were observed in the abilities of the various MEK1 mutants
to bind ERK2 in this pull-down assay (Fig. 6B). Similar
results were obtained in co-immunoprecipitation experiments (data not
shown). These observations indicate that Ser212 mutations
are unlikely to alter the global three-dimensional structure of the
MEK1 enzyme. They also demonstrate that the inactivation of MEK1
observed upon mutation of Ser212 to a phosphomimetic
residue cannot be explained by inhibition of activating loop
phosphorylation or by interference with substrate binding.
Mechanism of MAPKK Inactivation by Phosphorylation Is Conserved in
Yeast--
To determine whether the inhibitory mechanism of MAPKK
regulation by phosphorylation has been conserved during evolution, we
extended our studies to the yeast S. cerevisiae
STE7 and PBS2 MAPKK genes (41). The
STE7 and PBS2 gene products, Ste7p and Pbs2p,
display significant amino acid sequence identity to mammalian MEK1/2.
Ser212 in MEK1 corresponds to Ser353 in Ste7p
and Ser508 in Pbs2p (Fig. 3). Mutations of the
corresponding serine residues in Ste7p and Pbs2p were made by
site-directed mutagenesis, and the resulting mutants were subcloned
into a low copy yeast shuttle plasmid vector by in vivo
recombination in yeast. The function of these alleles was tested in
yeast strain W303-1A
The mating ability of the yeast S. cerevisiae requires the
function of Ste7p. Strain W303-1A
Corresponding mutations were also made in the PBS2 gene. The
Pbs2p signaling pathway is required for the hyperosmolarity stress response, and cells defective in Pbs2p function are unable to grow on
hyperosmotic medium. The sensitive yeast strain TM260 was transformed
with different alleles of PBS2, and the transformants were
tested for their ability to grow on hyperosmotic medium. Wild-type
Pbs2p allowed the growth of the hyperosmolarity-sensitive cells on
medium containing 0.9 M NaCl, and the S508A mutant
displayed a similar phenotype (Fig. 7A). In contrast,
replacement of Ser508 with an aspartate residue blocked the
growth of TM260 cells on hyperosmotic medium, suggesting that the S508D
mutation, similar to the corresponding mutation in STE7,
results in a nonfunctional allele of the MAPKK protein. This loss of
function was not due to differences in expression levels, as both
wild-type and mutant Pbs2p-GST fusion proteins were expressed at
comparable levels (data not shown).
Pbs2p Negative Regulatory Phosphorylation Site Ser508
Acts in a Dominant Manner--
It has been shown that substitution of
Ser514 and Thr518 with phosphomimetic amino
acid residues (either Glu or Asp) leads to constitutively activated
forms of Pbs2p (42). We changed these two residues to aspartate
residues to obtain a constitutively activated Pbs2p kinase
(Pbs2p(S514D/T518D)). Unlike wild-type Pbs2p, whose activity requires at least one of the upstream activating kinases Ssk2p, Ssk22p,
or Ste11p, Pbs2p(S514D/T518D) was able to activate the HOG pathway
independent of these activating kinases. To assess the regulatory
effect of Ser508 on the constitutively activated
Pbs2p(S514D/T518D) mutant, substitution of Ser508 with
either Ala or Asp was made in combination with the S514D/T518D mutation. The resulting constructs were transformed into strains YCW340
(
To test whether mutation of the dominant inhibitory phosphorylation
site Ser508 to alanine is sufficient to render Pbs2p
constitutively activated, wild-type Pbs2p, Pbs2p(S508A), and
Pbs2p(S508D) were transformed into strains YCW340, YCW365, and YGJ208
and assayed for activation of the HOG pathway. As expected, no HOG
pathway activity was observed in any strain transformed with
Pbs2p(S508D) under all conditions tested (Fig. 7B). This is
consistent with previous observations that
SHO1-STE11/STE50 signaling is essential in
the absence of the SLN1 two-component osmosensor
branch that activates the MAPKKKs Ssk2p and Ssk22p (38, 43). However,
Pbs2p(S508A) displayed a significant increase in
SHO1-independent HOG pathway activity, as judged by the
ability of cells to grow in medium containing 0.9 M NaCl.
However, this activity was not observed when either STE11 or
STE50 was deleted in the absence of SSK2 and
SSK22. Thus, the HOG pathway activity observed was
SHO1-independent, but STE11- and
STE50-dependent.
Enzymatic activation of MEK1 requires phosphorylation of
Ser218 and Ser222 in the activation loop (7,
8). However, the mechanisms responsible for MEK1/2 inactivation remain
to be established. Our observation that sustained phosphorylation of
MEK1 at regulatory Ser218/Ser222 residues
contrasts with the transient nature of MEK1 activation in Rat1
fibroblasts led us to believe that mechanisms other than the simple
involvement of protein phosphatases are involved in MEK1 inactivation.
MEK1 is phosphorylated on multiple peptides in cells, suggesting that
phosphorylation of residues other than Ser218 and
Ser222 might be involved in other aspects of MEK1
regulation (17, 26). Here, we have reported that MEK1 is phosphorylated
at Ser212 in intact cells. Importantly, we have provided
biochemical and genetic evidence that phosphorylation of the equivalent
Ser212 residue in human MEK1 and MEK2 and in the yeast
MAPKKs Ste7p and Pbs2p negatively regulates enzymatic activity in
vivo. These findings suggest that both activation and inactivation
of MAPKK family members are mediated by common evolutionarily conserved mechanisms.
Replacement of Ser212 with acidic residues does not prevent
activating phosphorylation of MEK1 at
Ser218/Ser222, nor does it affect binding to
ERK2 substrate, thereby suggesting that Ser212
phosphorylation may directly interfere with the catalytic reaction. Consistent with this hypothesis, a previous study has shown that substitution of Ser212 with aspartate completely abolishes
the basal kinase activity of MEK1 for exogenous substrates in
vitro (40). Conversely, replacement of Ser212 was
alanine was shown to increase the rate of autophosphorylation of
recombinant MEK1 (44) and to enhance the basal phosphotransferase activity of MEK1-GST by 3-4-fold (8) in in vitro kinase
assays. We also observed that the equivalent S212A mutation
significantly increases the enzymatic activity of MEK1 and MEK2 in
intact cells (Fig. 4). It is noteworthy that Ser212 is
localized within the activation loop of MEK1, close to the activating
phosphorylation sites. Although Ser212 phosphorylation does
not interfere with phosphorylation of
Ser218/Ser222, the presence of an additional
phosphate group might compete for or establish undesirable
electrostatic interactions with one or more basic residues in the
catalytic domain. Thus, Ser212 phosphorylation may hinder
the correct positioning of the aspartate residue essential for
catalysis or perturb the conformation of the activation loop, blocking
access of the substrate to the active site. Given the evolutionarily
conserved nature of the MAPKK family, elucidation of the crystal
structure of MEK1 in the inactive and active conformations will add
greatly to our understanding of the mechanisms controlling both
activation and inactivation of this family of enzymes.
Studies by different groups have shown that MEK1 is also phosphorylated
at Thr292, Ser298, and Thr386
in vivo (9, 25-28, 30). However, the exact biological
consequences of these phosphorylation events remain to be established.
It has been suggested that the MAPKs ERK1 and ERK2 phosphorylate MEK1 at Thr292/Thr386 and inhibit its activation by
a negative feedback mechanism (26). In contrast, another study reported
that the MEK1(T292A) mutant is inactivated more rapidly than wild-type
MEK1 in serum-stimulated cells (17). We did not observe any effect of
the T292A mutation on MEK1 activity in exponentially growing 293 cells
(data not shown). In a more recent study, it was reported that Akt
phosphorylates MKK4 at Ser78 and negatively regulates its
activity by interfering with substrate binding (45). MKK4 is the only
member of the mammalian MAPKK family that has a consensus Akt
phosphorylation motif. It is likely that MAPKKs are regulated by
phosphorylation mechanisms common to all members as well as by more
subtle mechanisms that allow differential regulation of individual
isoforms. Identification of the physiological kinases and phosphatases
that control the phosphorylation level of Ser212 and other
regulatory sites will be necessary for a complete understanding of
MAPKK regulation.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-gal,
and 1 µg of MEK1 expression plasmids. The total DNA amount was kept
constant at 3 µg with the pRc/CMV vector. After 48 h, the cells
were harvested, and the activity of luciferase was assayed using a
luciferase reporter assay kit (Promega). Transfection efficiency was
normalized by measuring
-galactosidase activity.
ste7
(MATa ade2 leu2 trp1 his3 ura3
ste7::LEU2) (B. Errede, University of North Carolina, Chapel Hill, NC), TM260 (MATa
ura3 leu2 trp1
pbs2::LEU2) (H. Saito, Harvard
Medical School, Boston, MA), YCW340 (MATa
ura3 leu2 his3 trp1 ssk2::LEU2
ssk22::LEU2 ste11::KanR), YCW365
(MATa ura3 leu2 his3 trp1
ssk2::LEU2 ssk22::LEU2
ste50::TRP1) (38), and YGJ208
(MATa ssk2::LEU2 ssk22::LEU2 sho1::TRP1)
(this study). Yeast cells were transformed by the method described
(39), and the plasmid-containing cells were identified on selective
plates. Mating of
ste7 strains carrying the different
STE7 mutant alleles with the wild-type tester strain DC17
(MAT
his1) (laboratory collection) was performed for
7 h before replicating the cells onto plates selecting for
diploids. Cells with different PBS2 mutant alleles were
analyzed for osmosensitivity by transferring to rich medium containing
0.9 M NaCl and scoring growth after 3 days.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (20K):
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Fig. 1.
Time course of MEK1 activation and regulatory
loop phosphorylation in response to serum. A, quiescent
Rat1 fibroblasts were stimulated with 10% serum for the times
indicated. exp, exponentially proliferating cells. Cell
lysates were prepared, and the activity of endogenous MEK1 was measured
using an ERK2 reactivation assay. B, cell lysates were
analyzed by sequential immunoblotting with a phospho-specific antibody
to MEK1 activation loop residues Ser218 and
Ser222 and with anti-MEK1 antibody. The results are
representative of four different experiments.
View larger version (20K):
[in a new window]
Fig. 2.
MEK1 is phosphorylated at Ser212
in intact cells. A, HEK 293 cells transfected with
HA-MEK1 were serum-starved for 24 h and metabolically labeled with
[32P]phosphoric acid. The 32P-labeled HA-MEK1
protein was immunoprecipitated, resolved by SDS-gel electrophoresis,
and digested in-gel with trypsin. The resulting peptides were then
purified by reverse-phase HPLC. The radioactivity of each fraction was
determined by Cerenkov counting. The sequence of the radioactive
peptide eluting at 49 min was identified by automated Edman degradation
and analysis of phenylthiohydantoin-derivatives. B, the
purified tryptic peptide identified in A was further
digested with endoproteinase Asp-N, and the digestion products were
purified by HPLC. The sequence of the major 32P-containing
peptide was determined by automated amino acid sequencing.
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Fig. 3.
Conservation of Ser212 in the
MAPKK family. Shown is an alignment of amino acid sequences
between subdomains VII and VIII from MAPKK family members and related
protein kinases. Residues equivalent to Ser212 of MEK1 are
indicated. PKA, cAMP-dependent protein
kinase.
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Fig. 4.
Ser212 regulates the biological
activity of MEK1 and MEK2. A and B,
HEK 293 cells were transiently transfected with HA-tagged MEK1 or MEK2
constructs, respectively. After 48 h, the ectopically expressed
MEK protein was immunoprecipitated with anti-HA antibody
( HA), and phosphotransferase activity was measured using
an ERK2 reactivation assay. Expression of HA-tagged MEK1 and MEK2
proteins was analyzed by immunoblotting with anti-HA antibody.
C, HEK 293 cells were transfected with expression plasmids
for wild-type (wt) MEK1 or the indicated mutants in
combination with Gal4-Elk-1 and the Gal4-dependent
luciferase reporter gene. After 48 h, the activity of luciferase
was measured and normalized to that of
-galactosidase. Results are
presented as -fold activation over vector-transfected cells. All
results are representative of four different experiments. MEK1 mutants:
DD, S218D/S222D; AA, S218A/S222A; ADD,
S212A/S218D/S222D; AAA, S212A/S218A/S222A; and
DDD, S212D/S218D/S222D.
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Fig. 5.
Effect of Ser212 mutation on the
kinetics of MEK1 activation by serum. A, populations of
Rat1 cells stably expressing wild-type (wt) HA-MEK1 or
HA-MEK1 Ser212 mutants were made quiescent and restimulated
with 10% serum for the indicated times. The enzymatic activity of the
ectopically expressed MEK1 constructs was assayed as described above.
B, expression of MEK1 proteins was analyzed by
immunoblotting with anti-HA antibody ( HA). Results are
representative of three different experiments.
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Fig. 6.
Effect of Ser212 mutation on
activating phosphorylation of MEK1 and binding to ERK2 substrate.
A, HEK 293 cells were transiently transfected with
HA-MEK1 constructs. The cells were serum-starved for 24 h and then
restimulated with serum for 5 min. The ectopically expressed MEK1
protein was immunoprecipitated (IP) with anti-HA antibody
and analyzed by immunoblotting with
anti-phospho-Ser218/Ser222 MEK1/2 and
anti-HA antibodies. B, cell extracts from HEK 293 cells
transfected with HA-MEK1 constructs were incubated with immobilized
recombinant His6-ERK2 fusion protein. ERK2 complexes were
pulled-down with cobalt-agarose beads and analyzed by immunoblotting
with anti-HA antibody. Expression of the various MEK1 mutants in total
cell lysates was comparable. Results are representative of three
different experiments. wt, wild-type; AA,
S218A/S222A; AAA, S212A/S218A/S222A; DD,
S218D/S222D; ADD, S212A/S218D/S222D; DDD,
S212D/S218D/S222D.
ste7 for STE7-related functions and in yeast strain TM260 for PBS2-related functions.
ste7 has no functional
STE7 and therefore is unable to mate with a partner of
opposite mating type. Transformation of the wild-type STE7
gene into strain W303-1A
ste7 restores the mating ability
of the cells, whereas the empty vector does not. Mutation of Ste7p
Ser353 to alanine had no significant effect on mating
efficiency (Fig. 7A). However,
substitution of Ser353 with a phosphomimetic aspartate
residue led to a sterile phenotype, suggesting that Ste7p(S353D) is
nonfunctional. To rule out the possibility that Ste7p(S353D) is not
expressed or has decreased stability, the wild-type and mutant versions
of Ste7p were expressed in yeast as GST fusion proteins and analyzed by
immunoblotting. The results confirm that both the S353D and S353A
mutants have steady-state levels of expression similar to those of
wild-type Ste7p (data not shown).
View larger version (79K):
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Fig. 7.
Regulation of Ste7p and Pbs2p function
in vivo. A, left
panels, complementation assay of different alleles of Ste7p
in the mating pheromone-response pathway. ste7 strains
carrying different STE7 mutant alleles were mated with the
wild-type tester strain DC17, and diploids were selected. Right
panels, complementation assay of different alleles of Pbs2p in the
hyperosmolarity-response pathway.
pbs2 cells bearing
different PBS2 alleles were analyzed for growth on medium
containing 0.9 M NaCl. B, functional analysis of
the regulatory effect of Ser508 of Pbs2p and its
relationship with the upstream activator kinase. Yeast cells of strains
YCW340 (ssk2::LEU2
ssk22::LEU2 ste11::KanR;
no MAPKKK for Pbs2p), YCW365 (ssk2::LEU2
ssk22::LEU2 ste50::TRP1;
no activable MAPKKK for Pbs2p), and YGJ208
(ssk2::LEU2 ssk22::LEU2
sho1::TRP1; no membrane osmosensor) bearing
different PBS2 alleles were tested for osmosensitivity on
medium containing the indicated concentrations of NaCl. All MAPKK
constructs were expressed from their own promoters in low copy
centromere plasmids. The results are representative of three to
four different experiments. SD-ura, synthetic dextrose with
uracil; wt, wild-type; DD, S514D/T518D;
ADD, S508A/S514D/T518D; DDD,
S508D/S514D/T518D.
ssk2
ssk22
ste11), YCW365
(
ssk2
ssk22
ste50), and YGJ208 (
ssk2
ssk22
sho1) and
tested for activation of the HOG pathway. As shown in Fig.
7B, the S508D mutation completely blocked the ability of
Pbs2p(S514D/T518D) to activate the HOG pathway, whereas no significant
effect of the S508A mutation was observed on Pbs2p(S514D/T518D)
activity. These results indicate that the regulatory effect of
Ser508 is dominant over the effect of activating
phosphorylation of Pbs2p at Ser514 and
Thr518.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We thank J. Noel and M. Arcand for technical assistance; M. H. Cobb, K.-L. Guan, A. Nebreda, and J. Woodgett for reagents; and H. Saito and B. Errede for strains.
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FOOTNOTES |
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* This work was supported in part by grants from the Canadian Institutes of Health Research and the Cancer Research Society (to S. M.) and the National Research Council/Genome Health Initiative (to M. W.). This is National Research Council Publication 46140.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.
¶ Recipient of fellowships from the Deutsche Forschungsgemeinschaft and the National Research Council/NSERC.
** Investigator of the Canadian Institutes of Health Research. To whom correspondence should be addressed: Inst. de Recherches Cliniques de Montréal, 110 Pine Ave. West, Montreal, Quebec H2W 1R7, Canada. Tel.: 514-987-5783; Fax: 514-987-5536; E-mail: melochs@ircm.qc.ca.
Published, JBC Papers in Press, December 27, 2002, DOI 10.1074/jbc.M211870200
2 G. Jansen, C. Wu, B. Schade, D. Y. Thomas, and M. Whitney, submitted for publication.
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ABBREVIATIONS |
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The abbreviations used are: MAPK, mitogen-activated protein kinase; MAPKK/MKK, mitogen-activated protein kinase kinase; MAPKKK, mitogen-activated protein kinase kinase kinase; ERK, extracellular signal-regulated kinase; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; HEK, human embryonic kidney; HA, hemagglutinin; HPLC, high performance liquid chromatography; GST, glutathione S-transferase; HOG, high osmolarity glycerol.
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