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INTRODUCTION |
The mitogen-activated protein kinases
(MAPKs)1 are a superfamily of
enzymes that have been implicated in key cellular processes including
cell proliferation, differentiation, apoptosis, and stress responses
(1-3). A wide variety of stimuli have been found to activate MAPKs
through specific signaling cascades. The basic core of these pathways
consists of a three-kinase module that is evolutionarily conserved (4).
The first component of this signaling module is a MAPK kinase
kinase. This serine/threonine kinase, once activated,
phosphorylates and activates the second component in the module, a MAPK
kinase (MAPKK). Subsequently, the dual specificity MAPKK phosphorylates
the threonine and tyrosine residues of a Thr-X-Tyr
(TXY) motif within the activation loop of the MAPK.
Phosphorylation of both residues within the activation loop is required
and sufficient for full activation of MAPKs (5).
One subfamily of MAPKs, the extracellular signal-regulated kinases
(ERKs), is identified by the TEY (Thr-Glu-Tyr) sequence within the
activation loop. ERK1 and ERK2 were the first ERKs identified. They are
the most widely studied and perhaps best understood members of the ERK
family. ERK3 was cloned in the same cDNA library screen as ERK1 and
ERK2 and shares about 43% overall sequence identity with ERK1 and ERK2
(6). However, ERK3 has a SEG activation motif. ERK4 was identified in
immunoblots as a protein that is tyrosine-phosphorylated in response to
NGF and EGF in a Ras-dependent fashion (7). Little else,
however, is known about ERK4, including its sequence. ERK5 was
initially identified as a redox-sensitive kinase (8). More recently, it
has been implicated in growth factor signaling, as evidenced by
mediating early immediate gene expression through phosphorylation of
MEF2C (9) and EGF-induced cell proliferation (10). ERK6 is highly expressed in human skeletal muscle and promotes differentiation of
myoblasts to myotubes (11). Its TGY activation motif makes it a member
of the p38 family. ERK7 is the most recently cloned ERK with a TEY
activation motif (12). Unlike other members of the ERK family, it has
constitutive activity in serum-starved cells, and typical activators of
other MAPKs fail to further increase ERK7's activity. Interestingly,
exogenous expression of ERK7 can lead to growth inhibition, which is
also regulated by the C-terminal domain of ERK7 (12). Thus, only four
of the seven MAPKs designated ERK have the signature TEY activation motif.
Three of these ERKs, ERK1, ERK2, and ERK5, participate in signal
transduction pathways that originate from the cell surface receptors.
The signaling pathways leading to ERK1 and ERK2 activation have been
characterized most extensively. The MAPK kinases that activate ERK1 and
ERK2 have been identified and cloned (13-15). Termed MAPK/ERK kinase
(MEK1 and MEK2), these dual specificity kinases are the immediate
upstream activating kinases for ERK1 and ERK2. The MAPK kinase kinase
for this module, Raf, has been shown to activate MEK1 and/or MEK2
(16-18). Recently, MEKK3 has been identified as the upstream activator
of MEK5 (19), which is a potent activator of ERK5 (20).
Upstream activators of the ERK7 signaling cascade have not been
identified. Instead, there is evidence to suggest that
autophosphorylation plays a significant role in its activation (12).
Under conditions where the wild-type ERK7 is TEY-phosphorylated, the
kinase-inactive mutant, K43R, remains unphosphorylated. In contrast,
the equivalent catalytically inactive ERK2 mutant can be
TEY-phosphorylated by MEK1 (21). This finding suggests that
autophosphorylation is required for the activation of ERK7. However, it
is not clear whether autophosphorylation is sufficient or whether other
upstream activators are also required. In addition, removing the
C-terminal domain of ERK7 significantly reduces its TEY phosphorylation
and kinase activity, suggesting that the C-terminal region regulates ERK7 activity (12). One possible model is that the C-terminal domain
interacts with the kinase domain to induce activation; alternatively,
interaction of the C-terminal region with other proteins might be required.
To further examine the nature of ERK7's constitutive activity and the
role of autophosphorylation, we expressed ERK7 in bacteria. Not only
were bacterially expressed glutathione S-transferase (GST)-ERK7 and polyhistidine-tagged ERK7 found to be
TEY-phosphorylated, they also had significantly higher in
vitro kinase activity compared with similarly expressed GST-ERK1.
Furthermore, deletion analysis of the C-terminal region indicated that
multiple regions within the C-terminal domain are important in
regulating ERK7 activity. Our results indicate that autophosphorylation
is not only required but is sufficient for activation of ERK7 in the
absence of a putative MEK in vitro. In addition, regulation
of ERK7's activity by the C-terminal domain is complex and may involve
multiple interactions.
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EXPERIMENTAL PROCEDURES |
Materials--
Bovine serum albumin, imidazole, myelin basic
protein, peroxidase-conjugated goat anti-rabbit IgG, and
peroxidase-conjugated goat anti-mouse IgG were purchased from Sigma.
Dulbecco's modified Eagle's medium, FBS, trypsin, penicillin, and
streptomycin were purchased from Life Technologies, Inc. Anti-ERK7
rabbit antiserum was raised against a C-terminal peptide of ERK7
(Cocalico Biologicals, Reamstown, PA). TALON resin was purchased from
CLONTECH. Protein A-Sepharose, the unique site
elimination mutagenesis kit, and the GST purification system were
purchased from Amersham Pharmacia Biotech. Monoclonal antibody (12CA5)
against the hemagglutinin (HA) epitope was purchased from Babco
(Emeryville, CA). High affinity rat monoclonal antibody (3F10) against
the HA epitope and peroxidase-conjugated sheep anti-rat Fab Ig were
purchased from Roche Molecular Biochemicals. Affinity-purified
peroxidase-conjugated goat anti-rabbit IgG and affinity-purified
peroxidase-conjugated goat anti-mouse IgG were purchased from
Transduction Laboratories (Lexington, KY). Anti-ERK1 and
anti-phospho-ERK polyclonal antibodies were purchased from New England
BioLabs (Beverly, MA). Enhanced chemiluminescence reagents and
[
-32P]ATP (6000 Ci/mmol) were purchased from
PerkinElmer Life Sciences. BCA protein assay reagents and GelCode Blue
protein stain were purchased from Pierce. PCR and sequencing primers
were synthesized by the University of Chicago oligonucleotide facility.
Sequencing was performed by the University of Chicago Cancer Research
Center sequencing facility. The pRSV-
-gal expression vectors were a gift from V. Sukhatme. The pSFFV and pSFFV YopJ plasmids were generated
as previously described (22).
Plasmid Construction and Preparation--
The HA-tagged mouse
ERK2 and the HA-tagged rat ERK7 were constructed as previously
described (12). Unique site elimination mutagenesis (23) was performed
to generate ERK7 mutants (K43R, T175R, Y177F, T175A/Y177F, 379, 410, 435, 464, 525, and 535). The mutated sequences were verified by
sequencing. Plasmid DNAs were prepared by CsCl-ethidium bromide
gradient centrifugation or by purification through columns according to
the manufacturer's instructions (Qiagen, Chatsworth, CA).
Cell Culture--
COS cells (American Type Culture Collection,
Manassas, VA) were cultured in Dulbecco's modified Eagle's medium
(Life Technologies, Inc.) supplemented with 10% fetal bovine serum and
antibiotics (50 units/ml penicillin and 50 µg/ml streptomycin) at
37 °C in a 95% air, 5% CO2 atmosphere. Quiescent cells
were obtained by incubating cells in serum-free medium for at least
24 h.
Transient Transfections and Preparation of Cell
Extracts--
COS cells were transfected as previously described (12),
except 1 × 106 cells were seeded on 100-mm plates
24 h prior to transfection with a total of 10 µg of plasmid DNA
and 20 µl of TransIt LT-1 (PanVera Corp., Madison, WI). For all
transfections, 10% of the total plasmid DNA consisted of either the
pRSV-
-gal or pCMV-
-gal expression vector. For the YopJ
experiments, pSFFV or pSFFV-FLAG-YopJ and either pcDNA3, pcDNA3
HA-ERK7, or pcDNA3 HA-ERK2 at a 2:1 to 5:1 ratio were used.
-Galactosidase activity was used to normalize for transfection
efficiency between groups as previously described (12). Unless
otherwise noted, cells were quiesced in serum-free medium for
24 h prior to harvesting. Cell lysates from transfected cells were
prepared as previously described (12).
Western Analysis--
Cell extracts (10-60 µg of
protein/lane) were resolved on an 8 or 10% acrylamide separating gel
by SDS-PAGE. Proteins were transferred to a nitrocellulose membrane.
Membrane blocking, washing, antibody incubation, and detection by
enhanced chemiluminescence were performed as previously described
(12).
Immunoprecipitation and Immune Complex Kinase Assay--
Immune
kinase assays were performed using ectopically expressed epitope-tagged
ERK2 and ERK7 from COS cells as previously described (12). Proteins
were isolated by SDS-PAGE on an 8 or 15% acrylamide separating gel.
The gels were either stained with GelCode Blue (Pierce) and dried or
transferred to nitrocellulose and then subjected to autoradiography.
Quantification of substrate phosphorylation was determined either by
scintillation counter or by the STORM 850 system (Molecular Dynamics,
Inc., Sunnyvale, CA). The presence of HA-tagged or TEY-phosphorylated
proteins in the immunoprecipitates was verified by Western analysis
with 3F10 monoclonal and anti-phospho-ERK polyclonal antibodies,
respectively. Quantification of HA-tagged and TEY-phosphorylated
proteins was performed using densitometric scanning (SigmaScan Pro,
Jandel Corp., San Rafael, CA). Multiple exposures were utilized to
verify linearity of the samples analyzed. Immunoprecipitation assays were performed in a similar fashion; however, the immune complexes were
eluted prior to the kinase reaction and subjected to Western analysis.
GST Fusion Proteins--
Plasmids encoding
GST-c-Fos (positions 210-313) and GST-human
ERK1 (GST-hERK1) were generously provided by T. Deng and M. Cobb,
respectively. GST-HA-ERK7, GST-HA-ERK7 K43R, GST-HA-ERK7 T175A, and
GST-HA-ERK7 Y177F were constructed using PCR to insert a
BglII restriction site upstream of the HA tag. The PCR
product was verified by sequencing and subsequently cloned into pGEX-2T using the BglII/BamHI and EcoRI sites.
GST fusion proteins were prepared using a GST purification system
(Amersham Pharmacia Biotech) with the following modifications. Briefly,
10 ml of overnight culture was diluted 1:100 to a final volume of 1000 ml and incubated at 37 °C with shaking until an
A600 value between 0.4 and 0.9 was
reached. Isopropyl
-D-thiogalactosidase was added to a
final concentration of 0.2 mM, and the mixture was
incubated for an additional 3 h at 37 °C with shaking. The
bacteria were centrifuged at 2600 × g for 20 min at
4 °C. The pellet was frozen at
80 °C and suspended and lysed in
100 ml of STE (150 mM NaCl, 50 mM Tris-HCl, pH
7.4, 1 mM EDTA) containing 500 µg/ml lysozyme, 0.05%
Nonidet P-40, 10 µg/ml leupeptin, 10 µg/ml pepstatin A, and 1 mM phenylmethylsulfonyl fluoride. After 10 min on ice,
MgCl2 and DNase A were added to a final concentration of
2.5 mM and 50 µg/ml, respectively, and the lysate was
incubated on ice for an additional 30 min. The lysate was centrifuged
at 20,000 × g for 30 min at 4 °C. The supernatant was incubated with 1 ml of 50% (v/v) GSH-Sepharose beads (Amersham Pharmacia Biotech) for 90 min at 4 °C with gentle mixing. The beads
were washed four times with STE containing 0.05% Nonidet P-40, 10 µg/ml leupeptin, 10 µg/ml pepstatin A, and 1 mM
phenylmethylsulfonyl fluoride and twice with STE. The fusion proteins
were eluted with 500 µl of 10 mM reduced GSH and 50 mM Tris-HCl, pH 8.0. Free GSH was removed by dialysis
against STE for 1 h at 4 °C. Thrombin cleavage was performed to
remove the GST as specified by the manufacturer (Amersham Pharmacia
Biotech). Purified proteins were dialyzed against 50% ethylene glycol,
50 mM Tris-HCl, pH 7.4, and 50 mM NaCl. Both
eluted and thrombin-cleaved proteins were quantified using a BCA
protein assay as specified by the manufacturer (Pierce) or separated on
10% or 15% SDS-PAGE and quantified by densitometric scanning using
bovine serum albumin as a standard.
Polyhistidine-tagged ERK7--
A polyhistidine His6
tag was added to the C-terminal end of HA-ERK7 and HA-ERK7 K43R using
PCR. The PCR products were inserted into pETBlue-2 as specified by the
manufacturer (Novagen, Madison, WI). The PCR products were verified by
sequencing. The His6-tagged proteins were expressed in
Escherichia coli strain Tuner (DE3)pLysI (Novagen) and
purified using TALON resin (CLONTECH) as previously described (24) with the following modifications. Expression of the
recombinant protein was induced at an A600 of
0.4-0.6 with 0.5 mM isopropyl
-D-thiogalactosidase at 37 °C for 3 h. The cells were lysed in buffer A (0.05% Nonidet P-40, 50 mM sodium
phosphate, pH 7.0, 300 mM NaCl, 5 mM
-mercaptoethanol, 500 µg/ml lysozyme, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 10 µg/ml pepstatin A, and 1 mM
phenylmethylsulfonyl fluoride). MgCl and DNase A were used, and the
lysate was cleared as described above for the GST fusion proteins. The
supernatants were diluted 1:1 in buffer A, and imidazole was added to a
final concentration of 10 mM. The lysate was incubated with
TALON resin equilibrated with buffer A containing 10 mM
imidazole. After washing with 10 volumes of buffer A containing 10 mM imidazole, the proteins were eluted with 2 volumes of
200 mM imidazole in 50 mM sodium phosphate, pH
7.0, 300 mM NaCl. The elutions were concentrated using
Centricon-50 concentrators (Amicon, Inc., Beverly, MA) and dialyzed
against 50 mM Tris-HCl, pH 8.0. Proteins were quantified
using the Bradford protein assay or separated by SDS-PAGE and
quantified by densitometric scanning using bovine serum albumin as a standard.
Two-dimensional Electrophoresis--
For the first dimension,
GST-HA-ERK7, GST-HA-ERK7 T175A, or GST-HA-ERK7 Y177F (5 µg) was
applied to Immobiline Dry Strips (pH 3-10 L or 6-11 L) (Amersham
Pharmacia Biotech) in the rehydration buffer (8 M urea, 2%
CHAPS, 18 mM dithiothreitol, 0.5% IPG buffer (Amersham
Pharmacia Biotech), bromphenol blue) and subjected to flatbed
electrophoresis for 12 h at 30 V, 1 h at 500 V, 1 h at 1000 V, and 10-20 h at 8000 V. For the second dimension, proteins were
separated by SDS-PAGE on an 8 or 10% acrylamide separating gel.
Proteins were transferred to nitrocellulose for immunoblotting.
In Vitro Kinase Assay--
Purified GST fusion proteins or
His6-tagged proteins were incubated in kinase buffer (20 mM HEPES, pH 7.4, 10 mM MgCl2, 10 mM MnCl2, 1 mM dithiothreitol, 0.2 mM sodium vanadate, 10 mM
D-nitrophenylphosphate, 5 µCi of
[
-32P]ATP) with each substrate at 30 °C for varying
lengths of time. Reactions were terminated by adding 4× Laemmli sample
buffer and heating at 95 °C for 5 min. Samples were subsequently
processed as described above for the immune complex kinase assay.
Statistical Analysis--
When applicable, statistical
significance was assessed by one-way analysis of variance. Differences
identified by analysis of variance were quantitated by the
Student-Newman-Keuls multiple comparison test.
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RESULTS |
MEK Inhibitors Do Not Block ERK7 Activation--
ERK1, ERK2, and
ERK5 participate in signal transduction pathways originating from
receptor tyrosine kinases, and their immediate activating kinases have
been identified as MEK1 and -2 for ERK1 and -2 (13-15) and MEK5 for
ERK5 (20). U0126 is a potent MEK inhibitor that has been shown to
inhibit MEK1, MEK2, and MEK5 (25, 26). ERK7 is the only other known
mammalian MAPK that shares the TEY activation motif with these MAPKs.
To determine whether activation of ERK7 is regulated by a MEK with a
similar inhibitory profile, we tested the effect of U0126 on the
activation of ERK7. We pretreated cells with U0126, a specific MEK
inhibitor that blocked nearly 100% of ERK1 and ERK2 phosphorylation
following PMA treatment (25) and markedly reduced EGF-induced ERK5
activation in COS7 cells at a concentration of 10 µM
(26). We found that pretreatment with 30 µM of U0126
completely inhibited TEY phosphorylation of ERK2 with serum stimulation
but failed to inhibit TEY phosphorylation of ERK7 (Fig.
1). Similar results were obtained with
PD98059 (data not shown), a less potent inhibitor that also suppresses
ERK activation by MEK1, -2, and -5 (26, 27). These results indicate
that the putative MEK for ERK7 is not MEK1, MEK2, MEK5 or a novel MEK with a similar inhibitory profile.

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Fig. 1.
U0126 inhibits ERK2 but not ERK7. COS
cells were transfected with either the epitope-tagged ERK7 or
kinase-inactive ERK7 mutant, K43R. Proliferating transfected cells were
treated with either 30 µM of U0126 or an equivalent
volume of the vehicle, Me2SO, for 30 min prior to
harvesting. Lysates were prepared and analyzed by immunoblotting with
either the anti-HA 3F10 monoclonal antibody (left) or the
anti-phospho ERK antibody (right). The positions of TEY
phosphorylated ERK2 (pERK2) and HA-ERK7 (pHA-ERK7) are indicated on the
right. The data shown are representative of three
experiments.
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The MAPK Kinase Inhibitor YOPJ Does Not Block ERK7
Activation--
The virulence factor from Yersinia
pseudotuberculosis, YopJ, inhibits the ERK, c-Jun amino-terminal
kinase, and p38 MAPK pathways as well as the nuclear factor
B
pathway (22, 28). This 33-kDa protein was recently shown to function by
binding members of the MAPK kinase family, blocking their
phosphorylation and activation (22). We tested the effect of YopJ on
ERK7 to determine if a related member of the MAPK kinase family
activates ERK7. As expected, co-transfection of YopJ inhibited the TEY
phosphorylation of HA-tagged ERK2 following stimulation with EGF in
COS7 cells (Fig. 2A). On the
other hand, YopJ failed to inhibit TEY phosphorylation of HA-tagged
ERK7 in similarly transfected, serum-starved COS7 cells (Fig.
2B). These results indicate that, if there is an upstream activator for ERK7, it is distinct from other known members of the MAPK
kinase family, and they raise the possibility that ERK7 can be
constitutively TEY-phosphorylated in the absence of an upstream
activator.

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Fig. 2.
YOPJ inhibits ERK2, but not ERK7.
A, COS cells were cotransfected with either HA-ERK2 or a
control plasmid, pcDNA3, and either FLAG-YopJ or a control plasmid,
pSFFV. Following serum starvation for 24 h, the cells were
stimulated with 50 µg/ml of EGF for 5 min, and cell lysates were
prepared. The epitope-tagged ERK2s were immunoprecipitated with the
12CA5 monoclonal antibody and assessed by Western analysis with either
anti-HA 3F10 monoclonal antibody (top) or anti-phospho-ERK antibody
(bottom). B, COS cells were cotransfected with
either HA-ERK7 or a control plasmid, pcDNA3, and either FLAG-YopJ
or a control plasmid, pSFFV. Following serum starvation for 24 h,
the cells were stimulated with 50 µg/ml of EGF for 5 min, and cell
lysates were prepared. The epitope-tagged ERK2 were immunoprecipitated
with the 12CA5 monoclonal antibody and assessed by Western analysis
with either anti-HA 3F10 monoclonal antibody (top) or
anti-phospho-ERK antibody (middle). Whole cell lysate was
also assessed by Western analysis with the anti-FLAG M2 monoclonal
antibody (bottom). The data shown are representative of
three experiments.
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Bacterially Expressed ERK7 Is TEY-phosphorylated--
The findings
that both U0126 and YopJ do not inhibit TEY phosphorylation of ERK7 and
our previous finding that the K43R mutant of ERK7 is not
TEY-phosphorylated suggest that autophosphorylation is sufficient for
ERK7 activation. To address this possibility, we determined whether
ERK7 was phosphorylated in the TEY activation domain when expressed in
bacteria in the absence of an upstream activator. Thus, both HA-tagged
ERK7 and the K43R mutant were fused in frame at the C-terminal end of
GST. Following expression in E. coli and purification by
glutathione-Sepharose, Western analysis with anti-GST and anti-HA
antibodies (Fig. 3A) indicated that both proteins were partially degraded despite the extensive use of
protease inhibitors. Nevertheless, identification of the full-length
fusion proteins was possible using an anti-ERK7 antiserum that
recognizes the terminal 14 amino acids of ERK7 (Fig. 3A, anti-ERK7 panel). Western analysis using the
anti-phospho-ERK antibody, which specifically recognizes the dually
phosphorylated TEY motif, indicated that bacterially expressed
GST-HA-ERK7 was significantly more TEY-phosphorylated than the
similarly processed GST fusion protein of human ERK1 (GST-hERK1) (Fig.
3A, Anti-PhosphoERK panel). As observed for ERK7
deletion mutants in mammalian cells, the lower molecular weight
GST-HA-ERK7 degradation products (Fig. 3A,
Anti-GST and Anti-HA panels), which correspond to
truncated proteins lacking the C terminus, are not recognized by the
anti-phospho-ERK antibody (Fig. 3A, Anti-PhosphoERK
panel). In addition, the GST-HA-ERK7 K43R mutant was not
recognized by the anti-phospho-ERK antibody (Fig. 3A,
Anti-PhosphoERK panel), which is also consistent with previous studies in mammalian cells (12). The fidelity of the anti-phospho-ERK antibody for the dually phosphorylated TEY motif was
verified using comparable amounts of the TEY activation domain mutants
T175A, Y177F, and T175A/Y177F as previously described (data not shown)
(12).

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Fig. 3.
Bacterially expressed ERK7 is TEY
phosphorylated. A, GST, GST-HA-ERK7, GST-HA-ERK7 K43R,
and GST-human ERK1 were expressed in E. coli, purified using
glutathione-Sepharose, and assessed by Western analysis with either an
anti-GST antiserum (first panel), anti-HA 3F10
monoclonal antibody (second panel), anti-ERK7
antiserum (third panel), anti-ERK1 polyclonal
antibody (fourth panel), or anti-phospho-ERK
polyclonal antibody (fifth panel). The anti-ERK7
antiserum recognizes the last 14 amino acids of the C-terminal end. The
anti-ERK1 polyclonal antibody recognizes the last 14 amino acids of rat
ERK2 (residues 345-358). B, bacterially expressed and
purified GST-HA-ERK7, GST-HA-ERK7 T175A, and GST-HA-ERK7 Y177F were
resolved by two-dimensional electrophoresis. Separated proteins were
subjected to Western analysis with either an anti-HA 3F10 monoclonal
antibody (panels 2-4) or the anti-phospho-ERK
polyclonal antibody (panel 1). The data shown are
representative of three experiments.
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To determine the extent of TEY phosphorylation of bacterially expressed
ERK7, two-dimensional electrophoresis was performed. Western analysis
using the anti-HA antibody revealed a complex pattern of GST-HA-ERK7,
GST-HA-ERK7 T175A, and GST-HA-ERK7 Y177F species across a wide
molecular weight range and within a pI range much lower than the
theoretical pI value of 9.10 for GST-HA-ERK7 (data not shown). These
species presumably represent multiply phosphorylated full-length and
C-terminally degraded GST-HA-ERK7. Closer examination of the higher
molecular weight species corresponding to full-length or nearly
full-length GST-HA-ERK7 indicates that only a few species are not
recognized by the anti-phospho ERK antibody (Fig. 3B,
first and second panels). As expected,
species with dual phosphorylation of the TEY activation site are more acidic. On the other hand, the higher molecular weight species of the
GST-HA-ERK7 TEY mutants are more basic, consistent with only partial
TEY phosphorylation (Fig. 3B, third and
fourth panels). Together, these results indicate
that the majority of full-length or nearly full-length ERK7 expressed
in bacteria is dually TEY-phosphorylated in the absence of any upstream activator.
Bacterially Expressed ERK7 Is a Functional Kinase--
Since the
activation domain was phosphorylated, we tested whether the bacterially
expressed GST-HA-ERK7 fusion protein has kinase activity. GST,
GST-HA-ERK7, GST-HA-ERK7 K43R, and GST-hERK1 were expressed in E. coli and purified using glutathione-Sepharose. The kinase activity
was measured by an in vitro assay using MBP as a substrate.
Equal amounts of total protein were used for each reaction. As shown in
Fig. 4A, only GST-HA-ERK7
phosphorylated MBP to a significant degree. Additional in
vitro kinase assays were performed using GST-c-Fos as a substrate
(Fig. 4B). GST-HA-ERK7 phosphorylated GST-c-Fos in
vitro, whereas phosphorylation by GST-hERK1 was negligible.
Furthermore, GST-HA-ERK7 was found to autophosphorylate in
vitro. In contrast, although autophosphorylation of GST-hERK1 was
also observed, the level was barely detectable. Similar assays using
thrombin-cleaved GST-HA-ERK7 were performed to exclude the possibility
that autophosphorylation of GST-HA-ERK7 might be dependent on GST
oligomerization. Neither the recognition of HA-ERK7 by the
anti-phospho-ERK antibody nor HA-ERK7's ability to phosphorylate MBP
in vitro was affected by the presence or absence of GST
(Fig. 4C).

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Fig. 4.
Bacterially expressed GST-HA-ERK7 is a fully
functional kinase. A, GST, GST-HA-ERK7, GST-HA-ERK7
K43R, and GST-human ERK1 were expressed in E. coli, purified
using glutathione-Sepharose, and assessed by Coomassie-based staining
(top). Enzymatic activity of purified GST, GST-ERK7,
GST-HA-ERK7 K43R, and GST-hERK1 was measured using an in
vitro kinase assay with 10 µg of MBP as a substrate
(bottom). B, bacterially expressed and purified
GST-HA-ERK7, GST-HA-ERK7 K43R, and GST-hERK1 were used in in
vitro kinase assays with 2 µg of GST-c-Fos as a substrate
(top). The Coomassie-based staining of GST-ERK7, GST-HA-ERK7
K43R, and GST-hERK1 is shown (bottom). C,
bacterially expressed and purified GST-HA-ERK7 and GST-HA-ERK7 K43R
were thrombin-cleaved and assessed by Coomassie-based staining
(first panel). The thrombin-cleaved HA-ERK7 and HA-ERK7 K43R
were subjected to Western analysis with either an anti-HA 3F10
monoclonal antibody (second panel) or the
anti-phospho-ERK polyclonal antibody (third
panel). Enzymatic activity of the thrombin-cleaved HA-ERK7
and HA-ERK7 K43R was measured using an in vitro kinase assay
with 2 µg of MBP as a substrate (fourth panel).
D, bacterially expressed and purified
HA-ERK7-His6 was subjected to Western analysis with either
an anti-HA 3F10 monoclonal antibody (panel 1), an
anti-ERK7 antiserum (panel 2), or the
anti-phospho-ERK polyclonal antibody (panel 3).
The anti-ERK7 antiserum recognizes the last 14 amino acids of the
C-terminal end. The Coomassie-based staining of purified
HA-ERK7-His6 is shown in panel 4. The
data shown are representative of three experiments.
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Determination of an accurate specific activity was not possible, since
a significant portion of the purified GST-HA-ERK7 protein was degraded
and contaminating bacterial proteins were detected following removal of
GST by thrombin cleavage (Fig. 4C, first panel). However, comparison of the activity of both intact
and degraded GST-ERK7 with that of bacterially expressed GST-hERK1 at a
comparable protein concentration suggested that the specific activity
of ERK7 was more than 100-fold greater than that of ERK1 (data not
shown). Since Western analysis of whole bacterial GST-HA-ERK7 lysate
indicated that degradation occurred primarily from the C-terminal end
(data not shown), a His6 tag was placed at the C terminus
to enhance purification of full-length HA-ERK7. Bacterial protein
contamination remained a problem during purification of His6-tagged HA-ERK7; however, it was possible to capture
full-length HA-ERK7 (Fig. 4D, first and second
panels), which was readily recognized by the anti-phospho-ERK
antibody (Fig. 4D, third panel). Comparison of the activity of His6-tagged HA-ERK7 with that
of bacterially expressed GST-hERK1 indicated that the specific activity of bacterially expressed ERK7 was 1000-fold greater than the specific activity of ERK1 (data not shown). The 10-fold lower level of activity
seen with GST-HA-ERK7 compared with His6-tagged HA-ERK7 is
probably due to the presence of inactive, degraded GST-HA-ERK7. The
difference in activity is not likely to be due to the presence or
absence of GST, since it does not affect the specific activity of ERK1
to a significant degree (29, 30). The 1000-fold difference in activity
between His6-tagged HA-ERK7 and GST-hERK1 is similar to
that observed for ERK2 following activation by MEK1 or MEK2 (31) and is
greater than the difference between ERK7 and ERK2 in unstimulated
mammalian cells (12). Together, these results demonstrate that ERK7 can
autophosphorylate and that, unlike ERK1, it has significant kinase
activity in the absence of an upstream activator.
Autophosphorylation of ERK7 Is an Intramolecular
Interaction--
Autophosphorylation of ERK2 on Tyr-185 has been shown
to be intramolecular, based on concentration independence (32). To determine if ERK7 autophosphorylation is inter- or intramolecular, ERK7
in vitro kinase assays using an excess of the
kinase-defective ERK7 were performed. Purified recombinant GST-HA-ERK7
K43R was used after thrombin cleavage of the GST tag. Removal of the
28-kDa GST tag allowed us to separate the K43R mutant from the tagged GST-ERK7 by SDS-PAGE. As shown in Fig. 5,
the K43R ERK7 mutant was not phosphorylated by GST-ERK7, and a 10-fold
excess of this mutant did not inhibit autophosphorylation of GST-ERK7.
These results indicate that autophosphorylation of ERK7 is an
intramolecular reaction.

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Fig. 5.
GST-HA-ERK7 does not phosphorylate
(GST)-HA-K43R in vitro and an excess of
kinase-defective ERK7 does not inhibit the autophosphorylation
reaction. GST-HA-ERK7 and GST-HA-ERK7 K43R were expressed in
E. coli and purified using glutathione-Sepharose. HA-ERK7
K43R was purified from thrombin-cleaved GST-HA-ERK7 K43R. An in
vitro kinase assay was used with either GST-HA-ERK7 or GST-HA-ERK7
K43R and increasing amounts of HA-ERK7 K43R. The positions of
GST-HA-ERK7 and HA-ERK7 K43R are noted on the left. The data
shown are representative of three experiments.
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Generation of ERK7 C-terminal Truncations--
ERK7 TEY
phosphorylation and kinase activity are not only regulated by
autophosphorylation but also by the presence of its C-terminal region
(12). To further examine the role of the C-terminal region in the
regulation of ERK7 activity, mutants containing successive deletions at
the C-terminal tail of ERK7 were constructed using site-directed
mutagenesis (Fig. 6A). COS7
cells were transiently transfected with expression vectors encoding
HA-ERK7 and the various HA-ERK7 mutants (Fig. 6B). The
full-length HA-ERK7 is 556 amino acids long including the nine-amino
acid HA tag. The tail-truncated mutants of ERK7 were generated by
introducing a stop codon at the designated amino acid residues using
site-directed mutagenesis. Expression of the two shortest mutants, 379 and 410, was markedly lower than that of the other mutants, and
differences in transfection efficiency as assessed by
-galactosidase
activity could not fully explain this disparity (data not shown).

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Fig. 6.
Generation of ERK7 C-terminal
truncations. A, a schematic illustration of HA-ERK7
K43R and tail truncation mutants generated by site-directed
mutagenesis. The full-length HA-ERK7 is 556 amino acids long. The tail
truncation mutants are designated by the position of the introduced
stop codon. B, COS cells were transiently transfected with
either the epitope-tagged ERK7 or various ERK7 mutants. Lysates were
prepared and analyzed by immunoblotting with the anti-HA 3F10
monoclonal antibody. The data shown are representative of three
experiments.
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The C-terminal Region Can Regulate ERK7 Activity--
The
C-terminal tail of ERK7 has been shown to contain some protein binding
motifs such as putative SH3 binding domains (12). Therefore, to
determine if a specific region of the C-terminal tail is critical for
ERK7 activity, COS7 cells were transiently transfected with expression
vectors encoding HA-ERK7 and the various HA-ERK7 mutants. After 24 h, the cells were serum-starved for another 24 h and harvested,
and the transfected kinases were immunoprecipitated using the 12CA5
anti-HA mouse monoclonal antibody. Immunoprecipitates were subjected to
Western analysis with either the 3F10 high affinity anti-HA rat
monoclonal antibody (Fig. 7A,
top) or the anti-phospho-ERK antibody (Fig. 7A,
middle). Immunoprecipitated, transfected kinases were also
evaluated by in vitro kinase assays using c-Fos as a substrate (Fig. 7A, bottom). The amounts of total
cellular protein from the transiently transfected cells were adjusted
to yield comparable expression levels for the various truncated mutants following immunoprecipitation. Quantification of both the degree of TEY
phosphorylation (Fig. 7B) and kinase activity, as evidenced by c-Fos phosphorylation (Fig. 7C), revealed a complex
pattern upon progressive loss of the C-terminal region. Thus, loss of the most C-terminal 21 amino acids reduced ERK7's ability to
phosphorylate c-Fos by 35% (Fig. 7C). Although a smaller
decrease in TEY phosphorylation was seen (Fig. 7B), TEY
phosphorylation in general tended to correlate with c-Fos
phosphorylation for all of the mutants. A significant decrease in both
TEY phosphorylation and c-Fos phosphorylation was seen between the 515 and 464 mutants (p < 0.05) and between the 410 and 379 mutants (p < 0.05). On the other hand, an increase in
TEY phosphorylation and c-Fos phosphorylation was seen between the 435 and 410 mutants (p < 0.05). These results indicate
that one specific region of the C-terminal domain is not critical for ERK7 activation or activity. Instead, these results raise the possibility that multiple interactions between the catalytic domain and
the C-terminal tail are involved in the regulation of ERK7 activity.

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Fig. 7.
Multiple regions of the C-terminal tail
regulate ERK7's activity. A, COS cells were
transiently transfected with either the epitope-tagged ERK7 or various
ERK7 mutants, serum-starved for 24 h, and lysed with 1% TLB.
Following immunoprecipitation with anti-HA 12CA5 monoclonal antibody,
samples were assessed by either Western analysis with either the
anti-HA 3F10 monoclonal antibody (top) or anti-phospho-ERK
antibody (middle) or by an in vitro kinase assay
using 2 µg of c-Fos as a substrate (bottom). B,
graphical depiction of TEY phosphorylation quantified using
densitometric scanning and expressed as a ratio of TEY phosphorylated
ERK7 as determined by anti-phospho-ERK Western analysis to total ERK7
as determined by anti-HA 3F10 Western analysis. The ratio of
phosphorylated wild-type ERK7 to HA-ERK7 was empirically determined to
be 1. Results are reported as mean ± S.D. The data are
representative of more than six independent experiments. C,
graphical depiction of the relative kinase activity of ERK7 compared
with the ERK7 C-terminal mutants. Phosphorylation of the substrate
c-Fos was normalized to the amount of enzyme present as determined by
densitometric scanning of the anti-HA 3F10 Western analysis. The value
for wild-type ERK7 was empirically determined to be 1, and the kinase
activity of the K43R mutant was used as background activity. Results
are reported as mean ± S.D. The data are representative of five
independent experiments.
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DISCUSSION |
The basic MAPK signaling pathway consists of a three-kinase
module. The upstream MAPK kinase and MAPK kinase kinase have been identified and characterized for many of the MAPKs. Although MAPK pathways, as a whole, respond to an extremely diverse collection of
stimuli, individual MAPK modules respond to specific extracellular stimuli. To date, we have been unable to identify the upstream regulator of ERK7. Instead, we have evidence suggesting that activation and regulation of ERK7 are unique. We previously found that typical activators of ERK1, ERK2, and ERK5 fail to further activate ERK7 (12),
suggesting that MEK1, MEK2, and MEK5 do not play a role in ERK7
activation. In this study, we verified that MEK1, MEK2, MEK5, or a
related MEK are not involved in the activation of ERK7 using the MEK
inhibitors PD098059 and U0126. In addition, ERK7 activation was not
inhibited by YopJ, a virulence factor recently shown to bind to a wide
variety of MAPKKs and block their phosphorylation and activation (22).
This indicates that other known MAPKKs, such as MKK3 and MKK4, are not
likely to be involved in the activation pathway of ERK7. Finally,
mutating the region of ERK7 corresponding to the conserved docking
domain in MAPKs, which has been proposed to facilitate docking of their
specific MAPKK and subsequent activation (33), had no appreciable
effect on ERK7 activation (data not shown). Together, these results
indicate that the specific upstream regulation of ERK7 differs from
that known for the other MAPKs.
Although the upstream regulator of ERK7 remains to be identified, it is
clear that autophosphorylation plays a role in the activation of ERK7.
To clarify whether autophosphorylation occurs directly on the
activation loop or is required at a different site to facilitate
interaction with an upstream activator, we expressed ERK7 in bacteria.
Despite low levels of expression and a significant degree of
degradation, recovery of the full-length, wild-type GST-ERK7 and
kinase-dead mutant was possible. When compared with the similarly
isolated GST fusion protein of ERK1, ERK7 displayed significant TEY
phosphorylation as determined by Western analysis with the
anti-phospho-ERK antibody. Further analysis by two-dimensional electrophoresis revealed that the majority of GST-ERK7 appears to be
TEY-phosphorylated. Comparison of the activity of
His6-tagged HA-ERK7 and GST-hERK1 suggests that the
specific activity of bacterial expressed ERK7 is at least 1000-fold
greater than that of ERK1. This difference cannot be attributed to the
presence or absence of GST, since it does not affect the specific
activity of ERK1 to a significant degree (29, 30). In the bacterial
system, the upstream activating kinase and other associated mammalian proteins should be absent. Competition experiments with the ERK7 K43R
mutant indicate that autophosphorylation occurs as an intramolecular reaction. Therefore, these findings suggest that not only is
autophosphorylation of ERK7 directly on the tyrosine and threonine
residues within the TEY activation motif, but other interacting
mammalian proteins are not required for its activation. These findings
are in distinct contrast to ERK1 and ERK2.
While classically identified as serine/threonine kinases, ERK1 and ERK2
are capable of autophosphorylating on the tyrosine residue within the
activation loop (32, 34). This process is limited, however, to only a
very small percentage of the protein in vitro. In addition,
the kinase activity of the singly phosphorylated forms of ERK1 and ERK2
is not much higher than that of the unphosphorylated forms (31, 32).
Therefore, when ERK1 and ERK2 are expressed alone in bacteria, they
display minimal substrate phosphorylation (29, 35-37). However,
co-expression with activated MEK increases their specific activity
about 1000-fold (35, 38), which is comparable with the level of ERK7
activity seen when expressed alone in vitro. The degree of
ERK7 activity in vitro in the absence of an upstream MEK
raises the question as to how its activity is regulated.
Previously, we found that removal of the C-terminal domain of ERK7
significantly reduced its TEY phosphorylation and kinase activity. In
this study, we used several C-terminal deletion mutants of ERK7 to
determine if there is a specific regulatory region of ERK7 within this
domain. Rather than identifying a distinct region, our findings
indicate that multiple regions of the C-terminal domain may participate
in the regulation of ERK7's constitutive activity, suggesting that the
mechanism by which the C-terminal region regulates ERK7 activity is
complex. One possibility is that other cellular proteins interact with
ERK7 through the C-terminal domain to facilitate activation. However,
expression of ERK7 in bacteria strongly suggests that additional
mammalian proteins are not required for its activation. Analysis of the
inactive and fully activated crystal structures of ERK2 suggests that
stabilization of an activated ERK7 is required, since conformational
changes that occur upon activation result in an "energetically
unfavorable structure" (39, 40). Thus, another possibility is that
multiple regions of the C-terminal domain interact with regions within the kinase domain, thereby stabilizing the activated structure. Partial
truncation of the C-terminal domain could result in nonproductive interactions that might be eliminated upon further truncation. Such a
mechanism could explain the fluctuating activation profile of the
C-terminal truncation mutants.
Despite sharing sequence homology including the TEY activation motif
with the other known ERKs, ERK7 clearly displays several unique
features. Extracellular stimuli that typically activate ERK1, ERK2, or
ERK5 do not increase ERK7's activity (12). An inhibitor of the
upstream activator of these ERKs as well as a more generic MAPKK
inhibitor failed to block ERK7 activation. Bacterially expressed ERK7
is a highly active kinase that is capable of autophosphorylating as
well as phosphorylating substrates in vitro. Stepwise
truncations of ERK7's C-terminal domain result in a reduction in
kinase activity. However, the pattern seen is not consistent with a
single region within the C-terminal domain responsible for governing
its activity. Instead, it indicates that multiple regions play a role
in regulating ERK7's kinase activity. Together, these data indicate
that autophosphorylation is not only required but is sufficient to
activate ERK7. To definitively answer whether the C-terminal domain
regulates ERK7 activity through stabilizing interactions similar to
ERK2-ERK2 interactions following activation, the crystal structure of
ERK7 will need to be solved. Our findings do not rule out the
possibility that ERK7, like other MAPKs identified to date, is part of
its own unique three-kinase signaling module in vivo and can
be further activated by its putative MEK. However, our results raise
the possibility that ERK7 represents an entirely novel class of MAPK
with a unique form of regulation.