From the
Mitogen-activated protein (MAP) kinase lies at the convergence
of various extracellular ligand-mediated signaling pathways. It is
activated by the dual-specificity kinase, MAP kinase kinase or MEK. MAP
kinase inactivation is mediated by dephosphorylation via specific MAP kinase phosphatases (MKPs). One MKP
(MKP-1 (also known as 3CH134, Erp, or CL100)) has been reported to be
expressed in a wide range of tissues and cells. We report the
identification of a second widely expressed MKP, termed MKP-2, isolated
from PC12 cells. MKP-2 showed significant homology with MKP-1 (58.8% at
the amino acid level) and, like MKP-1, displayed vanadate-sensitive
phosphatase activity against MAP kinase in vitro.
Overexpression of MKP-2 in vivo inhibited MAP kinase-dependent
gene transcription in PC12 cells. MKP-2 differed from MKP-1 in its
tissue distribution and in its extent of induction by growth factors
and agents that induce cellular stress, suggesting that these MKPs may
have distinct physiological functions.
Mitogen-activated protein kinases
(MAP
Activation of MAP kinase
involves specific phosphorylations on threonine and tyrosine residues
within the Thr-Glu-Tyr motif (10) by MAP kinase kinase (MAP kinase and ERK kinase or
MEK)(2, 11) . Phosphorylation of both these residues is
required for MAP kinase activation(11, 12) . It has been
suggested that the inactivation of MAP kinase is a critical
event that regulates the physiological response to MAP kinase
activation(13) . This inactivation is mediated, in part, by
dephosphorylation of MAP kinases by dual specificity phosphatases
called MAP kinase phosphatases (MKPs) that
dephosphorylate both the threonine and tyrosine residues phosphorylated
by MEK (13-16). The activation of MAP kinase appears to be
tightly regulated through the coordinate action of MEK and MKPs. By
regulating the extent of MAP kinase activation, these MKPs may dictate
the choice of differentiation or proliferation within a developing
cell(17) .
The prototype dual-specificity phosphatase, VH1,
was identified in vaccinia and showed similarity to cdc25, a protein
that controls cell entry into mitosis(18) . VH1 homologues from
human (PAC-1, CL100, and most recently B23), mouse (MKP-1 (3CH134 or
Erp)), and yeast (Yop51, MSG5) have also been
isolated(19, 20, 21, 22, 23, 24) .
All are dual-specificity phosphatases that specifically dephosphorylate
MAP kinase in vitro(25) and in
vivo(13, 15, 26) . MKP-1 (also called
3CH134 or Erp) was discovered as an immediate early gene whose rapid
transcription and subsequent translation are suggested to provide a
feedback loop to terminate growth factor
signals(13, 19, 26) . Overexpression of mouse
MKP-1 was shown to dramatically inhibit fibroblast proliferation,
suggesting that the inactivation of MAP kinase in vivo by
MKP-1 has a profound negative effect on cellular
proliferation(25, 26) .
MAP kinase activation by
growth factors has been extensively studied in PC12 cells(27) .
PC12 cells originate from a rat pheochromocytoma and retain many
features of neural crest-derived cells, most notably the ability to
undergo neuronal differentiation upon stimulation by NGF (28).
Transfection with activated forms of the oncogenes ras, raf-1, and src into PC12 cells is sufficient for
differentiation in the absence of NGF
stimulation(6, 8, 29) . As each of these genes
has been shown to converge on MAP kinase activation, this implies that
components of the MAP kinase cascade are required for neuronal
differentiation. More recently it has been shown that the activation of
MAP kinase kinase is required and sufficient for PC12 cell
differentiation(3) . Despite our understanding of MAP kinase
activation in neuronal differentiation, we know relatively little about
MAP kinase inactivation. Initial studies presented here characterize a
family of MKPs expressed in PC12 cells.
We report the cloning of a
novel MKP, MKP-2, that is highly expressed in PC12 cells. MKP-2 mRNA is
expressed at moderate levels in nearly all tissues and cells and
encodes a phosphatase that inactivates MAP kinase in vitro and
MAP kinase-dependent gene transcription in vivo. Its
distribution in the central nervous system and regulation in the
hippocampus suggest a potential role for this phosphatase in neuronal
signaling pathways.
These reverse transcriptase PCR partial
fragments of the novel MKP-2 cDNA were labeled and used as probes to
screen an oligo(dT)-primed PC12 cDNA library in
The putative 393-amino
acid MKP-2 protein shares 58.8% identity to the 367-amino acid MKP-1
protein, 62.3% identity to the 314 amino acid mPAC protein, and 33%
identity to the 397-amino acid human B23 protein (Fig. 2). The
similarities are greatest at the 3` end near the catalytic domain. In
contrast, however, the 5` end has significant sequence differences
compared with the other members of this family. The N-terminal half of
MKP-2 (amino acids 1-187) shares only 33% identity to MKP-1 while
the C-terminal half(188-393) shows much greater homology (76%)
with nearly 100% identity around the catalytic core (Fig. 2). The
complete coding region of MKP-2 was subcloned into Bluescript to
generate a full-length cDNA (MKP2FL-6) to allow transcription from the
T7 promoter. In vitro transcription and translation of MKP-1
and MKP-2 cDNA revealed the expected 39.4- and 42.6-kDa proteins,
respectively (Fig. 3A). Antisera directed to an MKP-1
C-terminal peptide sequence (amino acids 348-366) detected
proteins of sizes similar to MKP-1 and -2 from PC12 cell lysates (Fig. 3B). This cross-reactivity is likely the result of
the significant homology between MKP-1 and -2 in this region (Fig. 2).
To determine
whether MKP-2 mRNA is serum-inducible, total RNA was isolated from PC12
cells that were serum-starved overnight and then stimulated with media
containing 15% serum for various times. Ten µg of RNA from each
treatment was analyzed by Northern analysis using an MKP-2-specific
riboprobe as described above. Both MKP-1 and MKP-2 were expressed in
unstimulated PC12 cells (Fig. 7, A and B, and Fig. 9). Serum stimulation caused a biphasic stimulation of MKP-2
with a gradual increase by 1 h followed by a transient decrease and
then a subsequent increase in expression of MKP-2 by 4 h. The same blot
was stripped and reprobed with MKP-1, and the MKP-1 transcript followed
a similar but less robust stimulation (Fig. 9, A and B, left panel). All quantitations were normalized to
the 18 S ribosomal RNA as an internal control.
We have identified a second widely-expressed MAP kinase
phosphatase we term MKP-2. It is co-expressed with MKP-1 in a large
number of tissues but also shows distinct differences. In contrast, the
MKP PAC-1, is expressed only in lymphoid cells(23) . Like MKP-1
and PAC-1, MKP-2 shows homology to other tyrosine phosphatases. Its
sequence is most conserved with MKP-1 and PAC-1 within the C terminus
and less so within the N terminus (Fig. 2). All three share
identity within the catalytic core (VHCQAGISR) and display
phosphotyrosine phosphatase activity against synthetic peptides and
purified MAP kinase. The dual specificity of MKP-2 toward a
phosphothreonine has yet to be proven. However, we have shown that both
MKP-2 and MKP-1 block MAP kinase-dependent gene transcription in
vivo, as been shown for PAC-1(15) . All four MKPs contain
the CH2 domains (cdc25 homology 2), which
are regions present in members of the cdc25 family that flank the
catalytic domain(42) . In MKPs, the catalytic domain is situated
at the C terminus and not at the N terminus where the CH2 domains are
found (Fig. 2). Whether these CH2 domains are functional has yet
to be proven, but they have been speculated to either increase
substrate selectivity or to be involved in localizing proteins to
nuclear or cytoskeletal locations(42) .
The large (>3 kb)
3`-untranslated region in MKP-2 differs from the shorter (697 bp)
3`-untranslated region present in MKP-1 and may play a regulatory role
in post-transcriptional events such as transcript stability. Several
AUUUA motifs are found in the 3`-untranslated region of both MKP-1 (19, 26) and MKP-2 (Fig. 1B). MKP-2 also
has a 25-nucleotide stretch of AU sequences, which might also
contribute to posttranscriptional control. These AU sequence motifs
have been implicated in the short half-life (
In contrast to the expression of PAC-1,
which is limited to lymphoid cells(23) , MKP-1 and MKP-2 show
expression in a broad range of tissues with distinct differences. These
distinct tissue distribution profiles may dictate unique roles of the
members of this family in the regulation of MAP kinases and might
reflect their co-expression with certain MAP kinase isoforms. MKP-1,
ERK-1(42) , and MKP-2 mRNA are expressed in discrete areas of
the brain. ERK2 expression is also prominent in neuronal cell bodies
and dendrites particularly within the superficial layer of the
neocortex, the hippocampal CA3 region, dendate gyrus, and cerebellar
Purkinje cells(44) . The co-localization of MKP-2 and the ERK
isoforms in certain discrete areas of the brain suggests that ERK1 and
ERK2 may be physiological substrates for MKP-2. However, MKP-1 and
MKP-2 also show overlapping expression, which suggests that
co-expression of MKPs and ERKs is not the only criterion for substrate
specificity.
The function of MAP kinase in postmitotic neuronal
cells is unclear. MAP kinases within the developing and adult central
nervous system (45) are activated by both neurotrophic growth
factors and neurotransmitters. For example, activation of the N-methyl-D-aspartate receptor leads to increased
tyrsoine phosphorylation of an ERK isozyme in hippocampal
cultures(46) . Kainate is an N-methyl-D-aspartate receptor antagonist that induces
seizures and immediate-early genes within the
hippoocampus(47, 48) . The induction of CL100 (human
MKP-1) and B23 mRNA by oxidative stress and heat shock has been
reported(22, 24) , and the induction of MKP-1 and -2
observed with kainic acid treatment may represent a regulatory role
that these genes might play in response to cellular stress. It is not
known whether the stress-activated kinases are substrates for the
MKPs(49) .
Basal expression of MKPs may be important for the
resting cell. Vanadate stimulates MAP kinase activity in resting PC12
cells (data not shown). This suggests that a constitutive tyrosine
phosphatase activity may inhibit basal MAP kinase activity. In
addition, the introduction of dominant negative mutants of MKP-1 into
unstimulated COS cells activates MAP kinases in the absence of
serum(13) . Therefore MKPs may be active in resting cells and
might function to minimize the level of basal MAP kinase activity in
the resting cell.
In PC12 cells, both NGF and EGF stimulate a
receptor-tyrosine kinase to phosphorylate and activate similar
intracellular substrates including MAP kinase, whose action is required
for both proliferative and differentiating
responses(3, 4, 50) . It has been suggested that
the duration of MAP kinase activation determines the biological
response to growth factor stimulation(5, 51) .
Proliferation is associated with a transient MAP kinase
activation(5, 51, 52) , while agents that induce
a differentiating response produce a sustained activation. It is not
known whether regulation of MKPs establishes the time courses of MAP
kinase inactivation. We demonstrate that both NGF and EGF induced a
rapid increase in MKP-1 and MKP-2 mRNA levels with a more substantial
increase in MKP-2 compared with MKP-1. Whether this induction of MKPs
is responsible for the rapid inactivation of MAP kinase by EGF is not
known. A recent report demonstrates that inactivation of MAP kinase by
EGF is independent of MKP-1 induction(53) . Our results would
agree with their findings as NGF, which sustains the level of active
MAP kinase for a longer duration than EGF, also resulted in elevated
levels of MKP-1 and -2 for at least 4 h of drug exposure. Therefore, it
is possible that the MKPs are regulated post-transcriptionally by these
agents. The differences in transcriptional regulation of MKP-1 and
MKP-2 by NGF and EGF suggest that they have different mechanisms of
regulating MKP activity.
In conclusion, PC12 cells express at least
two related MKPs, MKP-1 and MKP-2. The identification of a family of
MKPs that are expressed within the same cell suggests distinct roles
for each member of this expanding family. Discrimination of the actions
of these MKPs may occur through the divergent amino termini of these
proteins. Further studies are required to identify the physiological
roles of each member of this unique family of phosphatases in order to
gain a better understanding of the mechanisms involved in cellular
proliferation, differentiation, and stress.
We thank Marty Mortrud and Drs. James Douglass, Pastor
Couceyro, Malcolm Low, and Richard A. Maurer for providing scientific
support and Sheri Medford for excellent secretarial assistance.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(
)kinases) mediate multiple cellular
pathways regulating growth (1) and
differentiation(2, 3) . In neuronal cells, MAP kinase
activity mediates the actions of growth factors like EGF that stimulate
cellular proliferation as well as factors like NGF that maintain
neuronal survival and differentiation(4, 5, 6) .
Such ligand-activated signal transduction pathways involve activation
of receptor-tyrosine kinases, which initiates a series of
phosphorylation events that activate a cascade of serine/threonine
kinases converging on the MAP kinase (also called extracellular signal-regulated kinase (ERK))
isoforms, ERK1 and ERK2 (7-9).
Materials
Restriction and modification enzymes
were purchased from New England Biolabs (Beverly, MA), Boehringer
Mannheim, and Promega. Superscript reverse transcriptase was from Life
Technologies, Inc.; Taq DNA polymerase from Perkin-Elmer and
Sequenase from U. S. Biochemical Corp. All enzymes were used according
to the instructions from the manufacturer.
[-
P]dATP (3000 Ci/mmol),
S-dATP,
S-UTP (1500 Ci/mmol),
[
-
P]ATP (800 Ci/mmol),
[
S]cysteine (1075 Ci/mmol), and
[
-
P]UTP (800 Ci/mmol at 40 mCi/ml) were
purchased from Dupont NEN. Oligonucleotides were synthesized by a core
facility at Oregon Health Sciences University. Antisera to MKP-1 was
purchased from Santa Cruz Biotechnology Inc. An anitibody to
phosphotyrosine (clone 4G10) was kindly provided by Brian Druker (OHSU,
Portland, OR)(30) .
Reverse Transcriptase PCR Amplification
One µg
of total RNA from PC12 cells was used to generate first strand cDNA
after an initial annealing reaction to 0.1 µg of random hexamers at
70 °C for 10 min. Following equilibration to ambient temperatures,
a buffer containing 50 mM Tris-HCl, pH 8.3, 75 mM KCl, 3 mM MgCl, 10 mM dithiothreitol, 500 µM of each of four dNTPs, and 200
units/µg of Superscript reverse transcriptase was added, and the
mix was incubated at 37 °C for 1 h. The reaction was terminated by
placing the tubes on ice, and the cDNA was recovered by ethanol
precipitation. The pellet was washed with 70% ethanol and resuspended
in 100 µl of 5 mM Tris, 0.5 mM EDTA mix. Five
µl of this cDNA was used as a template for PCR amplification.
Initially, two degenerate oligonucleotides were synthesized that
generated a 204-bp cDNA fragment. The 5` primer corresponded to the
conserved WFNEAI sequence present in MKP-1 (26) and PAC-1 (23) (5`-TGG-TT(T/C)-AA(T/C)-GA(G/A)-GC(G/A/T/C)-AT-3`), while
the 3` primer corresponded to the conserved NFSFMG sequence present in
MKP-1 (26) and PAC-1 (23) (5`-C-CAT-(A/G)AA-(G/A/T/C)(G/C)(A/T)-(A/G)AA-(A/G)TT-3`)
(see Fig. 2). Two additional oligonucleotides were made to
confirm the novelty of MKP-2. The 5` primer was a degenerate primer
corresponding to the conserved YDQGGP sequence
(5`-TA(T/C)-GA(T/C)-CA(A/G)-GG(G/A/T/C)-GG(G/A/T/C)-CC-3`), while the
3` primer was specific for MKP-2 (5`-ATGAAGAAACGGGTGCGG-3`)
corresponding to MKKRVR sequence (Fig. 1B and 2). This
set of primers generated a 336-bp cDNA fragment corresponding to
nucleotides 628-963 (Fig. 1B). The PCR reaction
consisted of 50 mM KCl, 1.5 mM MgCl
, 0.2
mM dNTPs, 15 mM Tris-HCl, pH 8.4, and 0.5 µg of
each primer pair. PCR was allowed to proceed for 30 cycles. Each cycle
consisted of 1 min at 94 °C, 1 min at 55 °C, and 1 min at 72
°C in a thermocycler (Perkin-Elmer Cetus). The PCR products were
purified and subcloned into pBluescript (SK-) (Stratagene) using
restriction enzymes engineered at the ends of each of the primers.
Figure 2:
Amino acid homology between MKP-2, MKP-1,
PAC-1, and B23. The amino acid sequences of rat MKP-2, mouse MKP-1,
mouse PAC-1, and human B23 are aligned with each other, and the areas
of homology are shown as shaded boxes. The catalytic domain is boxed. Dots represent spaces put in for alignment.
The dark gray boxes represent the two CH2 domains present in
all MKPs. Arrows correspond to the primers used in the reverse
transcriptase PCR screening strategy for cloning
MKPs.
Figure 1:
Restriction map
and sequence of MKP-2 cDNA. A, the 4.8-kb MKP-2 cDNA was
digested with various restriction enzymes, and a schematic
representation of some of these sites is shown. The following
abbreviations are used: RI, EcoRI; Pst, PstI; A, ApaI; H, HindIII; RV, EcoRV; Sma, SmaI; Bam, BamHI; 5` UT, 5`-untranslated region; 3`
UT, 3`-untranslated region; ATG, translation start site; TAG, translation stop site. The coding region of MKP-2 is
shown as a rectangularbox with two different domains
highlighted. The stippled box represents CH2 domains (42)
while the hatched box represents the catalytic domain. B, nucleotide sequence and the encoded amino acid sequence of
rat MKP-2 cDNA is shown. The translation start site is denoted as +1. The consensus catalytic site, the AU-sequence motifs
in the 3` untranslated region, and the putative polyadenylation signal
are underlined. The 5`- and 3`-untranslated regions are
depicted in lower case letters.
Screening of the PC12 cDNA Library and Isolation of the
Full-length Clone
The 336-bp PCR fragment generated by using the
specific MKP-2 primer (described above) was labeled by random primed
synthesis and used to screen a PC12 oligo(dT)-primed cDNA library in
gt10 with 5
10
individual recombinants that
had been size-selected prior to ligation for clones greater than 2 kb.
The library was plated onto 20 LB plates and allowed to grow at 42
°C to a concentration of 50,000 recombinants/plate. The plaques
were then transferred onto nitrocellulose filters in duplicate. The
filters were soaked in prehybridization/hybridization buffer (6
SSC, 5
Denhardt's solution, 1% SDS, 0.01 M EDTA,
50% formamide, 100 µg/ml denatured salmon sperm DNA) at 42 °C
for 1-2 h with gentle agitation. Random primed probe was made as
described in the Boehringer Mannheim kit. One to two million cpm/ml of
boiled MKP-2 specific probe was added directly to the
prehybridization/hybridization mix, and hybridization was allowed to
proceed at 42 °C for 24 h. The filters were washed in 2
SSC
and 1% SDS for 2 h at 65 °C with frequent changes in the wash
solution. The final wash was in 1
SSC and 1% SDS after which
the filters were air dried and put on film. After the tertiary screen,
three positive plaques were obtained. Phage DNA was isolated by
standard methods and digested with EcoRI to release the
insert. The inserts obtained were cloned into pBluescript (SK-)
and subjected to restriction enzyme mapping and sequencing.
Sequencing
MKP-2 cDNA inserts obtained were
sequenced on both strands by the method of Sanger (31) using
Sequenase reagents (U.S. Biochemical Corp.) according to the protocol
suggested by the manufacturer. Multiple internal primers were made to
allow sequencing of the complete 800-bp insert. The 4-kb insert was
also sequenced using multiple internal primers on both strands through
the termination codon and partially into the 3`-untranslated region.
Sequences proximal to the polyadenylation signal were also obtained as
shown (Fig. 1B).
Cell Culture and Drug Treatments
PC12-GR5 cells
(courtesy of Rae Nishi, OHSU, Portland, OR) were grown at 5% CO in Dulbecco's modified Eagle's medium containing 5%
fetal calf serum, 10% horse serum, and L-glutamine. Prior to
drug treatments, the cells were serum-starved for 24 h with
Dulbecco's modified Eagle's medium containing no serum and
treated with either 100 ng/ml NGF or 20 ng/ml EGF for the indicated
times.
RNA Isolation
Total cellular RNA was isolated
using RNAzol B (Biotecx Lab, Inc.) according to the
manufacturer's protocol. Briefly, cells were grown to
30-50% confluency in a 100-mm plate, rinsed with cold
phosphate-buffered saline, and scraped into 1 ml of RNAzol B. After
vortexing, 0.1 ml of chloroform was added and incubated on ice for 15
min. The suspension was centrifuged, and the RNA was precipitated from
the upper aqueous layer with an equal volume of isopropyl alcohol.
After pelleting, the RNA was resuspended in water, quantitated at A
, and used directly for reverse transcriptase
PCR or Northern analysis.
Northern Blot Analysis
Ten µg of total RNA was
electrophoresed through a 1.2% agarose formaldehyde gel and transferred
onto Magna NT filter (MSI, Westboro, MA) using standard methodology in
6 SSC. Filters were prehybridized in 3 ml of hybridization
buffer (5% SDS, 400 mM NaPO
pH 7.2, 1 mM EDTA, 1 mg/ml bovine serum albumin, 50% formamide) at 65 °C
for 4 h in a rotating hybridization oven. 2-5
10
cpm/ml of the antisense riboprobe was then directly added to the
hybridization buffer, and hybridization was allowed to proceed for 24
h. The next day, filters were initially washed in 1
SSC at room
temperature for 15 min and then washed in (0.05
SSC, 0.1% SDS,
5 mM EDTA, pH 8) at 70 °C for 3-4 h. Filters were
autoradiographed at -70 °C on Kodac XAR-5 film using Dupont
intensifying screens. Quantitations were performed using a Molecular
Dynamics PhosphorImager 445 SF, and all signals were normalized to the
18 S and cyclophilin signals respectively.
Riboprobe Synthesis
The 336-bp cDNA fragment
generated by using the specific MKP-2 primer (described under
``Reverse Transcriptase PCR Amplification'') was subcloned in
pBluescript (SK-) and was used to synthesize antisense riboprobes
by linearizing the plasmid with SalI that was engineered into
the 5` primer. Full-length MKP-1 cDNA (kindly provided by Nicholas
Tonks, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY) was
subcloned into pBluescript (SK-) and then linearized with BamHI to generate a 1.9-kb MKP-1 riboprobe. Rat cyclophilin
(pSP65 1B15) and 18 S ribosomal RNA (18SpSP65) plasmids (kindly
provided by James Douglass, Vollum Institute, Portland, OR) were
linearized using PstI and HindIII respectively to
generate linear antisense riboprobes. Antisense riboprobes were
synthesized as described previously(32) . Briefly, 1 µg of
template DNA was incubated in transcription reaction mix (40 mM Tris-HCl, pH 7.5, 6 mM MgCl, 2 mM spermidine, 10 mM NaCl, 10 mM dithiothreitol, 20
units of RNasin, 0.5 mM each of rATP, GTP, and CTP, 12
µM rUTP, 50 µM
[
-
P]UTP (800 Ci/mmol), and 15 units of the
appropriate RNA polymerase) at 37 °C for 60 min. The reaction was
stopped by the addition of 2 units of RNase-free DNase and incubated at
37 °C for 15 min. 25 mM EDTA was then added, samples were
phenol-chloroform extracted and ethanol-precipitated. Antisense
riboprobes were resuspended in water at a concentration of 1-2
10
cpm/µl.
In Situ Hybridization
Male Sprague-Dawley rats
(200-300 g) were anesthetized and perfused with 1000 ml of 4%
paraformaldehyde in borate buffer, pH 9.5, at 4 °C (fixation
buffer). Brains were dissected and incubated in fixation buffer for 8 h
and then incubated overnight in fixation buffer with 10% sucrose.
Brains were sectioned serially into five series of 30-µm slices
with a sliding microtome. Sections were prepared and hybridized as
described previously(33) . The 336-bp MKP-2 specific cDNA
fragment was used to synthesize antisense and sense riboprobes.
Sections were hybridized with S-labeled riboprobes
(10
cpm/ml) in 66% formamide, 0.26 M NaCl, 1.32
Denhardt's solution, 13.2 mM Tris, pH 8.0, 1.32
mM EDTA, 13.2% dextran sulfate, pH 8.0, at 65 °C for 24 h.
Slides were washed in 4
SSC, digested with RNase A (20
µg/ml for 30 min at 37 °C), and then rinsed in a stringent wash
containing 0.1
SSC at 65 °C for 30 min. Sections were
dehydrated, dipped in NTB-2 emulsion (Kodak), and developed after 21
days. Light- and darkfield photomicrographs were taken with a Dialux 22
EB at 32
magnification.
In Vitro Transcription and Translation
Reactions
Full-length MKP-1 and MKP-2 cDNAs were used in a
coupled in vitro transcription and translation reaction using
TNT coupled reticulocute lysate system (Promega) as per the
manufacturer's instructions. Briefly, 1 µg of circular DNA
was incubated with 27.5 µl of TNT rabbit reticulocyte lysate, 2
µl of TNT reaction buffer, 1 µl of T7 RNA polymerase, 1 µl
of 1 mM amino acid mix minus cysteine, 2 µl of
[
S]cysteine (1075 Ci/mmol at 11 mCi/ml), and 1
µl of RNAsin at 40 units/µl in a final volume of 50 µl. The
reaction was incubated at 30 °C for 1-2 h. The synthesized
proteins were separated by SDS-polyacrylamide gel electrophoresis and
analyzed by autoradiography.
PC12 Transfection Assays
PC12 cells were grown to
approximately 60% confluence prior to transfection. For transient
transfection experiments, 3 µg of 5 Gal4-E1B luciferase and
3 µg of cytomegalovirus (CMV) Gal4-Elk1 transactivation
domain
(
)were used in combination with either 6
µg of Rous sarcoma virus promoter coupled to globin (control) or
constitutively active form of Raf kinase (Raf BxB) (34) and 30
µg of either pCDNA3 (Invitrogen, Inc.) containing the CMV promoter,
CMV MKP-1, or CMV MKP-2. Cells were transfected by calcium
phosphate-mediated DNA transfer as described previously(35) .
Cell lysates were prepared 20-24 h following transfection, and
luciferase activity was determined as described previously(36) .
Western Blotting of PC12 Proteins
PC12 cells were
lysed in 200 µl of a 1% Nonidet P-40 lysis buffer (25 mM Tris, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1.5
mM MgCl, 10% glycerol, 1 mM EGTA, 1
mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, and
2 mM vanadate). Protein concentrations were determined by the
method of Bradford(37) . One hundred µg of total protein was
resolved on a 12% SDS-polyacrylamide gel and transferred onto Immobilon
P membrane. The membranes were probed with an MKP-1 antibody (diluted
at 1:2000) (Santa Cruz Biotech., Inc.). A horseradish
peroxidase-conjugated secondary antibody was used to allow detection of
the appropriate bands using enhanced chemiluminescence (Amersham
Corp.).
Bacterial Expression of MKP-2
The catalytic domain
of MKP-2 encoded within a carboxyl-terminal 690-bp fragment (C-MKP-2),
extending from amino acids 163-393, was subcloned into the PET
23b vector (Novagen) using specific PCR oligonucleotides. This vector
provides an amino-terminal epitope tag derived from the T7 capsid
protein (T7 tag) that can be detected using specific antibodies (T7
antibody, Novagen). The frame of the resultant cDNA was confirmed by
sequencing. This plasmid and the vector alone were used to transform
BL21 bacteria (Novagen). A protein of the expected size (28 kDa) was
induced upon incubation with 0.4 mM
isopropyl-1-thio--D-galactopyranoside and was detected
using anti-T7 capsid antibodies. Prior to phosphatase assays, bacterial
extracts were prepared in lysis buffer (50 mM Tris-HCl, pH
8.0, 2 mM EDTA) and sonicated. Insoluble debris was pelleted
and the supernatant assayed directly.
ERK2 Dephosphorylation Assay
Activated ERK2 was
prepared by incubating 10 ng of recombinant ERK2 (kindly provided by
Dr. Edwin Krebs, University of Washington, Seattle, Washington) with
0.1 µg of active MAP kinase kinase (MEK) (Santa Cruz Biotechnology,
Inc.) in 1 MEK buffer (25 mM Hepes, pH 7.5, 10 mM MgCl
, 1 mM dithiothreitol, and 50
µM [
-
P]ATP) at 30 °C for
30 min. The activation of ERK2 was confirmed by Western blot analysis
using an antibody directed against phosphotyrosine(30) . Ten ng
of activated ERK2 was incubated with 10 µg of bacterial lystates
from MKP-2 expressing and nonexpressing cells in 1
phosphatase
buffer (50 mM Tris-HCl, pH 7.5, 1 mM EDTA, 10 mM dithiothreitol) for 15 min at 30 °C. The reaction was stopped
by the addition of an equal volume of 2
Laemmli sample buffer,
and the samples were separated by 13% SDS-polyacrylamide gel
electrophoresis. The dephosphorylation of ERK2 was confirmed by Western
blotting with the phosphotyrosine antibody using enhanced
chemiluminescence for detection of the signal.
Phosphotyrosine Phosphatase Assay
The synthetic
peptide Raytide (Oncogene Sciences) was phosphorylated on a unique
tyrosine using Src tyrosine kinase activity immunoprecipitated from
C3H10T1/2 cells (kindly provided by Sally Parsons, University of
Virginia) as described(38) . Bacterial extracts containing
10-60 µg of bacterial proteins were incubated at 30 °C
for 30 min with labeled peptide (10 cpm) in 100 µl of 1
phosphatase buffer (described above). Additional reagents (10
µM vanadate, 20 nM microcystin-leucine-arginine,
and 1 µM okadaic acid) were added without prior
incubation. The reaction was terminated by the addition of 0.75 ml of
stop solution (2 mM NaHP0
, 90 mM sodium
pyrophosphate, 0.9 M HCl, 4% (v/v) Norit A). Following brief
centrifugation, 400 µl of the supernatant was added to 2.5 ml of
scintillant, and the released counts from phosphatase activity were
measured on a scintillation counter. All phosphatase assays were
performed in duplicate.
PC12 Cells Express Multiple MKPs: Identification and
Cloning of a Novel MKP
To identify potential MKPs that are
expressed in PC12 cells, a screening strategy involving reverse
transcriptase PCR amplification was employed. Alignment of the
sequences of the known members of the MKP family (human PAC-1, mouse
PAC-1, mouse 3CH134, and VH1) showed areas of high sequence homology
particularly surrounding the catalytic core consensus site
(HCXAGXXR, where X is any amino acid) (Ref.
23 and Fig. 2). Degenerate primers were designed to the conserved
amino acid sequence WFNEAI (5` primer) and the conserved amino acid
sequence NFSFMG (3` primer) surrounding the catalytic core site (Fig. 2). Reverse transcriptase PCR on total RNA from PC12 cells
with these two degenerate primers revealed the expected 204-bp product
that contained a representative population of MKPs expressed in PC12
cells (data not shown). This PCR product was gel-purified, digested
with restriction enzymes engineered at the primer ends, and subcloned
into the appropriate sites in Bluescript. Five positive clones were
obtained that were analyzed by sequencing. One clone was identified as
the rat homolog of MKP-1 or 3CH134(19) . The remaining four
clones were identical and showed some unique features in comparison
with the other known MKPs. To analyze this clone further, two
additional primers were designed. The 3` primer was directed to a
unique stretch in the cloned novel sequence (MKKRVR) (Fig. 2).
The 5` degenerate primer was designed to another stretch of conserved
sequence between the MKP family members (YDQGGP), 5` to the sequence
already obtained (Fig. 2). Subsequent amplification using these
two primers resulted in a 336-bp amplicon (data not shown). Sequencing
this fragment confirmed the novelty of this cDNA sequence (Fig. 1B and 2). These results demonstrate that PC12
cells express at least two potential MKPs, MKP-1 and the novel cDNA
that we have called MKP-2.
gt10. This library
contained 5
10
individual recombinants that had
been size-selected prior to ligation for clones greater than 2 kb. The
three positive clones obtained with this screen were plaque-purified,
and the phage DNA obtained was digested with EcoRI to release
the insert. Upon digestion, two of the three positive clones each
revealed two insert fragments of approximately 4 kb and 800 bp, while
the other clone contained a single 3-kb insert fragment. The 3-kb
insert was never successfully subcloned, and therefore was not analyzed
further. The two contiguous insert fragments (4 kb and 800 bp) were
cloned separately into Bluescript and characterized by restriction
analysis (Fig. 1A) and sequencing (Fig. 1B). The 5`-untranslated region and the first 135
amino acids of the novel MKP were contained within the 800-bp fragment,
while the remaining 3` end of the clone was contained in the 4-kb
fragment. The cDNA encoding MKP-2 contained at least 377 bp of
5`-untranslated region and a translation start site with a 11/13 match
with the Kozak consensus sequence (Fig. 1B). The open
reading frame extends 1182 bp and encodes a protein product of 393
amino acids with a predicted molecular mass of 42.6 kDa and an
isoelectric point of 7.86. The open reading frame was followed by an
unusually large 3`-untranslated region of greater than 3.0 kb. In
comparison, the size of full-length mouse MKP-1 (3CH134, Erp) and its
human homolog (CL100) is only 2.4
kb(19, 22, 26) , similar to the recently cloned
member of this family, B23, which is 2.5 kb(24) . The large
3`-untranslated region of MKP-2 contains several AU sequence motifs (Fig. 1B) that are thought to regulate mRNA stability
and are also present in MKP-1(26) .
Figure 3:
In
vitro translation of MKP-1 and MKP-2 cDNAs. A,
autoradiogram of [S]cysteine-labeled products of
a coupled in vitro transcription-translation reaction are
shown using cDNAs encoding MKP-1 (lane1) and MKP-2 (lane2). B, Western blot analysis of PC12
cell lysates (100 µg of total protein) using antisera directed to a
MKP-1 peptide with significant identity to the C-terminal residues of
MKP-2 (Santa Cruz Biotech). A 46-kDa protein molecular mass marker is
indicated.
The MKP-2 Protein Contains Phosphotyrosine Phosphatase
Activity
A carboxyl-terminal fragment of MKP-2 (amino acids
163-393) comprising the entire catalytic domain was subcloned
into the bacterial expression vector PET-23b and expressed in the Escherichia coli strain BL21 (LysS). Induction of bacterially
expressed truncated MKP-2 by
isopropyl-1-thio--D-galactopyranoside was confirmed by
Western blotting using an antibody directed to the T7 capsid epitope
(data not shown). Bacterial extracts expressing the truncated MKP-2
fusion protein displayed phosphotyrosine phosphatase activity that was
inhibited by vanadate but not by okadaic acid nor microcystin-LR,
inhibitors of serine/threonine phosphatases(39, 40) (Fig. 4A). These extracts, but not extracts
expressing vector alone, could dephosphorylate activated ERK2 protein in vitro as monitored by phosphotyrosine Western blotting (Fig. 4B). This dephosphorylation was blocked by
vanadate (Fig. 4B).
Figure 4:
MKP-2 contains phosphotyrosine phosphatase
activity. A, dephosphorylation of the synthetic peptide
Raytide by bacterial lysates expressing a truncated MKP-2 protein
(amino acids 163-393). U, untreated extracts from
bacteria expressing MKP-2; V, vanadate-treated extracts; O, okadaic acid-treated extracts; M,
microcystin-treated extracts; C, control extracts from
bacteria expressing vector alone. The activity is represented as
percent of maximal stimulation. B, dephosphorylation of ERK2
by MKP-2 is shown. Activated ERK2 (pp42) (see
``Experimental Procedures'') was incubated with phosphatase
buffer alone (lane1), bacterial lysates expressing
vector alone (lane2), bacterial lysates expressing
truncated MKP-2 protein (lane3), bacterial lysates
espressing MKP-2, assayed in the presence of vanadate (lane4). Phosphorylation was assayed by Western blotting with
a phosphotyrosine antibody as described.
MKP-2 Blocked MAP Kinase-dependent Activation of the
Elk-1 Transactivation Domain
To demonstrate biologically
significant activity toward ERKs in vivo, the full-length cDNA
encoding MKP-2 (MKP-2FL-6 cDNA) was subcloned into a CMV-driven
expression vector (CMV-MKP-2) and expressed in PC12 cells with a
Gal4-Elk-1 fusion protein used to direct expression of a
Gal4-luciferase reporter. Transcriptional activation by the Elk-1
transactivation domain requires activated MAP kinase(41) . In
these cells, a constitutively active Raf-1 mutant, Raf BxB, induced
Elk-1-dependent transcription of the luciferase reporter by greater
than 100-fold (Fig. 5). Co-transfection with a MKP-1 expression
vector under the control of a CMV promoter (CMV-MKP-1) blocked the
activation by greater than 60% of maximally stimulated levels.
Co-transfection with similar amounts of CMV-MKP-2 reduced expression by
greater than 90% of maximal levels (Fig. 5). These studies
demonstrate that MKP-2 can potently inhibit transcriptional activation
that is dependent on MAP kinase. Unstimulated activation of Elk-1 was
assayed in the presence of serum. Both MKPs inhibited this
``basal'' expression of luciferase activity to undetectable
levels, presumably by blocking serum-induced MAP kinase activity.
Figure 5:
MKP-2 blocks MAP kinase-dependent gene
transcription. PC12 cells were transfected with 6 µg of either Rous
sarcoma virus-globin (control) (white bars) or Raf BxB
(constitutively active Raf) (gray bars) in the presence of 30
µgs of vector alone (CMV), CMV-MKP-1 (MKP-1) or CMV-MKP-2 (MKP-2). In
addition, all cells received 3 µg of the reporter 5xGal4-E1B
luciferase and 3 µg of Gal4-Elk-1. Activity is shown as light
units/100 µg of protein. Note the activities of cells transfected
with Rous sarcoma virus-globin and either MKP are below the limits of
detection.
MKP-2 Is Expressed in Most Tissues and Cell Lines
Examined: Distinct Expression Compared with MKP-1
To determine
the distribution of MKP-2 in various tissues, Northern blot analysis
using a 336-bp MKP-2 specific riboprobe (described under
``Experimental Procedures'') revealed expression in most
tissues obtained from 10-12-week-old rats (Fig. 6A). MKP-2 mRNA was detected in most tissues
including brain, spleen, and testes with the highest expression in the
heart and lung and lower expression in skeletal muscle and kidney. No
MKP-2 expression was detected in the liver. The same blot was stripped
and reprobed with a riboprobe made to the entire coding region of MKP-1 (Fig. 6B). This probe did not cross-react with the 6-kb
MKP-2 transcript. A 2.4-kb mRNA corresponding to MKP-1 was detected in
all tissues but testes. The highest expression of MKP-1 was seen in the
lung, as previously reported(19, 26) , and in the heart
(similar to MKP-2). In the testes, MKP-2 is abundantly expressed, but
MKP-1 is not. In the liver, the inverse expression pattern is found.
These opposite expression patterns in two different tissues suggests
different physiological roles for the members of the MKP family. These
results show a fairly abundant basal expression of MKP-1 and MKP-2 in
most tissues with distinct tissue distribution patterns between MKP-1
and MKP-2. MKP-2, as expected from the 4.8-kb cDNA, which was missing a
portion of the 5`-untranslated region, encodes an approximate 6-kb
transcript distinct from the 2.4-kb MKP-1 mRNA detected in the same
tissues.
Figure 6:
Expression of MKPs in rat tissues. A
filter containing 2 µg of Poly(A) mRNA isolated
from the indicated tissues (Clonetech Lab, Inc., Palo Alto, CA) was
probed with a MKP-2-specific riboprobe (panel A),
stripped, and reprobed with a MKP-1-specific riboprobe (panel B). The molecular mass markers are indicated
to the right.
RNA was also isolated from cell lines of different
lineages, and 10 µg of total RNA from various cell lines was
analyzed by Northern analysis using the 336-bp MKP-2 specific riboprobe (Fig. 7A). A single 6-kb MKP-2 transcript was detected
in all rat- and mouse-derived cell lines of different lineages, while a
single 4-kb MKP-2 transcript was detected in all cells of human
origin. The same blot was stripped and reprobed with an MKP-1 riboprobe (Fig. 7B). No MKP-1 was detected in cells of human
origin. This may reflect the inability of the mouse MKP-1 riboprobe to
hybridize across species to cells of human origin under the stringent
conditions used. A rat MKP-1 probe was also used with similar results
(data not shown).
Figure 7:
Expression of MKPs in cell lines. 10
µg of total RNA was isolated from each of the cell lines indicated
and probed with an MKP-2 specific riboprobe (panelA), stripped, and reprobed with a MKP-1 specific
riboprobe (panelB). The migration of the
ribosomal bands is indicated to the right. hMKP-2 refers to an
MKP-2 specific transcript detected only in cells of human origin.
Abbreviations for the cell lines include: RIN, rat insulinoma; GHC1, rat pituitary tumor; AtT20,
mouse pituitary tumor; W2, rat medullary thyroid carcinoma; LTK, mouse fibroblasts; PC12, rat adrenal medullary
tumor; MiaPACA, human pancreatic carcinoma; MOLT48,
human lymphoblasts; HepG2, human hepatoblastoma; SKNMC, human neuroblastoma.
Distribution of MKP-2 in the Central Nervous
System
It has been shown that ERK1 mRNA is expressed in all
areas of the brain with the strongest expression in the hippocampus and
piriform cortex while there was no overlapping expression of CL100
(human MKP-1) in those same areas(42) . In order to determine
the distribution of MKP-2 mRNA in the brain, we performed in situ hybridization analysis on rat brain sections with an S-labeled antisense and sense MKP-2 riboprobe. MKP-2
appeared to be expressed in many areas of the brain with very strong
staining of the hippocampus, piriform cortex, and the suprachiasmatic
nucleus (Fig. 8). The sense riboprobe did not show any specific
staining (data not shown). These results suggest the co-localization of
an ERK isoform, ERK1, and a MKP isoform, MKP-2, in specific areas of
the brain where MKP-1 is not expressed.
Figure 8:
Localization of MKP-2 mRNA in the rat
brain using in situ hybridization. Lightfield (A, C, and E are thionin counterstains) and darkfield (B, D, and F) photomicrographs showing
representative distribution of MKP-2 mRNA (32). DG,
dentate gyrus of the hippocampus; Pir, piriform cortex; 3V, third ventricle; Sch, suprachiasmatic nucleus of
the hypothalamus. Sense MKP-2 riboprobe did not hybridize (data not
shown).
Regulation of MKP-1 and MKP-2 in PC12 Cells
MKPs
have been shown to be immediate early genes and are transcriptionally
regulated by a variety of agents. For example, MKP-1 mRNA appears to be
induced by bombesin, EGF,
12-O-tetradecanoylphorbol-13-acetate, cAMP, and fibroblast
growth factor to different extents and with different kinetics,
suggesting the involvement of this phosphatase in various signaling
pathways(26) . A recent member of the MKP family, B23, has also
been shown to be regulated by serum in human skin
fibroblasts(24) . In order to determine the involvement of MKP-2
in different neuronal signal transduction cascades in PC12 cells,
Northern blot analysis was performed to determine the levels of MKP-1
and -2 mRNA following growth factor stimulation.
Figure 9:
Effect of serum and growth factors on
MKP-1 and MKP-2 mRNA. A, PC12 cells were serum-starved for 24
h (U, untreated control) and were treated with medium
containing 15% serum for the indicated times (left panel) or
with NGF (100 ng/ml) or EGF (20 ng/ml) for the indicated times (right panel). 10 µg of total RNA from each treatment was
analyzed by Northern blot analysis using an MKP-2-specific riboprobe,
the filter was stripped and reprobed with MKP-1, stripped again, and
reprobed with a 18 S ribosomal RNA probe. The results from each probe
are shown. B, quantitative representation of MKP-1 and -2 mRNA
levels normalized to the 18 S ribosomal RNA from an average of two
independent experiments, one of which is shown in A. Presented
values for all treatments represent -fold induction compared with
untreated cells (laneU), which was normalized as 1.
The columns are aligned so as to be directly underneath the treatments
indicated in A. Black boxes represent MKP mRNA levels in PC12
cells serum stimulated for the indicated times; gray boxes represent MKP mRNA levels from serum-starved PC12 cells treated
with NGF for the indicated times; and the white boxes represent MKP mRNA levels from serum-starved PC12 cells treated
with EGF for the indicated times.
To address whether
differences in the induction of distinct MKPs may account for the
differences observed in the kinetics of MAP kinase activation seen
following NGF and EGF treatment of PC12 cells(5) , PC12 cells
were serum-starved and treated with NGF or EGF for various times. Ten
µg of total RNA from each of these treatments was analyzed by
Northern blot analysis for MKP-1 and -2 expression (Fig. 9A, right panel). All quantitations were
normalized to 18 S ribosomal RNA, which was used as an internal control
for the amount of RNA loaded (Fig. 9B, right
panel). NGF induced expression of MKP-1 and MKP-2 by 1 h.
Continued stimulation by NGF resulted in a maximum of 5-fold increase
in MKP-2 expression by 2 h and a 3-fold increase in MKP-1 expression by
1 h. Both MKP-1 and -2 were maintained at slightly elevated levels upon
NGF stimulation. 1 h of EGF also induced expression of MKP-1 by
2.7-fold and MKP-2 by 4-fold. In contrast to NGF, this induction
however was inhibited after 2 h of EGF treatment in both cases.
Additional incubation with EGF resulted in a second induction of both
MKP-1 (2.6-fold) and MKP-2 (5.7-fold) by 4 h. This biphasic kinetic
pattern was also seen following stimulation with serum, with a minimum
RNA level seen at 2-2.5 h, as well (Fig. 9B).
These results suggest that growth factors that activate members of the
MAP kinase cascade also result in the transcriptional activation of
MKPs. The significance of the different kinetic pattern of MKP
induction by NGF and EGF has yet to be determined.
Regulation of Hippocampal Expression
Due to the
high expression of MKP-2 mRNA in the hippocampus (Fig. 8), we
analyzed MKP-2 expression after adult rats (250-300 g) had been
subjected to the global seizure inducing drug, kainic acid (8 mg/kg).
RNA from the hippocampus of rats subjected to kainic acid was extracted
0.5 and 1 h after drug treatment and analyzed by Northern blot analysis (Fig. 10A). Quantitations were normalized to the amount
of cyclophilin in each case. MKP-2 mRNA was induced 5.1-fold by 1 h of
kainic acid treatment, while MKP-1 mRNA was induced 3.2-fold (Fig. 10B). This kainic acid induced transcriptional
stimulation of MKPs suggests the involvement of these genes in the
stress-induced pathways in the brain.
Figure 10:
Induction of MKPs in the hippocampus by
Kainic acid. 10 µg of total hippocamal RNA was isolated from
control rats (C) or rats treated with 8 mg/kg kainic acid for
the indicated times (kindly provided by Drs. James Douglass and Pastor
Couceyro). A, northern blot analysis was performed with a
MKP-2-specific riboprobe, the filter was stripped and probed with
MKP-1, stripped again, and reprobed with cyclophilin as an internal
control. B, the blot was quantitated, and the results were
normalized to cyclophilin and are shown graphically. The black box is the amount of MKP-1 mRNA, and the gray box represents
the levels of MKP-2 mRNA.
10 min) of MKP-1
mRNA(19) . Such runs of AU sequences occur in the
3`-untranslated regions of lymphokines, cytokines, and proto-oncogenes
and are thought to be recognition signals for selective mRNA
degradation(43) . The role of these motifs in MKP-2 regulation
has yet to be determined.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.