Differential Regulation of Mitogen-Activated Protein/ERK Kinase (MEK)1 and MEK2 and Activation by a Ras-Independent Mechanism

Shuichan Xu, Shih Khoo1, Alphonsus Dang, Sarah Witt, Vuong Do, Erzhen Zhen, Erik M. Schaefer and Melanie H. Cobb

University of Texas Southwestern Medical Center (S.X., S.K., A.D., S.W., V.D., E.Z., M.H.C.), Department of Pharmacology, Dallas, Texas 75235-9041,
Promega Corporation (E.M.S.), Madison, Wisconsin 53711


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Mitogen-activated protein (MAP)/ERK kinase (MEK) 1 and MEK2 are the upstream activators of the MAP kinases, ERK1 and ERK2. MEK1 and MEK2 are ~85% identical in sequence but have unique inserts in their C-terminal domains. MEK isoform-specific antibodies were used to examine expression and regulation of each enzyme. MEK1 and MEK2 were expressed in approximately equal amounts in several cell lines; in some, MEK1 was present in slight excess. Activation of tyrosine kinase-containing receptors, heterotrimeric G proteins, and protein kinase C enhanced the activities of both MEK isoforms in 293 and PC12 cells. AlF4- stimulated both MEK1 and MEK2 in PC12 cells expressing a dominant interfering Ras mutant that prevents nerve growth factor-dependent activation of the cascade. Carbachol also stimulated the pathway in these cells. Thus, in addition to their ability to activate Ras/Raf and the downstream ERK pathway, heterotrimeric G proteins also appear to trigger a Ras-independent mechanism to regulate this kinase cascade. In U373, Chinese hamster ovary (CHO), and INS-1 cells, MEK1 was activated by regulators of ERKs, while MEK2 was not. These data suggest that, like the MAP kinases ERK1 and ERK2, in some cell settings the two similar MEK isoforms are differentially regulated.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The mammalian mitogen-activated protein (MAP) kinase or ERK pathway (1, 2, 3, 4) has been implicated in many hormone-dependent regulatory events including cell differentiation and proliferation. In addition to mediating acute events in the cytoplasm and at the membrane, the MAP kinases ERK1 and ERK2 are thought to alter gene expression, with the best documented effects on transcription from the serum response element (5). Tyrosine kinase receptors are believed to activate the ERK pathway via Ras. Ras·GTP interacts with isoforms of the protein kinase Raf (6, 7, 8, 9), causing its association with the membrane where it is activated (6, 7, 8, 10). Raf-1 activates the MAP kinase kinases (11, 12, 13, 14, 15, 16, 17) also known as MAP kinase/ERK kinases MEK1 and MEK2, which in turn phosphorylate and activate ERK1 and ERK2. MEK1 and MEK2 are also activated by other MEK kinases, including the product of the c-mos protooncogene (18, 19) and MEK kinase 1 (MEKK1) (20, 21), further illustrating the complexity of this signaling cascade.

MEK1 and MEK2 are 85% identical overall and greater than 90% identical in their catalytic cores. The least similar regions of the molecules lie near their N termini and in an approximately 40-amino acid, proline-rich insert between subdomains IX and X. Identity between MEK1 and MEK2 in this insert region is only approximatley 40%. The insert is not present in any of the other known MEK family members, mammalian or yeast, suggesting that it serves functions unique for the ERK pathway. Although MEK1 and MEK2 are both activated by serum, Weber and co-workers (22) observed that MEK1, but not MEK2, forms complexes with Raf-1 (22). Deletion of the insert impaired its ability to form complexes with Raf (23). Thus, distinct mechanisms, perhaps arising from the unique catalytic domain inserts, may differentially regulate these two MEK isoforms.

In this study we examined the regulation of MEK1 and MEK2 in several cultured cell lines. Earlier studies suggested that these protein kinases co-chromatograph on ion exchange resins (17), necessitating a different approach to examine the regulation of each. Thus, isoform-specific antibodies were used to immunoprecipitate each kinase individually to measure regulation by activators of the MAP kinase cascade.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Detection of MEK1 and MEK2 by Immunoblotting
MEK isoform-specific antisera were prepared to peptides from the unique ~40-amino acid inserts near the C termini of the kinase domains of MEK1 and MEK2. We showed previously that each antiserum immunoblotted only a single MEK isoenzyme without cross-reactivity, and both antibodies are equally sensitive (21). Using these antisera, we found that MEK1 and MEK2 were expressed in PC12 and Chinese hamster ovary (CHO) cells, but MEK1 was in slight excess (Fig. 1AGo). The same was true of 3T3 cells (not shown). In 293, INS-1, and U373 cells, MEK1 and MEK2 were both expressed approximately equally. Compared with the other cell types, INS-1 cells expressed much less of either MEK (Fig. 1Go, A and B). Both MEK isoforms were also found in rabbit muscle. As reported earlier for transfected MEK1 and MEK2, the endogenous rabbit muscle proteins cochromatographed on MonoQ (Fig. 1Go, C and D). The peak of ERK2 phosphorylating activity, in fractions 4–8, contained all of the MEK1 and MEK2 immunoreactivity.



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Figure 1. Detection of MEK1 and MEK2 in Different Cells

A, Lysates of different cell lines were subjected to SDS-PAGE. MEK1 and MEK2 were visualized on duplicate blots with the MEK1-specific antibody A2227 or the MEK2-specific antibody A2228 in the absence or presence of peptide antigen. B, 293 cell lysates were blotted with A2227 or A2228 in the presence or absence of peptide antigen. C, MEK1 and 2 in rabbit skeletal muscle cochromatographed on Mono Q. {diamond}, MBP phosphorylation; {square}, ERK2 activating activity. D, Immunoblots of the Mono Q fractions with A2227 and A2228.

 
Activities of Both MEK1 and MEK2 Are Increased by Ligands to Multiple Receptor Types
The regulation of endogenous MEK1 and MEK2 by stimuli that work through different mechanisms was examined. Activities of MEK1 and MEK2 were measured in immune complexes using isoform-specific antibodies to immunoprecipitate MEK1 and MEK2 separately from lysates of untreated, stimulated, and transfected 293 cells. An ERK2 mutant, K52R ERK2, which has very low intrinsic protein kinase activity, was used as substrate. Epidermal growth factor (EGF), carbachol, AlF4-, and phorbol ester enhanced the activities of MEK1 (21) and MEK2 in serum-deprived 293 cells (Fig. 2Go). All of these stimuli increased activity nearly equally, with EGF being the most effective and phorbol ester the least.



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Figure 2. MEK2 is Activated by MEKK1 and Ligands for Multiple Receptor Types in 293 Cells

293 cells transfected with HA-MEKK1 or empty vector were stimulated with EGF, carbachol, phorbol 12-myristate 13-acetate, and A1F4- as described. Endogenous MEK2 was immunoprecipitated with A2228. MEK2 activity was assayed by using recombinant K52R ERK2 as substrate. Fold activation, compared with MEK2 activity in untransfected cells without stimulation, is shown under each lane.

 
MEKK1 was compared as a stimulus of the endogenous MEK activities. In MEKK1-transfected cells, both MEK1 (21) and MEK2 were activated even in serum-deprived cells. MEKK1 overexpression had no significant effect on the stimulated activities of MEK1 and MEK2; HA-MEKK1 is not activated by these stimuli (not shown). Expression of MEKK1 in 293 cells also increased the phosphorylation and activities of cotransfected, epitope-tagged MEK1 and MEK2 (21). These data indicated that stimuli activating protein kinase C, tyrosine kinase receptors, and heterotrimeric G proteins activated both MEK1 and MEK2 in 293 cells.

AlF4- and Carbachol Enhance MEK Activity in PC12 17N-1 Cells
We measured activation of the enzymes in PC12 cells to examine the requirement for H-Ras in the regulation of the ERK cascade by heterotrimeric G proteins. In PC12 cells MEK1 and MEK2 were both activated by nerve growth factor (NGF) (Fig. 3Go) and AlF4- (24). In NGF-stimulated cells, activity of MEK1 was greater than that of MEK2. Kinase activity was increased by AlF4- and to a lesser extent by carbachol (see Fig. 6Go) even in the 17N-1 clone of PC12 cells that overexpresses a dominant-interfering Ras mutant, S17N H-Ras. In earlier studies we demonstrated that NGF was unable to activate ERK1, ERK2, or a partially purified MEK activity in the 17N-1 PC12 line, although these activities were stimulated by AlF4- (24). These findings were confirmed below.



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Figure 3. Activation of MEK1 and MEK2 in PC12 and PC12 17N-1

Endogenous MEK1 and MEK2 were immunoprecipitated from NGF-stimulated PC12 cells (upper panel) or A1F4--treated PC12 17N-1 cells (lower panel). MEK activities were assayed as indicated.

 


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Figure 6. Detection of ERK Activation by an Antibody That Recognizes the Active Forms

A, Cell lysates (15 µg for 3T3 and 25 µg for all other) were analyzed by Western blotting. Duplicate blots were probed with Y691, an ERK antibody (upper panel) and 20291, an antibody that selectively recognizes active ERK (lower panel). B, Immunoblots of ERKs (upper) or active ERKs (lower) in 20 µg of lysate protein from PC12 or PC12 17N-1 cells treated as indicated.

 
MEK1 but Not MEK2 Is Activated by Secretagogs in INS-1 Cells
In INS-1 cells the effect of glucose plus forskolin, which promotes insulin secretion, was examined. As demonstrated previously (25, 26), ERK1 activity, detected in immune complexes with the ERK1-specific antibody X837 (27), was strongly increased by glucose plus forskolin (Fig. 4Go). Although the signaling pathways that link glucose to activation of ERKs are undefined, glucose metabolism and calcium uptake are required to control not only secretion but also activation of the protein kinase cascade (26). Both MEK1 and MEK2 are present in INS-1 cells (Fig. 1AGo); however, glucose plus forskolin stimulate the activity of MEK1 but not that of MEK2 (Fig. 4Go).



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Figure 4. MEK1, not MEK2, Is Activated by Glucose and Forskolin in INS-1 Cells

INS-1 cell lysates were prepared as described in Materials and Methods. Endogenous ERK, MEK1, and MEK2 were immunoprecipitated by antisera X837, A2227, and A2228, respectively. The activities of ERK and MEK were assayed as described.

 
Selective Activation of MEK1 in U373 and CHO Cells
Regulation of MEK1 and MEK2 in several other cell types was examined to determine whether the selective activation of MEK1 was unique to INS-1 cells. In CHO, 3T3, and U373 cells, FBS-enhanced MEK1 activity was found, but none was attributable to MEK2 (Fig. 5Go). 3T3 (not shown) cells express more MEK1 than MEK2, perhaps accounting for the difficulty in detecting activation of MEK2; however, CHO, U373, and INS-1 cells contain approximately equal amounts of MEK2 as MEK1. Further, U373 cells express as much immunoreactive MEK2 per mg lysate protein as do 293 cells. Thus, in these cell types, unlike in 293 or PC12 cells, activation of MEK1 can be detected without a comparable activation of MEK2.



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Figure 5. Activation of MEK1 and MEK2 in 3T3, CHO, and U373 Cells

3T3, CHO, and U373 cells were stimulated with FBS for 5 min. Endogenous MEK1 and MEK2 were immunoprecipitated and their activities assayed as described.

 
Activation of MEK1 and MEK2 or MEK1 Alone Results in Activation of Both ERK1 and ERK2
To compare activation of MEKs to their downstream targets, activation of ERK1 and ERK2 by a variety of stimuli was also examined. ERK1 and ERK2 must be phosphorylated on two sites, a tyrosine and a threonine, to be in the high activity state. Dephosphorylation of either residue inactivates the enzymes. ERK activation was examined in lysates from 3T3, CHO, PC12, and U373 cells by immunoblotting with an antibody raised to a doubly phosphorylated peptide that corresponds to the active state of ERK1 and ERK2. The antibody selectively detects the activated forms of these two MAP kinases; once either of the two activating phosphorylation sites is dephosphorylated, their reactivity with the anti-active ERK antibody is greatly decreased (28). Equal amounts of protein were also immunoblotted with antibody 691 that recognizes ERK1 and ERK2 equally to confirm that equivalent amounts of ERK1 and ERK2 were analyzed in lysates from stimulated and unstimulated cells (Fig. 6Go).

Both AlF4- and carbachol increased the amounts of immunoreactive active ERK1 and ERK2 in wild type PC12 cells and the 17N-1 line that overexpressed S17N Ras (Fig. 6Go, A and B). The effect of carbachol was smaller than that of AlF4-, but the effect was clearly detectable with the anti-active ERK antibody. Previously, protein kinase assays showed a 5- to 10-fold effect of AlF4- on total ERK activity in 17N-1 cells, measured using myelin basic protein as substrate (24). Activation of ERK1 and ERK2 by both an activator of all heterotrimeric G proteins and a muscarinic receptor agonist suggests that there is a Ras-independent mechanism through which heterotrimeric G proteins activate ERKs in PC12 cells.

In 3T3 cells, both ERK1 and ERK2 were activated by insulin, serum, and EGF, as well as carbachol. Again the effects of carbachol were the weakest. Serum also potently activated both ERK1 and ERK2 in CHO cells. U373 cells express more ERK2 than ERK1, but both isoforms were activated by serum under conditions that cause activation of a single MEK isoform. Both ERKs were also activated by glucose in INS-1 cells (26).


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The ERK cascade is activated by tyrosine kinases, certain heterotrimeric and small G proteins (24, 29), stimulation of protein kinase C (24, 30, 31, 32), and perhaps by other mechanisms. Both Ras-dependent and Ras-independent inputs (24, 29, 33, 34, 35) from tyrosine kinases, heterotrimeric G proteins, and protein kinase C have been reported. We compared the regulation of MEK1 and MEK2 by all three types of upstream signals and in various cell types. To measure the activities of these two MEKs separately, we developed isoform-specific antibodies for use in immune complex kinase assays. With these antibodies we found that both MEK1 and MEK2 were activated by all three types of stimuli.

AlF4-, an activator of all heterotrimeric G proteins, increased the activity of both MEK1 and MEK2 in wild type PC12 cells and the 17N-1 line that overexpresses a dominant negative mutant of Ras. S17N Ras blocks activation of the ERK cascade by NGF, which acts through a tyrosine kinase receptor (24, 36, 37). Furthermore, carbachol, a muscarinic agonist, also activated the cascade in PC12 cells overexpressing S17N Ras. These data support the conclusion that heterotrimeric G proteins are able to work through a Ras-independent input to this pathway.

An alternative explanation for our first results with AlF4--treated PC12 cells could be suggested by the finding that cAMP synergizes with NGF to activate the ERK pathway in PC12 cells (38). AlF4- will generate active species of not only G proteins normally coupled to this pathway (e.g. Gi, Gq) but also Gs, which will lead to accumulation of cAMP. Any residual Ras activity (downstream of Gi or Gq) may be sufficient in the presence of elevated cAMP to elicit increased kinase activity from MEKs and ERKs. The muscarinic agonist carbachol, however, does not increase Gs activity and is, therefore, unlikely to work by the same mechanism. It could potentially activate ERKs through Gi- and Gq-dependent pathways. Effects on the ERK pathway of phorbol ester, a protein kinase C activator, and bradykinin, which activates Gq, are blocked in the 17N-1 cell line, suggesting that the Ras-independent effect of carbachol is not mediated through Gq or by increasing protein kinase C activity. Thus, the Gi subfamily may be the most likely to be able to couple to the ERK cascade without activating Ras.

In 293 and PC12 cells, as well as in rabbit muscle, activation of both MEK isoforms was detected, supporting the conclusion that in many cell types MEK1 and MEK2 are coordinately regulated. However, in U373 and CHO cells, only MEK1 was activated in response to serum, a strong activator of both MEKs in many other cell types, in spite of ample expression of both MEK1 and MEK2. Further, in INS-1 cells glucose, which induces insulin secretion and causes the activation of ERK1 and ERK2 (25, 26), leads to activation of only MEK1, not MEK2. Thus, selective activation of one of the two MEK isoforms may occur even in cell types that express significant quantities of both. This conclusion is consistent with two other studies in the literature that examined regulation of endogenous MEK1 and 2. The only study to examine their activation in multiple cell types identified no differences in their regulation under the conditions examined (39). However, in mouse macrophages, which express both MEK isoforms, only MEK1 is responsive to tumor necrosis factor-{alpha} (40).

Activation of a single MEK is not coupled to activation of a single ERK isoform. Activation of both ERK1 and ERK2 was detected with antibodies that selectively recognize their active forms in INS-1, CHO, and U373 cells. In all cell types examined here, both ERK1 and 2 were activated by the agonists tested.

In certain cell settings, such as serum-stimulated U373 or CHO cells or glucose-treated INS-1 cells, coupling or specificity factors may distinguish between MEK1 and MEK2, resulting in ligand-selective activation of a single MEK isoform. Alternately, the specificity may be entirely attributable to upstream regulators. In this regard, it has recently been reported by Guan and co-workers that A-Raf phosphorylates and activates MEK1 but not MEK2 (41). However, in INS-1 cells Raf-1, which activates both MEKs, is present, suggesting that some other mechanism must account for the selective activation of MEK1. Future studies are aimed at determining the mechanisms underlying the selective activation of MEK1 reported here.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Plasmids and Proteins
A mammalian expression vector encoding hemagglutinin (HA) epitope-tagged MEKK1 (the C-terminal 682 amino acids) was as described (21). Recombinant His6-K52R ERK2 was expressed and purified as described (42).

Cell Culture, Transfection, and Preparation of Extracts
293 (human embryonic kidney), CHO (Chinese hamster ovary), 3T3 (mouse fibroblast), U373 (human astrocyte), and PC12 (rat pheochromocytoma) cells and the 17N-1 clone PC12 cells (24, 43) were grown in DMEM containing 10% FBS and 1% L-glutamine. The rat insulinoma cell line INS-1 was grown in RPMI 1640 containing 11 mM glucose, 10% FBS, 50 µM ß-mercaptoethanol, and 1 mM sodium pyruvate as described (44). In some experiments 293 cells at no less than 80% confluence were transfected with DNA by calcium phosphate coprecipitation (45) and maintained for 24–48 h after transfection. Before stimulation, 293, CHO, U373, and 3T3 cells were deprived of FBS for 16–18 h and then treated with EGF (50 ng/ml), carbachol (1 mM), AlF4- (30 mM NaF plus 10 µM AlCl3), phorbol ester TPA (100 nM), or FBS (10%), or left untreated. PC12 cells were exposed to fresh FBS-containing medium ~18 h before treatment with NGF (50 ng/ml) or FBS, carbachol (1 mM), or AlF4- (30 mM NaF plus 10 µM AlCl3). INS-1 cells were preincubated in the absence of glucose in Krebs-Ringer-bicarbonate-HEPES (KRBH) buffer for 1–2 h at 37 C and then exposed to 15 mM glucose plus 10 µM forskolin in KRBH without serum at 37 C for 30 min. Cells were lysed as described (25). After washing with cold PBS, 293 cells were lysed on ice for 10 min in 0.5 ml lysis buffer per 60-mm dish (20). Lysates were collected and sedimented at 14,000 x g for 10–15 min at 4 C. Other cells were homogenized in ERK lysis buffer, and supernatants were prepared from them as described (46). Supernatants were transferred to new tubes and stored at -80 C or assayed immediately.

Immunoprecipitation and Immunoblotting
The monoclonal antibody to the HA epitope was purchased from Berkley Antibody Co. (Berkley, CA). Anti-ERK1 antiserum 691, which recognizes both ERK1 and ERK2, was raised as described (27). Antibodies to MEK1 (A2227) and MEK2 (A2228) were produced by immunizing rabbits with peptides from rat MEK1 (MEK1–1, CQVEGDAAETPPR) and MEK2 (MEK2–1, AIFGRPVVDGEEGEPHSIS) (21). These peptides were derived from the unique inserts in MEK1 and MEK2 not contained in other known MEK family members. Specificity of immunoprecipitation with antibodies to MEK1 or MEK2 was confirmed using autophosphorylated recombinant His6-MEK1 and GST-MEK2 and was comparable to the specificity of immunoblotting (21). It was shown previously that these antibodies are approximately equally sensitive; under similar blotting conditions, amounts of MEK1 or MEK2 detected are comparable. Antibodies that selectively recognize the activated forms of ERK1 and ERK2 (20291) were from Promega, and their selective recognition of the high activity forms of ERK1 and ERK2 was confirmed as described elsewhere (28). To immunoblot endogenous MEK1 or MEK2, soluble lysates (80 µg protein) were loaded on gels, transferred to nitrocellulose membranes, and blotted with A2227 or A2228 in the absence or presence of the corresponding peptide (MEK1–1 or MEK2–1) at 50 µg/ml. For immune complex kinase assays, supernatants were incubated with the indicated antibodies and protein A-Sepharose as described (21) with rotation for 2 h at 4 C. The beads were washed with cold 0.25 M Tris-HCl (pH 7.6) plus 0.1 M NaCl and resuspended for kinase assay. Immunoblots probed with the above antisera were developed with the Amersham enhanced chemiluminescence (ECL) kit (Arlington Heights, IL).

Kinase Assays
Activity of MEK1 or MEK2 was measured in 50 µl of 10 mM HEPES (pH 8.0), 10 mM MgCl2, 1 mM benzamidine, 1 mM dithiothreitol, 100 µM ATP (1–15 cpm/pmol) containing 50 µg/ml recombinant K52R ERK2 at 30 C for 60 min using immunoprecipitated kinase associated with 10 µl protein-A-Sepharose beads. The reactions were stopped by sedimenting the beads and adding an equal volume of 2x SDS-loading buffer to the supernatant.


    ACKNOWLEDGMENTS
 
We would like to thank Clark Garcia for preparation of bacterial proteins, Jessie English and Lori Christerson for critical reading of the manuscript, and Kim McKinney for its preparation.


    FOOTNOTES
 
Address requests for reprints to: Melanie H. Cobb, University of Texas Southwestern Medical Center, Department of Pharmacology, 5323 Harry Hines Boulevard, Dallas, Texas 75235-9041.

This work was supported by NIH Research Grant DK-34128 and a fellowship from the Juvenile Diabetes Foundation (to S.X.).

1 In partial fulfillment of requirements for the Ph.D. Back

Received for publication December 23, 1996. Revision received July 24, 1997. Accepted for publication July 25, 1997.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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