(Received for publication, September 25, 1995)
From the
MKP-1 (also known as CL100, 3CH134, Erp, and hVH-1) exemplifies a class of dual-specificity phosphatase able to reverse the activation of mitogen-activated protein (MAP) kinase family members by dephosphorylating critical tyrosine and threonine residues. We now report the cloning of MKP-3, a novel protein phosphatase that also suppresses MAP kinase activation state. The deduced amino acid sequence of MKP-3 is 36% identical to MKP-1 and contains the characteristic extended active-site sequence motif VXVHCXXGXSRSXTXXXAYLM (where X is any amino acid) as well as two N-terminal CH2 domains displaying homology to the cell cycle regulator Cdc25 phosphatase. When expressed in COS-7 cells, MKP-3 blocks both the phosphorylation and enzymatic activation of ERK2 by mitogens. Northern analysis reveals a single mRNA species of 2.7 kilobases with an expression pattern distinct from other dual-specificity phosphatases. MKP-3 is expressed in lung, heart, brain, and kidney, but not significantly in skeletal muscle or testis. In situ hybridization studies of MKP-3 in brain reveal enrichment within the CA1, CA3, and CA4 layers of the hippocampus. Metrazole-stimulated seizure activity triggers rapid (<1 h) but transient up-regulation of MKP-3 mRNA in the cortex, piriform cortex, and some amygdala nuclei. Metrazole stimulated similar regional up-regulation of MKP-1, although this was additionally induced within the thalamus. MKP-3 mRNA also undergoes powerful induction in PC12 cells after 3 h of nerve growth factor treatment. This response appears specific insofar as epidermal growth factor and dibutyryl cyclic AMP fail to induce significant MKP-3 expression. Subcellular localization of epitope-tagged MKP-3 in sympathetic neurons reveals expression in the cytosol with exclusion from the nucleus. Together, these observations indicate that MKP-3 is a novel dual-specificity phosphatase that displays a distinct tissue distribution, subcellular localization, and regulated expression, suggesting a unique function in controlling MAP kinase family members. Identification of a second partial cDNA clone (MKP-X) encoding the C-terminal 280 amino acids of an additional phosphatase that is 76% identical to MKP-3 suggests the existence of a distinct structurally homologous subfamily of MAP kinase phosphatases.
A wide range of cell-surface stimuli, including growth and
differentiation factors and cytokines as well as ultraviolet radiation
and osmotic shock, trigger rapid and powerful activation of
mitogen-activated protein (MAP) ()kinase family
members(1, 2, 3, 4, 5) .
Currently, three major subclasses of MAP kinase can be identified, and
these comprise the ERK, SAPK/JNK, and p38/HOG1
families(2, 3, 6) . Full activation of MAP
kinase requires phosphorylation on critical tyrosine and threonine
residues, and several upstream dual-specificity kinases catalyzing this
modification have now been
identified(1, 2, 3, 6) . Once
activated, MAP kinases phosphorylate and regulate several cellular
proteins, including additional protein kinases, cytoskeletal elements,
stathmin, phospholipase A
, and transcription factors,
notably Myc, Elk-1, Jun, and
ATF-2(1, 7, 8, 9, 10, 11) .
This range of substrates indicates a pivotal role for MAP kinases in
cellular signal transduction, suggesting that mechanisms regulating the
extent and duration of their activation will play a key role in
controlling cell function. This is illustrated by mutational activation
of the MAP kinase kinase MEK, which leads to constitutive activation of
ERK2 accompanied by cellular transformation in fibroblasts or neuronal
differentiation in PC12 cells (12) . By contrast, inhibition of
ERK2 phosphorylation by interfering mutants of MEK suppresses growth
factor-stimulated proliferation, reverts oncogene-dependent
transformation, and blocks PC12 differentiation by NGF(12) .
Chemical inhibition of MEK similarly inhibits ERK phosphorylation as
well as PC12 cell differentiation(13) . These experiments
emphasize the critical importance of MAP kinase phosphorylation and
activation state in regulating cellular responsiveness and function.
MAP kinase phosphorylation is a reversible process, indicating that
protein phosphatases play a crucial role in controlling cellular
activities. Among the large number of protein-tyrosine phosphatases
currently identified(14, 15, 16) , an
emerging class of dual-specificity phosphatase may regulate directly
and specifically MAP kinase family members. The prototypic member of
this class is the vaccinia virus VH1
phosphatase(17, 18) , although mammalian homologs of
this gene have now been identified. The dual-specificity phosphatase
family is exemplifed by MKP-1 (also known as CL100, 3CH134, Erp, and
hVH-1), which dephosphorylates MAP kinases at both the Tyr and Thr
residues necessary for enzymatic
activity(19, 20, 21, 22, 23, 24) .
Activity toward phosphorylated Tyr and Thr is abolished when a single
active-site cysteine is mutated, suggesting a common catalytic
domain(23) . Other mammalian dual-specificity phosphatases
include VHR(25) , PAC-1(26, 27) , B23 (also
termed hVH-3)(28, 29) , hVH-2 (also known as MKP-2 and
TYP-1) (30, 31) , ()and hVH-5(32) .
Interestingly, many members of this gene family display rapid induction
by growth factors and cellular stresses, indicating an important
transcriptional mechanism for controlling MAP kinase activities (22,
23, 26, 28, 29, 30, 32-34). The significance of such
transcriptional regulation is unclear, although constitutive
overexpression or microinjection of MKP-1 blocks G
-specific
gene expression and S phase entry in fibroblasts(35) ,
suppresses normal and oncogene-driven
proliferation(19, 22, 23, 36) ,
inhibits neurite outgrowth in differentiating PC12 cells(37) ,
and blocks MAP kinase-dependent mesoderm formation in Xenopus embryo(38) . Antisense oligonucleotides that block
angiotensin II-dependent MKP-1 induction in vascular smooth muscle
cells also lead to a prolonged state of MAP kinase
activation(39) . Moreover, in the budding yeast Saccharomyces cerevisiae, pheromone triggers induction of the
dual-specificity phosphatase MSG5, which, when inactivated, leads to
increased activity of the MAP kinase FUS3 and diminished adaptation to
mating factor(40) . Clearly, these observations indicate a
critical role for dual-specificity phosphatases in the control of MAP
kinase activation state and associated cell functions.
In this report, we describe the cloning of a new dual-specificity phosphatase, MKP-3. Several structural features of this gene family are conserved in MKP-3, including an extended active-site sequence motif, VXVHCXXGXSRSXTXXXAYLM (where X is any amino acid), and two N-terminal domains displaying homology to Cdc25 phosphatase. MKP-3 blocks mitogen-stimulated activation of ERK2 and, unlike other dual-specificity phosphatases, is localized within the cytosol. This, together with a distinct tissue and brain distribution, suggests that MKP-3 plays an important and specific role in regulating MAP kinase activities. Isolation of a partial cDNA clone encoding a second phosphatase (MKP-X) 76% identical to MKP-3 indicates the existence of a distinct subfamily of structurally homologous MAP kinase phosphatases.
Specific antisense oligodeoxynucleotide
probes (40-mers) corresponding to rat MKP-3 and CL100 mRNAs were
synthesized (Applied Biosystems Model 394 synthesizer), purified by gel
electrophoresis, and diluted to a working concentration of 0.3
pmol/µl in double-autoclaved sterile water. Probes were end-labeled
using S-dATP(42) . In situ hybridization
was performed using fixed brain sections (12-µm thickness) that
were prewarmed to room temperature for 15 min.
S-Labeled
oligonucleotide probes (3
10
cpm/slide) were
diluted in 100 µl of hybridization buffer (50% (v/v) formamide, 4
SSC, 5
Denhardt's solution, 25 mM Na
PO
, 1 mM NaHPO
, 10%
(w/v) dextran sulfate, 10 µg of hydrolyzed salmon sperm DNA, 5
µg of polyadenylic acid) containing 10 mM dithiothreitol.
Sections were covered with 50
24-mm coverslips, and
hybridization was performed in a humidified chamber for 16-24 h
at 42 °C. After hybridization, coverslips were removed under 1
SSC at room temperature, and slides were washed first for 30
min in 1
SCC at 52 °C and then for 1 min in 1
SSC
and for 1 min in 0.1
SSC at room temperature. Slides were then
dehydrated by sequential immersion in 70% (v/v) and 100% (v/v) ethanol
for 3 min, after which they were air-dried and exposed to Amersham
Hyperfilm at room temperature for 1-6 days.
Nucleotide sequence analysis of the MKP-3 clone indicates that
it contains 226 bp of 5`-untranslated region and a translation start
site in good agreement with the Kozak consensus sequence (Fig. 1). Sequencing the 3`-untranslated region revealed a
poly(A) signal sequence and a poly(A) tail (data not shown). The open
reading frame extends 1146 bp and encodes a protein of 381 amino acids
with a predicted molecular mass of 42.3 kDa (Fig. 1). The
partial clone MKP-X does not contain a predicted translation initiation
site, but encodes the C-terminal 280 amino acids of a protein
displaying 76% amino acid identity to MKP-3 (Fig. 2). Alignment
of the deduced amino acid sequences of MKP-3 and MKP-X with the
GenBank/EMBL Data Bank revealed the greatest homology to
the dual-specificity phosphatases MKP-1, PAC-1, B23, hVH-2, and VHR.
The overall predicted amino acid sequence identity between this gene
family and MKP-3 is 32-37%, although this value masks greater
homology within C-terminal regions (Fig. 3A). Within
this region, MKP-X displays 83% primary amino sequence identity to
MKP-3 (Fig. 3A). As with all other dual-specificity
phosphatases, the C-terminal domains of both MKP-3 and MKP-X contain
the extended active-site sequence motif
VXVHCXXGXSRSXTXXXAYLM
(where X is any amino acid) (Fig. 3B). This
motif contains Cys-293 and Ser-300 (numbering according to MKP-3),
which, together with Asp-262, are cognate to Cys-124, Ser-131, and
Asp-92 of VHR and are likely to participate in the catalytic mechanism
underlying dual-specificity phosphatase
activity(47, 48) . Indeed, Cys-124 of VHR and
presumably also Cys-293 of MKP-3 are likely to serve as the active-site
nucleophile that forms a covalent thiol-phosphate intermediate during
catalysis(48) . Despite lower homology within N-terminal
regions, MKP-3 does contain stretches, termed CH2 domains, conserved
with two segments flanking the active site within the Cdc25 phosphatase (Fig. 3C). MKP-X also possesses one CH2 domain within
the most N-terminal sequence hitherto identified (Fig. 3C). These CH2 domains have also been identified
within the N-terminal regions of other dual-specificity
phosphatases(49, 50) , although their functional
significance is currently unknown.
Figure 1: Nucleotide and encoded amino acid sequences of MKP-3 cDNA. Amino acids are indicated in single letter code. Nucleotides and amino acids are numbered at the end of each sequence line, starting at the first nucleotide of the cloned cDNA and at the putative initiator methionine, respectively. The extended catalytic active-site motif conserved in dual-specificity phosphatases is indicated by the solid box. Residues in two domains (CH2) conserved within the Cdc25 phosphatase are boxed. Arrows indicate the positions of the degenerate primers used for reverse transcription-PCR to identify dual-specificity phosphatases, including the 210-bp product MKP-X used for screening the lung cDNA library.
Figure 2: Amino acid homology between MKP-3 and the C terminus of MKP-X. The amino acid sequences of MKP-3 and MKP-X encoded by the partial cDNA clone 310 are shown aligned using the GAP routine of the Wisconsin Genetics Computer Group sequence analysis software package 7. Identical residues are boxed.
Figure 3:
MKP-3 homology to other dual-specificity
phosphatases. A, schematic representation of MKP-3 amino acid
identity to other members of the dual-specificity phosphatase family.
Predicted amino acid sequences (MKP-1, GenBank/EMBL
accession number X84004 (M. Muda, unpublished data);
hVH-2(30) ; PAC-1, GenBank
/EMBL accession number
L11329(26) ; B23, GenBank
/EMBL accession number
U15932(28) ; and VHR, GenBank
/EMBL accession
number L05147(25) ) were compared with MKP-3 using the GAP
routine of the Wisconsin Genetics Computer Group sequence analysis
software package. Values shown are percentage identities (boxed) for independent comparisons of N- and C-terminal
regions based on residue numbers as indicated. N- and C-terminal
domains were defined based on a division on either side of the highly
conserved PV(E/Q)IL residues. B, conservation of the extended
active-site motif shared between dual-specificity phosphatase enzymes.
Amino acids used for comparison are indicated on the left of the
sequence, and identical residues are boxed. C,
alignment of two N-terminal homology domains (CH2-N and CH2-C) conserved between dual-specificity phosphatase family
members and human Cdc25 phosphatase (GenBank
/EMBL
accession number P30307) (49, 50) . Amino acid numbers
used for comparison are indicated, and identical residues are boxed.
Figure 4:
MKP-3 expression in COS-7 cells. COS-7
cells were transfected with empty plasmid (pMT-SM) or plasmid
containing MKP-3/Myc, MKP-3 (untagged), or TYP-1/Myc using
Lipofectamine. Following growth for 24 h and serum starvation for a
further 18 h, cells were incubated either with or without EGF (10
nM) for 10 min. A, COS-7 cell homogenates were
analyzed by Western blotting using a 10% gel and monoclonal antibody
9E10 detecting the Myc epitope. Immunoreactive bands of the expected
size were seen in cells expressing MKP-3/Myc (major band at 42 kDa) or
TYP-1/Myc (43 kDa). Numbers to the left indicate the positions
of molecular mass markers (in kilodaltons). B, COS-7 cell
homogenates were used for immunodetection of endogenous p42 ERK2 using a 10% gel and specific antibody 122. In control cells,
EGF induced a clear shift in ERK2 electrophoretic mobility, reflecting
increased phosphorylation state. This was blocked in cells expressing
MKP-3/Myc and untagged MKP-3. TYP-1/Myc expression resulted in partial
suppression of ERK2 phosphorylation state.
Mitogen-stimulated ERK2 phosphorylation results in a shift in its
electrophoretic mobility on SDS-polyacrylamide
gels(12, 51) . Consistent with this, when ERK2
endogenous to COS cells was studied by Western analysis, EGF induced a
clear retardation in gel migration (Fig. 4B). More
important, this effect on the ERK2 band shift was abolished in COS
cells either expressing MKP-3/Myc or transfected with plasmid carrying
an untagged MKP-3 (Fig. 4B). The EGF-stimulated shift
in ERK2 electrophoretic mobility was also inhibited, although not
completely abolished, in COS cells expressing TYP-1/Myc (Fig. 4B). To test whether suppressed EGF-stimulated
ERK2 phosphorylation correlates with inhibition of enzymatic activity,
immune complex assays were performed using myelin basic protein (MBP)
and ERK2 immunoprecipitated from transfected COS cells (see
``Experimental Procedures''). While ERK2 from cells
transfected with control plasmid displayed powerful EGF-stimulated MBP
phosphorylation, this was inhibited considerably in cells transfected
with either MKP-3/Myc or untagged MKP-3 (Fig. 5, A and B). Consistent with partial suppression of ERK2
phosphorylation state by TYP-1/Myc (Fig. 4B), this
dual-specificity phosphatase inhibited ERK2 activity by 60% (Fig. 5, A and B). Differences in MBP
phosphorylation reflect altered ERK2 activation state as similar levels
of ERK2 were immunoprecipitated in these comparative experiments
between transfected COS cells (Fig. 5C). In additional
experiments (data not shown), we have shown that MKP-3/Myc expression
also results in near complete inhibition of ERK2 activation by 15%
serum and the phorbol ester phorbol 12-myristate 13-acetate at 100
nM. Identical results were also obtained when we performed
immune complex assays on ERK2/Myc cotransfected in COS-7 cells together
with untagged MKP-3 (data not shown). Together, these experiments
demonstrate that MKP-3 displays functional activity expected for a
dual-specificity phosphatase in its ability to abolish
mitogen-stimulated activation of ERK2.
Figure 5: MKP-3 blocks EGF-stimulated ERK2 activation. Immune complex assays were performed using MBP and ERK2 immunoprecipitated from transfected COS-7 cells with antibody 122. Where indicated (+), cells were pretreated with EGF (10 nM) for 10 min prior to homogenization and extraction. A, shown is an autoradiogram of a 15% gel showing powerful MBP phosphorylation by ERK2 from control cells (pMT-SM) following treatment with EGF. In cells expressing MKP-3/Myc or transfected with untagged MKP-3, ERK2 showed little stimulated activity following EGF stimulation. Basal ERK2 activity was also diminished in these extracts. EGF-stimulated ERK2 activation was also suppressed in cells expressing TYP-1/Myc. Blank indicates activity associated with immunoprecipitates from homogenization buffer alone. Arrows indicate the positions of MBP bands phosphorylated by activated ERK. B, shown is the quantitation of immune complex assay data. Bands from the autoradiogram in A were excised and counted by scintillation spectrometry. Blank activity (not subtracted) was 264 dpm. C, immunoprecipitates used to perform the immune complex assay in A were analyzed for ERK2 protein content by Western blotting with antibody 122. All extracts displayed similar levels of ERK protein.
Figure 6:
Expression of MKP-3 mRNA in rat tissues.
Poly(A) mRNA (2 µg) obtained from tissues as
indicated was separated on a 1.2% agarose gel, transferred to nylon
membranes, and fixed by ultraviolet irradiation (CLONTECH).
Hybridization with a random-primed
P-labeled probe
corresponding to the 5`-end of the MKP-3 clone revealed a single 2.7-kb
mRNA species. Numbers represent the positions of molecular
mass markers (in kilobases).
Figure 7: Subcellular localization of MKP-3/Myc and TYP-1/Myc in sympathetic neurons. Sympathetic neurons from superior cervical ganglia of newborn rats were cultured for 5-7 days with NGF and microinjected with plasmid pMT-SM expressing either MKP-3/Myc (A and B) or TYP-1/Myc (C and D). Neurons were fixed, permeabilized, and incubated with monoclonal antibody 9E10 followed by fluorescein isothiocyanate-conjugated goat anti-mouse antibody. Nuclei were stained with Hoechst dye (B and D). Ultraviolet fluoresence showed MKP-3/Myc to be localized exclusively in the cytosol (A), whereas TYP-1/Myc was restricted to nuclei (C).
Figure 8: Northern blot analysis of the expression of MKP-3 mRNA in NGF-treated PC12 cells. PC12 cells were primed overnight in medium containing 1% horse serum and stimulated with NGF (50 ng/ml), EGF (150 ng/ml) or dibutyryl cAMP (0.5 mM) for the indicated periods of time. Total cellular RNA was extracted, and 5 µg was electrophoresed and used for Northern blot analysis using an antisense riboprobe specific for MKP-3. Film exposure corresponded to 68 h. Methylene blue staining to visualize ribosomal RNA revealed equal amounts of RNA electrophoresed per well.
Figure 9:
Localization of MKP-3 and MKP-1 mRNAs in
rat brain by in situ hybridization. Shown are film
autoradiograms of adult rat coronal brain sections hybridized with
MKP-3 (A) or MKP-1 (B) S-end-labeled
antisense oligodeoxynucleotides. Brain sections were prepared from rats
1 h following either saline (Control) or Metrazole (40 mg/kg
intraperitoneal injection) treatment. The localization of the following
brain regions is indicated: layers of the hippocampus (CA1 and CA3-4), piriform cortex (Pir), amygdala (A), and thalamus (T).
In summary, we have identified a novel member of the
dual-specificity phosphatase family which we have called MKP-3.
Structurally, MKP-3 displays both an extended active-site sequence
motif as well as regions of homology to Cdc25 phosphatase (CH2 domains)
shared by all known members of this gene family. MKP-3 also exhibits
functional properties expected of a dual-specificity phosphatase in
that it blocks mitogen-stimulated activation of the MAP kinase ERK2.
Identification of a second partial cDNA clone (MKP-X) encoding an
additional phosphatase that is 76% identical to MKP-3 indicates the
existence of a distinct subfamily of structurally homologous MAP kinase
phosphatase genes. This is also consistent with identification of the Xenopus dual-specificity phosphatase X17c, which blocks MAP
kinase-dependent embryonic mesoderm formation (59) and is 88%
identical to MKP-3. ()MKP-3 is the first dual-specificity
phosphatase to show an exclusively cytosolic localization, indicating a
unique regulatory role perhaps in inactivating MAP kinases targeting
cytoplasmic substrates or blocking nuclear translocation. A novel role
for MKP-3 is also supported by a distinct tissue distribution and
regulated expression in PC12 cells. An important conclusion from this
report as well as other recent publications(23, 27, 28, 29, 30, 31, 32) is
that molecular diversity within the dual-specificity phosphatase family
now parallels the number of ERK, SAPK/JNK, and p38/HOG1 MAP kinases
providing the opportunity for highly specific regulatory interactions.
Mechanisms underlying such specificity remain undefined, although they
could include enzymatic substrate selectivity, time course of
stimulus-dependent activation and induction, cell type-specific
coexpression, or subcellular compartmentalization.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) X94185 [GenBank]and X94186[GenBank].
Note Added in Proof-The predicted amino acid sequence is identical with rVH6(61) .