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Previous Article | Next Article 
Molecular and Cellular Biology, November 2002, p. 7593-7602, Vol. 22, No. 21
0270-7306/02/$04.00+0 DOI: 10.1128/MCB.22.21.7593-7602.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.
Huifen Chen,2 Eric B. Fauman,2 and David T. Dudley1*
Cell Biology,1 Structural Bioinformatics, Discovery Technologies, Pfizer Global Research and Development, Ann Arbor Laboratories, Ann Arbor, Michigan 481052
Received 27 June 2002/ Accepted 30 July 2002
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ABSTRACT |
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INTRODUCTION |
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MEK itself, is subject to regulation and activation by Raf phosphorylation on two serine residues, Ser218 and Ser222 (2, 33), which lie in a regulatory loop between conserved kinase subdomains VII and VIII (16). Substitution of these serine residues with negatively charged amino acids, such as aspartate or glutamate, partially mimics the phosphorylation modification and results in a constitutively active kinase, presumably through stabilization of the regulatory loop, allowing the enzyme to retain an active conformation (2).
Another regulatory feature of MEK is a proline-rich region, located carboxy terminal to the regulatory loop, which appears to be critical for Raf association (6), and may play an important role in the ability of MEK to efficiently activate MAP kinase (8). Additionally, this polyproline region contains phosphorylation sites that are phosphorylated in vitro by a number of other kinases, including MAP kinase, which may further regulate MEK activity (14).
The region of MEK amino-proximal to the catalytic core (approximately amino acids 1 to 67) has also been shown to play an important role in regulating kinase activity. The putative MAP kinase docking site is found within the first 32 amino acids of MEK, and these residues alone are sufficient for in vitro binding of MAP kinase (15). In particular, sequences of two to four positively charged residues in the extreme amino terminus of MEK (Lys3, -4, and -5 in MEK1) are conserved in all MEK family members (MKK1 to MKK7) and are considered essential for interaction with MAP kinases (3, 31). Additionally, the amino termini of MEK1 and -2 contain a recognition sequence for anthrax lethal factor, a proteolytic component of the Bacillus anthracis virulence factor. Lethal factor cleaves the first seven amino acids from MEK1, which impairs MEK enzymatic activity both in vitro and in vivo (12). Finally, amino acids 44 to 51 of MEK have been characterized as an autoactivation domain, since deletion of these residues results in an 80-fold increase in MEK activity relative to wild-type enzyme (20). When this deletion is coupled with the activating serine substitutions (Ser218Asp/Ser222Asp), the increase in activity is 640-fold over that of wild-type enzyme.
A key role for MEK in the development of tumors has been described. We have reported that a small molecule inhibitor of MEK, PD 184352, is capable of inhibiting up to 80% of tumor growth of human and murine colon carcinomas in mice (26). The compound is selective for MEK1 and -2 and is noncompetitive for ATP and MAP kinase (26). PD 184352 can block the activation of MEK by Raf, as well as inhibiting the active (Raf phosphorylated or mutationally activated) form of the kinase. However, in the present study, we show PD 184352 does not block Raf phosphorylation of MEK either in vitro or in vivo. In order to gain insight into the molecular mechanism of PD 184352-mediated inhibition of MEK kinase activity, we devised a genetic approach by using the budding yeast Saccharomyces cerevisiae to identify amino acids involved in the interaction of PD 184352 and MEK. Here we report the identity of a structural motif that interacts with the MEK specific inhibitor and describe a novel autoactivation domain within the kinase catalytic core that regulates overall MEK activity.
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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NT. Human MEK1 cDNA (GenBank no. L11284) was modified via PCR by the addition of a 5' BamHI site (underlined), a yeast Kozak sequence (italicized), and codon changes for yeast tRNA bias (boldface) with the following oligonucleotide: 5'-GGATCCACACATAAATAAACAAAATGCCAAAGAAGAAGCCAACGCCAA-3'. In addition, a sequence corresponding to the FLAG epitope (italicized), followed by an SstI site (underlined) was added to the 3' terminus of MEK with the following oligonucleotide: 5'-AAGCTTTTACTTGTCATCGTCGTCCTTGTAGTCGACGCCAGCAGCATGG. The BamHI/SstI PCR product encoding the modified MEK cDNA was inserted into pRS425 3' of the GAL1/10 promoter, generating pRS425GALMEK. For expression of recombinant protein in bacteria, modified MEK cDNA was subcloned into the NdeI and HindIII sites of pET21b plasmid (Novagen/CN Biosciences). Site-specific mutagenesis (QuikChange Mutagenesis kit; Stratagene, Inc.) was used to generate various point mutations in the MEK coding sequence in pET21b. For mammalian cell transfection assays, MEK cDNA was subcloned into the BamHI/HindIII sites of pCMV4A (Stratagene) by standard techniques, with a 5' mammalian Kozak sequence (-3ACC) added by PCR.
Generation of ste11 ste7 ste5 mutant yeast strain.
S. cerevisiae haploid strain SY2002 (MATaHIS3::FUS1::HIS3 mfa2-1::FUS1-lacZ ade1 leu2-3,112 trp1-DH1 ura3-52), a generous gift from G. F. Sprague, Jr., was modified by standard yeast genetic protocols (19) to disrupt the mating response pathway genes STE11, STE7, and STE5, generating a ste11 ste7 ste5 mutant strain. Unless otherwise noted, we used the genetic procedures and media described by Sherman et al. (28). Disruption of the loci was confirmed by PCR, growth on appropriate nutrient deficient media, and histidine auxotrophy conferred by disruption of the mating response pathway. Complementation of the disrupted pheromone response pathway was confirmed by transforming the ste11 ste7 ste5 strain with the Raf and MEK constructs and restoring the ability of the yeast to propagate on medium lacking histidine.
Random mutagenesis of MEK.
Escherichia coli XL-1 Red (Stratagene, Inc.) were used to generate random mutations in MEK cDNA due to deficiency in three primary DNA repair pathways (muts, mutD, and mutT) that results in a mutation rate 
5,000-fold higher than the wild-type strain. Plasmid pRS425GALMEK1 was transformed into E. coli XL-1 Red by standard methods and propagated on Luria-Bertani agar medium containing 100 µg of ampicillin/ml at 37°C for 24 h, allowing mutations to accumulate in the plasmid DNA. Colonies were scraped from the agar plates and transferred to 2 liters of Luria-Bertani liquid culture containing 100 µg of ampicillin/ml and allowed to expand for 18 h at 37°C. Cells were harvested by centrifugation, and plasmid DNA prepared by using standard reagents (Qiagen, Inc.).
MEK mutant library transformation and identification of PD 184352-resistant variants.
The ste11 ste7 ste5 FUS1::HIS3 strain harboring pRS314GALRafNT was transformed with 300 µg of randomly mutagenized MEK DNA by using the lithium acetate method of Ito et al. (17) and plated onto synthetic medium lacking tryptophan and leucine (SD-Trp,Leu). Yeast cells containing functional MEK were selected by growth in yeast nitrogen base (YNB) lacking Trp, Leu, and His (YNB-Trp,Leu,His), supplemented with 2% galactose to induce expression of both RafNT and MEK. PD 184352-resistant clones were selected by propagating histidine prototrophs in YNB-Trp,Leu,His supplemented with 2% galactose and containing 1 µM PD 184352. From this population, 275 separate yeast colonies were obtained. These were restreaked on YNB-2% galactose-Trp,Leu,His plus 1 µM PD 184352 to confirm resistance. Mutant-MEK plasmid DNA from these colonies was isolated (Bio 101) and rescued by transformation into E. coli KC8 (Clontech Laboratories, Inc.) grown on minimal medium lacking leucine and supplemented with 100 µg of ampicillin/ml. Mutations in the MEK coding sequence were identified by sequence analysis by using an automated sequencer (Perkin-Elmer Biosystems).
Expression and purification of recombinant MEK mutants. Plasmid pET21b (Novagen) encoding a MEK His6-tagged fusion was transformed into E. coli BL21(DE3) by standard methods. Expression of recombinant His6-tagged MEK in bacterial culture was induced with IPTG (isopropyl-ß-D-thiogalactopyranoside; 200 µM) and purified by using IMAC methods (Clontech Laboratories, Inc.). The molecular mass and protein concentration were estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Coomassie blue staining.
In vitro kinase assays.
The kinase activities of recombinant MEK(His)6 proteins were determined by using a glutathione S-transferase (GST) fusion protein of kinase-inactive ERK1 (GSTERK1K71R) as a substrate. Recombinant MEK protein (50 ng) was incubated with various concentrations of PD 184352 in 20 mM HEPES (pH 7.5), 10 mM MgCl2, 1 mM EGTA, 10 µM ATP, and 5 µCi of [
-32P]ATP in a total volume of 25 µl. Raf-1 (0.5 U; Upstate Biotechnology) was added, and the reaction initiated with the addition of GSTERK1(K71R) fusion protein (1 µg). Working within the linear range of the reaction, the samples were incubated at 27°C for 20 min and then quenched with 5 µl of 6x Laemmli sample buffer. The proteins were resolved by SDS-PAGE, and phosphoproteins were visualized by autoradiography. Radiolabel incorporated into ERK was determined by excision of the corresponding protein band and quantitated by using a standard scintillation counter (Beckman Instruments, Inc.).
3H-PD 184352 binding assay. A scintillation proximity assay (SPA) was utilized to evaluate direct interaction of PD 184352 with the mutant MEK proteins. Recombinant MEK(His)6 proteins were prepared as described. 3H-PD 184352 was custom synthesized (Amersham) with a specific activity of 22 Ci/mmol. MEK proteins were incubated with 3H-PD 184352 in binding buffer (10 mM K2HPO4, 10 mm KH2PO4, 50 mM NaCl, 2 mM CHAPS {3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate}, 10% glycerol; pH 7.6) in 96-well flat-bottom tissue culture plates in a final volume of 100 µl. The plates were shaken for 30 min at room temperature, followed by the addition of 50 µl of YSI-Copper-His TAG SPA beads (Amersham) in binding buffer containing 1.25 mg of bovine immunoglobulin G (IgG; Sigma)/ml. Samples were shaken an additional 45 min, and the amount of 3H-PD 184352 bound to the MEK-SPA bead complex was determined in a Wallac MicroBeta plate counter.
Analysis of phospho-MEK levels in cultured cells. KBALB cells were cultured in Dulbecco modified Eagle medium supplemented with 10% newborn calf serum, 25 mM HEPES (pH 7.5), and 1x Glutamax (Life Technologies, Inc.). Cells were serum deprived for 24 h, treated with PD 184352 at various concentrations for 0.5 to 1 h, and then stimulated with 50 ng of platelet-derived growth factor (PDGF)/ml for 5 min at 37°C. Lysates were prepared in 10 mM HEPES (pH 7.4), 50 mM ß-glycerophosphate, 1% Triton X-100 (Sigma-Aldrich Co.), 70 mM NaCl, 1 mM phenylmethylsulfonyl fluoride, 1 mM sodium vanadate, and 1 µM leupeptin (Sigma-Aldrich Co.) and then clarified by centrifugation at 30,000 x g. Protein concentrations in the lysates were determined by BCA assay (Pierce; Perbio). Extracts (15 to 20 µg) were subjected to SDS-PAGE electrophoresis, transferred to nitrocellulose and probed with anti-phospho-MEK (New England Biolabs, Inc.), anti-active MAP kinase (Promega Corp.), and anti-p44MAPK1/p42MAPK2 (Santa Cruz Biotechnology, Inc.) antibodies. Immunoreactive proteins were visualized with ECL reagents (Amersham International, plc.).
Expression of MEK mutants in mammalian cells. HEK293T cells were cultured in Dulbecco modified Eagle medium supplemented with 10% fetal bovine serum, 25 mM HEPES (pH 7.5), and 1x Glutamax (Life Technologies, Inc.). Transient transfection of 1 to 2 µg of DNA (pCMV4A clones) into HEK293T cells was performed by using Lipofectamine (Life Technologies, Inc.). At 24 h posttransfection, cell extracts were prepared as described above, subjected to SDS-PAGE electrophoresis, and transferred to nitrocellulose. The immunoreactive proteins were visualized by Western blotting with anti-FLAG-M2 (Sigma-Aldrich Co.) and anti-active MAP kinase antibodies (Promega Corp.).
Homology modeling. Homology models of MEK1 were constructed by using either the modeling package of Look (Molecular Applications Group) or the Homology module of InsightII (MSI). The N-terminal region prior to the kinase domain and an internal proline-rich loop region (residues 268 to 308) were not included for modeling. The crystal structure (PDB: 1CDK) of the active form of cyclic AMP-dependent protein kinase A was used as a template for homology modeling (4). Structure-based multiple sequence alignment of representative protein kinases and multiple alignment of all known MEK sequences were used to generate the final alignment. The final model was minimized by using the Discover module of InsightII and was evaluated by using the Procheck and Profile3D modules of InsightII.
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RESULTS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A possible explanation for the elevated levels of MEK phosphorylation in the presence of PD 184352 is the existence of negative feedback loops, whereby the inhibition of MAP kinase leads to hyperactivation of Raf-1 or some other signaling component upstream of Raf (27). However, while the dose dependency of PD 184352-stimulated phospho-MEK accumulation and MEK inhibition are distinct, it is clear that this compound does not inhibit Raf kinase activity.
An S. cerevisiae genetic screen to identify the residues of MEK that are involved in PD 184352-mediated inhibition.
Since PD 184352 is noncompetitive for ATP and exerts unexpected effects on MEK phosphorylation by Raf, we devised a screen to identify interacting residues in the protein. Homology between the mammalian MAP kinase signaling module and the budding yeast pheromone response pathway allows functional complementation of some members of this yeast signaling pathway with mammalian homologues. Genes for the yeast homologues of the mammalian MAP kinase cascade, ste11 (Raf), ste7 (MEK), and ste5 (scaffolding protein), were disrupted in a strain harboring a FUS1::HIS3 reporter, stably integrated at the FUS1 locus (SY2002). Strain SY2002 is auxotrophic for histidine, and growth on histidine-deficient medium is dependent on an intact pheromone response pathway. Disruption of any member of the pheromone response cascade renders this strain incapable of growing on a medium lacking histidine (30). However, transformation of the ste11 ste7 ste5 mutant strain with the mammalian counterparts of the MAP kinase pathway, specifically MEK1 and a constitutively active form of Raf (RafNT), restored the integrity of the signaling pathway and rendered the yeast prototrophic for histidine. Complementation of ste11 and ste7 required high-level expression of both MEK and RafNT, and this was accomplished by placing MEK and RafNT under the control of a galactose-inducible promoter. However, when yeast expressing RafNT and MEK1 were propagated on histidine-deficient medium containing 1 µM PD 184352, complementation was blocked, suggesting that PD 184352 was efficiently inhibiting MEK activity. PD 184352 was not merely toxic to the yeast, since the parental strain (SY2002) was able to propagate in the presence of 1 µM PD 184352. This control also demonstrates that PD 184352 did not inhibit the yeast MEK homologue ste7, since the wild-type (STE7+) strain grew normally on histidine-deficient medium containing PD 184352 (data not shown).
Isolation of PD 184352-resistant MEK mutants.
Random mutagenesis was performed on MEK cDNA by transforming pRS425GALMEK into E. coli XL-1 Red, a bacterial strain defective in three major DNA repair mechanisms (mutD, mutS, and mutT). The subsequent pool of mutagenized MEK plasmids was then transformed into the ste11 ste7 ste5 FUS1::HIS3 strain harboring pRS314GALRafNT. Mutations generating inactive forms of MEK, such as premature stop codons, were removed from the screen through the inability of the defective kinase to complement the ste7 deletion and return SY2002 to histidine prototrophy. Transformants capable of growing in the absence of histidine were then screened for their ability to grow on histidine-deficient medium containing 1 µM PD 184352.
Seven different mutant alleles were identified from the screen, all of which were single-base-pair mutations in the MEK coding sequence, summarized schematically in Fig. 2. Interestingly, five of the seven mutations were clustered in the region of the enzyme spanning kinase subdomains III and IV (Fig. 2). When the isolated mutant alleles were transformed into a naive ste11 ste7 ste5 strain, the MEK variants allowed growth on media containing PD 184352 (data not shown), confirming that a spontaneous mutation of the yeast host had not conferred PD 184352 resistance to this strain.
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MEK homology model.
A homology model of the active form of MEK1 was generated based on the crystal structure of the active form of cyclic AMP-dependent protein kinase A (4) and multiple sequence alignment of several protein kinases, including the known MEK family members. All of the mutated residues, except Phe53 and Glu203, form a cluster in the model (Fig. 5), suggesting that these residues could be part of a binding pocket for the MEK1-specific inhibitor. The clustered residues are located in the interface region between the 
-helix C and five-stranded ß-sheet of the N-terminal lobe of the kinase domain. This pocket is distinct from the ATP binding pocket and is accessible from the rear face of the kinase domain. Analysis of the putative inhibitor binding pocket suggests it can accommodate a molecule the size of PD 184352. The conserved residues Lys97 and Glu114 form a hypothetical salt bridge in the model and are part of the wall separating the ATP binding pocket and the pocket formed by the identified mutant residues. Despite the clustering of the mutated residues in an apparent pocket for PD 184352, only the Leu115Pro substitution showed complete resistance to PD 184352, indicating that this residue may be critical for inhibitor binding. The model suggests the side chain of Leu115 lines the putative PD 184352 binding pocket; hence, altering this residue could disrupt the conformation of the pocket and prevent PD 184352 binding. To test this hypothesis, Leu115 was substituted with alanine, arginine, glutamate, or tryptophan in order to characterize the effects of different amino acid side chains at this position on inhibitor activity. The recombinant proteins were expressed in E. coli and assayed in vitro for their ability to phosphorylate GSTERK1(K71R) in the presence of increasing concentrations of PD 184352 (0 to 3 µM). As shown in Fig. 6, only the conservative leucine-to-alanine (Leu115Ala) substitution was significantly inhibited by PD 184352, and this construct was still quite resistant to inhibition (IC50 = 0.68 µM versus IC50 = 0.067 µM for wild-type). Other Leu115 substitutions exhibited IC50 values of >3 µM, indicating that the side chain of this residue is critical for PD 184352 interaction with MEK. Furthermore, similar to what was observed with the variants obtained from the genetic screen, each amino acid substitution was coupled with an increase in basal activity. Resulting basal specific activities were 197.1, 478.8, 882.3, 1,581 and 2,739 pmol min-1 mg-1 for L115R, L115P, L115W, L115E, and L115A, respectively. These ranged from 1.6- to 22.5-fold higher than wild-type MEK basal kinase activity (121.8 pmol min-1 mg-1).
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DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Interestingly, the screen also identified Phe53, which falls outside of the usual kinase domain. The only significant homology between MEK and cyclic AMP-dependent protein kinase is within the kinase domain; thus, the MEK homology model can only be reasonably constructed for this domain. Phe53 is roughly 15 residues N-terminal to this domain and is not included in the modeling effort. However, Phe53 is very close to an N-terminal region comprising residues 32 to 51 that was identified by Ahn's group as acting to possible stabilize an inactive state of MEK (20, 21). Deletion of the region from residues 32 to 51 markedly activates MEK, as does the Phe53 mutation. Clearly, the N-terminal region of MEK also plays an important regulatory role in addition to substrate recognition (15).
Of the mutants identified in the yeast screen, the Leu115Pro mutation exerted the greatest effect on inhibitor activity. In the homology model of MEK1, Leu115 is part of -helix C and its side chain is located inside the putative PD 184352 binding pocket. Proline is not favored in -helices due to the lack of an amide hydrogen bond donor and to the steric constraints imposed upon the -helical structure by the proline side chain. Thus, the Leu115Pro mutation could disrupt a region of the -helix C and change the conformation of the pocket to prevent the binding of PD 184352 to MEK. Additionally, the site-specific mutagenesis and biochemical analysis of residues Ile111, Leu118, and Ile139, which were suggested by the model to be part of the inhibitor binding pocket, lend further support that this region is where PD 184352 interacts with MEK.
A possible explanation for the increased basal activity of the mutants is that a more active conformation is obtained due to changes in localized subdomains within the kinase core. X-ray crystal structures of several protein kinases have revealed catalytic activity of these enzymes involves two primary conformational changes (7). The first occurs due to phosphorylation of residues that lie within the activation lip of the kinase, which helps to stabilize the kinase in an active form. Second, the two lobes that comprise the kinase core domain rotate with respect to each other to close the catalytic cleft, thereby bringing the essential residues into the correct conformation to complete catalysis (7). However, other studies have indicated that subtle dynamic motion involving various secondary structures may also be important for catalysis. Specifically, Ahn's group have shown by deuterium exchange that significant dynamic movement and flexibility in the N-terminal lobe of the kinase core was observed in the mutationally activated form of MEK (24). In particular, the region of the kinase containing five of the seven mutated residues identified in the screen was shown to undergo the most movement when the enzyme was activated, with the exception of the activation segment.
We believe that the apparent dynamic nature of this region has profound implications for regulation of this enzyme. Thus, perturbations introduced by the mutations revealed by the screen could result in a loss of ordering in this region resulting in an increase in flexibility and hence catalytic activity. This could possibly also lead to elevated autophosphorylation at the activation loop, which would increase catalytic activity. Indeed, preliminary experiments suggest that Ile103Asn has a substantially higher level of autophosphorylation than with wild-type or the other mutants (data not shown). Conversely, any imposition on flexibility could have a significant impact on the catalytic activity of MEK. Binding of PD 184352 may lock the enzyme into an inactive conformation, preventing catalysis but still allowing phosphorylation of Ser218 and Ser222 in the activation segment by Raf. Additionally, this might interfere with recognition by MEK-specific phosphatases (27), possibly explaining the MEK hyperphosphorylation observed in KBALB cells treated with PD 184352.
The reported MEK inhibitors, PD 98059, PD 184352, and U0126, have proven to be exceptionally specific. Interestingly, they do not inhibit related MKK family members MKK3, MKK4, MKK6, or MKK7, although they do inhibit the closely related MEK2 (9, 26). Recently, these compounds have been reported to also show activity, albeit weaker, against MKK5 (18, 22, 23). Of the MKK family members, MKK5 exhibits the closest homology to MEK1 and MEK2, with ca. 46% amino acid identity in the catalytic domain (22). Alignments of the MKK family reveal that all of the mutants found here, as well as the targeted residues chosen from the homology model, are identical in MEK1, MEK2, and MKK5 and diverge in the other members. It is possible that a similar binding pocket exists in other MKK members and the MKK5 pocket is close enough to be recognized by the MEK1 inhibitors.
It is interesting that basal MEK kinase activity seen in vitro with some of the variants did not correlate with the activity of these mutants when overexpressed in HEK293T cells. Combined with the hyperphosphorylated state of endogenous MEK observed in cells treated with PD 184352, it is tempting to speculate that there are additional cellular components involved in regulation of MEK activity, as well as PD 184352-mediated inhibition of MEK. Further investigation to identify these unknown components, as well as the effects of other mutations in the proposed binding pocket on PD 184352 inhibition of MEK will be required to more fully understand the molecular mechanism of MEK kinase inhibition by PD 184352.
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FOOTNOTES |
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Present address: AstraZeneca Pharmaceuticals, Alderley Park, United Kingdom.
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), relevant kinase subdomains (
), Raf phosphorylation sites (P), and the lysine residue essential for phosphoryl transfer (K).
), Leu115Ala (•), Leu115Glu (
), Leu115Pro (
), Leu115Arg (
), and Leu115Trp (
) constructs. These data represent two independent experiments. The 100% control value reflects the Raf-activated, untreated enzyme activity.
