(Received for publication, October 2, 1996, and in revised form, November 19, 1996)
From the Geneva Biomedical Research Institute, Glaxo
Wellcome Research and Development S.A., CH-1228 Plan-les-Ouates,
Geneva, Switzerland and the § Cancer Research Campaign
Centre for Cell and Molecular Biology, Chester Beatty Laboratories,
The Institute of Cancer Research, Fulham Road,
London SW3 6JB, United Kingdom
Extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK), and p38/RK/CSBP (p38) mitogen-activated protein (MAP) kinases are target enzymes activated by a wide range of cell-surface stimuli. Recently, a distinct class of dual specificity phosphatase has been shown to reverse activation of MAP kinases by dephosphorylating critical tyrosine and threonine residues. By searching the expressed sequence tag data base (dbEST) for homologues of known dual specificity phosphatases, we identified a novel partial human sequence for which we isolated a full-length cDNA (termed MKP-4). The deduced amino acid sequence of MKP-4 is most similar to MKP-X/PYST2 (61% identity) and MKP-3/PYST1 (57% identity), includes two N-terminal CH2 domains homologous to the cell cycle regulator Cdc25 phosphatase, and contains the extended active site sequence motif VXVHCXAGXSRSXTX3AYLM (where X is any amino acid) conserved in dual specificity phosphatases. MKP-4 produced in Escherichia coli catalyzes vanadate-sensitive breakdown of p-nitrophenyl phosphate as well as in vitro inactivation of purified ERK2. When expressed in COS-7 cells, MKP-4 blocks activation of MAP kinases with the selectivity ERK > p38 = JNK/SAPK. This cellular specificity is similar to MKP-3/PYST1, although distinct from hVH-5/M3-6 (JNK/SAPK = p38 >>> ERK). Northern analysis reveals a highly restricted tissue distribution with a single MKP-4 mRNA species of approximately 2.5 kilobases detected only in placenta, kidney, and embryonic liver. Immunocytochemical analysis showed MKP-4 to be present within cytosol although punctate nuclear staining co-localizing with promyelocytic protein was also observed in a subpopulation (10-20%) of cells. Chromosomal localization by analysis of DNAs from human/rodent somatic cell hybrids and a panel of radiation hybrids assign the human gene for MKP-4 to Xq28. The identification and characterization of MKP-4 highlights the emergence of an expanding family of structurally homologous dual specificity phosphatases possessing distinct MAP kinase specificity and subcellular localization as well as diverse patterns of tissue expression.
Extracellular signal-regulated kinase (ERK),1 c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) and p38/RK/CSBP (p38) are distinct classes of mitogen-activated protein (MAP) kinase activated by a range of growth factors as well as pro-inflammatory cytokines and cellular stress (1-10). MAP kinases in turn phosphorylate diverse target proteins in membrane or cytosolic fractions (e.g.. kinases, cytoskeletal elements, phospholipase A2, and stathmin) as well as a number of nuclear transcription factors, indicating a critical role orchestrating many short and long term changes in cell function (8, 11-16). This has been confirmed recently using specific chemical kinase inhibitors or by expressing mutant versions of different MAP kinases or their upstream activators. These studies show that ERK kinases play a pivotal role mediating neuronal differentiation in PC12 cells as well as growth factor-stimulated proliferation and oncogenic transformation in fibroblasts (17-21). Such approaches also support the view that JNK/SAPK and p38 MAP kinases are critical to processes mediating platelet aggregation and secretion, in generation of inflammatory cytokines, and in pathways leading to apoptotic death in a number of cell types (7, 10, 22-25).
Full MAP kinase activation requires phosphorylation on both tyrosine and threonine residues by selective upstream dual specificity kinases (13, 26, 27). Since MAP kinase activation is a reversible process, control of cellular protein phosphatases is likely be an important regulatory mechanism. Recently, an emerging family of dual specificity phosphatases has been shown to inactivate MAP kinase through dephosphorylating both threonine and tyrosine residues crucial for enzymatic activity (28). Currently eight distinct dual specificity phosphatases have been identified. These include MKP-1/CL100 (identical to 3CH134) (29-32), VHR (33), PAC1 (34, 35), hVH-2 (also cloned as MKP-2, TYP-1) (36-38), hVH-3 (same as B23) (39, 40), hVH-5 (orthologue of M3-6) (41, 42), MKP-3 (identical to rVH6 and orthologue of PYST1) (43-45), and MKP-X (orthologue of PYST2) (43, 45). Among these family members, MKP-3/PYST1 and hVH-5/M3-6 appear exceptional insofar that they exhibit highly selective inactivation of either ERK or JNK/SAPK and p38 MAP kinases, respectively (45, 46). MKP-3/PYST1 is also the first dual specificity phosphatase to display an exclusively cytosolic rather than nuclear localization (43, 45). Interestingly, several dual specificity phosphatases undergo powerful regulated expression following cell stimulation by growth factors and/or exposure to stresses, suggesting one major mechanism for control of MAP kinases (29, 32, 34, 36-40, 42-45). One critical unanswered question is whether the existence of multiple ERK, JNK/SAPK, and p38 kinase genes and splice variants (1-8, 13, 26, 27) demands a similarly diverse range of dual specificity phosphatases to allow specific and potentially highly compartmentalized control of MAP kinase signaling.
As part of an investigation of dual specificity phosphatase diversity and cell function, we have screened the expressed sequence tag data base (dbEST) to identify new dual specificity phosphatases. MKP-4 is one novel family member for which we have isolated a full-length cDNA. MKP-4 is most similar to MKP-X/PYST2 (61% identity) and MKP-3/PYST1 (57% identity) and contains two N-terminal Cdc25 homology domains as well as an extended active site motif characteristic of this gene family. MKP-4 inactivates MAP kinase both in vitro and when expressed in mammalian cells, where it displays selectivity for ERK family members. This enzymatic selectivity, together with a distinct subcellular localization and highly restricted pattern of tissue expression, suggests a specific regulatory role for MKP-4.
Restriction and DNA modifying enzymes were
purchased from New England Biolabs Inc. or Life Technologies, Inc., and
Taq DNA polymerase was from Perkin Elmer.
[35S]Methionine (1000 Ci/mmol),
[-32P]dCTP (3000 Ci/mmol), [
-33P]ATP
(1000 Ci/mmol), and streptavidin-coated SPA beads were obtained from
Amersham International (Buckinghamshire, United Kingdom), while
[
-32P]ATP (5000 Ci/mmol) was from DuPont de Nemours
International S.A. (Regensdorf, Switzerland). Dulbecco's modified
Eagle's cell culture medium was purchased from Life Technologies, Inc.
(Basel, Switzerland), Protein A-Sepharose 4 fast flow from Pharmacia
Biotech Inc. (Uppsala, Sweden), and murine EGF was from Promega
(Madison, WI). Anti-Myc 9E10 monoclonal antibody was purchased from Dr. Glaser AG (Basel, Switzerland), anti-HA monoclonal antibody HA.11 was
from Rowag Diagnostics (Zurich, Switzerland), biotinylated HA
monoclonal antibody 12CA5 from Boehringer Mannheim (Rotkreuz, Switzerland), while horseradish peroxidase conjugates of avidin, goat
anti-mouse IgG, and goat anti-rabbit IgG were all from Bio-Rad Laboratories (Glattbrugg, Switzerland). Biotinylated anti-rabbit antibody and fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse second antibody were from Vector Laboratories.
Avidin-conjugated Cy3 was from Sigma (Buchs,
Switzerland). Sulfo-NHS-LC-Biotin and protein A/G-horseradish
peroxidase conjugate were from Pierce (Zurich, Switzerland). All other
chemicals were obtained from Sigma. Rabbit polyclonal
antibody detecting promyelocytic protein was a kind gift from Dr. Anne
Dejean (Pasteur Institute, Paris). The following plasmids were generous
gifts obtained as follows: pcDNA1-HA-p44 ERK1 from J. Pouyssegur
(CNRS, Nice, France); pMT2T-HA-p54-SAPK
and pGEX-c-Jun-(5-89) from
J. R. Woodgett (Ontario Cancer Institute, Canada); pcDNA3-HA-p38
and pGEX-ATF2-(1-96) from J. S. Gutkind (NIDR, National Institutes of
Health, Bethesda, MD); pGEX-c-Jun-(1-79) from E. Bettini (Glaxo
Wellcome, Verona, Italy); pGEX-ATF2-(19-96) from N. Jones (Imperial
Cancer Research Fund, London, United Kingdom (UK)); pGEX-MAPKAP kinase
2
3B from M. Gaestel (Max-Delbrück-Centrum für
Molekulare Medizin, Berlin, Germany); and pGEX-ERK2, pGEX-MEK EE, and
pEXV3-Myc-p21ras (G12V) from C. J. Marshall (Chester Beatty
Laboratories, Institute of Cancer Research, London, UK).
By screening the
expressed sequence tag data base (dbEST) for sequences similar to dual
specificity phosphatases, we identified a partial human cDNA
(accession number R51175[GenBank]) highly homologous to the C terminus of MKP-3
(43). This clone (ID 38872) was obtained from Research Genetics, Inc.
(Huntsville, AL) I.M.A.G.E. Consortium (Lawrence Livermore National
Laboratory (LLNL), Livermore, CA) and used to prepare a random
[32P]dCTP-radiolabeled 428-bp StyI fragment
for screening an oligo(dT)-primed human placenta gt10 cDNA
library (provided by Dr. G. Buell, Geneva Biomedical Research
Institute), which was performed as described previously (43). Three
positive clones were isolated and EcoRI inserts subcloned
into pBluescript SK(
) (Stratagene) for sequencing. One clone was
2,303 kilobase pairs in length and contained an open reading frame of
1152-bp with a poly(A) signal sequence preceding a 3
poly(A) tail. The
nucleotide sequence is 94.5% identical with the original 474-bp EST
clone (R51175[GenBank]). This clone was termed MKP-4.
Full-length
MKP-4 in pBluescript SK() was translated in vitro using a
TNT T7 RNA polymerase-coupled reticulocyte lysate system (Promega) in
25-µl incubations for 2 h at 30 °C in the presence of
[35S]methionine as described by the manufacturer.
Reactions were stopped by addition of Laemmli sample buffer (47) and
resolved using a 12% SDS-polyacrylamide gel, which was then soaked in
AmplifyTM (Amersham) to reveal labeled protein.
For bacterial
expression the MKP-4 open reading frame was subcloned into pGEX4T3
(Pharmacia) as follows. A StyI 1152-bp fragment was first
isolated, Klenow-treated, and ligated to a SmaI-digested pBluescript SK(). A construct with the correct orientation was then
identified, digested with BamHI and NcoI,
Klenow-treated, and religated. The MKP-4 insert was then subcloned as a
BamHI-XhoI fragment in frame with the GST coding
sequence. pGEX/MKP-4 was used to transform E. coli BLR
(Novagen). Cells were grown overnight to saturation in LB medium
containing 100 µg/ml ampicillin, after which growth was resumed by
diluting the culture 1:50 and incubating at 37 °C for 1 h.
Following transfer to 20 °C for 1 h, IPTG was then added to a
final concentration of 100 µM and cells cultured for
another 9 h. Cells were harvested, resuspended in
phosphate-buffered saline containing 1% (v/v) Triton X-100, 5 mM dithiothreitol, 2 mM EDTA, 5 mM
benzamidine, and 1 mM PefablocTM (Boehringer Mannheim) and
lysed by passing three times through a French press at 1000 p.s.i.
The extract was then centrifuged at 10,000 × g for 15 min at 4 °C, and the supernatant incubated with
glutathione-Sepharose (Pharmacia, Uppsala), washed, and eluted as
described by the manufacturer.
GST/MKP-4 fusion protein was assayed for intrinsic phosphatase activity as described (29, 45) with minor modifications. Briefly, 8-40 µg of GST/MKP4 was incubated for 1 h at 37 °C in a reaction volume of 800 µl containing 20 mM p-nitrophenyl phosphate (pNPP), 50 mM imidazole (pH 7.5), and 5 mM dithiothreitol. Reactions were stopped by addition of 1 M NaOH and pNPP hydrolysis measured by absorbance at 410 nm.
In Vitro ERK2 Inactivation by MKP-4Constitutively
activated rabbit MAP kinase kinase (MEK1 S218E,S222E) and mouse ERK2
were generated as GST fusion proteins in E. coli and
purified using glutathione-Sepharose and nickel-agarose columns as
described (48, 49). Human stathmin (50) expressed in E. coli
was purified using a Q-Sepharose column2
and biotinylated using sulfo-NHS-LC-biotin (Pierce) at 4 °C for 2 h according to the manufacturers instructions. Biotinylated stathmin was then dialyzed against phosphate-buffered saline and 20 mM Tris-HCl (pH 7.5). ERK2 enzymatic activity was based on measurement of stathmin phosphorylation by SPA in 96-well sample plates.2 Briefly, in a final volume of 50 µl, 0.1 µg of
MEK EE, 0.5 µg of ERK2, and 1 µg of biotinylated stathmin were
mixed with 0.02-1.6 µg of purified GST/MKP4 in buffer SPA (15 mM MOPS (pH 7.0), 10 mM MgCl2, 0.5 mM EGTA, 50 µM NaF, 1 mM
dithiothreitol, 3 µM [-33]ATP (~2000
dpm/pmol)). Following incubation at 37 °C for 45 min, reactions were
terminated by adding 200 µl of streptavidin-coated SPA beads
(Amersham) (2.5 mg/ml) in phosphate-buffered saline containing 0.1%
Triton X-100, 5 mM EDTA, and 50 µM ATP and
left to incubate for 1 h at room temperature. Plates were then
centrifuged at 1800 × g for 5 min and counted in a
1450 MicrobetaPlus liquid scintillation counter (Wallac).
-MKP-4 was tagged at the C terminus with the Myc epitope and subcloned into pMT-SM (43, 51) as follows. A 1152-bp StyI fragment containing the entire open reading frame of MKP-4 was blunt-ended using mung bean nuclease and ligated with two sets of double-stranded oligonucleotide adaptors, which simultaneously added SalI and EcoRI restriction sites together with sequence encoding the Myc epitope EQKLISEEDLN followed by a stop codon at the C terminus. The SalI-EcoRI fragment was then subcloned into the corresponding sites of pMT-SM.
Cell Culture, Transfection, and StimulationCOS-7 cells
were grown under 7.5% CO2 in Dulbecco's modified
Eagles's medium containing 10% (v/v) fetal calf serum and 2 mM glutamine. Cells were grown in 6-well plates (35 mm
diameter) to 80% confluence and transfected using LipofectAMINE (Life
Technologies, Inc.) according to the manufacturer's instructions.
Transfections were performed using the following plasmid combinations:
1.0 µg of pcDNA1-HA-p44 ERK1, pMT2T-HA-p54-SAPK, or
pcDNA3-HA-p38 together with 0.01, 0.05, 0.1, 0.5, or 1.0 µg of
pMT-SM-Myc-MKP4. Total plasmid concentration was maintained constant by
supplementing with pMT-SM vector. Following 6 h of incubation with
LipofectAMINE and plasmid DNA, cells were washed and grown for 40 h before starvation by incubation in serum-free medium for 2 h.
Cells were then exposed to EGF (10 nM), anisomycin (10 µg/ml), or H2O2 (0.5 mM) at
37 °C for 10-30 min. Where indicated, cells were triple-transfected with 0.25 µg of pEXV3-Myc-p21ras (G12V), 1.0 µg of
pcDNA1-HA-p44 ERK1, together with 0.01-1.0 µg of
pMT-SM-Myc-MKP4; under these conditions, starvation was for the last
16 h of growth.
Following stimulation cells were washed twice in 2 ml of
ice-cold phosphate-buffered saline and scraped into Eppendorf tubes with 300 µl of buffer TP (50 mM Tris-HCl, pH 7.5, containing 150 mM NaCl, 1% (v/v) Nonidet P-40, 10 µg/ml
aprotinin, 10 µg/ml leupeptin, 1 mM benzamidine, 1 mM phenylmethylsulfonyl fluoride, 10 mM NaF, 1 mM sodium pyrophosphate, 1 mM sodium vanadate,
1 mM EDTA, 10 nM calyculin, and 25 mM -glycerophosphate). Cells were then homogenized using
a sonicator probe at full power for 3 s on ice. Aliquots (150 µl) of the COS-7 cell homogenate were mixed with 850 µl of buffer
TP and rotary mixed for 1 h at 4 °C, after which time they were
centrifuged at 100,000 × g for 20 min at 4 °C. The
supernatant (800 µl) was then mixed by rotary mixing for 2 h at
4 °C with 75 µl of a preformed immunoprecipitating complex (100 µl of HA-epitope specific HA.11 monoclonal antibody preincubated with
900 µl of 50% (v/v) protein A-Sepharose beads in 10 mM
Tris-HCl, pH 7.5, for 2 h at 4 °C). Beads were then sedimented
by centrifugation at 10,000 × g for 3 min and washed
twice in 1.0 ml of ice-cold buffer TP and once in 1.0 ml of buffer K
(50 mM HEPES, pH 7.4, containing 20 mM
MgCl2, 200 µM sodium vanadate, 2 mM dithiothreitol, and 10 mM
-glycerophosphate) followed by final resuspension in 50 µl of
buffer K. Immune complex assays were performed by mixing 10 µl of
bead suspension with 10 µl of 6 µM
[
-32P]ATP (~300,000 dpm/pmol), 10 µl of substrate
protein (15 µg of MBP, 10 µg of GST-ATF-2, or 10 µg of GST-MAPKAP
kinase 2
3), and 30 µl of buffer K followed by incubation for 30 min at 30 °C. Reactions were terminated by adding 15 µl of 10 × Laemmli sample buffer (47) and heating for 5 min at 95 °C.
Following centrifugation at 10,000 × g for 5 min,
supernatants were analyzed by SDS-polyacrylamide gel electrophoresis
and autoradiography as described previously (43).
For immunodetection of MAP kinases
precipitated for immune complex assay, 10 µl of immunocomplex beads
(see above) were diluted with 20 µl of 10 × Lammli sample
buffer and heated for 5 min at 95 °C, followed by centrifugation at
10,000 × g for 5 min. Western analysis was then
performed using supernatant fractions (20 µl) by
SDS-polyacrylamide gel electrophoresis and electrotransfer to
nitrocellulose membranes as described (43). Immunoprecipitated HA-ERK1,
HA-p54-SAPK, and HA-p38 MAP kinases were detected using biotinylated
HA-epitope-specific monoclonal antibody 12CA5 (Boehringer Mannheim)
together with avidin-horseradish peroxidase conjugate and enhanced
chemiluminescence. To detect levels of Myc-MKP-4 and Myc-p21ras
(G12V) expression in transfected COS-7 cells, Western analysis was
performed on cell homogenates (20 µg of protein) using anti-Myc epitope monoclonal antibody 9E10 and goat anti-mouse IgG horseradish peroxidase conjugate with chemiluminescence.
Northern analysis was performed
using ~2 µg of human tissue poly(A)+ RNA separated on
formaldehyde-agarose (1.2%) gels, transferred to nylon membranes, and
fixed by ultraviolet irradiation (Clontech Multiple Tissue Northern
Blots, catalogue nos. 7750-1, 7754-1, 7755-1, 7759-1, and 7760-1). A
random [32P]dCTP-radiolabeled 428-bp StyI
fragment corresponding to the 3-untranslated sequence of MKP-4 was
hybridized to membranes and exposed as described (43).
Rat sympathetic neurons from superior cervical ganglia of newborn rats were prepared, cultured, and microinjected with pMT-SM-Myc-MKP4 or pMT-SM-Myc-MKP-3 as described (43). COS-7 or NIH 3T3 cells were transfected with the same MKP-4 plasmid using LipofectAMINE as described above. Immunocytochemistry was performed using monoclonal antibody 9E10 with FITC-conjugated goat anti-mouse second antibody. In double labeling experiments, nuclear bodies were labeled with a rabbit polyclonal antibody detecting promyelocytic protein (PML), which was detected using a combination of biotinylated anti-rabbit antibody and avidin-Cy3 conjugate. Microscopic analysis of cells was performed using ultraviolet fluorescence.
Somatic Cell Hybrid AnalysisPCR primers corresponding to
the sequences 5-CAACGATGCCTATGACCTGG-3
(sense; nucleotides
1052-1071, Fig. 1) and 5
-GAAGGCGCCATCACTGGTGG-3
(antisense;
nucleotides 1231-1250, Fig. 1) were used to amplify a 200-bp fragment
of the 3
coding sequence of MKP-4 from a panel of human/rodent somatic
cell hybrid DNAs (obtained from the UK Human Genome Mapping Project
Resource Center). DNA amplification was performed with 32 cycles of
denaturation (94 °C, 30 s), annealing (52 °C, 30 s),
and extension (72 °C, 1 min), with a final extension (72 °C, 5 min). The products were analyzed by electrophoresis in 4% Metaphor
(FMC Bioproducts) agarose gels. Chromosomes retained by the hybrids are
summarized schematically in Fig. 11. The same primers and PCR
conditions were used to amplify MKP-4 from a panel of radiation hybrid
DNAs (52).
Southern Blot Analysis
Human male and female genomic DNAs
(10 µg) were digested overnight with EcoRI or
HindIII and subjected to agarose gel electrophoresis followed by transfer to Hybond N+ (Amersham). Membranes
were hybridized with a random [32P]dCTP-radiolabeled
428-bp StyI fragment corresponding to part of the
3-untranslated region of MKP-4. Hybridization was performed as
described (53).
To identify novel dual
specificity phosphatases, we performed BLAST computer searches of the
expressed sequence tag data base (dbEST) for partial cDNAs showing
similarity with MKP-3 (43). One human sequence (accession number
R51175[GenBank]) was found to be highly homologous to the C terminus of MKP-3
and, moreover, encoded the extended active site sequence motif
VXVHCXAGXSRSXTXXXAYLM conserved in dual specificity phosphatases (28, 42, 43). This clone (ID
38872) was obtained from Research Genetics, Inc. (Huntsville, AL)
I.M.A.G.E. Consortium (LLNL) and used to prepare a probe to screen a
human placental cDNA library (see "Experimental Procedures").
One of the positive clones contained an insert of 2,303 bp, and
nucleotide sequence analysis revealed 113 bp of 5-untranslated
sequence upstream of a translation start site CGCCCATGG compatible with
the Kozak consensus sequence (Fig. 1). This is followed
by an open reading frame of 1152 bp and a poly(A) signal sequence
preceding a 3
poly(A) tail (Fig. 1). Over the relevant region, the
nucleotide sequence displays 94.5% identity with the original 474-bp
EST clone (R51175[GenBank]), indicating that we had cloned the corresponding
full-length cDNA, which we called MKP-4.
The MKP-4 open reading frame encodes a protein of 384 amino acids, and
comparison of this sequence with the GenBankTM/EMBL Data Bank revealed
greatest homology with other members of the dual specificity
phosphatase family (see Introduction), with identities ranging from
61% for MKP-X/PYST2 down to 35% for hVH-5/M3-6. Direct comparison
between MKP-4 and its closest full-length homologue, MKP-3/PYST1 (57%
identity), indicates that regions of greatest similarity include, and
fall either side of, the extended active site sequence motif
VXVHCXAGXSRSXTX3AY(L/I)M
(where X is any amino acid) (Fig. 2,
solid boxes). This is also true for all other members of the
dual specificity phosphatase family, with which MKP-4 displays significantly greater sequence identity within the catalytic C-terminal half of the molecule (Fig. 3). Importantly, this region
in MKP-4 includes Asp-259, Cys-290, and Ser-300 (Fig. 1), which are
cognate to Asp-92, Cys-124, and Ser-131 of the dual specificity
phosphatase VHR and are likely to represent critical residues
underlying enzymatic activity (54, 55). According to a model of VHR
catalysis, Cys-290 of MKP-4 may function as an active-site nucleophile
forming a covalent thiol-phosphate intermediate, while Asp-259 acts as a general acid to donate a proton to the leaving group (54, 55).
Despite lower homology within the N-terminal half of the dual
specificity phosphatases (Figs. 2 and 3), this region of MKP-4 contains
two stretches containing residues conserved with two segments flanking
the active site of the Cdc25 phosphatase (Fig. 1, shaded
boxes). These regions of homology are termed CH2 domains and are
also found in other dual specificity phosphatases, although their
functional significance is currently unknown (56, 57).
MKP-4 Expression, Phosphatase Activity, and MAP Kinase Inactivation
MKP-4 is predicted to encode a protein of 41.8 kDa,
and this is in agreement with the size of a single
35S-labeled protein band generated upon in vitro
transcription and translation using a T7 RNA-polymerase-coupled
reticulocyte lysate system (Fig. 4). To establish
whether MKP-4 possesses endogenous catalytic activity, we overexpressed
full-length MKP-4 as a GST fusion protein in E. coli and
tested the purified protein using pNPP as substrate. Increasing
concentrations of GST/MKP-4 resulted in a linear rise in pNPP
hydrolysis (up to 40 µg of protein), and this catalytic activity was
effectively inhibited by the protein-tyrosine phosphatase inhibitor
sodium vanadate (Fig. 5A).
Given the close homology between MKP-4 and MKP-3/PYST1, which is highly selective for inactivating ERK family MAP kinases (45, 46), we next established whether GST/MKP-4 is able to inactivate ERK2 in vitro. Using a combination of purified recombinantly expressed proteins, ERK2 enzymatic activity was assessed by measuring phosphorylation of the MAP kinase substrate stathmin (50). Phosphorylation of stathmin is dependent upon ERK2 co-incubation with the constitutively active MAP kinase kinase MEK1 (S217E,S221E) (19). This phosphorylation reflects ERK2 activation as MEK1 (S217E,S221E) is alone unable to phosphorylate stathmin directly (data not shown). Inclusion of GST/MKP-4 in the reaction mix results in efficient blocking of ERK2 enzymatic activity (Fig. 5B). Importantly, GST/MKP-4 is unable to dephosphorylate stathmin directly (control, Fig. 5B), indicating highly effective ERK2 inactivation under these assay conditions.
To establish whether MKP-4 displays similar enzymatic activity within
intact cells, we next co-transfected the HA-tagged MAP kinases p44
ERK1, p54-SAPK, or p38 together with Myc-tagged MKP-4 in COS-7
cells. To obtain a clear impression of the relative effectiveness of
MKP-4 to inactivate each MAP kinase, cells were transfected using a
range of plasmid concentrations (0.01-1.0 µg/well). These concentrations were chosen to give a reproducible
dose-dependent increase in the levels of immunodetectable
MKP-4 protein (at ~44 kDa), while expression of each MAP kinase is
unaltered (Fig. 6). Thus, although with higher plasmid
concentrations levels of MKP-4 protein are likely to represent a
considerable overexpression, these conditions allow a direct comparison
of MKP-4-dependent inactivation of different MAP kinase
family members. Using this approach MKP-4 displays moderate selectivity
for p44 ERK1 when compared with p54 SAPK
and p38 MAP kinases. Hence,
EGF-stimulated p44 ERK1 activation is inhibited by ~50% when cells
were transfected with only 0.01 µg of plasmid, conditions under which
MKP-4 protein was not detected by Western analysis (Fig. 6). This level
of MKP-4 expression had little effect on anisomycin-stimulated p54
SAPK
or H2O2-dependent
activation of p38 MAP kinase (Fig. 6). Inhibition of p44 ERK1
activation is increased to ~80% following tranfection with 0.05 µg
of plasmid, while under identical conditions stress-induced activation
of p54 SAPK
and p38 MAP kinases was suppressed by <50% (Fig. 6).
Full blockade of p54 SAPK
and p38 MAP kinases required maximal
expression of MKP-4 using 1.0 µg of plasmid (Fig. 6). Together, these
studies confirm that as observed in vitro (Fig.
5B), MKP-4 is able to inactivate ERK family MAP kinases when
expressed in mammalian cells. Importantly, these results also indicate
that MKP-4 selectivity (ERK > p38 = JNK/SAPK) is similar to
its close homologue MKP-3/PYST1, which is significantly more effective
against ERK family isoforms (ERK
JNK/SAPK ~ p38) (45, 46). In
contrast to this, MKP-4 enzymatic specificity is quite different from
hVH-5/M3-6 (JNK/SAPK ~ p38
> ERK) (46), MKP-2/hVH-2/TYP-1 (ERK = JNK/SAPK > p38) (58), and PAC-1
(ERK = p38 > JNK/SAPK) (58). It is of note that, despite
these observations, we cannot exclude the possibility that additional
cellular proteins, possibly unrecognized MAP kinases, also represent
targets for MKP-4 action particularly at more physiological levels of
expression. Notwithstanding this caveat, the existence of multiple dual
specificity phosphatases with clear selectivity for inactivation of
known MAP kinases strongly suggests specific functional roles for
different family members.
Mutated constitutively activated p21ras (G12V) stimulates ERK
MAP kinases, and this may underlie mitogenesis and cellular
transformation induced by this oncogene (19, 59, 60). To test whether
MKP-4 is able to block oncogenic p21ras-dependent
MAP kinase activation, COS-7 cells were triple-transfected with
constitutively active Myc-tagged p21ras (G12V), p44 HA-ERK1,
and varying concentrations of Myc-MKP-4 plasmid. This experiment
reveals that, as with acute exposure to EGF (Fig. 6), MKP-4 blocked
completely p44 ERK1 activation by p21ras (G12V), although this
required higher concentrations of plasmid and expressed protein (Fig.
7). MKP-4-dependent blockade of growth factor and oncogene-stimulated MAP kinase activation as observed here
appears functionally equivalent to previous observations of ERK
inhibition following expression of a dominant negative mutant form of
MAP kinase kinase MEK1 (19), or in cells treated with the MEK1
inhibitor PD098059 (61). Since inhibition of ERK activity using these
approaches also blocks growth factor-stimulated proliferation and even
reverts oncogene-driven transformation (19, 61), one important function
for MKP-4 could be to inhibit cellular proliferation and possibly act
as a tumor suppressor.
MKP-4 Tissue Distribution
To establish which cells and
tissues may be major sites for MKP-4 action, we performed Northern blot
analysis on poly(A)+ RNA isolated from a range of human
cell and tissue types. Using a probe from the 3-untranslated sequence
of MKP-4, we detected a single band at 2.5 kilobases expressed only in
placenta and kidney (Fig. 8) and fetal liver (not
shown). We failed to detect any MKP-4 mRNA in the following adult
cells and tissues: heart, lung, liver, skeletal muscle, pancreas,
adrenal medulla, adrenal cortex, testis, ovary, thyroid, colon,
intestine, stomach, appendix, thymus, spleen, lymph node, leukocytes,
bone marrow, prostate, or any of 15 brain regions tested. This
distribution pattern may be similar to the human dual specificity
phosphatase hVH-2/TYP-1, which is also enriched in placenta while being
expressed in other tissues at low or undetectable levels (36, 38). In
contrast, MKP-4 expression differs substantially from PYST1, CL100, and hVH-3 (also human genes), which although detected in placenta, are also
found in a number of other cell and tissue types at similar or higher
levels (39, 40, 45, 57). Three other human dual specificity
phosphatases, PAC-1, PYST2, and hVH-5, display a highly restricted
expression pattern, although these are distinct from MKP-4 insofar that
they are enriched in hematopoietic tissues, liver, or brain and
skeletal muscle tissue, respectively (34, 42, 45). Together, these
studies highlight significant differences in the distribution of MKP-4
and other dual specificity phosphatase family members, indicating
important roles regulating MAP kinases in a limited complement of cell
and tissue types. High level MKP-4 expression in placenta, for
instance, appears to parallel the distribution ERK1 and ERK2 outside of
the nervous system (1) and supports the notion of regulatory
interaction under physiological conditions.
MKP-4 Subcellular Localization
Sustained activation of MAP
kinases has been reported to trigger their translocation to the nucleus
(62, 63). This relocalization places MAP kinases in close proximity
with several nuclear target proteins, including the transcription
factors Elk-1, c-Jun, ATF-2, and CHOP (11-16). ERK isoforms are also
associated with microtubules (64), and this could indicate sites of
specific subcellular anchorage as demonstrated for MAP kinases in
Saccharomyces cerevisiae, which bind the scaffold protein
Ste5 (65). Such an emerging picture of MAP kinase compartmentalization
could suggest that dual specificity phosphatases responsible for their
regulation will be localized to overlapping subcellular sites. For
instance, recently, we and others have reported that MKP-3/PYST1 is
exclusively cytosolic, indicating a role inactivating MAP kinases in
non nuclear compartments (43, 45). This contrasts with MKP-1/CL100,
PAC-1, hVH-2/MKP-2/TYP-1, and hVH-3/B23, which are localized entirely within the nucleus (34, 36, 39), while another dual specificity phosphatase family member, hVH-5/M3-6, is cytosolic or nuclear depending on the cellular environment (41). We have studied the
subcellular localization of Myc-tagged MKP-4 following expression in
rat sympathetic neurons as well as in COS-7 and NIH 3T3 cell lines. In
sympathetic neurons both MKP-3 and MKP-4 were immunodetectable within
the cell body cytoplasm, while MKP-4 protein was also clearly detectable within neurites (Fig. 9, A and
B). Interestingly, in some neurons, MKP-4 protein appeared
to concentrate in regions resembling synaptic swellings (Fig.
9B, indicated by arrows). By electron microscopy
these regions possess a high content of vesicular membranes
characteristic of premature synaptic
structures.3 MKP-4 was also localized
within the cytosol of NIH 3T3 (Fig. 9, C and D)
and COS-7 cells (Fig. 9, E and F), although a
subset of transfected cells (10-20%) also displayed punctate staining within the nucleus (Fig. 9, D and F). Confocal
microscopy confirms an intra-nuclear localization for MKP-4 (data not
shown). Although we have not yet been able to test whether localization
of heterologously expressed protein reflects exactly compartmentation
of endogenous MKP-4, this pattern of subcellular
localization is clearly different from other dual specificity
phosphatases also expressed as epitope-tagged proteins (34, 36, 39, 41,
43, 45).
Together with MKP-4, several dual specificity phosphatases have now been described as nuclear (see above), although none possess a clear bipartite nuclear targeting motif (66). Despite this, two N-terminal charged clusters exemplified by RRAR-(15)-RAR in hVH-3/B23 (39, 40) can be identified also in the primary amino acid sequences of MKP-1/CL100 (29), hVH-2/TYP-1 (36, 38), and PAC-1 (34) and could contribute to their presence in the nucleus. MKP-4 also contains two clusters of basic amino acids, although these are separated by a longer spacer (RRLRR-(30)-RRRR) (Fig. 1). It remains to be established whether this could account for its distinctive subcellular localization.
MKP-4 Co-localization with Promyelocytic Leukemia (PML) ProteinPunctate nuclear staining as seen for MKP-4 has also been
reported for PAC-1 (34), although not for CL100, hVH-2, or hVH-3 (36,
39), and may be a distinctive property of a subset of dual specificity
phosphatases. Punctate nuclear localization is also reminiscent of the
PML protein, which concentrates in discrete subnuclear regions known as
nuclear bodies or PML oncogenic domains (67). To test directly for
co-localization, we used a rabbit polyclonal antibody to PML and
performed double immunocytochemistry on MKP-4-transfected COS-7 cells.
This approach revealed that MKP-4 and PML display patterns of nuclear
staining that are exactly overlapping (Fig. 10,
A-C). Since PML has been reported as a phosphoprotein (68),
this nuclear staining pattern indicates a potential role for MKP-4 as a
direct or indirect regulator of kinases phosphorylating PML. Such a
regulatory interaction is currently under investigation in our
laboratories.
MKP-4 Chromosomal Localization
To determine the chromosomal
localization of MKP-4, DNAs from human/rodent somatic cell hybrids were
analyzed for the presence of the human MKP-4 gene by PCR. An
MKP-4-specific human fragment of 200 bp was distinguishable from
rodent-specific fragments. This human fragment was present in hybrids
GM07299, MCP6BRA, 1aA9607+, GM10478, THYB1.3, and HORL9, all of which
retain the long arm of human chromosome X (Fig. 11).
PCR analysis of DNAs from a panel of radiation hybrids (52) confirmed
this, assigning the gene for MKP-4 to Xq28 (data not shown). Southern
blot analysis of human male and female genomic DNAs using a fragment
from the 3-untranslated region of MKP-4 as a probe identified a single
band (Fig. 12). A stronger signal was detected in the
lanes containing female DNA, again indicating that the gene for MKP-4
is X-linked (Fig. 12). No other dual specificity phosphatase genes have
been found to map to the Xq28 gene (69,
70),4 and, based on tissue expression in
adult, there are no obvious candidate human diseases assigned to this
region that may be caused by mutations in MKP-4.
In this paper we report the identification and initial characterization
of MKP-4 as a novel dual specificity phosphatase. Although structurally
homologous to other family members, including an extended active-site
consensus as well as two N-terminal CH2 domains, MKP-4 is unique in
terms of both function and distribution. Our studies indicate that
MKP-4 inactivates MAP kinases with the selectivity ERK > p38 = JNK/SAPK. This enzymatic specificity is similar to its close
homologue MKP-3/PYST1 (ERK JNK/SAPK = p38) (45, 46),
although distinct from hVH-5/M3-6 (JNK/SAPK = p38 >>> ERK)
(46), MKP-2/hVH-2/TYP-1 (ERK = JNK/SAPK > p38) (58), and
PAC-1 (ERK = p38 > JNK/SAPK) (58). In addition to blockade of acute MAP kinase activation by MKP-4, inhibition of chronic ERK
activation by oncogenic p21ras (G12V) could indicate a tumor
suppressor function. MKP-4 displays a distinctive distribution insofar
that, among a large range of cell and tissue types, mRNA was
detected only in placenta, kidney, and embryonic liver. Its subcellular
localization is also striking in that in three distinct cell types
MKP-4 is clearly cytosolic with additional punctate nuclear staining
observed in a subset of cells. These observations highlight the
emergence of a growing family of dual specificity phosphatase
possessing many distinctive properties indicative of a gene family
performing specific functional roles in different cellular and
physiological contexts.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) Y08302[GenBank].
We are grateful to the following for generous
gifts: Dr. Anne Dejean for antibody detecting promyelocytic protein,
Dr. J. Pouyssegur for pcDNA1-HA/p44 ERK1, Dr. J. Woodgett for
pMT2T-HA/p54 SAPK, Dr. J. S. Gutkind for pcDNA3-HA/p38 HOG and
pGEX-ATF2-(1-96), Dr. N. Jones for pGEX-ATF2-(19-96), Dr. E. Bettini
for pGEX-c-Jun-(1-79), Dr. M. Gaestel for pGEX-MAPKAP kinase-2
3B,
and Professor C. Marshall for pGEX-ERK2, pGEX-MEK EE, and
pEXV3-Myc/p21ras (G12V). We also acknowledge our appreciation
of the support and encouragement provided by Drs. J. Knowles, J. Delamarter, and J.-C. Martinou.