ERK7 expression and kinase activity is regulated by the ubiquitin-proteosome pathway.

ERK7 is a unique member of the extracellular signal-regulated kinase (ERK) subfamily of MAP kinases. Although ERK7 shares a TEY motif in the activation loop of the kinase, it displays constitutive activation, nuclear localization, and growth inhibitory properties that are regulated by its C-terminal domain. Because ERK7 is expressed at low levels compared with ERK2 and its activity is dependent upon its expression level, we investigated the mechanism by which ERK7 expression is regulated. We now show that ERK7 expression is regulated by ubiquitination and rapid proteosomal turnover. Furthermore, both the kinase domain and the C-terminal tail are independently degraded at a rate comparable with that of the intact protein. Analysis of a series of chimeras between ERK2 and ERK7 reveal that the N-terminal 20 amino acids of the kinase domain are a primary determinant of ERK7 degradation. Fusion of the N-terminal 20 amino acids is both necessary and sufficient to cause proteolytic degradation of both ERK2 and green fluorescent protein. Finally, ERK7 is stabilized by an N-terminal mutant of Cullin-1 suggesting that ERK7 is ubiquitinated by the Skip1-Cullin-F box complex. These results indicate that ERK7 is a highly regulated enzyme whose cellular expression and kinase activation level is tightly controlled by the ubiquitin-proteosome pathway.

MAP 1 kinases are an evolutionarily conserved family that regulate cell growth, differentiation, survival, and a variety of other cellular responses (reviewed in Ref. 1). These kinases, which consist of the extracellular signal-regulated kinases (ERKs), the Jun kinases (JNKs), and the p38 kinases, are classically activated via dual phosphorylation by a MAP kinase kinase. MAP kinase kinases, which phosphorylate an activation loop on MAP kinase consisting of a TXY motif, are in turn phosphorylated by MAP kinase kinase kinases that respond to extracellular stimuli. Although this paradigm applies to the general MAP kinase family, recent evidence suggests that ac-tivation of more distant members of this family may be regulated by other mechanisms.
The ERK family of MAP kinases can be divided into at least two classes: 1) the classic MAP kinases that consist primarily of a kinase domain such as ERK1 and ERK2; and 2) the big MAP kinases such as ERK3, ERK5, ERK7, and ERK8 that consist of both a kinase domain and a C-terminal domain and range in size from 60 to over 100 kDa. In contrast to ERK1 and ERK2 that require discrete scaffolding proteins such as KSR for maximal activation, the big MAP kinases have C-terminal regions that can function as protein interaction domains that regulate kinase localization (2), activation (3), and transcriptional activity (4). The C-terminal domain of ERK7 has been shown previously to be required for ERK7 constitutive activation and nuclear localization as well as growth inhibition by ERK7 (2,3). However, a role for the C-terminal domain in regulating protein expression has not previously been described.
From an evolutionary perspective, ERK7 is a unique member of the MAP kinase family. Unlike ERKs, JNKs, and p38s that share over 90% homology between human and rodents, ERK7, which was originally cloned in rat, shares less than 70% homology with its closest human counterpart identified in the human genome, ERK8. Furthermore, ERK8 differs in its mechanism of activation, localization, and substrate specificity (5). Thus, although ERK8 may be considered the human orthologue of ERK7, its evolutionary divergence that derives in large part from the C-terminal domain raises the possibility that the two enzymes may have unique as well as overlapping functions. The physiological roles of ERK7 and ERK8 are currently under investigation. Initial studies revealed that overexpression of ERK7 can lead to suppression of DNA synthesis that was dependent on the presence of the C-terminal domain of ERK7 but independent of its kinase activity (2). A recent study suggested that exogenously expressed rat ERK7 can regulate the proteolytic turnover of the estrogen receptor in human cells (6). However, in light of the significant sequence and functional differences between ERK7 and ERK8, the physiological relevance of these observations requires further investigation.
One of the striking features of ERK7 and ERK8 are the extremely low levels of endogenous protein expression in cultured cells and tissues. The present study was undertaken to determine the mechanism by which ERK7 is regulated. Whereas no significant difference in transcript levels were detected, our results indicated that the ERK7 protein is rapidly ubiquitinated and degraded by the proteosome, and this process is regulated in part by the N terminus of the kinase domain.

EXPERIMENTAL PROCEDURES
Materials-A rabbit polyclonal antibody against green fluorescent protein (GFP) (GFP (FL) sc-8334) was purchased from Santa Cruz Biotechnology (San Diego, CA). The anti-GFP antibody was used at a 1:1000 dilution in 0.5% milk/TBST buffer (25 mM Tris-HCl, pH 8.0, 125 mM NaCl, and 0.1% Tween 20). Antibody against human Cullin-1 protein (CUL-1 (H-213)) was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Proteasome inhibitor I, lactacystin, MG-132, and E64 were purchased from Calbiochem (San Diego, CA). Protease Inhibitor Mixture Set III from Calbiochem was used at a 1:200 dilution in lysis buffer for immunoblotting and a 1:2000 dilution in washing buffer for immunoprecipitation. Protease Inhibitor Mixture Set III includes 4-(2-aminoethyl)benzenesulfonylfluoride, HCl (100 mM), aprotinin (80 M), bestatin (5 mM), E64 (1.5 mM), leupeptin (2 mM), and pepsin A (1 mM). Anti-HA antibodies used were HA.11 purchased from Covance (Berkeley, CA) for immunoprecipitation, and 3F10 purchase from Roche Diagnostics for immunoblotting. Anti-galactosidase antibody was used at 1:2000 dilution. Cycloheximide purchased from Sigma was prepared in 50% methanol at 30 mg/ml and added to cells at a final concentration of 50 g/ml. Oligonucleotides were ordered from Integrated DNA Technologies, Inc. (Coralville, IA), and DNA sequencing was done by the DNA Sequencing Facility at the University of Chicago Cancer Research Center.
Cell Culture-COS and HEK293 cells were cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum (Invitrogen) and 1% penicillin and streptomycin (10,000 units and 10,000 g of stock, respectively) at 37°C in a 5% CO 2 incubator.
ERK7 and ERK2 Chimeric Constructs-Unique site mutagenesis was used to generate new restriction enzyme sites on HA-ERK7 and HA-ERK2 expression vectors. New restriction enzyme sites were created for domain swapping between ERK7 and ERK2. An EcoRV site was created in ERK7 by mutating T to C at nucleotide position 423 using the oligonucleotide 5Ј-agggatatCtacctgtgt-3Ј. Similarly, a new EcoRV site was created in ERK2 using an oligonucleotide with the sequence 5Ј-aaagataAtCtatatagta-3Ј. The slash in the sequence RDI/ YIV indicates the junction between ERK7 and ERK2 in the ERK7/2(VI) construct, and the slash in the sequence KDI/YLV indicates the junction between ERK2 and ERK7 in the ERK2/7(VI) construct. A KpnI site was created in HA-ERK2 at nucleotide position 634 with the primer 5Ј-tggtacCgagctcca-3Ј. The slash in the sequence WY/RAP indicates the junction between ERK7 and ERK2 in both the ERK2/7(VIII) and ERK7/ 2(VIII) constructs. Other ERK7/2 and ERK2/7 chimeras and ERK7/GFP chimeric constructs were generated using the overlap extension PCR approach (8). The final PCR product was subcloned into a mammalian expression vector, pCR3.1 (Invitrogen), using the T-A cloning method. The outside primers and the specific, chimeric primer pairs are listed in Table I. The coding regions of all constructs were verified by DNA sequencing.
Transfection of Cells-Cells transfected with TransIT-LT1 reagent (Mirus, Madison, WI) were plated 1 day before transfection and harvested between 36 and 48 h after transfection.
COS or HEK293T cells were co-transfected with test constructs along with transfection control vectors such as pCMV ␤-galactosidase or pGreen Lantern-1 using Trans-LT1 reagent (1:3; DNA to TransLT1). The total amount of DNA used for transfection was 3-5 g per 100-mm dish and the ratio of the transfection control vector to test constructs was 1:6 to 1:10. About 24 h after transfection, cells were pooled and split into smaller dishes for a time course of CHX treatment or for proteasome inhibitor treatments.
Preparation of Cell Lysate-Cells in culture were washed once with ice-cold phosphate-buffered saline and lysed with RIPA buffer (1% Triton X-100, 0.1% SDS, 0.5% sodium deoxycholate, 20 mM Tris-HCl, pH 7.5, 10% glycerol, 1 mM ␤-glycerophosphate, 137 mM NaCl, 10 mM NaF, 5 mM EDTA, 1 mM sodium vanadate, 1 mM EGTA, 40 mM pnitrophenyl phosphate, and protease inhibitor mixture as described above). The cell lysate was centrifuged at 14,000 rpm in an Eppendorf bench top centrifuge for 15 min at 4°C to clear cell debris. Protein concentration was determined by the Bradford method using a Bio-Rad protein assay reagent (Bio-Rad).
Western Blot Analysis-Cell extracts (10 -60 g of protein per lane unless otherwise noted) were resolved by 8 or 10% SDS-PAGE. Proteins were transferred to a nitrocellulose membrane. The membrane was blocked in 5% nonfat dry milk in 1ϫ TBST for 1 h, incubated with primary antibodies at appropriate concentrations for 1 h, washed for 30 min, and then incubated with secondary antibodies for 1 h. All incubations and washes were done at room temperature. The signal was detected with enhanced chemiluminescence (ECL) reagent (PerkinElmer Life Sciences) and analyzed by autoradiography or digital imaging. To reprobe membranes with a new antibody, membranes were incubated in a stripping buffer (62.5 mM Tris-HCl, pH 6.8, 2% SDS, and 0.1 M 2-mercaptoethanol) at 50 -55°C for 30 min and then washed extensively with 1ϫ TBST.
Northern Blot Analysis-Cells were transfected as described above and total RNA was isolated using TRIzol reagent according to the manufacturer's protocol (Invitrogen). 20 g of total RNA was loaded in each lane, separated, and transferred as previously described (2). A 32 P-labeled ␤-galactosidase probe was prepared using the Megaprime DNA Labeling System (Amersham Biosciences), and a 32 P-labeled antisense-HA oligo probe was prepared as previously described (2).
Immunoprecipitation-Cell lysates were diluted into 0.5ϫ RIPA and incubated with specific antibodies on a rotator for 4 h or overnight at 4°C. Sepharose-Protein A or Sepharose-Protein G was added to each tube, and the tubes were reincubated for another 2 h. The immunocomplexes were washed 3 times with 0.5ϫ RIPA. After washing, 20 l of 2ϫ loading dye was added to the beads, and the tubes were boiled for 5 min to elute proteins from the beads. Eluted proteins were separated by 8 or 10% SDS-PAGE, transferred to a nitrocellulose, and the signals were detected by Western blotting and ECL as described above. 600 g of protein extract from each sample was used for immunoprecipitation and detection of ubiquitinated protein, and 200 -300 g was used for The junction of case change in each primer indicates the position where two different genes are fused.
ERK7 Proteolytic Turnover serum induction. The presence of epitope-tagged proteins in the immunoprecipitates was verified by Western analysis with a 3F10 monoclonal antibody against the HA epitope.
Pulse-Chase Analysis-Cells transfected with HA-ERK7 were pooled and re-plated to normalize for transfection efficiency. Following incubation in medium deficient in methionine and cysteine, cells were incubated with Tran 35 S-label (MP Biomedicals, Aurora, OH) for a 30min pulse. Cells were then incubated in growth medium containing an excess of cold methionine and cysteine until harvested at the indicated time points. HA-ERK7 was immunoprecipitated from cleared lysates using an anti-HA antibody and separated by SDS-PAGE. The gel was incubated in an autoradiographic enhancer, dried, and analyzed using the STORM 850 system (Amersham Biosciences).
Virus Production and Infection of PC12 Cells-The full-length ERK7 and ERK7 tail consisting of the last 193 amino acids were subcloned into the pCLE retroviral vector (9) to generate pCLE-ERK7 and pCLE-ERK7 TAIL, respectively. These constructs were co-transfected into a 293T-derived packaging cell line (Phoenix-gp) along with pHCMV-G, which expresses the VSV-G protein. Phoenix-gp cells were transfected at ϳ65% confluency in 75-cm 2 flasks by calcium phosphate precipitation using 12.5 g of each retroviral construct and 14 g of pHCMV-G DNA. Virus-containing supernatant was harvested at 24 h and used to infect PC12 cells that were plated 24 h earlier at 7 ϫ 10 5 cells per 100-mm dish. Ͼ90% of PC12 cells were infected based on staining (alkaline phosphatase substrate kit IV; Vector Laboratories, Inc.) for the human placental alkaline phosphatase gene contained in the pCLE vector.
pCLE-ERK7 and pCLE-ERK7 TAIL Turnover Assay-Cells stably expressing either pCLE-ERK7 or pCLE-ERK7 TAIL were plated at 1 ϫ 10 6 cells per 60-mm dish. After 24 h, the medium was replaced with fresh complete Dulbecco's modified Eagle's medium containing 5% horse serum and 50 g/ml cycloheximide. Cells were harvested at the time points indicated and 20 g of each lysate was used for Western analysis with an anti-ERK7 antibody as previously described (2).

RESULTS
To compare the expression levels of ERK2 and ERK7, COS cells were transfected with ␤-galactosidase and either HA-ERK2 or HA-ERK7 on CMV expression vectors. As shown in Fig. 1, ERK2 protein levels were 10 -100 times higher than ERK7 at comparable transfection efficiencies. Co-expression of ERK2 and ERK7 in the same cell did not change the relative ERK expression levels. Similarly, co-expression of HA-ERK7 with GFP on CMV expression vectors did not alter the levels of GFP protein (data not shown) suggesting that the difference in expression levels is a property of ERK7 itself and not an indirect effect of ERK7 signaling. However, it is also independent of the state of activation of ERK7, as shown by the kinase-minus (K43R) mutant.
To determine whether the regulation of ERK7 expression occurred at the transcriptional level, COS cells transfected with ␤-galactosidase and HA-ERK2, HA-ERK7, or HA-ERK7(K43R) were lysed and analyzed for mRNA transcripts by Northern blot analysis. As shown in Fig. 2, ERK7 mRNA levels were comparable with those of ERK2. These results indicate that the regulation of ERK7 was likely occurring at a posttranscriptional level.
The previous results suggest that protein rather than mRNA stability might be the source of differential ERK expression. To test this possibility, COS cells transfected with HA-ERK2 or HA-ERK7 were treated with cycloheximide to block new protein synthesis, and the stability of the remaining protein determined by immunoblotting with an anti-HA antibody (Fig.  3A, top). The results indicate that ERK7 is degraded with a t1 ⁄2 of about 2 h. In contrast to HA-ERK7, HA-ERK2 is a stable protein and its t1 ⁄2 is longer than 6 h (Fig. 3A, top). Pulse-chase 35 S-labeled HA-ERK7 confirmed the short t1 ⁄2 of ϳ2 h. (Fig. 3A, middle and bottom panels). To determine whether degradation of ERK7 was mediated by the proteosome, COS cells transfected with HA-ERK7 were pretreated with proteosome inhibitors prior , and HA-ERK2 constructs were co-transfected with pCMV ␤-galactosidase (␤-gal) into COS cells, and total RNA was isolated from the cells. Twenty micrograms of total RNA was loaded into each lane, and the RNA was separated in a formaldehyde gel by electrophoresis. Northern blot analyses were performed, and the membranes were probed with 32 P-labeled HA antisense oligonucleotides and ␤-galactosidase antisense DNA fragments. A, analysis of Northern blots by autoradiography. B, plot of transcript levels. Northern blots were scanned with a phosphorimager, and the radioactive bands were quantitated. The levels of HA-ERK7 and HA-ERK7(K43R) transcripts were normalized to the level of HA-ERK2 transcripts. The data plotted represent the average of three independent experiments Ϯ S.D.
to cycloheximide treatment. ERK7 degradation was blocked by a number of proteosome inhibitors including lactacystin, proteosome inhibitor 1, or MG132, whereas E64, a cysteine protease inhibitor not specific to the proteosome pathway, had no effect (Fig. 3B). Similar results were obtained when HEK293T cells were used for the experiments (data not shown).
To directly confirm that ERK7 is ubiquitinated, HEK293T cells were transfected with HA-ERK7 and His 6 -Myc-ubiquitin. Cells were then either untreated or pretreated with proteosome inhibitor 1 to block degradation of ubiquitinated protein. The cell lysates were immunoprecipitated with anti-HA, anti-Myc, or anti-ERK7 antibodies, and the immunoprecipitated complexes were analyzed by immunoblotting with anti-ubiquitin or anti-HA antibodies (Fig. 4). Analysis of immunoprecipitated HA-ERK7 with anti-ubiquitin antibody shows a series of bands corresponding to protein with different amounts of conjugated ubiquitin. Detection of HA-ERK7 in the Myc-ubiquitin immunoprecipitate and the ERK7 immunoprecipitate indicates that most of the ubiquitinated ERK7 is less conjugated. These results clearly demonstrate that ERK7 is ubiquitinated prior to protein degradation.
One of the three major classes of ubiquitin ligases that recognize and ubiquitinate substrates are the Cullin-based E3 ligases (reviewed in Ref. 10). Among these E3 ligase complexes are the anaphase promoting complex (APC), the von Hippel Landau tumor suppressor/Elongin BC complex (CBC), and the SCF (Skp1/Cull/F-box protein) that contains Cullin-1 (Cul-1).
To determine whether the degradation of ERK7 occurred via the SCF complex, we expressed ERK7 in HEK293T cells in the presence or absence of a mutant human Cullin-1 (hCul-1) comprising the N-terminal 452 amino acids of Cullin-1 that blocks ubiquitination of substrates (11). Cells were then pretreated with cycloheximide to monitor the rate of protein turnover. The addition of mutant hCul-1 stabilized ERK7 (Fig. 5). The stabilizing effect of the dominant-negative mutant hCul-1 on HA-ERK7 was similar to the stabilizing effect of MG132, a proteasome inhibitor (Fig. 5). These results provide additional evidence that ERK7 is degraded by the proteosome and suggest that ERK7 is degraded by a multisubunit Cullin-based SCF complex.
To identify the region(s) in ERK7 that are responsible for its ubiquitination, we generated chimeras between ERK2 and ERK7. Initially, we analyzed a set of mutants and chimeras in which the kinase domains I-V or I-VII were switched (Fig. 6). The kinase domains were analyzed either alone or with the addition of the ERK7 C-terminal tail. To monitor stability, the mutant and chimeric proteins were expressed in either COS or HEK293 cells along with a GFP expression vector to control for transfection efficiency. As shown in Fig. 6C (right panel), the kinase domain of ERK7 (ERK7⌬T) is highly unstable relative to ERK2. Transfer of the N-terminal kinase domains I-V or I-VII from ERK7 to ERK2 resulted in significantly lower expression of the resultant chimeric proteins ERK7/2(VI) and ERK7/2(VIII) (Fig. 6C, right panel). Conversely, substituting kinase domains I-V or I-VII of ERK2 for the comparable domains in the ERK7 kinase lacking the C-terminal tail (ERK2/ 7(VI)⌬T and ERK2/7(VIII)⌬T) dramatically stabilized the proteins (Fig. 6C, left panel). These findings indicate that a signal promoting the turnover of ERK7 exists within N-terminal kinase domains I-V of ERK7.
In addition, it appears that a region in the C terminus of ERK7 can confer instability. Thus, the ERK2/7(VI) chimera that contains kinase domain I-V of ERK2 is expressed at a lower level than its counterpart lacking the C-terminal region of ERK7, ERK2/7(VI)⌬T (Fig. 6C, left panel). However, the ERK2/7(VI) chimera is less stable than the ERK2/7(VIII) chimera that also has ERK2 domains VI and VII (see chimera ERK2/7(VIII) in Fig. 6C, left panel), suggesting that domains VI and VII in the ERK7 kinase may also contribute to the instability conferred by the tail. This possibility is supported by the observation that addition of only the C-terminal ERK7 tail to ERK2 causes some degradation but little loss in overall protein expression (Fig. 6C, right panel). Surprisingly, the isolated C-terminal tail of ERK7 has a turnover rate that is at least as high as that of ERK7 (Fig. 7), suggesting that the tail is more stable when associated with the kinase domain. In this figure, the band with the slower mobility in the gel probably represents phosphorylated ERK7/T protein. Neither serum stimulation, nor serum starvation made a difference in the relative expression levels (data not shown). Taken together, these results indicate that the N terminus of ERK7 is a major site of instability in the protein and regions(s) within the Cterminal tail also contribute.
To narrow the region of instability in the kinase domain further, additional chimeras on the N terminus consisting of domains I, I-II, I-IV, and I-V from ERK7 fused to the remaining domains of ERK2. As shown in Fig. 8B, all of the ERK7/2 chimeric proteins were expressed at a level much lower than that of ERK2 and similar to the level of ERK7 expression (Fig.  8B). These results indicate that domain I of ERK7 is sufficient to impart instability to ERK2. ERK7/2(VIa) and ERK7/2(VIb) are two chimeric constructs that differ by one residue. Both constructs contain domains I-V from ERK7, but ERK7/2 (VIa) ends with residues IYLV and ERK7/2(VIb) with residues IYL. The expression levels of ERK2/7(VIa) and ERK7/2(VIb) were comparable, confirming that the different levels of protein expression were not because of the process of subcloning or subtle differences in amino acids at the sites of fusion. Conversely, the addition of the N-terminal domains I or I-II of ERK2 to the kinase domain of ERK7 enhanced the stability of the ERK7 kinase chimeras ERK2/7(II)⌬T and ERK2/7(III)⌬T (Fig. 8C).
To determine whether the instability of the ERK7/2 chimera was regulated by the proteasome pathway, one of these constructs, ERK7/2(II), was co-transfected with the dominant negative hCul1-N452 plasmid into HEK293 cells. Reduced expression of both the wild-type ERK7 and ERK7/2(II) chimera was seen following treatment with cycloheximide. However, the levels of both were stabilized following treatment with cycloheximide when co-expressed with the dominant negative hCu-lin1-N452 (Fig. 9). The results showed that the hCul1-N452 mutant stabilized the ERK7/2(II) chimera containing Domain I from the N terminus of ERK7 (Fig. 9). Taken together, these results indicate that the N-terminal 20 amino acids of ERK7 are both necessary and sufficient for the SCF-mediated ubiquitination and rapid proteolytic turnover of the ERK7 kinase domain.
One possible explanation for the results obtained is that the ERK7/ERK2 chimeric proteins are misfolded. To ensure that these chimeric proteins are not structurally altered, we determined whether the ERK7/2 chimeric protein that contains domains I and II of ERK7 is an active kinase. As shown in Fig. 10, the TEY motif in the activation loop of the chimeric protein is phosphorylated in response to serum stimulation similar to that of the wild type ERK2. These results indicate that the chimeric protein is functionally active and therefore not denatured.
To determine whether the 20-amino acid N-terminal domain is sufficient to confer instability on proteins unrelated to ERK7, we fused these N-terminal amino acids to GFP. As shown in Fig. 11, the addition of this N-terminal ERK7 domain significantly decreased the stability of GFP. Furthermore, the addition of proteasome inhibitor I increased the stability of ERK7(I)/GFP proteins (Fig. 11B), suggesting that the regulation of ERK7(I)/GFP was through the proteasome pathway. These results confirm that the N-terminal 20 amino acids of ERK7 are necessary and sufficient as a signal for ubiquitination and proteosomal degradation of ERK7.

DISCUSSION
ERK7 is expressed at extremely low levels in tissues and cultured cells. The results presented here demonstrate that ERK7 is rapidly degraded by the ubiquitin-proteosome pathway. Whereas the ERK7 mRNA levels are comparable with those of ERK2, the stability of the ERK7 and ERK2 proteins are significantly different. Although the C-terminal tail also contributes to instability, the main determinant of ERK7 protein turnover is the first 20-amino acid region (Domain I) at the N terminus. Because ERK7 is constitutively activated, its kinase activity is dependent upon the level of ERK7 expression. We have also previously shown that the C-terminal domain of ERK7 can confer a number of properties including inhibition of DNA synthesis. The results shown here now demonstrate that FIG. 6. Both the N-and C- , and the other chimeric constructs are similarly named C. Expression of mutant and chimeric ERK7 and ERK2 constructs. Chimeric constructs of ERK7 and ERK2 were generated by site-directed mutagenesis and a subcloning approach as described under "Experimental Procedures." The constructs were transfected into either HEK293T or COS cells with pCMV-␤-gal or pGreen-Lantern-1 as controls for transfection efficiency. Cells were lysed, and extracted proteins were resolved by 10% SDS-PAGE. Western blotting was performed using an anti-HA antibody. The membrane was stripped and re-probed with either an anti-␤-galactosidase or an anti-GFP antibody. C, left panel, ERK7 and ERK2/7 chimeric protein expression in cells. Each chimeric protein was loaded in duplicate lanes. ⌬T refers to lack of a C-terminal ERK7 tail. ␤-Galactosidase (␤-gal) is a control for transfection and protein loading. C, right panel, the level of ERK2/T, ERK7⌬T, and ERK7/2 chimeric protein expression in cells. GFP is a transfection and protein loading control. the N terminus of the kinase domain and the C-terminal domain control ERK7 stability and provide a mechanism for the regulation of both ERK7 kinase activity as well as the kinaseindependent activity of the ERK7 C terminus.
Interestingly, the members of the ERK subfamily that are "big" MAP kinases characterized by extended C-terminal domains are expressed at significantly lower levels than ERK 1 and 2. It has recently been shown that ERK3, which has a different activation loop motif (SEG instead of TEY) but shares homology with the ERK subfamily, is also regulated by ubiquitination and proteosome turnover. In the case of ERK3, two regions within its N terminus were identified as responsible for its turnover with a t1 ⁄2 of 30 min (12). In contrast, ERK7 is regulated by one 20-amino acid region at the N terminus of ERK7 as well as a region within the C-terminal tail, and the t1 ⁄2 for turnover occurs between 1 and 2 h. Analysis of ERK2/ERK7 chimeras shows that the N-terminal amino acids are both necessary and sufficient to regulate protein turnover but an interaction between the C terminus and domains V-VII chimeras can also confer instability to the full-length enzyme. It is possible that, in the three-dimensional structure of ERK7, there is an interaction between the N terminus and C-terminal tail with the same E3 ligase. However, resolution of this question must await ERK7 crystallization and identification of the regions of instability within the C-terminal tail as well as the nature of the E3 ligase. Taken together, these results indicate that there is a subset of MAP kinases that have an alternative mechanism for regulation consisting of protein turnover. Given the low level of ERK7 expression, the tight regulation and rapid turnover suggests that ERK7 expression rapidly responds to cellular signals.
Other kinases are also regulated by a similar mechanism. One in particular, SGK, is a serine/threonine protein kinase related to Akt (13). Interestingly, the amino acid residues that are responsible for SGK turnover are also located at the N terminus. However, analysis of the sequences reveals no obvious homology. It has been suggested that this region might regulate SGK localization, bringing the kinase in proximity to the ubiquitination complex. However, although the kinase domain of ERK7 is localized in the cytoplasm and the intact ERK7 is nuclear, both proteins are turned over at similar rates. Similarly, the ERK7 C-terminal domain when expressed alone is localized in the nucleus but also degraded at a comparable rate. FIG. 7. Turnover of ERK7 and the ERK7 tail in stably transfected PC12 cells. Both ERK7 and the ERK7 C-terminal tail domain (ERK7/T) were subcloned into a pCLE retroviral vector, and then transduced into PC12 cells. Clones stably expressing the ERK7 genes were selected, and two of these stable lines were used for assaying ERK7 protein turnover. One million cells were seeded onto each 60-mm dish, and, 24 h later, the cells were treated with CHX (50 g/ml). Cells were harvested at different time points, lysed, and analyzed for ERK7 and ERK7/T protein by SDS-PAGE and Western blotting as described under "Experimental Procedures." An ERK7 antibody raised against a Cterminal peptide was used to probe ERK7 and ERK7/T proteins. Membranes were stripped and reprobed with an anti-tubulin antibody as a control for protein loading. These results are representative of three independent experiments.
FIG. 8. The N-terminal domain I is responsible for rapid turnover of the ERK7 kinase. A, schematic of domains I to V from both ERK7 and ERK2. B, the N terminus of ERK7 contributes to rapid protein turnover of the kinase. Chimeric constructs were made as described under "Experimental Procedures." ERK7/2(II) represents a chimera containing domain I from ERK7 fused to the remaining domains of ERK2. The ERK7/2(III) chimera contains domains I-II from ERK7, the ERK7/2(V) contains domain I-IV from ERK7, and both ERK7/2(VIa) and ERK7/2(VIb) contain domains I-V from ERK7. The chimeric constructs were co-transfected with GFP into HEK293T cells, and cell lysates were analyzed by SDS-PAGE, Western blotting with anti-HA and anti-GFP antibodies, and chemiluminescence as described under "Experimental Procedures." The protein amount in the first ERK2 lane (B) was only one-tenth of the amount loaded in each of other lanes. HA-ERK7, HA-ERK2, and GFP proteins are marked by arrows. The GFP protein is a control for transfection efficiency. C, ERK7 protein is stabilized by swapping the N-terminal domain I of ERK7 with the N-terminal domain I from ERK2. Construction of ERK2/7(II)⌬T and ERK2/7(III)⌬T was described under "Experimental Procedures." ERK2/7(II)⌬T represents the fusion of domain I from ERK2 to the remaining domains of ERK7 without the C-terminal tail region. The constructs were transfected into HEK293T cells, and cycloheximide (CHX) was added and incubated with cells as indicated. Cell lysates were analyzed by SDS-PAGE, Western blotting, and chemiluminescence as described under "Experimental Procedures." These results are representative of at least three independent experiments. The stabilization of ERK7 by expression of dominant-negative Cullin-1 suggests that the proteolytic recognition complex is an SCF complex. SCF is one of the best characterized ubiquitin ligase complexes and consists of a number of subunits including Cullin-1. Functioning as a scaffold protein, Cullin family proteins bind both to the ubiquitin E3 ligase complex and Roc 1, a RING finger protein that binds to the ubiquitinconjugating E2 enzyme (14). The N terminus of Cullin-1 interacts with the adapter protein Skp1, which complexes to a number of different F-box proteins such as Skp2 to assemble different SCF-Roc1 E3 ligases. The E3 ligase complexes then recruit and ubiquitinate specific substrates including many nuclear cell cycle regulatory proteins such as p27 (15) and p21 (16,17). The dominant-negative Cullin-1 that we used to stabilize ERK7 expression lacks the C terminus that interacts with the Roc1 protein, and thus does not ubiquitinate its substrates (11). The result we obtained suggests that ERK7 is ubiquitinated by an SCF E3 ligase, but does not identify a specific recognition protein. Because ERK7 is localized in the nucleus and can inhibit DNA synthesis (2), proteolytic regulation similar to that of cell cycle proteins would not be surprising.
Proteolytic turnover of ERK7 was demonstrated using exogenously expressed ERK7 that was either transiently or stably expressed in cells. Similar approaches have been used to show proteolytic turnover of other unstable kinases such as SGK (13). Endogenous levels of ERK7 are extremely low and difficult to detect above nonspecific background levels, even with the addition of proteosome inhibitors, preventing accurate quantitation of turnover rates. Thus, we cannot rule out the possibility that regulation of ERK7 also occurs at a transcriptional as well as a post-transcriptional level. Expression of ERK7/ERK2 chimeras enabled us to localize one main site of proteolytic recognition to a region at the N terminus of ERK7. The ability to destabilize other proteins such as ERK2 and GFP after transferring this region to them confirmed the identity of this domain. Two types of experiments were included to ensure that the proteolytic turnover observed was not a result of misfolding or overexpression of a cytotoxic protein. First, we demonstrated that the ERK7/ERK2 chimera containing only the N terminus of ERK7 was activated by serum indicating that the basic kinase structure was not altered. Second, we showed that an unrelated and benign protein such as GFP was also rapidly degraded by the addition of the N-terminal ERK7 sequence. Taken together, these results clearly identify a region in ERK7 that is recognized by a Cullin-1-dependent, proteosome degradation complex.
The tight regulation of ERK7 expression raises the possibility that ERK7 is cytostatic or cytotoxic to the cell. The previous observation that overexpression of ERK7 leads to an inhibition of DNA synthesis in CV-1 cells is consistent with this interpretation (2). Similarly, stabilization and the resulting higher expression of ERK3 results in G 1 arrest (12). In both cases, FIG. 11. ERK7 domain I is sufficient to convert GFP protein to a rapid turnover protein. ERK7 domain I was fused to the N terminus of the GFP genes as described under "Experimental Procedures." The ERK7(I)/GFP and pGreen-Lantern-1 constructs were transfected into HEK293T cells. The plasmid pCMV-␤-galactosidase was included as a control for transfection efficiency. At 36 after transfection, cells were treated with proteasome inhibitor I (PSI) (right panel) or with cycloheximide (CHX) (left panel) as described in the legend to Fig. 9. Cell lysates were prepared and analyzed by SDS-PAGE, Western blotting, and chemiluminescence as described under "Experimental Procedures." An anti-GFP antibody was used to probe GFP and HA-ERK7(I)/ GFP proteins. These results are representative of at least three independent experiments.
FIG. 9. HA-ERK7/2(II) protein is stabilized by disrupting SCF complexes with a Cullin-1 mutant. HA-ERK7 and HA-ERK7/2(II) constructs were co-transfected with GFP and a mutant hCullin-1 construct (hCul-1-N452) or a control vector into HEK293T cells as described in the legend to Fig. 5. At 48 h after transfection, cycloheximide (CHX) was added to cells, and cells were lysed at different time points as indicated. Cell lysates were analyzed by SDS-PAGE, Western blotting, and chemiluminescence as described under "Experimental Procedures." hCul-1-N452 was detected with an anti-hCul-1 antibody, and HA-ERK7 and HA-ERK7/2(I) proteins were detected with an anti-HA antibody. GFP protein was included as a transfection control. These results are representative of at least three independent experiments.
FIG. 10. ERK7/2 chimeric proteins are active kinases. HA-ERK2 and HA-ERK7/2(III) constructs were transfected into HEK293T cells. At 36 h after transfection, cells were grown in serum-free medium for 12 h and then were either untreated or treated with 10% serum for 10 min. Cell lysates were prepared and HA-tagged proteins were immunoprecipitated with an anti-HA antibody. The amount of the immunoprecipitates loaded in each lane from HA-ERK2 was one-sixth of that from HA-ERK7. The immunoprecipitates were resolved by 10% SDS-PAGE, Western blotting with a dual phospho-specific antibody was performed to detect phosphorylated HA-ERK2 and HA-ERK7/(III) proteins (bottom panel). Membranes were stripped and reprobed with an anti-HA antibody to detect total HA-tagged proteins (top panel). HA-ERK2, HA-ERK7/2(III), and GFP proteins are marked with arrows. These results are representative of at least three independent experiments. inhibition of proteolytic turnover would enable rapid suppression of cell cycle progression similar to other regulators of the cell cycle such as p53. Although we have identified a major N-terminal domain that promotes turnover of ERK7, other regions also contribute to ERK7 instability. Once all the regions of E3 ligase recognition have been identified and mutated, it should be possible to determine the effect of stable ERK7 expression on cell growth and integrity.