ERK7 Is an Autoactivated Member of the MAPK Family*

Mark K. AbeDagger , Kristopher T. KahleDagger , Matthew P. SaelzlerDagger , Kim Orth§, Jack E. Dixon§, and Marsha R. Rosner||

From the Dagger  Department of Pediatrics and Ben May Institute for Cancer Research and the  Department of Neurobiology, Pharmacology and Physiology, University of Chicago, Chicago, Illinois 60637 and the § Department of Biological Chemistry, University of Michigan, Ann Arbor, Michigan 48109

Received for publication, January 2, 2001, and in revised form, April 3, 2001


    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Extracellular signal-regulated kinase 7 (ERK7) shares significant sequence homology with other members of the ERK family of signal transduction proteins, including the signature TEY activation motif. However, ERK7 has several distinguishing characteristics. Unlike other ERKs, ERK7 has been shown to have significant constitutive activity in serum-starved cells, which is not increased further by extracellular stimuli that typically activate other members of the mitogen-activated protein kinase (MAPK) family. On the other hand, ERK7's activation state and kinase activity appear to be regulated by its ability to utilize ATP and the presence of its extended C-terminal region. In this study, we investigated the mechanism of ERK7 activation. The results suggest that 1) MAPK kinase (MEK) inhibitors do not suppress ERK7 kinase activity; 2) intramolecular autophosphorylation is sufficient for activation of ERK7 in the absence of an upstream MEK; and 3) multiple regions of the C-terminal domain of ERK7 regulate its kinase activity. Taken together, these results indicate that autophosphorylation is sufficient for ERK7 activation and that the C-terminal domain regulates its kinase activity through multiple interactions.


    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 [gamma -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-beta -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-beta -gal or pCMV-beta -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. beta -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 beta -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 beta -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 beta -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 [gamma -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.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.

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 kappa 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.

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.

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.

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.

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 beta -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.

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.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.

    ACKNOWLEDGEMENTS

We thank M. Hershenson for a critical review of the manuscript; S. Gomes for technical assistance; and M. Cobb, T. Deng, and V. Sukhatme for generous provision of reagents.

    FOOTNOTES

* This work was supported by National Institute of Health Grants HL 03867 (to M. K. A.), NS 33858 (to M. R. R.), DK 18024 (to J. E. D.), and HL 56399 (to M. R. R.); grants from the American Lung Association of Metropolitan Chicago (to M. K. A), the Dorothy and Gaylord Donnelley Foundation (to M. K. A.), and the Walther Cancer Institute (to J. E. D. and K. O.); and a gift from the Cornelius Crane Trust Fund for Eczema Research (to M. R. R.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

|| To whom correspondence should be addressed: Ben May Institute for Cancer Research, University of Chicago, 5841 S. Maryland Ave., MC 6027, Chicago, Illinois 60637-1470. Tel.: 773-702-0380; Fax: 773-702-4634; E-mail: mrosner@ben-may.bsd.uchicago.edu.

Published, JBC Papers in Press, April 3, 2001, DOI 10.1074/jbc.M100026200

    ABBREVIATIONS

The abbreviations used are: MAPK, mitogen-activated protein kinase; MAPKK, MAPK kinase; ERK, extracellular signal-regulated kinase; EGF, epidermal growth factor; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; GST, glutathione S-transferase; HA, hemagglutinin; PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis; hERK1, human ERK1; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid.

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RESULTS
DISCUSSION
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