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INTRODUCTION |
The mitogen-activated protein
(MAP)1 kinases are essential
participants in signal transduction pathways from the cell membrane to
the nucleus (1-3). There are three major MAP kinase cascades: the
extracellular signal-regulated protein kinase (ERK) pathway, which
responds to stimuli that induce proliferation, differentiation, and
oncogenic transformation; the c-Jun N-terminal protein kinase pathway;
and the p38 kinase pathway, the last two of which are activated
in response to environmental stresses and cytokines that mediate
inflammation. MAP kinase activity is tightly regulated by
phosphorylation and dephosphorylation. Activation of MAP kinase activity requires dual phosphorylation of both Thr and Tyr residues in
the activation loop motif TXY. One mechanism for MAP
kinase inactivation involves the action of MAP kinase phosphatases
(MKPs). The MKPs are dual specificity phosphatases that are capable of catalyzing the removal of the phosphoryl group from Tyr(P) as well as Thr(P). MKPs are unrelated to the serine/threonine protein phosphatases but belong to the protein tyrosine phosphatase (PTPase) superfamily (4).
At least nine mammalian MKPs have been identified (5, 6). These MKPs
share two common structural features: a conserved catalytic domain that
contains the PTPase active site signature motif
(H/V)CX5R(S/T) and an N-terminal domain
containing two conserved motifs that are also found in the cell cycle
regulator Cdc25 phosphatases. Interestingly, however, each MKP
appears to have distinct specificity for its substrate MAP kinase. For
example, MKP3 is predominantly localized in the cytoplasm and is highly
specific in dephosphorylating and inactivating ERK1/2 (7-10). The
N-terminal domain of MKP3 can physically associate with ERK1/2 (11),
and purified recombinant ERK2 stimulates the phosphatase activity of
MKP3 (12). We sought to understand how these findings contribute to the
specificity of MKP3 to ERK2.
To gain insight into the mechanism of ERK2-induced MKP3 activation, we
have carried out a detailed kinetic analysis of the MKP3-catalyzed aryl
phosphate hydrolysis both in the absence and presence of ERK2 (13). Our
data suggest that, like PTPases, MKP3 can exist either in a general
acid loop open or closed conformation. Normally, MKP3 is in the open
state in which the active site residues (Cys293 and
Arg299) are misaligned and the general acid
(Asp262) is positioned away from the active site (14). For
PTPases, the loop that contains the general acid is brought into the
active site as a result of the interactions between the phosphoryl
moiety in the substrate and the side chain of the active site Arg,
which in turn enables the Arg residue to interact with a hinge residue in the general acid loop (15, 16). A similar mechanism for loop closure
could also occur in MKP3 that allows the repositioning of catalytic
groups and the reorienting of the electrostatic environment at the
active site. However, unlike PTPases, the rate of this substrate-induced conformational change in MKP3 depends on the nature
of the substrates. It appears that with p-nitrophenyl
phosphate (pNPP) as a substrate, the interaction between the
substrate and MKP3 is not sufficient to allow the attainment of optimal
alignment of active site residues with respect to the substrate.
Because ERK2 is a physiological substrate of MKP3, it can engage in
specific protein-protein interactions with MKP3 and elicit full
activation of MKP3 activity by facilitating the repositioning of active
site residues and general acid loop closure in MKP3 (13, 17).
Catalytic activation by MAP kinases has also been observed with other
MKPs. In addition, catalytic activation mirrors the substrate
selectivity of MKPs; an MKP is only activated by its physiological
substrates (5, 6, 12). These observations suggest a general mechanism
by which all members of the MKP family are regulated; the MKPs exist in
latent, inactive states, and upon association with specific MAP kinase
substrates, the MKPs are activated, leading to selective inactivation
of MAP kinases. The molecular basis for the specific MKP activation by
MAP kinases is not understood. Our hypothesis is that molecular
determinants responsible for the specific MAP kinase-MKP interactions
are contained within the interacting proteins themselves. The goal of
the current study is to determine the structural features in MKP3 that
are important for ERK2 binding as well as ERK2-induced activation. Our
results provide a molecular basis by which MKP3 recognizes ERK2 showing
how specific interactions between ERK2 and MKP3 modulate the intrinsic
catalytic activity and possibly the substrate specificity of MKP3.
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EXPERIMENTAL PROCEDURES |
DNA Construction of MKP3 and Its Mutants--
The coding
sequence for the wild type MKP3 with a C-terminal His6 tag
was produced by PCR using the oligonucleotides
GGCCATATGATAGATACGCTCAGACCCG (NdeI site
underlined) and
GCCGAATTCTCAGGTGGTGGTGGTGGTGCGTAGATTGCAGGGAGTC (EcoRI site and His6 tag underlined) as the 5'
and 3' primers, respectively, using pGEX-4T3-MKP3 (a generous gift from
Dr. Marco Muda, University of Michigan) as a template. The PCR product, containing a 5' NdeI site and 3' EcoRI site and
nucleotides encoding MKP3 and a His6 tag immediately before
the stop codon, was subcloned into the pET21a vector (Novagen).
MKP3/C293S was produced by the same procedure using pGEX-4T3-MKP3/C293S
as the PCR template.
To produce C-terminal truncations of MKP3, the oligonucleotide
GGCCATATGATAGATACGCTCAGACCCG (NdeI site
underlined) was used as the 5' primer, and
GCCGAATTCTCAGTGGTGGTGGTGGTGGTGCAGGGTCCTTTCAAAGTC (for MKP3/1-347),
GCCGAATTCTCAGTGGTGGTGGTGGTGGTGAGAGTCCTTGGCACAGC (for MKP3/1-222), and
GCCGAATTCTCAGTGGTGGTGGTGGTGGTGCTGGCTGTTGGACAG (for MKP3/1-203) were used as the 3' primers (EcoRI site
and His6 tag underlined). To produce the N-terminal
truncations of MKP3 by PCR, the oligonucleotides
GCCCATATGGGCTCGTGCAGCAGCAGCT (for MKP3/152-381),
GCCATATGTCCTTCCCGGTGGAGATTTTG (for MKP3/204-381) were used
as the 5' primers (NdeI site underlined), and
GCCGAATTCTCAGTGGTGGTGGTGGTGGTGCGTAGATTGCAGGGAGTC was used as the 3' primer (EcoRI site and His6
tag underlined). To produce MKP3/204-347, the oligonucleotides
GCCATATGTCCTTCCCGGTGGAGATTTTG (NdeI site
underlined) and
GCCGAATTCTCAGTGGTGGTGGTGGTGGTGCAGGGTCCTTTCAAAGTC (EcoRI site and His6 tag underlined) were
used as the 5' and 3' primers, respectively. The coding sequence of
these truncated versions of MKP3, containing a 5' NdeI site
and 3' EcoRI site and nucleotides encoding a
His6 tag immediately before the stop codon, were amplified
by PCR from pGEX-4T3-MKP3 and were subcloned into the pET21a vector.
To produce MKP3/
KIM, in which amino acids 61-75 were deleted
from the MKP3 sequence, oligonucleotides
GCCCGGGTCTATTCACGCGCTGCGAGGA (SmaI site
underlined), and
GCCGAATTCTCAGTGGTGGTGGTGGTGGTGCGTAGATTGCAGGGAGTC (EcoRI site and His6 tag underlined) were
used to PCR-amplify the MKP3 sequence from residue 76 to 381 from
pGEX-4T3-MKP3 and to introduce SamI and EcoRI
restriction sites and a C-terminal His6 tag immediately
flanking the stop codon at the C terminus. Then the DNA fragment
encoding the sequence of MKP3 from 76-381 amino acids was ligated into
the plasmid pET21a-MKP3-His6 fragment containing residues
1-60 of MKP3 after digesting with SmaI and EcoRI.
The point mutations of MKP3 were generated by PCRs according to the
standard procedure of the QuickChangeTM site-directed
mutagenesis kit (Stratagene) using the pET21a-MKP3-His6 plasmid (or pET21a-MKP3/152-381-His6,
pET21a-MKP3/
KIM-His6) as a template. All MKP3 mutants
were verified by DNA sequencing.
Protein Expression and Purification--
Full-length MKP3 with a
C-terminal His6 tag was expressed and purified using
standard procedures of rapid affinity purification with the pET His tag
system (Novagen) with minor modifications. Briefly,
pET21a-MKP3-His6 was transformed into BL21(DE3) competent cells by standard procedure. A single colony was selected and grown in
LB medium (supplemented with 100 µg/ml ampicillin) at 37 °C
overnight. A 10-ml overnight culture was transferred to 1 liter of LB
medium (supplemented with 100 µg/ml ampicillin) and allowed to grow
at 37 °C until the absorbance at 600 nm was between 0.6 and 0.8. Following the addition of
isopropyl-thio-
-D-galactopyranoside to a final
concentration of 1 mM, the culture was incubated at room
temperature with shaking for an additional 6 h. The cells were
harvested by centrifugation at 5,000 rpm (Sorvall Centrifuge, SS-34
rotor) for 5 min. The cell pellets from 1 liter of culture were
resuspended in 30 ml of lysate buffer (20 mM Tris, pH 7.9, 500 mM NaCl, 5 mM imidazole). The cells were
lysed by passage through a French pressure cell press at 1,200 p.s.i.
twice. Cellular debris was removed by centrifugation at 16,000 rpm for
30 min. A 4-ml 50% slurry of Ni2+-nitrilotriacetic acid
agarose (Qiagen; preequilibrated by lysate buffer) was added to the
supernatant. After incubating with gentle agitation at 4 °C for
2 h, the matrix was transferred to a column and washed by 20 bed
volumes of lysate buffer and 20 bed volumes of 20 mM Tris,
pH 7.9, 500 mM NaCl, 20 mM imidazole. The
His6-tagged protein was eluted by washing the column with
10 bed volumes of 20 mM Tris, pH 7.9, 500 mM
NaCl, 100 mM imidazole. The elute was concentrated with a
Centriprep-30 filtration unit (Amicon), and the buffer was changed to
25 mM Tris, pH 7.5, 1 mM EDTA, and 2 mM dithiothreitol. The mutants of MKP3 were expressed and
purified by the same procedures as the wild-type MKP3. Protein
concentration of MKP3 and its mutants was determined using the Bradford
dye binding assay (Bio-Rad) diluted according to the manufacturer's recommendations with bovine serum albumin as standard.
pGEX-2T-ERK2 (a generous gift from Dr. Chris Marshall of the Institute
of Cancer Research, London) was used to transform BL21(DE3). GST-ERK2
fusion protein was expressed and purified as described (18). After
thrombin cleavage at room temperature for 1 h, ERK2 was eluted
from the GST-ERK2-bound glutathione-Sepharose 4B resin. The elute was
concentrated with a Centriprep-30 filtration unit (Amicon). Protein
concentration of ERK2 was determined using the absorbance at 280 nm
using an extinction coefficient of 1.10 ml mg
1 cm
1. All of the
purified proteins were made to 20% glycerol and stored at
80 °C.
Steady-state Kinetics--
The phosphatase activity of
MKP3 or its mutants was assayed using pNPP as a substrate at
30 °C in 50 mM 3,3-dimethylglutarate buffer, pH 7.0, containing 1 mM EDTA with an ionic strength of 0.15 M adjusted by the addition of NaCl. The reaction was
initiated by the addition of the enzyme to a reaction mixture (0.2 ml)
containing various concentrations of pNPP and quenched after
60 min by the addition of 0.05 ml of 5 N NaOH. The range of
substrate concentration used was 0.2-5 Km. The
nonenzymatic hydrolysis of the substrate was corrected by measuring the
control without the addition of enzyme. After quenching, 0.2 ml of
reaction mixture was transferred to a 96-well plate. The amount of
product p-nitrophenol was determined from the absorbance at
405 nm detected by a Spectra MAX340 microplate spectrophotometer
(Molecular Devices) using a molar extinction coefficient of 18,000 M
1 cm
1.
The Michaelis-Menten kinetic parameters were determined from a direct
fit of the velocity versus substrate concentration data to
Michaelis-Menten equation using the nonlinear regression program KinetAsyst (IntelliKinetics, State College, PA).
Determination of Dissociation Constants--
The dissociation
constants of MKP3 and its mutants for ERK2 were determined by the
activation assay or competitive binding assay at 30 °C and pH 7.0, in 50 mM 3,3-dimethylglutarate buffer, containing 1 mM EDTA with an ionic strength of 0.15 M
adjusted by the addition of NaCl. For MKP3 and its mutants possessing
phosphatase activity, the activation assay was used to determine the
binding affinity of MKP3 or its mutants for ERK2. In this assay, the
reaction was initiated by the addition of MKP3 in a reaction mixture
(0.2 ml) containing 40 mM pNPP and various
concentration of ERK2 and quenched after 60 min by the addition of 0.05 ml of 5 N NaOH. The enzymatic and nonenzymatic hydrolysis
of pNPP were measured as described above. The dissociation
constant Kd was calculated by fitting the absorbance
at 405 nm versus ERK2 concentration data to Equation 1,
where A is the absorbance at 405 nm of the sample in
the presence of ERK2; A0 is the absorbance at
405 nm in the absence of ERK2; A
is the
absorbance at 405 nm when the concentration of ERK2 is infinite;
CM is the MKP3 concentration, which is fixed at 0.1 µM; CE is the ERK2 concentration
during titration; and Kd is the dissociation
constant for ERK2 binding to MKP3.
For peptides corresponding to the kinase interaction motif (KIM) and
the N-terminal fragments of MKP3, which lack phosphatase activity, the
competitive binding assay was used to determine the binding affinity
for ERK2. In this assay, the reaction was initiated by the addition of
0.1 µM MKP3 in a mixture (0.2 ml) containing 20 mM pNPP, 1.2 µM ERK2, and various
concentration of the KIM peptide or the MKP3 mutant. The reaction was
stopped after 60 min by the addition of 0.05 ml of 5 N
NaOH. The enzymatic and nonenzymatic hydrolysis of pNPP were
measured as described above. The data were fitted to Equation 2 (19) by
nonlinear regression analysis,
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(Eq. 2)
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where the KIM peptide (or MKP3 mutant) and MKP3 are assumed to
bind ERK2 competitively, and MKP3 concentration is much smaller than
ERK2 concentration. A is the absorbance at 405 nm in the presence of KIM peptide or MKP3 mutant. A0 is
the absorbance at 405 nm in the absence of KIM peptide or MKP3 mutant.
A
is the absorbance at 405 nm when the
concentration of KIM peptide or MKP3 mutant is infinite.
C
is ERK2
concentration, which is fixed at 1.2 µM.
C
is peptide or MKP3
mutant concentration during titration.
K
is the
dissociation constant for MKP3 binding to ERK2 with a value of 0.17 µM (Table I), and
K
is the
dissociation constant of the peptide (or MKP3 mutant) for ERK2.
Isothermal Titration Calorimetry--
All isothermal titration
calorimetry experiments were performed using an MCS Isothermal
Titration Calorimetry System from Microcal Inc. (Northampton, MA).
Experiments at pH 7.0 were conducted at 25 °C, in 50 mM
3,3-dimethylglutarate buffer. The ionic strength of the buffer was
adjusted to 0.15 M by the addition of NaCl. The ERK2
concentration in the cell of the calorimeter was 90 µM, while the KIM peptide in the syringe was 1 mM. High
concentration stock solutions were prepared for the peptides with 50 mM 3,3-dimethylglutarate buffer. The stock was diluted to 1 mM with the buffer before titration. The binding data were
analyzed using the Origin software (20). The binding constant
K and the enthalpy change
H were used to calculate the free energy change
G and the entropy change
S according to
RTlnK =
G =
H
T
S, where R is the gas constant and
T is the absolute temperature.
GST Pull-down and Western blot Analysis--
The binding
interactions between ERK2 and MKP3 or its mutants were examined by GST
pull-down and Western blot assay. GST-ERK2 (10 µg) in 1 ml of
phosphate-buffered saline (140 mM NaCl, 2.7 mM
KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, 2 mM
dithiothreitol, pH 7.3) was immobilized on 20 µl of
glutathione-Sepharose 4B beads (Amersham Pharmacia Biotech) by
incubating with gentle agitation at 4 °C for 2 h. MKP3 or its
mutants (1 µg) in 200 µl of phosphate-buffered saline were
incubated with 20 µl of GST-ERK2 bound beads with gentle agitation at
4 °C for 2 h. After washing the beads with phosphate-buffered
saline buffer, 25 µl of 2× SDS sample buffer was added to the beads.
The proteins were released by boiling the beads for 5 min. The sample
was microcentrifuged at 10,000 rpm for 2 min. The supernatant (10 µl)
was loaded on 12.5% SDS-polyacrylamide gel. When the electrophoresis
was complete, the proteins on the gel were transferred to
nitrocellulose membrane using a Trans-Blot SD semidry electrophoretic
transfer cell (Bio-Rad) at 150 mA and room temperature for 1 h.
After blocking in 15 ml of 5% nonfat dry milk in TBS-T (20 mM Tris, 150 mM NaCl, 0.1% Tween 20, pH 7.6)
for 1 h at room temperature, the membrane was incubated with mouse
anti-His6 monoclonal antibody (sc-8036; Santa Cruz
Biotechnology, Inc.) with gentle agitation overnight at 4 °C. The
membrane was washed three times with TBS-T and incubated with goat
anti-mouse monoclonal antibody conjugated with horseradish peroxidase
(sc-2005; Santa Cruz Biotechnology) in 5% nonfat dry milk in TBS-T
with gentle agitation for 1 h at room temperature. The
immunocomplexes were detected by chemiluminescence upon incubation with
ECL reagents (Amersham Pharmacia Biotech). The membrane was immediately
exposed to Kodak BioMax Light Film for 10 s to 2 min to visualize
the bound MKP3.
Peptide Synthesis and Characterization--
Peptides were
synthesized on Rink amide resin (Advanced Chemtech, Louisville, KY)
using a standard protocol for
2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate/N-hydroxybenzotriazole/N-methylmorpholine activation of Fmoc-protected amino acid derivatives (Advanced Chemtech,
Louisville, KY). Side chains of Asp, Glu, Ser, and Thr were
tert-butyl-protected; Asn, Gln, and His were
trityl-protected; Lys was tert-butyloxycarbonyl-protected;
and Arg was protected with the
4-methoxy-2,3,6-trimethyl-benzenesulfonyl group.
The coupling reaction was performed in
N,N-dimethylformamide for 1.5 h using a
3-fold excess of amino acid. Fmoc removal was performed with 20%
piperidine in N,N-dimethylformamide. The N terminus of the peptides was acetylated with 2.5% acetic anhydride and
5% N,N-diisopropylethylamine in
N,N-dimethylformamide for 15 min. Final cleavage
and side chain deprotection was achieved with 1 M
trimethylsilyl bromide, 1 M thioanisole in trifluoroacetic acid with 5% 1,2-ethanedithiol and 1% m-cresol for 15 min
under a blanket of nitrogen at 0 °C. The resin was removed by
filtration, and the remaining trifluoroacetic acid solution was
concentrated under nitrogen flow. Dry diethyl ether was added, and the
precipitated peptides were collected by centrifugation. The peptides
were resuspended, washed twice with ether, dissolved in water, and
purified by semipreparative reverse phase HPLC. All peptides were
obtained in high yield (>80%) and in high purity (>95%, as analyzed
by matrix-assisted laser desorption ionization/time of flight mass
spectrometry and analytical HPLC).
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RESULTS AND DISCUSSION |
An Activation-based Assay for the Determination of Binding Affinity
between MKP3 and ERK2--
The catalytic activity of MKP3 against
pNPP increases when it binds to ERK2 (12). To determine the
stoichiometry of MKP3 binding to ERK2, the dependence of the
MKP3-catalyzed pNPP hydrolysis on the ratio of MKP3 to ERK2
was measured, while keeping the total concentration of MKP3 and ERK2
constant. Fig. 1 shows that maximum pNPP hydrolysis is reached at an MKP3 to ERK2 ratio of 1:1,
indicating a 1:1 binding stoichiometry between MKP3 and ERK2. We have
found that the activation of MKP3 by ERK2 is dose-dependent
and saturable (13). From the concentration dependence of ERK2 on the
kinetic parameters kcat/Km
(determined at low [pNPP], e.g. 0.1 mM, ~0.08 Km) or
kcat (determined at saturating
[pNPP], e.g. 40 mM, ~31
Km) for the MKP3-catalyzed reaction, we can
determine the dissociation constant (Kd) for
ERK2-MKP3 and ERK2-MKP3·pNPP complexes,
respectively. The Kd value of ERK2 for the
MKP3·pNPP complex determined by this activation-based assay is 0.17 ± 0.04 µM at pH 7.0 and 30 °C
(Fig. 2), which is similar to the
affinity of ERK2 for free MKP3 (data not shown). In addition, we can
also determine the maximal level of MKP3 activation upon the addition
of ERK2. The kcat increases 9-fold and the
Km decreases 8-fold for the MKP3-catalyzed
pNPP hydrolysis, resulting in a nearly 70-fold increase in
the second order rate constant, kcat/Km (Table
I). Thus, the concentration dependence of
activation of MKP3 pNPP phosphatase activity by ERK2
provides a powerful means to quantitatively assess the importance of
structural features in MKP3 for ERK2 recognition, both in terms of
binding affinity for ERK2 and the ability to be activated by ERK2.

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Fig. 1.
Job-plot for the binding of MKP3 to ERK2 at
pH 7.0 and 30 °C. pNPP concentration was 40 mM. The total concentration of MKP3 and ERK2 was 10 µM. The absorbance at 405 nm reached the maximum at a
MKP3/ERK2 ratio of 1:1.
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Fig. 2.
Concentration dependence of ERK2 on the
MKP3-catalyzed pNPP reaction. The concentration
of pNPP was 40 mM, and the concentration of MKP3
was 0.10 µM. The absorbance at 405 nm monitored the
amount of p-nitrophenol produced. The data were fitted to
Equation 1 to determine the Kd value of ERK2 for the
MKP3·pNPP complex (13). The Kd value
determined (0.17 ± 0.04 µM) is slightly lower than
that reported previously (13), due to differences in protein
concentration determination (Bradford dye binding assay for the
GST-ERK2 fusion protein in the early measurements and absorbance at 280 nm for ERK2 derived from thrombin cleavage of the GST fusion protein in
the current study).
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Table I
Binding affinities of MKP3 and its mutants with ERK2 and kinetic
parameters for MKP3 and its mutants in the presence and the absence
of ERK2
All experiments were performed at 30 °C and pH 7.0.
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MKP3 Contains a "Kinase Interaction Motif" Responsible for High
Affinity ERK2 Binding--
Because the N-terminal 1-221 of MKP3
(MKP3/1-221) is able to bind ERK2 (11), we initially focused on this
region to identify residues/regions important for ERK2 recognition.
Examination of the sequence alignment of all MKPs (Fig.
3) led to the identification of a short
sequence motif (residues 61-75 in MKP3) that resembles the KIM found
in PTP-SL and STEP (21), PTP-ER (22), HePTP (23), and possibly in the
yeast Ptp3 (24). The KIM sequences found in these PTPases are critical
for ERK2 binding and efficient dephosphorylation of Tyr(P) in the TEY
activation loop of ERK2. As this work was in progress, putative KIM
sequences characterized by a cluster of two or three positively charged
Arg or Lys residues have also been found in MAP kinase activators
(e.g. MEKs) and substrates (e.g. RSK1), in
addition to the MAP kinase inactivators (e.g. MKP3 and
HePTP) (25-28). It was suggested that all ERK2-interacting proteins
bind ERK2 through a common electrostatic interaction between the
positively charged KIM motif and the negatively charged Asp316 and Asp319 in ERK2 (27). This model
would predict a simple mutually exclusive competition among various MAP
kinase regulators and substrates for MAP kinases. It is not obvious how
specificity can be maintained in this model, which is, therefore, not
sufficient to explain the available biochemical data.

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Fig. 3.
Amino acid sequence alignments of the nine
MAP kinase phosphatases. Amino acid sequences of MKP3 (Q64346),
MKPX (Q99956), MKP4 (Q16829), MKP1 (X68277), MKP2 (S80632), PAC1
(L11329), hVH3 (I38890), hVH5 (U27193), and MKP5 (AB026436) were
aligned using ClustalW (1.7) (European Molecular Biology Laboratory)
(accession numbers of MKPs are given in parentheses). Amino acids of
MKP3 are numbered on the right. Residues in the black
boxes represent absolutely invariant residues among MKPs,
and residues in shaded boxes indicate conserved
substitutions.
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Although the KIM sequence has been shown, based mostly on GST pull-down
and/or immunoprecipitation experiments, to be important for ERK2
binding, the exact contribution of the KIM sequence in MKP3 to ERK2
binding and the ERK2 binding-induced MKP3 activation has not been
established. Furthermore, it is not known whether additional regions in
MKP3 are also required for specific ERK2 recognition and activation. To
further evaluate the importance of the KIM sequence in ERK2 recognition
and to identify additional residues/regions in MKP3 that are important
for ERK2 binding and activation, we have generated various MKP3 mutants
via site-directed, deletion, and truncation mutagenesis (Fig.
4). The effects of structural alterations
in MKP3 on ERK2 binding and activation have been determined using
purified MKP3 mutants and ERK2 and the activation-based assay outlined
above.
To determine the contribution of the KIM sequence in MKP3 to ERK2
binding and activation, we first deleted the putative KIM sequence
(residues 61-75) to generate MKP3/
KIM (Fig. 4). Using the
activation-based assay, the Kd of MKP3/
KIM for
ERK2 was determined to be 22.7 µM, which is 135-fold
higher than that of the wild-type MKP3 (Table I). This supports the
notion that this KIM motif is important for high affinity ERK2 binding.
When residues 1-151 or 1-203 were removed from MKP3 (Fig. 4), the
resulting MKP3/152-381 and MKP3/204-381 displayed
Kd values for ERK2 that were similar to that of
MKP3/
KIM (Table I). These results indicate that the KIM sequence is
a major determinant in the N-terminal domain of MKP3 responsible for
high affinity ERK2 binding. We then determined the importance of
several conserved residues in the KIM sequences of MKPs with preference
for ERK2 (Fig. 3, MKP3, MKPX, and MKP4). As shown in Table I, when the Arg residues at positions 64 and 74 were replaced by an Ala, the affinity of MKP3/R64A and MKP3/R74A for ERK2 decreased 7.3- and 3.4-fold, respectively. However, when Arg65 was replaced by
an Ala, the affinity of MKP3/R65A for ERK2 decreased 150-fold, which
was similar to that of the KIM deletion mutant MKP3/
KIM. Finally,
substitution of Leu71 with an Ala reduced the affinity of
MKP3/L71A for ERK2 by 2.6-fold. Thus, the most important residue in the
KIM sequence for ERK2 binding is Arg65.
To visualize MKP3 binding to ERK2 directly, we also performed GST
pull-down experiments. In these experiments, GST-ERK2 was used to bind
His6-tagged MKP3 and its mutants. The amount of MKP3 associated with ERK2 was estimated by anti-His6 antibody
(for details, see "Experimental Procedures"). As shown in Fig.
5A, the amount of MKP3 bound
to ERK2 in the GST pull-down assay correlates well with the relative
affinities of MKP3 for ERK2 determined by the activation assay.
However, the GST pull-down assay is not sensitive enough to detect
binding interactions with Kd values higher than 10 µM under our conditions.

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Fig. 5.
MKP3 and its mutants binding to ERK2 detected
by GST pull-down and Western blotting. A, effects of
mutations in the MKP3/KIM sequence on ERK2 binding. B,
effects of MKP3 N- or C-terminal truncations on ERK2 binding.
C, effects of mutations in residues 161-177 of MKP3 on ERK2
binding. His6-tagged MKP or its mutants were incubated with
GST-ERK2 bound to glutathione-Sepharose 4B resins. The amount of
MKP3 associated with ERK2 was visualized by anti-His6
monoclonal antibody (for details, see "Experimental
Procedures").
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It is noteworthy to point out that the KIM or N-terminal deletion MKP3
mutants and the MKP3 mutants with point mutations in the KIM sequence
all exhibited basal phosphatase activity similar to that of the
wild-type MKP3 in the absence of ERK2 (Table I). These results
indicated that the mutant proteins were folded correctly. Interestingly, these MKP3 mutants with decreased affinity for ERK2 can
still be fully activated by ERK2 at saturating ERK2 concentrations (Table I). This suggests that although the KIM sequence is important for high affinity ERK2 binding by MKP3, it is not essential for the
ERK2-induced MKP3 activation.
To study the binding of the KIM sequence to ERK2 further, we
synthesized peptides corresponding to the KIM sequences of MKP3, HePTP,
and RSK1 (Fig. 6). In addition, we made
mutant versions of the MKP3/KIM peptide, in which
Arg64/Arg65 or Leu71 were
substituted by an Ala. The binding affinity of ERK2 with the KIM
peptides was measured by isothermal titration calorimetry (ITC). ITC
allows a simultaneous determination of the binding constant
(K), stoichiometry, and the enthalpy change
(
H) associated with the binding of a ligand to a
macromolecule (20). From these parameters, the Gibbs free energy of
binding (
G) and the entropy change (
S) of
binding can also be derived from the expression
G =
RTlnK =
H
T
S. From curve fitting of ITC binding
isotherms, the stoichiometry for the binding of the peptides to ERK2
was determined to be 1:1 (Table II). When
Arg64 and Arg65 were substituted by Ala, the
Kd for MKP3/KIM/R64A/R65A increased to 1.77 mM, which was 40-fold higher than that of the wild-type
MKP3/KIM peptide (Table II). This supports the notion that the Arg
residues in the KIM sequence are important for high affinity binding to
ERK2. The substitution of Leu71 to Ala in MKP3/KIM did not
change the binding affinity of MKP3/KIM with ERK2. The affinities of
the KIM peptides for ERK2 are very different from each other, with a
Kd of 5.5 µM for HePTP/KIM, 80.6 µM for RSK1/KIM, and 43.8 µM for MKP3/KIM.
In addition, the thermodynamic parameters also differ for the binding
of the KIM peptides to ERK2 (Table II). Therefore, while the basic
residues in the KIM sequences are important for interaction with ERK2
(Refs. 25, 28, 29, and this study), additional determinants for ERK2
binding must exist within the individual KIM sequences.
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Table II
Thermodynamic parameters for the binding of KIM peptides with ERK2
determined by isothermal titration calorimetry
All experiments were performed at 25 °C and pH 7.0.
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Interaction between ERK2/D319N and MKP3--
Residue
Asp319 of ERK2 is conserved in all MAP kinases from yeast
to humans. A dominant gain-of-function mutation of the
rolled MAP kinase gene in Drosophila, termed
Sevenmaker (rlsevenmaker), contains a
single amino acid substitution of the analogous Asp334 by
an Asn (D334N) and activates several developmental pathways (30). The
same mutation in mammalian ERK2 (ERK2/D319N) appears to be resistant to
inactivation by MKPs in transfected cells (31, 32). Previous studies
indicate that ERK2/D319N displays reduced affinity for MKP3 and is
unable to activate the MKP3 phosphatase activity (12, 27). However,
using the quantitative activation-based assay, we were able to
determine the Kd of ERK2/D319N for MKP3 (14.8 ± 1.2 µM), which is 87-fold higher than that of the
wild-type ERK2. More importantly, we found that ERK2/D319N can activate
MKP3 to the same level induced by the wild-type ERK2 when a saturating
amount of ERK2/D319N is added to the reaction. Thus, like
Arg65 in MKP3, Asp319 in ERK2 plays a major
role in ERK2 binding to MKP3, but it is not essential for ERK2-induced
MKP3 activation.
A Competition-based Assay for the Determination of Binding Affinity
of Fragments/Domains of MKP3 Involved in ERK2 Recognition--
The
activation-based procedure described above is ideal for the
quantitative measurement of the binding affinity of MKP3 and its
catalytically active variants with ERK2. However, it is not suitable
for the analysis of MKP3 fragments/domains that lack phosphatase
activity. For example, although the N-terminal noncatalytic domain
(amino acids 1-221) of MKP3 binds ERK2 (11), it is not known how tight
this association is as compared with the wild-type MKP3. We have
developed a competition-based assay to quantitatively determine the
binding affinity of catalytically inactive MKP3s for ERK2. The
rationale for this approach is that peptides/fragments in MKP3
required for the ERK2-induced MKP3 activation should function as
inhibitors/competitors of the ERK2-induced MKP3 activation. Thus,
peptide ligands that compete with MKP3 for ERK2 binding should exhibit
a dose-dependent inhibition of the ERK2-induced MKP3
activation (Fig. 7). Nonlinear regression
curve fitting of the inhibition data by MKP3/1-222 and MKP3/1-203 to
Equation 2 yields a Kd value of 2.60 ± 0.28 and 7.03 ± 0.70 µM, respectively, for ERK2 binding
(Table III). The relative binding affinities of MKP3/1-203 and MKP3/1-222 as estimated by the GST pull-down assay parallel those determined by the competition-based assay (Fig. 5B). Similarly, the Kd value
of the catalytically inactive active site Cys293 to Ser
mutant MKP3 (MKP3/C293S) for ERK2 was determined to be 0.13 ± 0.01 µM, which is similar to that of wild-type MKP3 for ERK2. As a control, the catalytically inactive Cdc25A/C430S,
which contains two CH2 domains flanking its active site, is unable to inhibit the ERK2-induced MKP3 activation at 100 µM
concentration.

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Fig. 7.
Inhibition of ERK2-induced MKP3 activation by
competitive ligands. The figure shows the dose
dependence for the ability of MKP3/C293S (open
circle), MKP3/1-221 (open square),
and the KIM peptide from HePTP (solid circle) to
inhibit the ERK2-induced activation of MKP3-catalyzed pNPP
hydrolysis reaction. The data were fitted to Equation 2 (19) by
nonlinear regression analysis to obtain Kd values of
the competitive ligands (KIM peptides or MKP3 mutants lacking
phosphatase activity) for ERK2 (for details, see "Experimental
Procedures").
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Table III
Binding affinity of KIM peptides and MKP3 mutants lacking
phosphatase activity for ERK2 determined by the competitive binding
assay
All experiments were performed at 30 °C and pH 7.0. A /A0 corresponds to the ratio
of MKP3 activity in the presence of infinite concentration of an ERK2
binding ligand to MKP3 activity in the absence of any ERK2 binding
ligand (see Equation 2).
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Using the competitive binding assay described above, we have also
determined the binding affinities of the KIM peptides for ERK2. As
shown in Table III, the Kd values for MKP3/KIM, MKP3/KIM/L71A, and HePTP/KIM determined by the competitive assay are similar to those obtained from the ITC experiments (Table II).
Surprisingly, the binding of the KIM peptides with ERK2 does not
completely abolish the activation of MKP3 by ERK2 even at saturating
concentrations of the KIM peptides (Fig. 7 and Table III). This is in
contrast to MKP3/1-203, MKP3/1-222, and MKP3/C293S, which completely
suppress the ERK2-induced MKP3 activation (Fig. 7). No obvious
inhibition of the ERK2-induced MKP3 activation by MKP3/KIM/R64A/R65A
and RSK1/KIM peptides is observed. These data suggest that the KIM
sequence in MKP3 is not the sole determinant for high affinity ERK2 binding.
Ionic Strength Dependence of Binding between MKP3 and
ERK2--
It appears that the major driving force for the recognition
of ERK2 by MKP3 may involve electrostatic interactions between Arg65 of MKP3 and Asp319 of ERK2 (Refs. 12 and
27 and this study). If this charge-charge interaction is the only
determinant for MKP3 binding to ERK2, then binding affinity of ERK2
with MKP3 should decrease when solution ionic strength is increased.
This is in part due to the ability of ions to "screen"
electrostatic interactions. By contrast, higher salt concentrations
tend to favor hydrophobic interactions. Using the assays described
above, we have examined the dependence of binding of MKP3 with ERK2 on
salt concentration. We found that as the ionic strength of the solution
increased from 0.03 to 0.5 M, the affinities of MKP3,
MKP3/C293S, and MKP3/1-222 for ERK2 did not change (Table
IV). This suggests that both
electrostatic and hydrophobic interactions are involved in MKP3 (or
MKP3/1-222) binding to ERK2. Interestingly, the Kd
value of HePTP/KIM for ERK2 increased 3-fold, indicating the importance
of electrostatic interactions (Table IV). As expected, when the KIM
sequence or the Arg residues are removed from MKP3, the
Kd values of MKP3/
KIM and MKP3/R64A/R65A for ERK2
decrease 3-fold, suggesting the importance of hydrophobic interactions
(Table IV). Collectively, these results suggest that the association
between ERK2 and MKP3 is not a simple charge-charge interaction.
Besides the KIM sequence, other regions in the MKP3 sequence must also
be involved in ERK2 binding through hydrophobic interactions.
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Table IV
Binding affinity of ERK2 binding ligands in buffers of different ionic
strength
All experiments were performed at 30 °C and pH 7.0.
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The KIM Sequence in MKP3 Is Not the Only Region Involved in ERK2
Recognition--
Based on our quantitative analyses, we believe that
specific ERK2 recognition by MKP3 involves multiple regions of MKP3.
First, we have shown that deletion or mutation of the KIM sequence in MKP3 can lead to dramatic decrease in affinity of MKP3 for ERK2. However, it is important to note that these MKP3 mutants can still be
fully activated by ERK2 at saturating ERK2 concentrations (Table I).
Second, we have demonstrated that KIM peptides from HePTP, RSK1, and
MKP3 are able to bind to ERK2. However, unlike MKP3/C293S, the KIM
peptides were only able to suppress the ERK2-induced MKP3 activation by
30% at saturating peptide concentrations (Fig. 7). Third, we found
that the affinities of the KIM peptides for ERK2 decreased as the salt
concentration was raised, whereas the affinities of MKP3/
KIM and
MKP3/R64A/R65A for ERK2 increased as the salt concentration was raised.
By contrast, high salt concentrations have no effect on the
Kd values of MKP3 and MKP3/C293S for ERK2,
suggesting that both electrostatic and hydrophobic interactions are
involved. Finally, the N-terminal domain (residues 1-221) is known to
be able to bind ERK2. We have determined that the Kd
value of MKP3/1-222 for ERK2 is 2.6 µM (Fig. 7), which is 15-fold lower than that of the wild-type MKP3. This indicates that
the C-terminal domain of MKP3 also contributes to ERK2 binding. Collectively, these results suggest that the KIM motif is not the only
determinant for ERK2 binding, and specific recognition and activation
by ERK2 involves multiple regions of MKP3.
The C-terminal Domain of MKP3 Is Important for MKP3 Activation by
ERK2--
The fact that MKP3/1-222 binds ERK2 with a 15-fold lower
affinity than that of the wild-type MKP3 reveals the importance of the
C-terminal domain of MKP3 in ERK2 recognition. An examination of the
amino acid sequence alignment of the MKPs (Fig. 3) indicated the
presence of a putative FXFP motif in the C terminus of MKP3 (364FTAP367). The FXFP sequence has
been identified as a conserved docking site that mediates ERK binding
to its substrates in multiple protein families (33). Interestingly,
similar FXFP sequences can also be found in MKPs (MKP3,
MKP4, MKPX, MKP1, and MKP2) that are capable of inactivating ERK (Fig.
3). To establish the importance of the sequence
364FTAP367 in ERK2 binding and MKP3 activation,
we deleted the last 34 residues from MKP3, yielding MKP3/1-347. Using
the activation-based assay, we determined that MKP3/1-347 binds ERK2
with 7-fold lower affinity than the wild-type MKP3. Strikingly,
MKP3/1-347 could only be activated by ERK2 to 15% of the wild-type
MKP3 kcat/Km value at
saturating ERK2 concentrations (Table V).
Note that elimination of the C-terminal 34 residues in MKP3 does not
affect its ability to hydrolyze pNPP in the absence of ERK2
(Table V). This is very different from mutations in the N-terminal MKP3
domain (point mutations or deletions/truncations). Thus, the C terminus of MKP3 is important for the specific activation by ERK2.
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Table V
Binding affinity of MKP3 and its mutants with ERK2 and kinetic
parameters for MKP3 and its mutants in the presence and absence of ERK2
All experiments were performed at 30 °C and pH 7.0.
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We converted the four residues of the
364FTAP367 sequence to AAAA to determine the
contribution of 364FTAP367 to ERK2 binding and
MKP3 activation. The resulting mutant MKP3/A4 displays kinetic
parameters similar to those of the wild-type MKP3 in the absence of
ERK2. However, MKP3/A4 exhibits a 4-fold lower affinity toward ERK2 and
can only be activated to 32% of the wild-type MKP3
kcat/Km value by ERK2 (Table
V). The relative affinities of MKP3/1-347 and MKP3/A4 estimated by the
GST pull-down experiments are consistent with the Kd values determined by the activation-based assay (Fig. 5B).
Moreover, conversion of 364FTAP367 to AAAA in
MKP3/
KIM, MKP3/R64A/R65A, and MKP3/152-381 further reduces the
affinities of these proteins to ERK2 and drastically impairs their
ability to be activated by ERK2 (Table V). These results indicate that
the 364FTAP367 sequence in MKP3 plays an
important role for both ERK2 binding and binding-induced activation.
We also prepared MKP3/204-347, which corresponds to the catalytic
domain of MKP3. This is the same construct used in an early x-ray
crystal structure study (14). In the absence of ERK2, MKP3/204-347
displays similar activity as the wild-type MKP3. However, no
appreciable activation of MKP3/204-347 is observed in the presence of
ERK2 (Table V). Because MKP3/152-381/A4 has higher affinity for ERK2
than MKP3/204-347, and MKP3/152-381 has higher affinity for ERK2 than
MKP3/204-381, it is possible that residues between 153 and 204 may
also participate in ERK2 binding.
Residues 161-177 of MKP3 Are Also Involved in ERK2
Recognition--
When the sequence alignment of the MKPs was further
examined, we discovered that there is a sequence just after the second CH2 motif in MKP3, residues 161-177, which is also conserved in MKPX
and MKP4, but not in other MKPs (Fig. 3). MKP3, MKPX, and MKP4 are
cytosol-localized MKPs. In fact, residues 162-170 have previously been
suggested to serve as a nuclear export signal that may be responsible
for the cytoplasmic localization of MKP3 (10). Besides MKP3, MKPX, and
MKP4, a BLAST search of the GenBankTM, Protein Data Bank,
and SwissProt data bases using the MKP3 sequence 161PLPVLGLGGLRISSDSS177 yielded only one
protein, X17C, which also contains this sequence. Interestingly, X17C
is a Xenopus MKP found within the cytosolic fraction of the
oocytes (34). X17C is highly homologous to MKP3 with 88% sequence identity.
To investigate whether this sequence is involved in ERK2 binding, we
substituted the conserved Leu167 and Leu170 to
Ala and the conserved Asp175 to Asn. MKP3/D175N has the
same Kd and kinetic parameters as the wild-type MKP3
in the presence and the absence of ERK2 (Table V), indicating that
Asp175 does not interact with ERK2. However, the binding
affinity of MKP3 with ERK2 decreases 16-fold when both
Leu167 and Leu170 are replaced by Ala residues.
Similarly, MKP3/L167A and MKP3/L170A exhibit binding affinities for
ERK2 that are 10- and 15-fold lower, respectively, than that of the
wild-type MKP3 (Table V). Note that the relative affinities of these
MKP3 mutants determined by the GST pull-down experiments approximate
those measured by the activation-based assays (Fig. 5C).
Like the KIM deletion mutants, MKP3/L167A, MKP3/L170A, and
MKP3/L167A/L170A can still be activated to the wild-type MKP3 level by
ERK2 (Table V). These data indicate that Leu167 and
Leu170 play a role in ERK2 binding but they are not
required for the ERK2-induced MKP3 activation.
Summary--
Although the upstream signaling processes that
activate MAP kinases have been extensively characterized, considerably
less is known about how MAP kinase activity is down-regulated. MKP3 is
predominantly localized in the cytoplasm, forms a physical complex with
ERK2, and is highly specific for ERK2 inactivation. Furthermore,
purified recombinant ERK2 stimulates the phosphatase activity of MKP3
toward pNPP. To gain further insights into the molecular
mechanism of the specific ERK2 recognition by MKP3 and the ERK2-induced
MKP3 activation, we have carried out a systematic mutation and deletion
analysis of MKP3. The mutant MKP3s have been quantitatively analyzed
for their ability to bind ERK2 and for their propensity to be activated
by ERK2, using an activation-based and a competition-based assay. Our
results show that the recognition of and activation by ERK2 involves
multiple regions of MKP3.
We have shown that the KIM sequence (residues 61-75) in MKP3 is
important for high affinity ERK2 binding. Deletion of the KIM sequence
from MKP3 results in a 135-fold reduction in ERK2 binding affinity. We
have identified a unique sequence conserved in cytosolic MKPs (residues
161-177 in MKP3) that also contributes to ERK2 binding (15-fold).
Although both the KIM sequence and residues 161-177 are involved in
ERK2 binding, these two regions are not essential for ERK2-induced MKP3
activation. ERK2-induced MKP3 activation requires a third ERK2 binding
site localized in the C terminus of MKP3 (residues 348-381). We have
determined that although deletion of this region or mutation of the
putative ERK-specific docking sequence
364FTAP367 in this region reduces MKP3's
affinity for ERK2 by less than 10-fold, this region is absolutely
required for ERK2-induced MKP3 activation. Collectively, our data
indicate that the KIM sequence and residues 161-177 in MKP3 are
important for high affinity ERK2 binding, while the C terminus of MKP3
including the sequence 364FTAP367 is primarily
responsible for ERK2-induced MKP3 activation.
Our study documents that specific ERK2 recognition by MKP3 involves
multiple regions of MKP3. Data from mutagenesis studies of various
protein-protein interfaces suggest that the binding energy is not
evenly distributed over the entire protein interaction surface but
rather concentrated in energetic "hot spots," in which only a small
subset of amino acids accounts for most of the change in binding free
energy (35, 36). Point mutation analyses indicate that
Arg65 in the KIM sequence of MKP3 is the most important
residue for high affinity ERK2 binding. Because substitution of
Arg65 in MKP3 by an Ala or replacement of
Asp319 in ERK2 by an Asn decreases the affinity of MKP3 for
ERK2 by 2 orders of magnitude, the guanidinium side chain of
Arg65 in the KIM sequence may directly participate in
bidentate hydrogen bonds and/or charge-charge interactions with the
carboxylate side chain of Asp319 in ERK2. Thus,
Arg65 in MKP3 and Asp319 in ERK2 probably form
a hot spot for high affinity ERK2/MKP3 binding. Interestingly, both Arg
and Asp residues are enriched in hot spots observed in protein-protein
interactions (37). The energetically less important residues probably
surround the hot spot interactions and serve to occlude bulk solvent
from the hot spot. Together, the basic and large hydrophobic amino
acids in the KIM sequence, residues 161-177, and the C terminus of
MKP3 cooperatively promote specific association of MKP3 with ERK2
through a combination of hot spot electrostatic interactions and apolar surrounding hydrophobic interactions.