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INTRODUCTION |
The dual-specificity protein tyrosine phosphatases
(DS-PTPs)1 are members of a
large family of enzymes that catalyze the phosphomonoester hydrolysis
of protein substrates (1). The DS-PTPs were identified as
protein-tyrosine phosphatases (PTPs) that are capable of efficiently hydrolyzing phosphotyrosine as well as phosphothreonine/serine residues. Approximately 15-20 distinct DS-PTPs have been identified to
date. The first identified DS-PTP (VH1, for vaccinia open
reading frame H1) was discovered in the H1 open reading
frame of vaccinia virus (2) and was later found to be an essential gene
in the poxvirus (3). More recently discovered DS-PTPs have been
suggested to play a central role in the regulation of the cell cycle
and signaling pathways mediated by the families of MAP
(mitogen-activated protein) kinases (4). The MAP kinases facilitate
intracellular signaling events triggered by mitogens, growth factors,
and stress that result in cellular growth, differentiation, and death
(5, 6). The MAP kinases are activated by their specific upstream dual-specificity kinases (MAP-kinase kinases) through phosphorylation on both threonine and tyrosine residues in the TXY motif. In
response to extracellular stimuli, activated MAP kinases then
phosphorylate an array of cellular substrates, including nuclear
transcription factors. In mammals, three distinct MAP kinase families
have been identified. Members of one family, ERK1 and ERK2, are
primarily activated by growth and differentiation factors as well as by phorbol esters (7). The JNK family (or SAPK) and p38 MAP kinase family
are activated by proinflamatory cytokines and environmental stress.
Several DS-PTPs have been implicated in ERK regulation by
dephosphorylation of both Thr183 and Tyr185
(8-11). Studies have shown that such DS-PTPs, termed MAP kinase phosphatases (MKPs), harbor distinct substrate preferences for the
various MAP kinase families (11-14). All of the MKPs identified to
date, with the exception of Pyst1, are immediate-early genes that are
induced by various mitogens, growth factors, and stresses. The
induction of MKPs by distinct stimuli and their cell-specific expression suggest that there is a great diversity in the function of
the various MKPs. For example, MKP-1 is induced by stress, mitogens,
and phorbol esters and is localized to the nucleus (8, 15). PAC-1 is
induced by mitogens and T-cell activation and is found in the nucleus
of hematopoietic cells (9, 16). MKP-3/VH6 is predominantly cytosolic
and is induced by nerve growth factor (17, 18). While MKP expression
generally correlates with MAP kinase inactivation (8-14), numerous
reports have demonstrated inconsistencies with a model in which MKPs
act as the primary catalysts for dephosphorylation and inactivation of
ERK1 and ERK2 (19-26). In many cells, ERK1 and ERK2 inactivation is
rapid (within 15-20 min) and does not require new protein synthesis
(19-26). Accordingly, MKP RNA/protein expression is normally not
detectable until 30-60 min after stimulation (9, 10, 17, 18, 23, 27,
28), and expression of MKP-1 and MKP-2 is actually induced by the ERK1
and ERK2 cascade (28). With the exception of Pyst1 (11), MKPs are not
expressed in quiescent cells (29), and disruption of the
mkp-1 gene does not affect mouse development and normal MAP
kinase activity was observed in MKP-1-deficient fibroblasts (30).
Collectively, these observations argue that a phosphatase(s) distinct
from the MKPs is responsible for the rapid down-regulation of ERK.
It has been suggested that a constitutively expressed phosphatase is
directly involved in ERK1 and ERK2 inactivation. In rat mesangial
cells, sustained ERK2 activation by endothelin and EGF was regulated by
a vanadate-sensitive protein phosphatase but not by a transcriptionally
regulated protein (20). Rapid inactivation of ERK2 in 3T3-L1, PC12, and
PAE cells was attributed to the serine/threonine protein phosphatase
PP2A and an unknown PTP distinct from MKP-1 (22). The protein synthesis
inhibitor cycloheximide failed to affect the inactivation of MAP kinase
following induction with EGF in A431 and PC12 cells (21, 23),
suggesting that MKPs are not involved. Others have proposed that a
vanadate-sensitive and tyrosine-specific phosphatase is responsible for
the repression of ERK1 and ERK2 activity in the absence of serum and
that a tyrosine phosphatase regulates ERK1 and ERK2 activity in cells
transformed by upstream oncoproteins (19).
In the present study, we have utilized affinity trapping methods to
identify potential cellular substrates of the mammalian VHR phosphatase
(for VH1-related), a putative dual-specificity PTP that was previously identified by an expression cloning strategy (31) and has been the subject of detailed biochemical and structural analyses (1, 32-36). The physiological function has been uncertain since no in vivo substrates had been identified. The D92A
catalytic mutant of VHR was covalently coupled to a solid matrix which
then served as an affinity absorbent for specific
tyrosine-phosphorylated proteins from hydrogen peroxide-stimulated COS1
cells. We selected hydrogen peroxide (H2O2) as
a mitogen for two main reasons. We have previously demonstrated that
H2O2 will rapidly and reversible inactivate VHR
and other PTPs by selectively oxidizing the catalytic cysteine thiolate
(37). Knowing that endogenous VHR is highly expressed in COS-1 cells,
we surmised that treatment with H2O2 would
inactivate the endogenous VHR, leading to increased phosphorylation of
its authentic protein substrate(s). Extracts from
H2O2-treated COS-1 cells would then provide an
enriched fraction of the physiological substrate. Moreover, reactive
oxygen species such as H2O2 have been shown to
stimulate increases in relevant cellular tyrosine phosphorylation
(38-43), and intracellular generation of H2O2
has been shown to be required or involved in growth factor signaling pathways (44, 45), perhaps as a second messenger. Potent activation of
extracellular regulated protein kinase (ERK) is observed with H2O2 treatment (46-49).
The phosphorylated forms of ERK1 and ERK2 were specifically retained
and eluted from the VHR affinity columns. Kinetic and cellular
expression studies further demonstrated that ERK1 and ERK2 are
authentic in vitro and in vivo substrates for
VHR. Our data suggest that VHR is a constitutively expressed and
tyrosine-specific phosphatase localized to the nucleus and is
responsible for the repression of ERK1 and ERK2 in quiescent cells and
for their rapid inactivation following stimulation.
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MATERIALS AND METHODS |
Reagents and Mammalian Vectors--
Full-length cDNA
fragments of VHR or C124S mutant were cloned out of pT7-7-VHR (32)
using polymerase chain reaction and inserted into the BamHI
(5' end) and EcoRI (3' end) sites of pcDNA3 vector
(Invitrogen) to generate pcDNA3-VHR and pcDNA3-C124S,
respectively. Both constructs direct the expression of all 185 amino
acids, with no vector added sequences. Antisense VHR construct
(pcDNA3-anti-VHR) was generated by removing the XbaI and
EcoRI insert from pT7-7-VHR and inserting into the
XbaI and EcoRI sites of pcDNA3. The D92A mutant was generated using the Bio-Rad Muta-gene method as described previously (35). Recombinant ERK2 was purified and phosphorylated by
recombinant MEK1 mutant G7B (50). The c-myc-ERK2 pcEXV-n plasmid was a
gift from Dr. Phil Stork (14). All constructs were verified by DNA
sequencing. Rabbit polyclonal antibodies specific to VHR were
immunopurified from the serum of VHR-(full-length) immunized rabbits
(Cocalico). Chicken immunopurified anti-VHR antibody was generated by
immunizing chickens with the full-length VHR protein. The IgY fraction
was purified from egg yolks (Aves Labs). Both VHR antibodies were
affinity purified over a column containing recombinant VHR conjugated
to Affi-Gel 10. Chicken antibodies were eluted with 0.1 M
sodium phosphate (pH 2.5) and stored in PBS. Rabbit antibodies were
eluted with 0.1 M sodium phosphate (pH 2.5) and stored in
PBS.
Western Blotting--
Proteins were separated by
SDS-polyacrylamide gel electrophoresis and transferred to Immobilon-P
polyvinylidene difluoride (Millipore) membrane at 125 V for 45 min.
Membranes were rinsed with TBS containing 0.05% Tween 20 (TBST) then
blocked with TBST containing either 5% bovine serum albumin (BSA)
(Sigma) or non-fat dry milk for 1 h at 25 °C. Blots were then
incubated with primary antibodies diluted in TBST containing either 3%
BSA or non-fat dry milk overnight at 4 °C. The blots were then
rinsed with TBST (3 times) for 5 min at 25 °C and incubated in the
appropriate secondary antibody diluted in TBST containing either 3%
BSA or 5% non-fat dry milk for 1 h at 25 °C. The blots were
rinsed another 3 times for 5 min with TBST before detection by enhanced
chemiluminescence (ECL) (NEN Life Science Products Inc.). Blots were
stripped by incubating in 62.5 mM Tris-HCl, 2% SDS, 100 mM 2-mercaptoethanol (pH 6.7) for 30 min at 50 °C.
Stripped blots were then rinsed extensively with TBST and reprobed as
described above. The following antibodies were used: rabbit polyclonal
antibody (rAb) specific to p44/42 MAPK (New England Biolabs), rAb
specific to the phosphorylated forms of p44/42 MAPK
(Thr202/Tyr204) and p38 MAPK
(Thr180/Tyr182) (New England Biolabs), rAb
specific to the phosphorylated forms of SAPK/JNK
(Thr183/Tyr185) (Promega), rAb specific to VHR
that was immunopurified from the serum of VHR-(full-length) immunized
rabbits (Cocalico), mouse monoclonal antibody specific to
anti-phosphotyrosine (4G10) (Upstate Biotechnology), monoclonal
antibody-agarose conjugated and unconjugated specific to c-Myc (9E10)
(Santa Cruz Biotechnology), goat anti-rabbit IgG-horseradish peroxidase
conjugate (Bio-Rad), and horse anti-mouse IgG-horseradish peroxidase
conjugate (New England Biolabs).
Substrate Trapping--
Human VHR (C124S and D92A) proteins were
bacterially expressed and purified as described (35) and coupled to
Affi-Gel 10 (Bio-Rad). Affi-Gel 10 (2 ml) was washed 5 times with
ice-cold 50 mM MES (pH 7.0) and incubated with 2 ml of 0.5 mg/ml D92A or C124S VHR for 1 h at 4 °C. Unreacted activated
esters were capped with 200 µl of 1 M ethanolamine (pH
8.0) for 1 h at 4 °C and the coupled resin was washed
extensively with 50 mM Bis-Tris, 50 mM NaCl (pH
6.5) (wash buffer). Alternatively, Affi-Gel 10 was washed and capped as
above without protein coupling. COS-1 cells were grown to 80-90%
confluence in 150-mm plates with Dulbecco's modified Eagle's medium
containing low glucose, L-glutamine, sodium pyruvate, 10%
fetal bovine serum, penicillin at 1000 units/ml, and streptomycin at
1000 µg/ml (Life Technologies, Inc.) (growth media). Cells were then
treated with 200 µM H2O2 for 40 min at 37 °C. After treatment, cells were rinsed once with 50 mM Tris, 150 mM NaCl, 1 mM EDTA (pH
7.2), harvested, and frozen at
20 °C. When needed, cells were
lysed in 1 ml of ice-cold 50 mM Bis-Tris, 50 mM
NaCl, 1% Nonidet P-40, protease inhibitors (1 mM
phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, and 5 µg/ml
aprotinin) (pH 6.5), sonicated for 30 s on ice, Dounce homogenized
for 15 strokes, and centrifuged at 15,000 rpm for 10 min. Clarified
extract was applied to the affinity column, attached to a Beckman
BioSys 510 liquid chromatography system running at a flow rate of 1 ml/min. The column was washed with wash buffer (3 × column
volumes) and retained phosphoproteins were eluted with 10 ml of 0.2 mM sodium arsenate. The eluant was collected and the level
of tyrosine-phosphorylated proteins, ERK, and phosphorylated ERK were
measured by Western blot analysis.
Transient Transfection of COS-1 Cells--
COS-1 cells were
seeded at 1.0 × 106 in 100-mm plates and grown for
16-24 h. LipofectAMINE (Life Technologies, Inc.) (30 µl) and
purified plasmid DNA (at 5 µg of c-myc-ERK and 1 µg all others) were combined in 500 µl of Opti-MEM I (Life Technologies, Inc.) and
incubated at 25 °C for 20 min. The cells were rinsed once with
Opti-MEM I and the Lipid/DNA solution was added dropwise to the cells
which were then covered with Opti-MEM I (5 ml). After 6-8 h,
transfected cells were placed in growth media for 16-24 h. Cells were
then serum starved and left for 16-24 h before treatment with 100 nM EGF (Collaborative Biomedical Products) for 15 min at
37 °C. After treatment, cells were rinsed once with PBS, lysed with
500 µl of ice-cold 20 mM Tris, 137 mM NaCl,
10% glycerol, 1% Nonidet P-40, 2 mM, 1 mM
sodium vanadate, 10 mM sodium fluoride, 0.1 M
phenylmethylsulfonyl fluoride, 5 µg/ml leupeptin, and 2.5 µg/ml
aprotinin, sonicated on ice for 30 s, and centrifuged at 15,000 rpm for 10 min. Protein concentrations were determined by the method of
Bradford. Immunoprecipitation of c-myc-ERK was then performed on 500 ng
of cell lysate using 10 µl of the agarose-conjugated mAb (9E10)
specific to c-Myc in a total volume of 1 ml for 2 h at 4 °C.
Agarose beads were washed twice with 500 µl of 50 mM Tris, 0.5 M NaCl, 5% sucrose, 0.2% Nonidet P-40 (pH 7.3)
for 2 min at 4 °C and resuspended in 2 × Laemmli buffer.
Immunofluorescence--
Cells were seeded at 5 × 105 cells per well in two-well chamber slides and grown for
24 h. The cells were then rinsed twice with PBS, fixed with 3.7%
formaldehyde for 20 min at 25 °C, rinsed 3 times with PBS, 0.1%
Triton X-100, 0.2% BSA (I.F. wash solution), blocked and lysed with
PBS, 0.5% Triton X-100, 3% BSA, rinsed 3 times with I.F. wash
solution, and incubated with the rAb specific to VHR diluted 1:500 in
PBS, 0.1% Triton X-100, 3% BSA (antibody dilution solution) for
1 h at 25 °C. The cells were then rinsed with I.F. wash
solution 5 times, incubated with a goat anti-rabbit IgG-Alexa
488-conjugated antibody (Molecular Probes) diluted 1:400 in antibody
dilution solution, rinsed 5 times with I.F. wash solution, and mounted
with 50% glycerol, 0.2% p-phenylenediamine. Cells were
viewed using a Leica DMRB microscope with a 63X PL APO objective. As a
control, recombinant VHR was first preabsorbed to the primary antibody
and the immunofluorescence staining determined as described above.
Dephosphorylation in COS-1 Cell Extract--
Serum-starved COS-1
cells were treated with 200 µM
H2O2 for 40 min at 37 °C. Cells were rinsed
in PBS, harvested, and lysed as described above. Recombinant VHR, or an
equal volume of buffer, was added to a final effective concentration of
0-0.16 µM and incubated for 20 min at 30 °C. The
reactions were quenched by adding 5 × Laemmli sample buffer
containing SDS and subjected to Western analysis.
Dephosphorylation of Recombinant ERK2 by VHR--
Recombinant
VHR, or an equal volume of buffer, at a final effective concentration
of 0.1 µM was combined with 2.5 µM
recombinant phosphorylated ERK2 in 50 mM Tris, 50 mM Bis-Tris, 100 mM sodium acetate, 1 mM dithiothreitol, 0.01% BSA (pH 7.0) and incubated for up
to 40 min at 25 °C. Aliquots were withdrawn at the times indicated
and the reactions were terminated by adding 5 × Laemmli sample
buffer. The samples were then subjected to Western analysis to examine
the phosphorylation state of ERK using the phospho-ERK antibody.
Changes in the phosphorylation of ERK were quantified by densitometry
using Bio-Rad GS-700 Imaging Densitometer and the Molecular Analyst
Software. The identical samples were also coupled to a kinase assay to
directly examine the effect of VHR on the activity of phosphorylated
ERK2. Aliquots were withdrawn at the same times as above, mixed with 20 mM HEPES (pH 8.0), 10 mM MgCl2, 1 mM dithiothreitol, 1 mM
Na3VO4, 0.3 mg/ml myelin basic protein, 0.1 mM ATP, and 1000 cpm/pmol [32P]ATP, incubated
for 20 min at 25 °C, and the reaction was stopped by spotting onto
P81 phosphocellulose discs (Life Technologies, Inc.). Discs were washed
3 times for 10 min in 150 mM phosphoric acid to remove free
phosphate, and incorporation of 32P into myelin basic
protein was measured by liquid scintillation. Percent remaining
phosphorylation or kinase activity of ERK was calculated by dividing
the phosphorylation or activity at each time point by the control (no
addition of VHR). The amount of dephosphorylated ERK was determined by
subtracting the fraction remaining from 1 and multiplying by the
initial phosphorylated [ERK]. The data were then fitted to the
integrated Michaelis-Menten equation (33).
Immunodepletion of Endogenous VHR from COS-1
Lysates--
Serum-starved COS-1 cells were treated with 10 µg/ml
anisomycin for 30 min at 30 °C and lysed in the absence of
phosphatase inhibitors. Cell lysate was then combined with an equal
volume of ethanolamine capped Affi-Gel 10 or a polyclonal chicken
antibody specific to VHR coupled to Affi-Gel 10 (mock treated) and
incubated for 1 h at 4 °C. Lysates were then separated from the
beads by centrifugation and dithiothreitol was added to 1 mM. Samples were removed for zero time controls and
combined with 5 × Laemmli sample buffer containing SDS. The
remaining lysates were incubated at 30 °C for 20 min and combined
with 5 × Laemmli sample buffer containing SDS. All samples were
then subjected to Western blot analysis as described above.
Phosphoamino Acid Analysis--
VHR, MKP3, or an equal volume of
pH 7.0 buffer (0.1 M sodium acetate, 0.05 M
Tris, and 0.05 M Bis-Tris), was mixed with enzymatically active, diphosphorylated [32P]ERK2 at 30 °C for up to
1 h. Aliquots were removed and the reactions were terminated by
the addition of 5 × Laemmli sample buffer. Samples were resolved
by SDS-polyacrylamide gel electrophoresis and the proteins were
electrotransferred to polyvinylidene difluoride. The corresponding ERK
protein bands were excised and subjected to hydrochloric acid (6 M), 110 °C for 1 h. The samples were dried by
vacuum centrifugation and resuspended in 15 µl of phosphoamino acid
(1 mg/ml phosphotyrosine, phosphoserine, and phosphothreonine) standards. The samples were then spotted (2.5 µl) onto cellulose thin-layer plates. One-dimensional electrophoresis was then performed employing an glacial acetic acid/pyridine/water, 50:5:945 (v/v), buffer
for 45 min at 1000 V. Phosphoamino acids were visualized by
PhosphorImaging utilizing a Bio-Rad Molecular Imager System. Phosphoamino acid standards were detected with ninhydrin.
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RESULTS |
Affinity Trapping of ERK1 and ERK2 by the VHR Phosphatase--
In
the current study, we used affinity chromatography to identify cellular
substrates of VHR. The catalytic mutant D92A was covalently linked to a
solid phase and was employed to retain authentic substrates from
extracts of H2O2-stimulated COS-1 cells (Fig.
1). This method involved passing extract
over the affinity column, washing away nonspecific proteins, and
eluting high-affinity phosphorylated proteins with the competitive
inhibitor arsenate (Ki of 10 µM (33)).
Arsenate will only displace phosphorylated proteins that associate with
VHR through the active-site. Although VHR is a putative member of the
dual-specificity subfamily of PTPs, phosphopeptide substrate analysis
had indicated that VHR prefers phosphotyrosine over
phosphoserine/threonine by 1000-fold (33). Given this significant
preference for phosphotyrosine, we postulated that authentic
substrate(s) would be tyrosine-phosphorylated and could thus be
detected by anti-phosphotyrosine Western blot analysis.

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Fig. 1.
ERK1 and ERK2 are specifically retained from
COS-1 lysate by a D92A VHR affinity absorbant. Lysates made from
COS-1 cells treated with H2O2 for 40 min were
passed over a D92A VHR affinity column attached to an high performance
liquid chromatography system (Beckman BioSys 510) operating at a flow
rate of 1 ml/min. The column was washed with 3 column volumes of 50 mM Bis-Tris, 50 mM NaCl (pH 6.5) and
phosphoproteins were eluted in 10 ml of 0.2 mM sodium
arsenate in the same buffer. A, anti-phosphotyrosine Western
blot of H2O2-treated and untreated COS-1 cells.
B, D92A VHR affinity column elution. Anti-phosphotyrosine
Western blot of H2O2-treated COS-1 cell extract
(lane 1), arsenate elution of the D92A VHR affinity column
(lane 2), and arsentate elution of control column
(lane 3); C, anti-phospho-ERK Western blot from
B. The anti-phosphotyrosine blot from B was
stripped and reprobed with the phospho-ERK antibody ("Experimental
Procedures"). D, quantitation of an elution profile.
Fractions were analyzed for protein content (Bradford method)
(diamonds), anti-ERK (squares), or
anti-phospho-ERK (circles) immunoreactivity (densitometry of
fluorescence intensity). Within each data set, the strongest signal was
given a value of 1, and each point was determined relative this maximal
intensity.
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A major tyrosine-phosphorylated protein(s) of ~42 kDa was retained
and eluted specifically from the VHR affinity columns. Fig. 1,
B-D, is a representative example from three independent experiments employing D92A columns. A control column (Affi-Gel 10 resin
capped with ethanolamine) was run under the identical conditions to
confirm that binding of retained proteins was specific for VHR and not
the resin. Panel A is an anti-phosphotyrosine Western blot
demonstrating H2O2-induced tyrosine
phosphorylation in COS-1 cells. Panel B is an
anti-phosphotyrosine Western blot comparing COS-1 cell extract elutions
from D92A and control columns. Elution from the D92A column clearly
indicated the specific retention of a tyrosine-phosphorylated
protein(s) of ~42 kDa. Given the size of the observed protein, we
explored the possibility that this band(s) was the MAP kinase ERK. The
anti-phosphotyrosine Western blot was then reprobed with an antibody
specific to the active form (diphosphorylated on Thr183 and
Tyr185) of ERK1 and ERK2 (51-53). The ~42-kDa protein(s)
coincided exactly with the immunoreactive bands of ERK1 and ERK2 (Fig.
1C). To demonstrate that only the phosphorylated form of ERK
is retained, a detailed analysis of an elution profile was performed
(Fig. 1D). Fractions were analyzed for protein content
(Bradford method), anti-ERK, or anti-phospho-ERK immunoreactivity
(densitometry of immunofluorescence intensity). The majority of total
ERK (predominantly unphosphorylated) co-elutes with the general protein
peak. In contrast, almost half of the total phosphorylated ERK1 and
ERK2 is retained on the VHR column and rapidly eluted with arsenate.
These data suggest that although the majority of ERK protein is
unphosphorylated, only phosphorylated ERK can bind to VHR. A
significant portion of the phosphorylated ERK pool elutes with the
general protein peak, suggesting that the capacity of the column may
have been exceeded or that a fraction of the phosphorylated ERK is not
accessible to VHR.
ERK1 and ERK2 Are Rapidly and Specifically Dephosphorylated by the
VHR Phosphatase--
The VHR affinity columns demonstrated that active
ERK1 and ERK2 bind specifically to immobilized VHR. Whether ERK1 and
ERK2 were true substrates for the VHR phosphatase remained to be
verified. We therefore examined the dephosphorylation kinetics and
substrate specificity of wild type VHR for ERK1 and ERK2 from cellular
extracts of COS-1 cells stimulated with H2O2.
Low levels of H2O2 induce physiologically
relevant tyrosine phosphorylation and transient inactivation of
constitutively expressed PTPs. Purified recombinant human VHR
phosphatase was added in increasing concentrations to the stimulated
extract (Fig. 2, A and
B). Strikingly, a protein(s) of ~42 kDa was rapidly
dephosphorylated at low levels (0.08 µM) of VHR (Fig.
2A). The 42-kDa protein was almost completely
dephosphorylated before any significant dephosphorylation was detected
on the entire pool of tyrosine-phosphorylated proteins. Not unexpected,
with much higher levels of VHR there was a corresponding general loss of immunoreactivity toward other phosphotyrosine proteins. To verify
that the ~42-kDa protein(s) corresponded to ERK, the
anti-phosphotyrosine Western blot (Fig. 2A) was reprobed
with the anti-phospho ERK antibody and the anti-ERK antibody (Fig.
2B). The resulting immunoreactivity overlaps exactly with
the ~42-kDa protein(s), again indicating that the protein(s) is
indeed ERK (Fig. 2, A and B).

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Fig. 2.
ERK is rapidly and specifically
dephosphorylated by wild type VHR phosphatase. Lysates made from
COS-1 cells treated with H2O2 for 40 min were
incubated with 0-0.16 µM recombinant VHR for 20 min at
25 °C. A, anti-phosphotyrosine Western blot showing the
rapid tyrosine dephosphorylation of the ~42-kDa protein in COS-1 cell
extracts; B, Western blot from A was stripped and
reprobed with the anti-phospho-ERK antibody (upper panel in
B) and separately with the anti-ERK antibody (lower
panel in B); C, time-dependent
dephosphorylation of native ERK in COS-1 cell extracts by the
anti-phospho-ERK Western blot analysis. Lysates in C were
made from COS-1 cells treated with H2O2 for 40 min was incubated with 0.4 µM recombinant VHR, or an
equal volume of buffer, for 0-40 min at 25 °C.
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The kinetics of ERK1 and ERK2 dephosphorylation by VHR were then
investigated (Fig. 2C). VHR was capable of rapidly
dephosphorylating ERK1 and ERK2 from stimulated extracts. At 0.4 µM VHR, ERK1 and ERK2 were >80% dephosphorylated in 20 min, whereas the buffer control displayed no hydrolysis even after 40 min. In the buffer control samples, the lack of ERK dephosphorylation
by endogenous VHR or other ERK-specific phosphatases is consistent with
the transient inactivation of PTPs from
H2O2-treated cells (37, 46).
Recombinant ERK2 Is Efficiently Dephosphorylated by VHR--
In
order to firmly establish that VHR was responsible for the direct
dephosphorylation of ERK in cellular extracts, purified recombinant ERK
was utilized as a substrate for purified VHR. ERK2 and a
constituitively active form of MEK were overexpressed separately in
bacteria and purified using nickel-affinity chromatography (50, 54).
ERK2 was then stoichiometrically phosphorylated and purified to
homogeneity by anion exchange chromatography. The purified and
phosphorylated ERK2 was then examined as a substrate of VHR. With
recombinant ERK2, steady-state levels of phosphorylated ERK2 could be
used for the dephosphorylation assay and accurate kinetic analyses
could be performed. ERK2 (2.3 µM) was reacted with 0.1 µM VHR and the inactivation of ERK2 was assessed by the myelin basic protein kinase assay (Fig.
3). The progress curve was fitted to the
integrated Michaelis-Menten equation (33) and yielded a
kcat/Km second-order rate
constant of 40.4 × 103 ± 4.2 × 103
M
1 s
1 (Fig. 3). Being a
physiologically relevant parameter, the
kcat/Km value is the
second-order rate constant for the reaction of free enzyme with free
substrate and reflects both binding affinity and catalytic efficiency.
Among all substrates examined to date, this represents the largest
measured value (33). For comparison, the commonly used PTP substrate
p-nitrophenyl phosphate is catalyzed 80-fold less
efficiently.

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Fig. 3.
Rate of recombinant ERK2 inactivation by VHR
phosphatase. VHR (0.1 µM) was incubated with
phosphorylated ERK2 (2.3 µM) at 25 °C for 0-40 min
(pH 7). At the indicated times, VHR-catalyzed inactivation of MAP
kinase was quantified by measuring the kinase activity remaining
("Experimental Procedures"). The remaining activities were
converted to [dephosphorylated ERK] and fitted to the integrated
Michaelis-Menton equation.
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To establish that inactivation of ERK2 by VHR is the direct result of
dephosphorylation, the phosphorylation status of ERK2 was correlated
with kinase activity. VHR was reacted with active phosphorylated ERK2,
the phosphorylation state was quantitated by anti-active ERK Western
analysis, and the corresponding kinase activity was determined.
Quantitation of the initial velocities resulted in curves with
identical slopes (data not shown), indicating that VHR inactivated ERK
by direct dephosphorylation and demonstrating that immunoreactivity of
the phospho-specific ERK antibody accurately reflects changes in ERK activity.
Only the Native Structure of ERK2 Is Recognized by VHR--
Next,
we investigated whether the native structure of ERK is required for
efficient substrate recognition by VHR. It is predicted that the
specificity toward an authentic substrate would be mediated through the
recognition of the folded protein. If binding and catalysis are not
specific, rates of hydrolysis between folded and unfolded protein
should be comparable. We therefore examined the ability of VHR to
recognize and dephosphorylate unfolded ERK. Active ERK2 was either
maintained in its native conformation or unfolded by heat denaturation
and employed as a substrate for VHR (Fig.
4A). With excess VHR (0.87 µM) and 0.5 µM phosphorylated ERK2, no
dephosphorylation was observed over 40 min in the heat-denatured sample
(Fig. 4A, lane 3), whereas native phosphorylated ERK (Fig. 4A, lane 2) was hydrolyzed to more than 80% of the control
(Fig. 4A, lane 1). The remaining 20% of this particular
phosphorylated ERK preparation was resistant to dephosphorylation by
VHR (Fig. 4A, lane 2), suggesting that this 20% fraction
was unfolded. Heat-denatured ERK was found to be completely inactive as
a kinase, verifying loss of the native structure (data not shown).
These data suggest that only native ERK is recognized by VHR.

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Fig. 4.
Catalysis by VHR requires the native
structure of ERK and is specific for tyrosine 185 of ERK2.
A, anti-phospho-ERK Western blot of 0.5 µM
native phosphorylated ERK2 (lane 1), VHR (0.87 µM) with 0.5 µM native phosphorylated ERK2
(lane 2), or a heat denatured sample of 0.5 µM
ERK2 with active VHR (0.87 µM) (lane 3) for 40 min. B, phosphoamino acid analysis showing the
dephosphorylation of ERK by VHR. VHR (0.5 µM),
VH6 (0.5 µM), or a buffer control was mixed with
phorphorylated ERK2 (5.0 µM) at 30 °C (pH 7). Time
points were taken and subjected to phosphoamino acid analysis
("Experimental Procedures"). The levels of phosphotyrosine and
phosphothreonine were determined by separation on TLC and the resulting
audioradiograms were visualized using a GS-525 Molecular Imager system
(Bio-Rad). C, analysis of tyrosine dephosphorylation from
diphosphorylated ERK. VHR (0.5 µM), VH6 (0.5 µM), or a buffer control was mixed with phorphorylated
ERK2 (5.0 µM) at 30 °C (pH 7). At the times indicated,
the reactions were quenched by Laemmli sample buffer and subjected to
anti-phosphotyrosine Western analysis.
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VHR Is Specific for Phosphotyrosine 185 of ERK--
Phosphoamino
acid analysis of the VHR-catalyzed dephosphorylation of ERK indicated
that VHR specifically hydrolyzed Tyr185 but not
Thr183 (Fig. 4B). Catalytic amounts of VHR or
MKP3 (0.5 µM) were reacted with active recombinant ERK (5 µM) and the specific dephosphorylation at both tyrosine
and threonine was assessed by phosphoamino acid analysis. The
autoradiogram clearly demonstrates that VHR rapidly catalyzes the
specific dephosphorylation of tyrosine while no significant hydrolysis
at phosphothreonine was observed (up to 1 h). In contrast,
MKP3/VH6 (17, 55) rapidly hydrolyzed both phosphoamino acids (Fig.
4B). The buffer control demonstrated that nonenzymatic
phosphoester hydrolysis over the 1-h time course was insignificant. The
lower overall signal of phosphotyrosine (relative to phosphothreonine)
in control samples is due to the acid-labile phosphoester of tyrosine.
To corroborate these findings, the specific tyrosine dephosphorylation
was followed by anti-phosphotyrosine Western blot analysis (Fig.
4C). With both VHR (0.5 µM) and MKP3 (0.5 µM), rapid tyrosine dephosphorylation of ERK (5 µM) was observed. By 10 min, greater than 90% of the
phosphotyrosine was hydrolyzed. The rates of dephosphorylation by VHR
and MKP3 were comparable, with MKP3 reacting approximately 2-fold more
rapidly. No hydrolysis was observed in the buffer control. The rapid
dephosphorylation kinetics presented in Figs. 3 and 4, B and
C, are in excellent agreement, yielding
kcat/Km values between 20,000 and 40,000 M
1 s
1. The VHR
dephosphorylation data presented in this study were determined by four
independent approaches: 1) anti-phospho-ERK Western analysis (Fig.
2C); 2) ERK kinase activity assays (Fig. 3); 3) phosphoamino
acid analysis (Fig. 4B); and 4) anti-phosphotyrosine Western
blot analysis (Fig. 4C).
VHR Does Not Significantly Dephosphorylate p38 and JNK--
The
ability of VHR to discriminate between the different MAP kinases was
tested. Since H2O2 treatment (200 µM) of COS-1 cells leads to only modest activation either
of p38 or JNK stress MAP kinases (Fig. 5,
B and C), anisomycin was used to provide robust activation of these stress kinase pathways. Anisomycin, originally identified as an antibiotic, has been extensively utilized to induce
p38 and JNK MAP kinase pathways (56, 57). Interestingly, anisomycin and
H2O2 are equally potent stimulators of the ERK pathway (Fig. 5A). Recombinant VHR (0.16 µM)
or a buffer control were added to extracts generated from anisomycin
and H2O2-treated COS-1 cells. The
phosphorylation status of both p38 and JNK were then evaluated by
Western blotting with antibodies specific to the phosphorylated forms
of these proteins (Fig. 5, D and E). The
dephosphorylation of p38 and JNK is not appreciably affected by
exogenously added VHR. The dephosphorylation of p38 is rapid whether or
not VHR is added, although a slight increase is noted in the presence
of VHR. These data indicate the presence of a p38-specific phosphatase
which rapidly dephosphorylates the kinase and is insensitive to 200 µM H2O2. Although we cannot rule
out the possibility that endogenous VHR is responsible for this rapid p38 activity, it is unlikely since VHR would be rendered inactive by
the H2O2 treatment. The phosphorylation state
of JNK is unchanged in both the buffer control and in the sample with
recombinant VHR. These results are in contrast to our finding that VHR
rapidly and specifically dephosphorylates ERK from
H2O2 treated COS-1 cells (Fig. 2). In the above
experiment, COS-1 cells were treated with both anisomycin and
H2O2 to ensure that any PTPs present will be
transiently inactivated. When extracts from anisomycin-treated COS-1
cells were utilized, a significant amount of ERK dephosphorylation was
observed in the buffer control (see below). These results suggested
that endogenous VHR or another PTP is active in anisomycin-treated COS-1 cell lysates.

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Fig. 5.
Activation of ERK, p38, and JNK pathways and
analysis of VHR specificity. Activation of ERK, p38, and JNK
pathways (A, B, and C). COS-1 cells were grown to 80-90%
confluence and serum starved prior to treatment with 10 µg/ml
anisomycin or 100 µM H2O2 for 30 min. Cells were rinsed and then lysed in the presence of protease
inhibitors. Activation of each kinase by anisomycin or
H2O2 was determined by Western blots using
antibodies specific for the phosphorylated forms of ERK (A),
p38 (B), or JNK (C). Total MAP kinase levels were
determined from anti-ERK, anti-p38, and anti-JNK Western analysis,
shown in the lower panels of A-C. Analysis of VHR
specificity (D and E). Extracts from anisomycin-
and H2O2-treated COS-1 cells were combined with
0.16 µM recombinant VHR, or an equal volume of buffer,
and incubated at 30 °C for the indicated times. D,
Western blot analysis using an antibody specific to the phosphorylated
form of p38 in the presence of buffer (top panels) or wild
type VHR phosphatase (bottom panels); E, Western
blot analysis using an antibody specific to the phosphorylated form of
JNK, in the presence of buffer (top panels) or wild type VHR
phosphatase (bottom panels). Total p38 and JNK levels were
determined from anti-p38 and anti-JNK Western analysis and are included
(D and E)
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Immunodepletion of Endogenous VHR Eliminates ERK
Dephosphorylation--
To explore the idea that VHR is the ERK
phosphatase responsible for its rapid inactivation, endogenous VHR was
immunodepleted from extracts of anisomycin-treated COS-1 cells and the
time course of ERK dephosphorylation was assessed (Fig.
6). Extracts were incubated with either
capped Affi-Gel 10 (mock treated) or Affi-Gel 10 coupled to an antibody
(chicken polyclonal) raised against VHR. The lysates were then
separated from the beads and the dephosphorylation of ERK and p38 were
analyzed by Western blotting with antibodies specific to their
respective phosphorylated forms (Fig. 6). Immunodepletion of endogenous
VHR resulted in a marked decrease in the dephosphorylation of ERK over
time (Fig. 6A), but had no effect on the dephosphorylation of p38 (Fig. 6B). After 20 min, the mock-treated sample
showed nearly complete ERK dephosphorylation (Fig. 6A) while
the immunodepleted sample displayed only a slight drop in ERK
phosphorylation. VHR Western blot analysis indicated that >90% of the
endogenous VHR was removed by immunodepletion (Fig. 6C). The
slight ERK dephosphorylation observed after 20 min is consistent with
the small amount of residual VHR. These results suggest that VHR may
account for all the ERK phosphatase activity observed in COS-1 cell
extracts.

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Fig. 6.
Immunodepletion of endogenous VHR eliminates
the in vitro dephosphorylation of ERK. COS-1
cells were grown to 80-90% confluence and serum starved prior to
treatment with 10 µg/ml anisomycin for 30 min. Cells were then lysed
in the presence of protease inhibitors and incubated with either capped
Affi-Gel 10 (mock treated) or a specific chicken anti-VHR antibody
coupled to Affi-Gel 10. The beads were removed and depleted lysates
were then incubated at 30 °C for 20 min prior to Western blot
analysis (A-C). Western blot analysis using antibodies
specific to the phosphorylated forms of ERK (A), or p38
(B) for both mock-depleted or VHR-depleted lysates at 0 and
20 min. Total ERK and p38 levels were determined from anti-ERK and
anti-p38 Western analysis and are shown in the lower panels;
C, VHR Western blot analysis (immunopurified rabbit anti-VHR
antibody, "Experimental Procedures"), showing that VHR protein
levels are greatly diminished upon immunodepletion.
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VHR Phosphatase Is Widely Expressed in Mammalian Cells--
To
examine endogenous VHR expression levels, Western blotting was
performed on a variety of mammalian cell lines. Using an immunopurified
rabbit anti-human VHR antibody, Western blotting of whole cell extracts
revealed an immunoreactive protein band of 20,600 Da corresponding to
VHR in all cell lines tested (Fig. 7A). Using quantitative
Western blot analysis of recombinantly expressed VHR (data not shown),
we estimated that VHR constitutes as much as 0.1% of the Nonidet P-40
dissolved cellular protein. High levels of protein expression were
observed in COS-1, COS-7, CV-1, and A431 cell lines. Similar high
expression was also observed in human breast cancer cell lines BT474,
SKBR3, and T47D and breast cell line HBL-100. VHR thus appears to be
widely and highly expressed in cells, suggesting that it plays a
fundamental role in cellular processes. It should be noted that VHR
protein levels are not altered by serum, EGF, or other mitogenic
stimulation (data not shown). This is in direct contrast to the
expression of previously reported MKPs whose protein levels are only
detectable after 30 min of mitogenic stimulation. Immunostaining of
endogenous VHR in COS-1 cells revealed that VHR resides
primarily in the nucleus, however some cytoplasmic staining was also
observed (Fig. 7B).

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Fig. 7.
VHR protein expression in a variety of
mammalian cell lines and immunostaining of endogenous VHR protein in
COS-1 cells. A, VHR Western blot analysis of extracts
from CV-1 (1 µg), HeLa (2.5 µg), and SKBR-3 (2.5 µg of nuclear
fraction) cells showing immunoreactivity to the 20.6-kDa protein. High
levels of protein expression were also observed in COS-1, COS-7, A431,
PC-12, BT474, T47D, and HBL-100 cell lines. VHR constitutes as much as
0.1% of the Nonidet P-40 dissolved cellular protein. B,
immunostaining of endogenous VHR in COS-1 cells. Cells were fixed with
formaldehyde and stained using a polyclonal primary antibody (rabbit)
specific to VHR and a secondary goat anti-rabbit antibody conjugated to
the Alex 488 fluorophore. Similar nuclear staining was also observed in
A431 cells (data not shown). C, no significant
immunofluorescence was observed when the primary antibody was first
pre-absorbed to recombinant VHR. Cells were viewed using a Leica DMRB
microscope with a 63X PL APO objective.
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ERK Is an in Vivo Substrate of VHR--
To provide evidence for
the in vivo dephosphorylation of ERK by VHR, we investigated
whether ERK phosphorylation in EGF-treated COS-1 cells was dependent on
VHR protein levels. To accomplish this, we co-transfected COS-1 cells
with ERK2 (c-Myc tagged) and either a wild type VHR or an RNA antisense
VHR construct. Serum-starved cells were stimulated with EGF for 20 min,
myc-ERK2 was recovered from lysates via immunoprecipitation, and the
level of ERK2 phosphorylation was determined. Overexpression of wild
type VHR leads to almost complete ERK dephosphorylation after 20 min of
EGF stimulation relative to the antisense vector control (Fig.
8A, left panel), but did not
effect the initial activation of ERK (Fig. 8 and data not shown). To
verify protein levels, Western blots of VHR (Fig. 8B),
immunoprecipitated c-myc-ERK (Fig. 8C) and expressed
c-myc-ERK were performed (Fig. 8D). A 6-fold decrease in ERK
phosphorylation (Fig. 8A, left panel) and a >6-fold
increase in VHR protein levels (Fig. 8B) were calculated in
cells transfected with the wild type vector over cells transfected with
the antisense control. This indicates that there is a direct
correlation between the phosphorylation of ERK and the cellular levels
of VHR. We were aware of the possibility that transfection of VHR
antisense RNA could lower the expression of endogenous VHR in our
studies (58). Direct detection of endogenous VHR levels between the
pcDNA3 and pcDNA3-antisense-VHR transfected cells was difficult
because of the 10-30% transfection efficiency. However, VHR Western
blots of total cell lysates suggested there is a slight decrease of VHR
protein levels in cells transfected with pcDNA3-antisense-VHR
compared with pcDNA3. Although these small changes were within
experimental error, it is quite possible that transfection with the
antisense plasmid resulted in significant suppression of endogenous VHR
protein. Regardless, the pcDNA3-antisense-VHR plasmid is a suitable
control for comparisons with pcDNA3-VHR transfected cells.

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Fig. 8.
In vivo dephosphorylation of ERK
by VHR. Overexpression of VHR in COS-1 cells down-regulates
EGF-stimulated ERK phosphorylation. COS-1 cells were transfected with
myc-ERK2 and either pcDNA3-antisense VHR or pcDNA3-VHR.
Transfected cells were then serum starved, treated with 100 nM EGF for 20 min, and lysed in the presence of protease
and phosphatase inhibitors. Myc-ERK2 was then recovered via
immunoprecipitation with c-Myc-conjugated antibody. A,
Western blot analysis using the phospho-ERK antibody showing the level
of phosphorylated myc-ERK2 recovered from transfected cells.
B, Western blot of cell lysate using the VHR antibody
showing the level of VHR overexpression. C, the phospho-ERK
blot from A was then reprobed with a general ERK antibody in
order to determine the relative level of immunoprecipitated myc-ERK2
between lanes. D, the VHR blot from B was then
reprobed with the anti-ERK antibody in order to determine overall
myc-ERK2 expression levels. The protein levels in untreated transfected
cells was determined and is displayed in the right panels
(A-D).
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DISCUSSION |
Using a variety of approaches including affinity chromatography,
enzyme kinetics, and cellular transfection studies, we demonstrated that ERK1 and ERK2 are authentic in vitro and in
vivo substrates of VHR. Our data suggest a novel role for VHR in
down-regulating the ERK pathway, distinct from that proposed for the
MKPs. Identifying physiological substrates of DS-PTPs and PTPs is a
fundamental step toward defining their roles in regulating signaling pathways.
Affinity Chromatography--
A substrate trapping approach has
been recently employed using the protein-tyrosine phosphatases PTP1B
(59), YopH (60), and TCPTP (61). However, instead of identifying
substrates from immunoprecipitated complexes (59-61), we have used
affinity chromatography to absorb substrates to catalytically inactive
mutants of VHR. With this approach, phosphoproteins only interact with
the coupled phosphatase and the use of immune complexes is avoided.
Utilizing this technique, we have identified ERK1 and ERK2 as authentic substrates of the dual-specificity phosphatase VHR. With the D92A VHR
affinity absorbent, the phosphorylated forms of ERK1 and ERK2 were
specifically retained, and eluted with the strong competitive inhibitor
arsenate (Fig. 1). This method should have wide utility for determining
the physiological substrates of other dual-specificity phosphatases and PTPs.
In Vitro Dephosphorylation of ERK1 and ERK2 by VHR--
Detailed
biochemical and kinetic experiments were then performed to validate
ERK1 and ERK2 as physiological substrates of VHR. Our study
demonstrated that VHR is highly specific for ERK1 and ERK2 (Fig. 2),
that VHR recognizes the native structure (Fig. 4A), and that
VHR dephosphorylates ERK with a second-order rate constant of 40,000 M
1 s
1 (Fig. 3). VHR was shown
to have no effect on the dephosphorylation of JNK and only slight
effect on p38 dephosphorylation (Fig. 5, D and
E). In addition, immunodepletion of endogenous
VHR nearly eliminated dephosphorylation of cellular ERK but not of p38,
suggesting that VHR accounts for nearly all of the ERK phosphatase
activity in COS-1 cells. VHR catalyzes the specific hydrolysis of
phospho-Tyr185 from activated ERK (Fig. 4, B and
C), resulting in direct inactivation of kinase activity
(Fig. 3). The concentration of phosphatase used and the rate constants
for hydrolysis are important factors to consider when drawing
conclusions about physiological substrates. Low catalytic
concentrations of VHR (the ratio of VHR to active ERK was as low as
1:23) were used during the in vitro dephosphorylations of
recombinant ERK. To establish the validity of any particular phosphatase-substrate pair by in vitro dephosphorylation
studies, the amount of enzyme used and the value of the rate constants are critical. The enzyme concentrations used in the current study were
much lower than those used in other studies to validate DS-PTP substrates (9, 62). With these considerations, the rapid dephosphorylation (40,000 M
1
s
1) of ERK by low catalytic levels of VHR supports the
proposal that ERK1 and ERK2 are relevant substrates. We believe that a stringent kinetic analysis is critical to any investigation in which
the validation of physiological substrates of DS-PTPs and PTPs is required.
In Vivo Dephosphorylation of ERK by VHR--
We demonstrated that
the in vivo phosphorylation of ERK depends on VHR protein
levels in EGF-stimulated COS-1 cells (Fig. 8). These transfection
studies provide convincing evidence that ERK1 and ERK2 are in
vivo targets of the VHR phosphatase. These studies compliment the
detailed biochemical and kinetic investigations which provide direct
proof for the specific binding and efficient catalysis of ERK by VHR.
Overexpression of VHR resulted in a 6-fold decrease in the total level
of phosphorylated ERK following 20 min of stimulation, as compared with
a VHR antisense control vector (Fig. 8A). In addition, the
kinetics of VHR-dependent dephosphorylation indicate that
overexpression of VHR leads to rapid ERK inactivation following normal
activation (Fig. 8A and data not shown).
Western blot analysis demonstrated that total VHR protein levels do not
change upon EGF, H2O2, and serum stimulation,
consistent with a previous report of unaltered RNA transcription upon
mitogenic stimulation (10). It should be again noted that significant levels of endogenous VHR protein were observed in COS-1 cells. As a
consequence, transient overexpression of VHR in COS-1 cells simply
results in elevating the concentration of a normally expressed protein.
We estimate that overexpression of VHR results in a 6-fold or greater
increase above normal levels (Fig. 8B). It would be predicted that only the rate of VHR's normal function would
be accelerated by increasing the enzyme level. As predicted, the dephosphorylation rate of ERK is enhanced by higher in vivo
concentrations of VHR.
Role of VHR in ERK Signaling--
Constituitive expression and
nuclear localization of VHR is consistent with the proposal that
nuclear VHR is involved in the rapid inactivation of ERK by
dephosphorylating Tyr185. Our data would also suggest that
VHR may function in quiescent cells to maintain ERK in the inactive
state. In this capacity, VHR would ensure that spurious or below
threshold activation is maintained until appropriate stimulation
is communicated to the cell. As discussed earlier, a constitutively
expressed and tyrosine-specific phosphatase has been implicated in
direct inactivation of ERKs. In rat mesangial cells, sustained ERK2
activation by endothelin and EGF was regulated by a vanadate-sensitive
protein phosphatase but not by a transcriptionally regulated protein
(20). Rapid inactivation of ERK2 in a variety of cell lines was
attributed to the serine/threonine protein phosphatase PP2A and an
unknown PTP (22). The protein synthesis inhibitor cycloheximide failed to affect the inactivation of MAP kinase following induction with EGF
in A431 and PC12 cells (21, 23). Gopalbhai et al. (19) have
proposed that a tyrosine-specific phosphatase represses ERK1 and ERK2
activity in the absence of serum, and that a tyrosine phosphatase
regulates ERK1 and ERK2 activity in cells transformed by upstream
oncoproteins. The data presented in the current study strongly
suggests that VHR is the candidate phosphatase responsible for these
previously described phenomena.
Single and Dual-specificity Protein Phosphatases--
Upon
dual-phosphorylation by MEK, activated ERK is translocated to the
nucleus where it regulates expression of immediate-early genes
(63-66). Subsequent dephosphorylation of ERK is then thought to
mediate its exit from the nucleus. Our results are consistent with a
model in which the dephosphorylation of nuclear ERK by VHR may lead to
its removal from the nucleus. Because dual-phosphorylation is required
to maintain high activity (1000-fold activation) (65), the actions of
single-specificity phosphatases are sufficient to inactivate the MAP
kinases. The constitutive and serine/threonine-specific phosphatase
PP2A may act in concert with VHR by dephosphorylating Thr183 of ERK (21, 22, 67). The cell's use of
single-specificity phosphatases may reflect an additional level of
regulation for ERK activation, not yet fully realized. This type of
regulatory mechanism prompts the consideration of several interesting
areas for future study, including the regulation of ERK by
single-specificity phosphatases, the mechanism of subsequent
reactivation of monophosphorylated ERK by MEK or single specificity
kinases, the relevance of ERK autophosphorylation, and the subcellular
distribution of the various phosphorylated forms of ERK. Although
diphosphorylation of ERK promotes its nuclear translocation and
retention, the consequent trafficking of the monophosphorylated ERKs
has not been described. The fission yeast phosphatase Pyp1 inactivates
the stress-activated MAP kinase SpcI/StyI by
specific dephosphorylation of Tyr173 within the activation
motif (68), suggesting that MAP kinase regulation by single-specificity
phosphatases may be conserved throughout eukaryotic evolution.
Feedback regulation of the MAP kinase pathways is thought to be
mediated by the inducible MKPs which act to turn off the pathway by
dephosphorylating both threonine and tyrosine. The tyrosine specificity
of VHR distinguishes this phosphatase from these truly dual-specific
MKPs. The physical basis for the two distinct specificities has not
been established. However, the major structural differences between VHR
and the MKPs may hold the answer. VHR is a single domain structure
consisting of the catalytic core that is conserved among all DS-PTPs
(36). The MKPs harbor a catalytic domain, but also contain an
amino-terminal domain. In the case of MKP3, this amino-terminal domain
binds ERKs independently of phosphorylation and causes a 34-fold
activation of MKP3 phosphatase activity (69, 70). In the absence of
this activation MKP3 is a rather inefficient enzyme. The amino-terminal
domain is thus thought to provide high affinity binding to ERK. Using
small diphosphorylated peptides corresponding to the activation lip of
the MAP kinases, it has been demonstrated that both VHR and MKP3 prefer
hydrolysis at phosphotyrosine relative to phosphothreonine in the
context of these synthetic peptides (33, 55). For VHR, the preference for phosphotyrosine hydrolysis is 1000-fold higher than for
phosphothreonine. The phosphothreonine hydrolysis by MKP3 could not be
detected. The 1000-fold lower activity was concluded to reflect the
lower affinity for phosphothreonine. For the MKPs, like MKP3, the amino terminus may provide the basis for the efficient phosphothreonine hydrolysis observed against diphosphorylated ERK protein. Independent of phosphorylation, the high affinity binding of the MKP amino-terminal domain to ERK provides an efficient means to increase the "effective concentration" of both phosphomonoesters, leading to efficient catalytic rate enhancement. On the other hand, since VHR appears to
bind to ERK in classical enzyme/substrate fashion, there is no added
energetic basis to compensate for the 3-4 orders of magnitude difference in the intrinsic reactivity between phosphotyrosine and
phosphothreonine. We are currently exploring this model as a mechanism
for dual-specificity of the MKPs.
Regulation of VHR and PTPs--
The proposed function of VHR
raises an interesting question regarding constituitively expressed
PTPs. That is, how is a growth signal maintained if the PTPs that
function to down-regulate the pathway are present during mitogenic
signaling? It is likely that a negative regulatory mechanism exists to
turn off the PTPs. Support for negative regulatory mechanisms has
come from several investigations (37, 71-74).
Numerous reports have now indicated that cellular redox status plays an
important role in the mechanisms which regulate the function of growth
factors and tyrosine phosphorylation-dependent signal
transduction pathways (38-43). Hydrogen peroxide has been shown to
stimulate a complete program of mitogenic signal transduction. Hydrogen
peroxide-treated cells mimic the phosphorylation cascades that are
induced by several growth factors such as EGF (44) and platelet-derived
growth factor (45). Potent activation of ERK is observed after
H2O2 treatment (Refs. 46-49 and see
"Results"). Recent reports have indicated that the cellular
production of H2O2 is involved in normal
receptor-mediated signal transduction. In vascular smooth muscle cells,
platelet-derived growth factor transiently increased the intracellular
concentration of H2O2 (45). When growth factor
stimulation of intracellular H2O2 production was blocked, tyrosine phosphorylation, MAP kinase activation, DNA
synthesis, and chemotaxis were inhibited. Similarly, EGF treatment induced the transient intracellular generation of
H2O2 in human epidermoid carcinoma cells (75)
and the tyrosine kinase activity of EGF receptor was required for the
production of H2O2. There is now significant
evidence that H2O2 is generated by
receptor-mediated events and thereby may play a critical role in signal
transduction. We (37) and others (74) have proposed that PTPs are a
logical target of H2O2, leading to transient
and reversible inactivation of phosphatase activity by oxidizing the
catalytic cysteine to a sulfenic acid (37).
In support of this proposal, we have observed robust activation of ERK
by low levels (200 µM) of H2O2 in
COS-1 cells. Curiously, only slight activation of the stress MAP kinase
p38 and JNK was observed (Fig. 5, B and C).
Treatment of COS-1 cells with H2O2 eliminates
the dephosphorylation of ERK by endogenous VHR (Fig. 2). Addition of
reduced thiols recovers the ERK-specific phosphatase activity (data not
shown). In anisomycin-treated cells, VHR rapidly dephosphorylates ERK
(Fig. 6A), indicating that VHR is not inactivated during
anisomycin stimulation. Collectively, the results suggest that VHR is
transiently inactivated in H2O2-treated cells
and are consistent with a model of PTP inactivation in which the
catalytic cysteine is reversibly oxidized. These observations support a mechanism of ERK regulation whereby H2O2
negatively regulates the activity of VHR, allowing for full ERK
activation during mitogenic stimulation.