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Molecular and Cellular Biology, September 2003, p. 5979-5988, Vol. 23, No. 17
0270-7306/03/$08.00+0     DOI: 10.1128/MCB.23.17.5979-5988.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Extracellular Signal-Regulated Kinase 7, a Regulator of Hormone-Dependent Estrogen Receptor Destruction

Department of Microbiology,¹ Department of Pathology,³ Center for Cell Signaling, University of Virginia, Charlottesville, Virginia 22908,² Department of Biochemistry, Boston University School of Medicine, Boston, Massachusetts 02118⁴

Received 16 December 2002/ Returned for modification 31 January 2003/ Accepted 19 May 2003

	ABSTRACT

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Estrogen receptor alpha (ER) degradation is regulated by ubiquitination, but the signaling pathways that modulate ER turnover are unknown. We found that extracellular signal-regulated kinase 7 (ERK7) preferentially enhances the destruction of ER but not the related androgen receptor. Loss of ERK7 was correlated with breast cancer progression, and all ER-positive breast tumors had decreased ERK7 expression compared to that found in normal breast tissue. In human breast cells, a dominant-negative ERK7 mutant decreased the rate of endogenous ER degradation >4-fold in the presence of hormone and potentiated estrogen responsiveness. ERK7 targets the ER ligand-binding domain for destruction by enhancing its ubiquitination. Thus, ERK7 is a novel regulator of estrogen responsiveness through its control of ER turnover.

	INTRODUCTION

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Estrogen receptor alpha (ER) belongs to the superfamily of ligand-activated transcription factors (7). This superfamily shares a common modular structure, which consists of an N-terminal region, a DNA-binding domain, and a ligand-binding domain. ER regulates the expression of genes involved in growth and development and plays an important role in breast and endometrial cancers. The cellular response to estrogens in vivo is ER limited (33), and a key mechanism in regulating ER concentration is receptor degradation (2). Loss of ER is associated with aggressive breast tumors and poor clinical outcome, and it is essential, therefore, to understand the molecular basis for the control of ER turnover.

In response to estradiol, the rate of ER degradation through ubiquitination and the 26S proteasome pathway is increased by an unknown mechanism (2, 20, 22, 35). The 26S proteasome pathway is the major pathway of regulated proteolysis in eukaryotes and is responsible for the destruction of ubiquitinated substrates (32). Ubiquitin is a 76-amino-acid protein that can be covalently attached to a lysine residue on the substrate through the action of an enzyme cascade involving E1, E2, and E3 enzymes. E1 activates ubiquitin and transfers it to E2 enzymes, which transfer ubiquitin to the substrate by themselves or in cooperation with E3 enzymes. E2 and E3 enzymes both contribute to target specificity.

Parallel studies on other members of the nuclear receptor superfamily have suggested a role for mitogen-activated protein kinase (MAPK) (also referred to as extracellular signal-regulated kinase 1/2 [ERK1/2]) in regulating receptor turnover. For example, MAPK(ERK1/2) phosphorylation of Ser-294 in the human progesterone receptor increases the progesterone receptor degradation rate (18). MAPK(ERK1/2) is also known to phosphorylate ER, and we therefore investigated whether MAPK(ERK1/2) enhances ER turnover (4, 14). MAPK(ERK1/2) belongs to a kinase subgroup, the activity of which is regulated by phosphorylation and/or dephosphorylation of threonine and tyrosine residues present in a Thr-Glu-Tyr (TEY) motif within the activation loop. Other members of this subgroup include ERK5 and ERK7. MAPK(ERK1/2) and ERK5 are both activated in response to growth factor signals (6, 15). However, ERK7 is a unique member of the TEY subgroup in that it is constitutively active and does not appear to be further activated by agents known to stimulate MAPK(ERK1/2) activity (1). Very little is known about ERK7, and its biological function has not been established.

We now show that ERK7 is a specific regulator of ER degradation in human breast cells. ERK7 is expressed in normal human breast tissue, and loss of ERK7 is strongly correlated with breast cancer progression. Inhibition of ERK7 activity in human breast cells by a dominant-negative ERK7 mutant significantly enhances ER levels in the presence of hormone and increases estrogen responsiveness. Our studies have therefore revealed the existence of a new signaling pathway impinging on the 26S proteasome machinery, in which ERK7 regulates hormone responsiveness in breast cells by controlling the rate of ER degradation. Furthermore, the loss of this pathway appears to be correlated to the development of breast cancer.

	MATERIALS AND METHODS

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Reagents. The monoclonal antibodies, 12CA5 (antihemagglutinin [anti-HA]) and 9e10 (anti-MYC), were obtained from the University of Virginia Lymphocyte Culture Facility. The anti-active MAPK antibody (also called anti-pTEY antibody) was purchased from Promega. The cDNAs for human ER, human IB, human androgen receptor (AR), and mouse SF1 were provided by P. Chambon (30), D. Ballard (3), M. Weber (9), and K. Parker (21), respectively. MEKc was provided by N. Ahn (25), ERK5 by J. Dixon (36), and HA-ubiquitin by D. Bohmann (31). The peptides to the C-terminal end of rat ERK7 (CRSALGRLPLLPGPRA) and to the activation loop of rat ERK7 (CQALTEY) and the polyclonal antibodies, anti-ERK7 and anti-AL ERK7, were produced by Research Genetics.

Expression vectors. The cDNA for ERK7 was obtained from a rat testis cDNA library by using the PCR with primers specific to the published ERK7 sequence (1). The sequence was verified, and the cDNA was inserted in frame into pK3H (contains a triple HA tag) or PKR7-MYC (contains a MYC tag) (5) or modified pCMV-Tag5 (Stratagene). The vector pCMV-Tag5 (Stratagene) was modified by inserting the promoter and triple HA tag sequences from pKH3 into pCMV-Tag5. All ER and ERK7 mutants were produced by using PCR, and the sequences were verified by the University of Virginia Biomolecular Research Facility. Both wild-type and mutant ERs were subcloned into pSG5 (30).

Immunoblots. BHK cells were transfected in 10-mm-diameter dishes with calcium phosphate, 1 µg of wild-type or mutant ER or FLAG-IB or HA-SF1 or HA-AR construct, and 5 µg of a construct encoding a wild-type or mutant kinase or vector control. At 8 h posttransfection, the cells were washed with phosphate-buffered saline and placed in Dulbecco's modified Eagle's medium (DMEM) with 5% charcoal-stripped fetal calf serum (FCS). After 1 h the cells were washed with phosphate-buffered saline and serum starved for 15 h, and either estradiol or vehicle was added 6 h before lysis with boiling sodium dodecyl sulfate-polyacrylamide gel electrophoresis sample buffer (dithiothreitol [DTT]) (13).

For the rate experiments, transfected cells were trypsinized, aliquoted, and pretreated with 50 µM cycloheximide for 2 h in the absence of serum before the addition of estradiol. The cells were lysed at various times after estradiol treatment.

Breast lines were serum starved for 15 h before lysis with sample buffer (-DTT). Human tissue obtained from the University of Virginia Tissue Procurement Facility was pulverized in the presence of liquid N₂, and the powder was added to sample buffer (-DTT).

Total protein was determined by using DC protein assay (Bio-Rad), and 100 µg of protein was electrophoresed (DTT was added to each sample prior to loading) and transferred to nitrocellulose as described elsewhere (13). Immunoblots and densitometric analysis were as described (13), except that, for Fig. 6B, the 9e10 antibody was labeled directly with horseradish peroxidase.

View larger version (57K): [in this window] [in a new window]   FIG. 6. ERK7 enhances the ubiquitination of the ER ligand-binding domain. (A) BHK cells were cotransfected with vectors encoding a deletion mutant of ER and either HA-ERK7 or vector control (V). The serum-starved, transfected cells were treated with or without 10 nM E2 for 6 h and were then lysed. Equal amounts of total protein were electrophoresed and immunoblotted. (B) BHK cells were transfected with a vector encoding HA-ubiquitin and either MYC-ERK7 or vector control (V). They were also transfected with either additional vector control or a vector encoding MYC-ER(283-595). The transfected cells were pretreated with MG132 for 2 h before lysis. Immunoprecipitations (IPs) were performed by using anti-HA antibody, and the IPs were electrophoresed and immunoblotted (IB).

Generation of stable clones. MCF-7 cells were maintained in DMEM with 5% charcoal-stripped FCS and 5% CO₂. They were transfected with Lipofectamine (Life Technologies) according to the manufacturer's directions with 6 µg of the modified pCMV-Tag5 vector with or without the K43A-ERK7 cDNA. Stable clones were obtained by using G418 (600 µg/ml) selection. Isolated colonies were cloned and propagated, and where appropriate the lines were analyzed for expression of HA-K43A-ERK7.

Transcriptional analysis. MCF-7 stables were transfected and assayed for luciferase and ß-galactosidase as described previously (13).

Proliferation. Stable MCF-7 cells were seeded at 5 x 10⁴ per well in a 24-well dish containing DMEM and 5% charcoal-stripped FCS with 600 µg of G418/ml. After 15 h the cells were treated with or without 1 µM ICI 182,780. At various times the cells were lysed and the amount of ATP was determined by CellTiter-Glo Luminescent Cell Viability Assay according to the manufacturer's protocol (Promega).

	RESULTS

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ERK7 specifically enhances ER destruction. To test whether members of the MAPK(ERK1/2) family regulate ER turnover, we determined whether any of the family members could influence the steady-state levels of ER expression. ER and the indicated kinases were coexpressed in baby hamster kidney (BHK) cells, and the expression levels of ER and the kinases were determined by immunoblotting the lysates of the transfected cells. In these experiments BHK cells were chosen because they are easily transfected and support ER-mediated transcription when provided with ER cDNA (5). Ectopic expression of MAPK(ERK1/2) would not substantially increase cellular MAPK(ERK1/2) activity, because in order for MAPK(ERK1/2) to be active, it must be phosphorylated by MAPK kinase. Therefore, a constitutively active mutant of MAPK kinase (MEKc) was expressed to activate endogenous MAPK(ERK1/2). In this transient-transfection system, we did not observe any difference in the steady-state levels of ER between vehicle and estradiol treatments in the vector control (Fig. 1A and B). However, in the vector control, the amount of ER present in the more slowly migrating electrophoretic band increased in the presence of estradiol (Fig. 1A). It was previously determined that phosphorylation of Ser-118 in human ER results in a reduced electrophoretic mobility (13). Activation of endogenous MAPK(ERK1/2) by MEKc increased the amount of ER present in the more slowly migrating band, which can be most clearly observed by comparison with the vector control in the absence of estradiol. These data are consistent with observations that MAPK(ERK1/2) phosphorylates Ser-118 (4, 13). The anti-pTEY antibody (also referred to as the anti-active MAPK antibody) specifically recognizes the dually phosphorylated Thr and Tyr residues present in the activation loop of MAPK(ERK1/2) and ERK7 (1). Thus, as expected, the anti-pTEY immunoblot shows that the expression of HA-MEKc enhanced the activity of MAPK(ERK1/2) above that observed with the vector control. However, increasing MAPK(ERK1/2) activity did not alter ER protein levels compared to those found in the vector control. Remarkably, however, the ectopic expression of ERK7 decreased the steady-state levels of ER expression 2-fold in the absence and 3-fold in the presence of estradiol (Fig. 1B). The immunoblot with the anti-pTEY antibody demonstrates that similar levels of active ERK7 and endogenous MAPK(ERK1/2) were achieved. Expression of another member of the TEY kinase subgroup, ERK5, did not influence ER levels (Fig. 1A), further demonstrating that a decrease in the steady-state level of ER is a specific response to ERK7.

View larger version (50K): [in this window] [in a new window]   FIG. 1. Active ERK7 enhances ER destruction. (A) BHK cells were cotransfected with vectors encoding ER and either the indicated kinase or vector control (V). The transfected cells were serum starved and treated with or without 10 nM estradiol (E2) for 6 h before addition of boiling sample buffer. Equal amounts of total protein were electrophoresed and immunoblotted. (B) Relative amounts of ER were determined from the immunoblots by densitometry. The data are expressed as the percentage of ER divided by ER in the vector control in the absence of E2. The means ± standard errors are shown for n = 8. *, P < 0.05; **, P < 0.005 (Student's t test); values were obtained by comparing ER levels with coexpressed ERK7 to those obtained with the appropriate vector control. (C) BHK cells were cotransfected with vectors encoding ER and either HA-ERK7 or vector control (V). The transfected cells were then aliquoted and pretreated with 50 µM cycloheximide for 2 h before the addition of vehicle or 10 nM E2. Thereafter, at various time points over a 6-h period, the cells were lysed and immunoblotted and the relative amount of ER was quantitated by densitometry. The rate data were fitted by using a single exponential decay, and the rate constants were determined. The means ± standard errors are shown for two separate experiments. *, P < 0.05 (Student's t test); value was obtained by comparing the rate constant to that of the appropriate control.

View larger version (48K): [in this window] [in a new window]   FIG. 2. ERK7 specifically enhances the degradation of ER. (A) BHK cells were cotransfected with vectors encoding ER and either HA-ERK7 or vector control (V). The serum-starved, transfected cells were pretreated with or without 25 µM MG132 for 2 h before addition of vehicle or E2 and were then lysed after 6 h, and equal amounts of protein were electrophoresed and immunoblotted. (B) BHK cells were cotransfected with vectors encoding FLAG-IB and HA-ERK7 or HA-K43A-ERK7 or vector control (V). The serum-starved, transfected cells were treated with or without MG132 for 8 h and lysed, and equal amounts of protein were electrophoresed and immunoblotted. (C) BHK cells were cotransfected with vectors encoding HA-SF1 and HA-K43A-ERK7 or HA-ERK7 or vector control (V). The serum-starved, transfected cells were lysed, and equal amounts of protein were electrophoresed and immunoblotted. (D) BHK cells were cotransfected with vectors encoding ER or HA-AR and HA-ERK7 or MYC-ERK7 or vector control (V). The transfected cells were then aliquoted and pretreated with 50 µM cycloheximide for 2 h before the addition of 10 nM E2 or 100 nM dihydroxytestosterone (DHT). Thereafter, at various times after ligand addition the cells were lysed and immunoblotted.

ERK7 specifically enhances ER degradation. ER is degraded through the 26S proteasome pathway, and one possible explanation for the observed decrease in ER levels is that ERK7 enhances the rate of proteasome-mediated degradation. Therefore, we tested whether the competitive proteasome inhibitor MG132 could prevent ERK7 from decreasing ER levels. In agreement with the results of Nawaz et al., addition of MG132 increased ER protein levels in both the presence and absence of estradiol (Fig. 2A) (22). Treatment with MG132 also increased the amount of ER coexpressed with ERK7 and increased the abundance of higher-molecular-weight forms of ER induced by ERK7. In the presence of a proteasome inhibitor, the ubiquitinated product accumulates; thus, the higher-molecular-weight ER forms are likely due to ubiquitination. Similar results were observed with another proteasome inhibitor, lactacystin (data not shown). It is not possible to determine by immunoblotting whether these forms are a result of ubiquitination because antiubiquitin antibodies are extremely insensitive. Additionally, our anti-ER antibody does not immunoprecipitate the higher-molecular-weight ER forms. These results suggest that ERK7 decreases the abundance of ER by enhancing its degradation through the proteasome pathway.

To test the specificity of ERK7-mediated degradation, we determined whether ERK7 could enhance the destruction of IB, which is degraded by the 26S proteasome pathway (24). ERK7 did not reduce the protein levels of IB in comparison to the vector control (Fig. 2B). These results support the hypothesis that ERK7 is a specific regulator and is not a general activator of the ubiquitination and proteasome machinery.

To determine whether ERK7 can trigger the destruction of other nuclear receptor superfamily members, we used steroidogenic factor I (SF1), a distant, evolutionarily related relative of ER (19). ERK7 had no effect on the protein levels of SF1 compared to the vector control (Fig. 2C). These results suggest that ERK7 stimulates the degradation of only a subset of the nuclear receptor superfamily, which includes ER.

To further define the nuclear receptors that are targets of ERK7, we tested the ability of ERK7 to enhance the rate of degradation of AR, a close relative of ER. The rate of degradation was determined in the presence of cycloheximide in a similar manner to that performed with ER, except that dihydroxytestosterone was used. Remarkably, ERK7 did not enhance the degradation of the AR (Fig. 2D). Taken together, these data suggest that ERK7 specifically regulates ER turnover.

ERK7 is highly expressed in normal human breast cells. To understand the importance of ERK7 in regulating estrogen action, we determined whether ERK7 was expressed in breast cells, an estrogen target tissue. We generated a polyclonal antibody to the extreme C terminus of rat ERK7, which was able to specifically detect ERK7 but not a truncated mutant of ERK7 in lysates from transfected BHK cells (Fig. 3A, left). Note that deletion of the C-terminal tail of ERK7 also inhibits its constitutive kinase activity and that the truncation is therefore not detected by the anti-pTEY antibody. The anti-ERK7 antibody was used to immunoblot lysates of MCF-10A, a normal breast cell line, and the breast cancer cell lines MCF-7 and MDA-MB-231 (Fig. 3B). A band of 60 kDa was strongly detected by the anti-ERK7 antibody in MCF-10A cells, and this molecular mass is in agreement with the calculated molecular mass based on the rat ERK7 cDNA. The 60-kDa band recognized by anti-ERK7 colocalized with a band recognized by the anti-pTEY antibody (Fig. 3B). Together, these data strongly suggest that the 60 kDa-band observed in normal breast cells is human ERK7. It is not particularly surprising that the antibody raised to rat ERK7 recognizes human ERK7, because a number of kinases are virtually identical in their amino acid sequences between rats and humans, e.g., ERK2.

View larger version (50K): [in this window] [in a new window]   FIG. 3. ERK7 is expressed in normal human breast cells. (A) In the left panel BHK cells were transfected with vectors encoding an HA-tagged wild type or a deletion mutant of ERK7 or vector control (V). The serum-starved, transfected cells were lysed, and equivalent amounts of protein were electrophoresed and immunoblotted. In the right panel normal human breast tissue was electrophoresed and immunoblotted with anti-ERK7 in the presence (+) or absence (-) of the antigenic peptide (200 µM) used to produce anti-ERK7 (p). (B) Equivalent amounts of lysate from serum-starved MCF-10A, MCF-7, and MDA-MB-231 cells were electrophoresed and immunoblotted. (C) Normal human breast tissue, benign tumor tissue, and breast cancer tissue samples were solubilized, normalized for Ran expression, electrophoresed, and immunoblotted. The tumor grade was obtained from the pathologist's report. ER-positive samples are indicated by an asterisk.

To further understand the physiological significance of ERK7 in regulating estrogen action, we determined the expression levels of ERK7 in normal human breast tissue and breast cancer tissue (Fig. 3C). Detection of the 60-kDa band in normal human breast tissue was blocked by preincubating the anti-ERK7 antibody with the antigenic peptide (Fig. 3A, right). In total we examined 13 normal tissues, 5 benign tumors, and 66 breast tumors, and the results shown are representative. The samples are grouped by their tumor grade from the least to the most aggressive, according to the pathologist's report. The tissue samples were normalized to each other by immunoblotting for Ran, a housekeeping protein whose expression level is not known to change with any disease state. ERK7 was expressed in all of the normal and benign tissue samples, approximately half of the grade 1 samples, 20% of the grade 2 tumors, none of the grade 3 tumors, and 30% of the metastatic tumors (Fig. 3C). Thus, loss of ERK7 is correlated with breast cancer progression. These samples were also analyzed for active MAPK(ERK1/2), and in agreement with the literature (26, 27), we observed that MAPK(ERK1/2) activity was generally higher in breast cancers than in the normal or benign tumor samples. But, importantly, we did not observe a correlation between MAPK(ERK1/2) activity and loss of ERK7. These results demonstrate that the loss of ERK7 is not merely a reflection of a general decrease in the levels of various members of the MAPK(ERK1/2) family. Also in agreement with the literature (29), we observed that normal human breast mammary epithelial cells have very low levels of ER, which is consistent with our observations that normal breast tissue has significant amounts of ERK7. Additionally, cancer tissues that had detectable amounts of ER had undetectable levels of ERK7 expression. Thus, loss of ERK7 correlates with increased ER levels. These data further suggest that ERK7 is an important regulator of estrogen action in the breast.

ERK7 regulates hormone responsiveness in human breast cells. The concentration of ER is limiting for estrogen responsiveness in vivo, and ER synthesis and degradation, therefore, play a pivotal role in controlling the expression of ER-regulated genes (33). Taken together, our results argue that ERK7 may regulate hormone responsiveness in cells that endogenously express ER. To test this hypothesis, we attempted to produce MCF-7 clones that stably expressed HA-ERK7; however, we found that the majority of the ERK7 was not active (data not shown). These results suggest that ERK7 activity is regulated differently in breast cells from how it is regulated in BHK cells, in which 50% of ERK7 is active. Kinase-dead mutants are often able to act as dominant negatives; therefore, we produced MCF-7 clones that stably expressed K43A-ERK7. We hypothesized that MCF-7 cells contained extremely low levels of ERK7 and that we could inhibit its ability to degrade ER by using K43A-ERK7. In MCF-7 lysates we observed a faint band at 60 kDa upon extended exposure of the anti-ERK7 immunoblot (data not shown). The anti-ERK7 antibody is able to immunoprecipitate HA-ERK7 from lysates of transfected cells (Fig. 4A, left); therefore, we used this antibody to immunoprecipitate ERK7 from MCF-7 lysates. ERK7 was detected in the immunoprecipitate by use of another polyclonal antibody to ERK7 that recognizes residues in the activation loop of ERK7 (anti-AL ERK7) (Fig. 4A, right). The anti-AL ERK7 recognizes both the active and inactive forms of ERK7 (Fig. 4A, left). Thus, these results demonstrate that MCF-7 cells do contain ERK7.

View larger version (27K): [in this window] [in a new window]   FIG. 4. ERK7 regulates endogenous ER degradation in human mammary cells. (A) In the left panel BHK cells were transfected with vector encoding HA-ERK7 or the vector control (V). The serum-starved, transfected cells were lysed, and the HA-ERK7 was immunoprecipitated (IP) with either anti-HA or anti-ERK7 antibodies. The IPs were electrophoresed and immunoblotted. In the right panel serum-starved MCF-7 cells were lysed and either immunoglobulin G or anti-ERK7 antibody was added to the lysate. The immunoprecipitates were electrophoresed and immunoblotted. (B) Serum-starved MCF-7 stable lines expressing either vector alone (V) or HA-K43A-ERK7 were immediately lysed after the addition of 50 µM cycloheximide (CHX) or after a 4-h pretreatment with CHX with or without 10 nM E2. Equivalent amounts of protein were electrophoresed and immunoblotted. (C) Relative ER levels were determined by densitometry, and the data were expressed for each stable line as the percentage of ER after incubation with CHX divided by ER before incubation with CHX. The means ± standard errors for two separate experiments are shown. *, P < 0.05; **, P < 0.005 (Student's t test); values were obtained by comparing the response obtained with the lines expressing K43A-ERK7 to the appropriately treated control. (D) MCF-7 stables were cotransfected with an ERE-regulated luciferase reporter and ß-galactosidase expression vectors. The serum-starved, transfected cells were treated with or without E2 and MG132. The luciferase and ß-galactosidase activity was determined 24 h after the indicated treatments. The luciferase data were normalized to ß-galactosidase activity to control for differences in transfection efficiency. To facilitate comparison between different experiments, the data were normalized so that, in the vector control, the response to vehicle addition was 0 and the response to E2 was 100. The means ± standard errors are shown (n = 3), each donein quadruplicate. **, P < 0.005 (Student's t test); this value was obtained by comparing the E2 response obtained with the vector control in the presence of MG132 to that obtained in its absence. (E) Stable MCF-7 lines were treated with or without 1 µM ICI 182,780. At various times the cells were lysed and growth was determined. ER-dependent growth was obtained by subtracting the growth in ICI 182,780 from the growth obtained in vehicle control. The results are taken from two experiments in which each time point was determined in triplicate and from two independent lines. , K43A-ERK7; , V. *, P < 0.05 (Student's t test); value was obtained by comparing the response obtained with the stable lines expressing HA-K43A-ERK7 to that found with the appropriately treated control.

To further test whether ERK7 is an important regulator of estrogen action, we determined the effect of K43A-ERK7 on ER-regulated transcription. The MCF-7 stable lines were cotransfected with a vector encoding the luciferase reporter under the control of estrogen-responsive elements (ERE), plus a control vector encoding ß-galactosidase. Cells expressing K43A-ERK7 had an 3-fold-greater transcriptional response to estradiol than the control cells (Fig. 4D). MG132 also increased the transcriptional response of the control cells by 3-fold. However, the transcriptional response in the K43A-ERK7 cells was not affected by MG132. These results suggest that K43A-ERK7 and MG132 influence ER-mediated transcription through a similar mechanism, by inhibiting the ER degradation rate. In our experiments, a TATA box and a dual ERE regulate luciferase reporter expression. Therefore, we next tested whether ERK7 also regulates ER-regulated transcription of endogenous genes in MCF-7 cells. As a readout for broad responses to ER, we used the ability of estrogen to stimulate MCF-7 cell proliferation, because the proliferation response is known to be regulated by ER transcriptional activation (8, 28). It is likely that ERK7 regulates the activity of molecules in addition to ER and that these other molecules may influence proliferation. Therefore, to examine only the effects of K43A-ERK7 on ER-mediated proliferation, we determined growth in the presence of vehicle or ICI 182,780, a pure estrogen antagonist (10). To examine only the ER-dependent growth, we subtracted the growth in the presence of ICI 182,780 from that obtained with the vehicle. K43A-ERK7 enhanced the rate of ER-dependent proliferation 2-fold (Fig. 4E). These data suggest that ERK7 regulates ER-mediated transcription from both simple and complex promoters. Furthermore, the ability of K43A-ERK7 to promote proliferation suggests that ERK7 regulation of the ER degradation rate is important in determining estrogen responsiveness.

ERK7 phosphorylation of ER does not influence ER stability. Because turnover is commonly regulated by phosphorylation of the target protein, it seemed possible that ERK7 regulates ER degradation by direct phosphorylation (17). ERK7 is most likely a proline-directed serine/threonine kinase because the catalytic domain is highly related to MAPK(ERK1/2) (1); thus, there are four putative ERK7 phosphorylation sites in ER, Ser-104, Ser-106, Ser-118, and Ser-294. It was previously determined that Ser-118 is the major site of phosphorylation in response to estradiol binding (12); therefore, we determined whether ERK7 could phosphorylate ER in vivo. Lysates from cells transfected with wild-type ER or S118A-ER in the presence or absence of ERK7 were normalized for total ER and were immunoblotted with anti-ER. The lysates were normalized to ER rather than total protein so that we could directly compare the intensities of the various ER bands between the ERK7 and vector control samples. The appearance of Ser-118 phosphorylation can be observed as a sharp band, and ERK7 enhances the amount of this phosphorylation in both the absence and presence of estradiol (Fig. 5A). The kinase-dead mutant, K43A-ERK7, diminishes the intensity of this more slowly migrating ER band (Fig. 1A). Taken together, these data suggest that ERK7 can regulate either directly or indirectly the level of Ser-118 phosphorylation. ERK7 also increases the number of other higher-molecular-weight ER forms that are observed as diffuse bands both in wild-type ER and the S118A-ER mutant. The identity of these bands will be discussed later.

View larger version (30K): [in this window] [in a new window]   FIG. 5. Ser-118 phosphorylation does not influence ER turnover. (A) BHK cells were cotransfected with vectors encoding ER or S118A-ER and HA-ERK7 or vector control (V). The serum-starved, transfected cells were treated with or without 10 nM E2 for 6 h before lysis. Lysates containing equivalent amounts of ER were electrophoresed and immunoblotted. (B) BHK cells were cotransfected with vectors encoding S118-ER and either HA-ERK7 or vector control (V). The transfected cells were then aliquoted and pretreated with 50 µM cycloheximide for 2 h before the addition of 10 nM E2. At various time points after E2 addition, the cells were lysed and immunoblotted and the relative amount of S118A-ER was quantitated by densitometry. The rate constants were determined as described in the Fig. 1C legend, and the data shown are the means ± standard errors for two separate experiments.

The ligand-binding domain targets ER for ERK7-mediated destruction. ERK7 targets the wild-type ER for degradation but not a deletion mutant lacking the ligand-binding domain. Therefore, we determined whether ERK7 enhanced the destruction of the ligand-binding domain. In agreement with the literature (20), we observed that estradiol enhanced the level of ER(283-595) coexpressed with the vector control (Fig. 6A). Nonetheless, ERK7 was able to decrease the expression level of ER(283-595) in the presence and absence of estradiol. These results suggest that the ligand-binding domain plays an important role in the ability of ERK7 to regulate ER protein levels.

Inhibitors of the 26S proteasome pathway are able to decrease ERK7-mediated ER degradation (Fig. 2A), and ERK7 enhances the formation of higher-molecular-weight ER forms (Fig. 2A and 5A). Therefore, we suspected that these high-molecular-weight forms consist of ubiquitinated ER. However, as mentioned before, it is not possible to directly determine whether these higher-molecular-weight forms are the result of ubiquitination. Therefore, to test our hypothesis that ERK7 enhances ER ubiquitation, BHK cells were cotransfected with constructs encoding HA-ubiquitin plus either MYC-tagged ER(283-595) or vector control. Additionally, the cells were transfected with either MYC-tagged ERK7 (MYC-ERK7) or vector control and were treated with MG132. To isolate ubiquitinated proteins, lysates were immunoprecipitated with anti-HA antibody and were then immunoblotted with either anti-MYC or Ab10, an antibody to the ligand-binding domain of ER. The amount of ER(283-595) containing HA-ubiquitin was greatly increased in the presence of ERK7 compared to that found with the vector control (Fig. 6B). The first arrow indicates a band that is at a molecular weight consistent with the addition of a single ubiquitin to MYC-ER(283-595), whereas the other bands are consistent with polyubiquitination. The results with Ab10 were similar to those shown with the -MYC antibody (data not shown). These results support the hypothesis that ERK7 enhances the level of ER ubiquitination.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

 
Human ERK7 was identified based on the ability of two different antibodies to recognize a protein of a molecular weight similar to that predicted for rat ERK7. Support for the physiological importance of ERK7 is provided by our observations that ERK7 is expressed in human breast tissue, an estrogen target, and that there is a decrease in ERK7 expression during breast cancer progression. Furthermore, in all tumor samples that are ER positive, the expression of ERK7 has been lost. This inverse correlation between ER and ERK7 expression levels was also observed in human breast cell lines. Further evidence for the physiological importance of ERK7 is provided by our observations that a dominant-negative ERK7 mutant is able to regulate hormone responsiveness in breast cells as measured by transcription and proliferation. A dominant-negative ERK7 mutant was able to enhance ER-mediated transcription to the same extent as treatment with the proteasome inhibitor, MG132, which indicates that endogenous ERK7 is the predominant pathway of ER degradation in breast cells.

Our results support a model in which ERK7 enhances hormone-dependent ER destruction through the 26S proteasome pathway by increasing ER ubiquitination. This model is supported by observations that a dominant-negative ERK7 mutant preferentially inhibits degradation of the hormone-bound ER compared to that of unbound ER. ERK7 enhances the level of ubiquitination of the ligand-binding domain, and this domain is required for ERK7-mediated destruction. In response to hormone binding, it is known that the ligand-binding domain undergoes a conformational change (23) and presumably this conformational change targets hormone-bound ER for destruction by ERK7.

It has been previously shown that, in response to hormone, the phosphorylation of Ser-118 increases (12). ERK7 enhances Ser-118 phosphorylation in vivo, but this phosphorylation is not important for ERK7-mediated degradation, because ERK7 is able to enhance the rate of degradation of ER and Ser-118-ER to the same extent. We were also unable to find any evidence that mutation of other putative ERK7 phosphorylation sites influenced ERK7-mediated ER degradation. It seems unlikely that phosphorylation of the ligand-binding domain plays a role in regulating turnover, because enhanced phosphorylation in the ligand-binding domain during use of [³²P]orthophosphate labeling and microsequencing of the radiolabeled ER peptides in the absence or presence of lactacystin has not been observed (12, 13; data not shown).

There are several cases known in which the destruction of specific targets is regulated by kinases. The kinases phosphorylate a residue within a specific sequence context, and this phosphorylation results in the interaction with particular E2 and E3 enzymes. For example, glycogen synthase kinase 3ß phosphorylates ß-catenin and IB kinase phosphorylates IB, which regulates their destruction through the Skp1-Cullin-F-box complex (11, 16, 24). There is also evidence that the destruction of some members of the nuclear steroid receptor superfamily is regulated by their phosphorylation (18, 34). However, our discovery of ERK7-mediated destruction of ER is mechanistically distinct. One possible mechanism is that ERK7 phosphorylates a component of the ubiquitin machinery, which increases its catalytic activity or affinity for ER. Another plausible mechanism is that ERK7 phosphorylates an ER-interacting protein, which exposes a surface on ER that targets it for destruction. Although we have not elucidated all the components of the signaling pathway leading from ERK7 to enhanced ER degradation, we have provided the first biologically relevant function for ERK7.

Remarkably, ERK7 expression is lost during breast cancer progression. Normal breast epithelial cells have extremely low levels of ER and are considered to be ER negative (29). Hormone-responsive breast cancers express 10-fold-higher or greater ER levels, but as the cancer becomes more aggressive, ER expression is frequently lost. In our studies all the ER-positive tumors also had lost ERK7 expression, and it may be that the increase in ER levels in hormone-responsive breast cancers is due to decreased ERK7 expression. However, not all tumors that had lost ERK7 expression were ER positive, suggesting that other factors in addition to ERK7 are involved in regulating ER levels. It may be that during tumor progression ERK7 downstream effectors have been inappropriately activated, resulting in reduced ER levels even in the absence of ERK7. We suggest that ERK7 is important in maintaining the homeostasis of a normal breast cell and that with high frequency ERK7 is lost at an early step in breast cancer progression.

	ACKNOWLEDGMENTS

 
We thank Ian Macara and Sarah Parsons for helpful discussions.

This work was supported by the Biotechnology Training Program no. T32 GM08715 (L.M.H.) and the University of Virginia Cancer Center Support Grant (D.A.L.).

	FOOTNOTES

 
* Corresponding author. Mailing address: Center for Cell Signaling, Box 800577, Health Sciences Center, University of Virginia, Charlottesville, VA 22908-0577. Phone: (434) 924-1144. Fax: (434) 924-1236. E-mail: dal5f{at}virginia.edu.

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