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• W2022135311 abstract "We have purified 3500-fold from rabbit skeletal muscle a 12,020-Da mitogen-activated protein kinase kinase (MEK)-enhancing factor (MEF) that stimulates both mitogen-activated protein kinase (MAPK) autophosphorylation and the rate (24-fold) at which the enzyme is phosphorylated by MEK in vitro. This was manifest by the finding that in the presence of MEF, molar equivalents of MEK to MAPK were sufficient to produce fully phosphorylated (2.1 ± 0.4 mol/mol; S.D., n = 3) and activated MAPK. This contrasted with the 40:1 molar excess ratio of MEK to MAPK required to produce fully phosphorylated and activated MAPK in the absence of MEF. Phosphoamino acid analysis revealed that in the presence of MEF, phosphorylation of MAPK by MEK was ordered, with Tyr-185 phosphorylation preceding Thr-183 phosphorylation. However, the rate at which Thr-183 was phosphorylated relative to Tyr-185 was greatly increased. The finding that MEF stimulated MAPK autophosphorylation and increased its ability to be phosphorylated by MEK suggests a mechanism of action in which MEF interacts with MAPK to alter its conformation. We have purified 3500-fold from rabbit skeletal muscle a 12,020-Da mitogen-activated protein kinase kinase (MEK)-enhancing factor (MEF) that stimulates both mitogen-activated protein kinase (MAPK) autophosphorylation and the rate (24-fold) at which the enzyme is phosphorylated by MEK in vitro. This was manifest by the finding that in the presence of MEF, molar equivalents of MEK to MAPK were sufficient to produce fully phosphorylated (2.1 ± 0.4 mol/mol; S.D., n = 3) and activated MAPK. This contrasted with the 40:1 molar excess ratio of MEK to MAPK required to produce fully phosphorylated and activated MAPK in the absence of MEF. Phosphoamino acid analysis revealed that in the presence of MEF, phosphorylation of MAPK by MEK was ordered, with Tyr-185 phosphorylation preceding Thr-183 phosphorylation. However, the rate at which Thr-183 was phosphorylated relative to Tyr-185 was greatly increased. The finding that MEF stimulated MAPK autophosphorylation and increased its ability to be phosphorylated by MEK suggests a mechanism of action in which MEF interacts with MAPK to alter its conformation. INTRODUCTIONThe mitogen-activated protein kinases (MAPKs) 1The abbreviations used are: MAPKsmitogen-activated protein kinasesMEKsmitogen-activated protein kinase kinasesMEKKsMEK kinasesI-MEKKinsulin-stimulated MEK kinaseMEFMEK-enhancing factorGSTglutathione S-transferasePAGEpolyacrylamide gel electrophoresisHPLChigh pressure liquid chromatography. p42MAPK (ERK2) and p44MAPK (ERK1) are thought to mediate the actions of numerous hormones controlling cellular events as diverse as growth, division, and metabolism (for reviews, see (1Robbins D.J. Zhen E. Cheng M. Xu S. Ebert D. Cobb M.H. Adv. Cancer Res. 1994; 63: 93-116Crossref PubMed Google Scholar, 2Sturgill T.W. Wu J. Biochim. Biophys. Acta. 1991; 1092: 350-357Crossref PubMed Scopus (328) Google Scholar, 3Davis R.J. J. Biol. Chem. 1993; 268: 14553-14556Abstract Full Text PDF PubMed Google Scholar, 4Crews C.M. Erikson R.L. Cell. 1993; 74: 215-217Abstract Full Text PDF PubMed Scopus (296) Google Scholar)). p42MAPK and p44MAPK belong to a conserved family of protein kinases that also includes p54MAPK(5Zheng C.F. Guan K.L. J. Biol. Chem. 1993; 268: 23933-23939Abstract Full Text PDF PubMed Google Scholar, 6Zheng C.F. Guan K.L. J. Biol. Chem. 1993; 268: 11435-11439Abstract Full Text PDF PubMed Google Scholar) and the stress-activated protein kinases JNKs/SAPKs and p38MAPK/RKs(7Freshney N.W. Rawlinson L. Guesdon F. Jones E. Crowley S. Hsuan J. Saklatvala J. Cell. 1994; 78: 1039-1049Abstract Full Text PDF PubMed Scopus (774) Google Scholar, 8Han J. Richter B. Li Z. Kravchenko V.V. Ulevich R.J. Biochim. Biophys. Acta. 1995; 1265: 224-227Crossref PubMed Scopus (72) Google Scholar, 9Derijard B. Raingeaud J. Barrett T. Wu I.-U. Han J. Ulevich R.J. Davis R.J. Science. 1995; 267: 682-684Crossref PubMed Scopus (1405) Google Scholar, 10Doza Y.N. Cuenda A. Thomas G.M. Cohen P. Nebreda A.R. 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Wigler M. Marcus S. Mol. Cell. Biol. 1993; 4: 107-120Crossref Scopus (125) Google Scholar).Much attention in recent years has focused on delineating the immediate steps from cell-surface receptors to the activation of p42MAPK and p44MAPK. To date, two tentative connections have been made. In one pathway, several laboratories, utilizing a combination of overexpression and antisense techniques, have identified p74raf-1 and possibly its homologs A-Raf and B-Raf as activators of MEK in vitro(23Dent P. Haser W. Haystead T.A.J. Vincent L.A. Roberts T.M. Sturgill T.W. Science. 1992; 257: 1404-1407Crossref PubMed Scopus (496) Google Scholar, 24Kyriakis J.M. App H. Zhang X. Banerjee P. Brautigan D.L. Rapp U.R. Avruch J. Nature. 1992; 358: 417-421Crossref PubMed Scopus (967) Google Scholar, 25Howe L.R. Leevers S.J. Gomez N. Nakielny S. Cohen P. Marshall C.J. Cell. 1992; 71: 335-342Abstract Full Text PDF PubMed Scopus (627) Google Scholar, 26Moodie S.A. Willumsen B.M. Weber M.J. Wolfman A. 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Cooper J. Cell. 1993; 74: 205-214Abstract Full Text PDF PubMed Scopus (1656) Google Scholar, 38Warne P.H. Vicinia P.B. Downward J. Nature. 1993; 364: 352-355Crossref PubMed Scopus (581) Google Scholar, 39Zhang C. Guan K.L. J. Biol. Chem. 1993; 268: 11435-11439Abstract Full Text PDF PubMed Google Scholar, 40Traverse S. Cohen P. Patterson H. Marshall C. Rapp U. Grand R.J.A. Oncogene. 1993; 8: 3175-3181PubMed Google Scholar, 41Fabian J.R. Vojtek A.B. Cooper J.A. Morrison D.K. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5982-5986Crossref PubMed Scopus (156) Google Scholar, 42Deleted in proofGoogle Scholar, 43Jaiswal R.K. Moodie S.A. Wolfman A. Landreth G.E. Mol. Cell. Biol. 1994; 14: 6944-6953Crossref PubMed Google Scholar). In yeast, an alternative pathway suggests that MEKs are activated by heterotrimeric G protein-coupled receptors via the activation of STE-11 homologs of MEK kinases (MEKKs)(19Errede B. Levine D.E. Curr. Opin. Cell Biol. 1995; 7: 197-202Crossref PubMed Scopus (217) Google Scholar, 20Kosako H. Nishida E. Gotoh Y. EMBO J. 1993; 12: 787-794Crossref PubMed Scopus (118) Google Scholar, 21Lange-Carter C.A. Plieman C.M. Gardner A.M. Blumer K.J. Johnson G. Science. 1993; 260: 315-319Crossref PubMed Scopus (869) Google Scholar, 22Neiman A.M. Stevenson B.J. Xu H.-P. Sprague G.F. Herskowitz I. Wigler M. Marcus S. Mol. Cell. Biol. 1993; 4: 107-120Crossref Scopus (125) Google Scholar, 44Blumer K.J. Johnson G.L. Lange-Carter C.A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4925-4929Crossref PubMed Scopus (48) Google Scholar). Other less well characterized activators of MEK have also been described, including c-mos(45Merrall N.W. Plevin R.J. Stoke D. Cohen P. Nebreda A.R. Gould G.W. Biochem. J. 1993; 295: 351-355Crossref PubMed Scopus (28) Google Scholar, 46Nebreda A.R. Hunt T. EMBO J. 1993; 12: 1979-1986Crossref PubMed Scopus (251) Google Scholar, 47Posada J. Yew N. Ahn N.G. Vande Woude G.F. Cooper J. Mol. Cell. Biol. 1993; 13: 2546-2553Crossref PubMed Scopus (335) Google Scholar), a 400-kDa MEK-activating factor in Xenopus(48Matsuda S. Gotoh Y. Nishida E. J. Biol. Chem. 1993; 268: 3277-3281Abstract Full Text PDF PubMed Google Scholar), and a 50-60-kDa insulin-stimulated MEK kinase (I-MEKK) in isolated adipocytes (49Haystead T.A.J. Dent P. Wu J. Haystead C.M.M. Sturgill T.W. FEBS Lett. 1992; 306: 17-22Crossref PubMed Scopus (124) Google Scholar) and NIH 3T3 cells(29Reuter C.W. Cattling A.D. Jelnek T. Weber M.J. J. Biol. Chem. 1995; 270: 7644-7655Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). The existence of multiple MEK-activating pathways has led to the hypothesis that MEK acts as a signaling convergence point, explaining how so many diverse cellular agonists can activate MAPKs.Our laboratory has had a long standing interest in the mechanism by which insulin both activates and then inactivates MAPK in vivo. We recently identified in isolated adipocytes a MEK kinase (I-MEKK) that, like MEK and MAPK, showed acute phasic activity kinetics in response to the hormone(50Haystead C.M. Gregory P. Shirazi A. Fadden P. Mosse C. Dent P. Haystead T.A.J. J. Biol. Chem. 1994; 269: 12804-12808Abstract Full Text PDF PubMed Google Scholar). Since this initial report, we have made several unsuccessful attempts to purify I-MEKK from insulin-treated adipocytes. Although I-MEKK remains stable over anion-exchange and gel filtration chromatography, further purification results in immediate loss of all activity. These findings led us to conclude that activation of MEK by MEK kinases in vitro requires additional factors that are lost in subsequent purification steps. Accumulating genetic evidence in yeast may support this hypothesis. In Saccharomyces cerevisiae, the conserved MAPK module, comprising STE-11 (MEKK), STE-7 (MEK), and FUS-3 (MAPK), requires a fourth protein, STE-5, to mediate pheromone responses ((51Choi K.-Y. Satterberg B. Lyons D.M. Elion E.A. Cell. 1994; 78: 499-512Abstract Full Text PDF PubMed Scopus (162) Google Scholar); see also (52Herskowitz I. Cell. 1995; 80 (– and references therein): 187-197,Abstract Full Text PDF PubMed Scopus (863) Google Scholar)). In the module, STE-5 has been proposed to tether these protein kinases together in order to bring about their sequential activation. If an STE-5-like protein exits to activate MEK in higher eukaryotes, this might also explain discrepancies we have observed in the mechanism by which MEK activates MAPK in vitro. We had reported earlier that in the presence of catalytic amounts of purified MEK, the Tyr-185 phosphorylated form of MAPK accumulated. To produce the fully activated and phosphorylated form of MAPK, repeated additions of purified MEK were required(49Haystead T.A.J. Dent P. Wu J. Haystead C.M.M. Sturgill T.W. FEBS Lett. 1992; 306: 17-22Crossref PubMed Scopus (124) Google Scholar). From this study, we concluded that phosphorylation of MAPK by MEK was ordered, with Tyr-185 phosphorylation preceding Thr-183 phosphorylation. Recently, Goldsmith and co-workers (53Zhang F. Strand A. Robbins D. Cobb M.H. Goldsmith E.J. Nature. 1994; 367: 704-711Crossref PubMed Scopus (532) Google Scholar) solved the apo structure of p42MAPK to 2.3-Å resolution. In the apo structure, Tyr-185 is buried in a hydrophobic pocket near the ATP-binding cleft, whereas Thr-183 is exposed on the surface of the enzyme. These findings are paradoxical since the apo structure would indicate that MEK is unlikely to phosphorylate MAPK on Tyr-185 first. Taken together, these observations strongly support the hypothesis that additional factors may be required for the activation of MAPK and possibly MEK. In this paper, we report the purification of a 12,020-Da protein that potently enhances the activation of MAPK by MEK. We propose that our findings have general implications for some of the models currently proposed for MAPK activation in vivo.EXPERIMENTAL PROCEDURESMaterialsRecombinant p42MAPK was purified from Escherichia coli strain BL21(DE3)[pET-MK](49Haystead T.A.J. Dent P. Wu J. Haystead C.M.M. Sturgill T.W. FEBS Lett. 1992; 306: 17-22Crossref PubMed Scopus (124) Google Scholar). Native MEK was purified from rabbit skeletal muscle as described previously(54Haystead C.M.M. Gregory P. Sturgill T.W. Haystead T.A.J. Eur. J. Biochem. 1993; 214: 459-467Crossref PubMed Scopus (67) Google Scholar). The pGEX-2T plasmids expressing wild-type recombinant MEK1, S218D/S222D MEK1, and KR52 MAPK were a gift from Dr. Andrew Cattling (Department of Microbiology, University of Virginia).MethodsPreparation of Recombinant GST-MEK Fusion ProteinsDH5α cells transformed with the respective pGEX-2T MEK plasmid were streaked onto LB plates containing ampicillin and incubated overnight at 37°C. Six 4-ml cultures containing LB broth, 50 μg/ml ampicillin, and a single colony of E. coli were grown overnight at 37°C. Six flasks containing 1 liter of LB broth and 50 μg/ml ampicillin were inoculated with the overnight culture and grown at 37°C to an absorbance of 600. Cells were induced with 30 μM isopropyl-β-D-thiogalactopyranoside for 10 h at room temperature and then centrifuged at 3600 × g for 20 min at 4°C. The pellet was stored overnight at −20°C. The thawed pellet was resuspended in 45 ml of lysis buffer (20 mM Tris, pH 8, 100 mM NaCl, 1 mM EDTA, 0.5% Nonidet P-40, 1 mM phenylmethanesulfonyl fluoride, and 1 mM benzamidine), lysed with 2 mg/ml lysozyme, incubated on ice for 15 min, sonicated for 3 min, and then centrifuged at 14,500 × g for 30 min at 4°C. 1.0 ml of glutathione-Sepharose 4B beads at 50% (v/v) beads/lysis buffer was added to the supernatant and rotated overnight at 4°C. The beads were centrifuged at 200 × g and washed four times with 25 ml of lysis buffer. The GST-MEK fusion protein was eluted with 6 ml of 5 mM glutathione, 50 mM Tris, pH 8. The protein was dialyzed overnight against 1 liter of Tris buffer (50 mM Tris, pH 7.3, 1.5 mM EGTA, 1 mM benzamidine) and applied to a Waters Protein-Pak Q AP-1 anion-exchange column equilibrated in buffer A (50 mM β-glycerophosphate, pH 7.3, 1.5 mM EGTA, 150 μM sodium orthovanadate, 1 mM dithiothreitol, 1 mM phenylmethanesulfonyl fluoride, and 1 mM benzamidine). The column was developed with a 400 mM linear salt gradient (200 ml), and 2.0-ml fractions were assayed for activation of MAPK(54Haystead C.M.M. Gregory P. Sturgill T.W. Haystead T.A.J. Eur. J. Biochem. 1993; 214: 459-467Crossref PubMed Scopus (67) Google Scholar). The peak of activity was pooled and concentrated under a vacuum to a 1-ml volume. Glycerol was added to 50% (v/v), and protein was stored at −20°C.Preparation of MEFA fed female New Zealand White rabbit was euthanized by lethal injection with phenobarbital via the marginal ear vein. The back and hind limb skeletal muscle (300 g, wet weight) were rapidly excised, minced, and homogenized at 4°C with 2.5 volumes of buffer B (50 mM β-glycerophosphate pH 7.3, 1.5 mM EGTA, 0.15 μM sodium orthovanadate, 1 mM dithiothreitol, 1 mM phenylmethanesulfonyl fluoride, and 1 mM benzamidine). All subsequent steps were carried out at 4°C. The homogenate was centrifuged at 6000 × g for 45 min, and the supernatant was applied to a FFQ-Sepharose column (20 × 50 cm; Pharmacia Biotech Inc.) equilibrated with buffer B. The column was washed extensively until the absorbance at 280 nm of the eluate was <0.005 and then developed with the indicated salt gradient (see Fig. 2). The most active fractions of MEF were pooled (1500 ml) and concentrated successively to 10 ml in an Amicon 400-ml stirred cell using a Diaflo YM-10 membrane (Amicon, Inc.) followed by vacuum concentration. Concentrated MEF was further purified by successive applications to a Waters SW300 gel filtration column equilibrated in buffer C (25 mM Hepes, pH 7.4, 1 mM EDTA, 1 mM EGTA, 1 μM microcystin, and 10 mM β-mercaptoethanol) containing 150 mM NaCl (see Fig. 3). The most active column fractions were pooled, heated at 90°C for 5 min, and centrifuged at 30,000 × g. Following concentration to 1.0 ml, the extract was applied to a Waters Nova-Pak C8 reverse-phase column (3.9 × 150 mm) equilibrated in 0.1% trifluoroacetic acid. The column was developed as described (see Fig. 4A). For further purification, the most active fractions were pooled and reapplied to a Waters Nova-Pak C18 reverse-phase column (3.9 × 150 mm) equilibrated in 0.1% trifluoroacetic acid, and the column was developed as described (see Fig. 4B).Figure 3:Gel filtration analysis of MEF activity. A concentrated sample (200 μl) of the column wash from anion-exchange chromatography containing MEF activity was applied to a Waters SW300 gel filtration column (8.0 × 300 mm), and the column was developed at 1.0 ml/min. Column fractions (200 μl) were assayed for MAPK activity in the presence (○) or absence (▵) of S218D/S222D MEK1. Molecular mass markers were as follows: 150 kDa, aldolase; 69 kDa, bovine serum albumin; 42 kDa, MAPK; 12 kDa, MEF; and 8 kDa, aprotinin. Data shown are from a single experiment; however, this was repeated with identical results.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 4:Reverse-phase chromatography of MEF. Heat-treated MEF was purified to homogeneity over C8 (A) and then C18 (B) reverse-phase columns developed (1.0 ml/min) with the indicated gradients of O.1% trifluoroacetic acid/water and acetonitrile. In A, MEF activity was measured on column fractions following microdialysis against buffer C. In B, peaks of absorbance were collected by hand, and MEF activity was measured following microdialysis against buffer C. Absorbance was measured by an on-line photodiode array detector at 214 nm.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Assay of MEF ActivityMEF activity was determined by its ability to enhance the activity of MEK toward MAPK. Two types of assay were utilized, either a two-stage robotic assay that measured activation of MAPK or direct phosphorylation of MAPK. In the two-stage robotic assay, 20 μl of column fraction was placed into microtiter plate wells, and the reaction was started by the addition of 40 μl of buffer C containing 0.6 μg of p42MAPK, 0.6 μg of MEK, 300 μM ATP, 7.5 mM MgCl2, and 1 μM microcystin. After 10 min at 22.5°C, the robot removed 20 μl and placed this into a second set of wells containing 5 μg of myelin basic protein, 300 μM [γ-32P]ATP (250 cpm/pmol), and 7.5 mM MgCl2 in buffer C. The reaction was terminated after 5 min by the addition of 20 μl of 100 mM H3PO4. The reactions (40 μl) were spotted onto P-81 paper (Whatman) and washed four times in 100 mM H3PO4, and the amount of radioactivity incorporated into myelin basic protein was determined by scintillation counting. To measure endogenous MEK activity, the first reaction was carried out in the absence of exogenous MEK. In the second type of assay, 20 μl of column fraction was placed into microtiter plate wells, and the reaction was started by the addition of 40 μl of buffer C containing 0.6 μg of p42MAPK, 0.6 μg of MEK, 300 μM [γ-32P]ATP (250 cpm/pmol), 7.5 mM MgCl2, and 1 μM microcystin. After 20 min at 22.5°C, the robot terminated the reaction by the addition of 20 μl of SDS sample buffer (20% glycerol, 62.5 mM Tris-HCl, pH 6.8 (25°C), 2% SDS, and 5% 2-mercaptoethanol). Again, endogenous MEK was measured by carrying out the assay in the absence of exogenous MEK. Following heating (100°C) for 5 min, the phosphorylated proteins were characterized by SDS-PAGE and autoradiography. Quantitation of phosphorylation was by PhosphorImager analysis. In assays in which the stoichiometry of MAPK phosphorylation was measured, reactions were terminated with 25% trichloroacetic acid, and the radioactivity incorporated into the precipitated proteins was determined by Cerenkov counting.RESULTSPhosphorylation of MAPK by MEKThe ability of recombinant p42MAPK to be phosphorylated by various concentrations of purified native rabbit skeletal muscle MEK1 was measured (Fig. 1). Fig. 1 shows that in order to achieve a stoichiometry close to 1 mol/mol within a 30-min period, a 3-4-fold (3.6 ± 0.19 S.D., n = 3) molar excess of MEK (~2 μM) over MAPK (0.5 μM) was required. At this time point and concentration of MEK, phosphoamino acid analysis of MAPK revealed ~80% of the phosphate to be incorporated into tyrosine, with the remainder on threonine (Fig. 1, inset). Increasing the concentration of MEK by an order of magnitude (40:1) achieved a stoichiometry of phosphorylation close to 2 mol/mol within the same time frame. Under these conditions, the phosphothreonine content of MAPK was equal to phosphotyrosine (Fig. 1, inset). When fully phosphorylated, recombinant MAPK displayed a specific activity toward myelin basic protein of 0.32 ± 0.08 μmol/min/mg (S.D., n = 3), approximating values previously estimated for the activated native enzyme(49Haystead T.A.J. Dent P. Wu J. Haystead C.M.M. Sturgill T.W. FEBS Lett. 1992; 306: 17-22Crossref PubMed Scopus (124) Google Scholar). Increasing the concentration of MAPK in the assay to 10 μM, with a fixed MEK concentration of 20 μM, resulted in an appreciable reduction in the overall stoichiometry of phosphorylation to >0.2 ± 0.05 mol/mol (S.D., n = 3). Correspondingly, phosphorylation of threonine was considerably reduced relative to tyrosine. These results suggest that the relative poor rate of phosphorylation of MAPK by purified MEK was not due to a suboptimal substrate concentration being utilized in our assays. In previous work, we had also noted that high concentrations of MEK relative to MAPK were required to activate the enzyme completely(49Haystead T.A.J. Dent P. Wu J. Haystead C.M.M. Sturgill T.W. FEBS Lett. 1992; 306: 17-22Crossref PubMed Scopus (124) Google Scholar). Also in this earlier study, it was observed that Tyr-185 phosphorylation increased relative to Thr-183, suggesting that phosphorylation of MAPK by MEK was ordered.Figure 1:Phosphorylation of MAPK by native MEK. Purified recombinant p42MAPK (0.5 μM) was incubated for 30 min in a 30-μl assay containing 300 μM ATP, 7.5 mM MgCl2, 25 mM Hepes, pH 7.4, 1 mM dithiothreitol, 1 mM EDTA, and the indicated concentrations of purified rabbit skeletal muscle MEK1. Reactions were terminated by the addition of ice-cold 25% trichloroacetic acid. Following centrifugation (14,000 × g for 5 min) and washing, the radioactivity in the precipitated protein was determined. Insets are the results of phosphoamino acid analysis (62van der Geer P. Luo K. Sefton B. Hunter T. Hardie D.G. Protein Phosphorylation: A Practical Approach. Oxford University Press, Oxford, United Kingdom1993: 31-59Google Scholar) of trichloroacetic acid-precipitated MAPK from reactions containing 1 or 10 μM MEK. Results are means ± S.D. of three separate experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Purification of MEFThe finding that in vitro an excess concentration of MEK over MAPK is required to achieve full activation of MAPK is some what surprising since both enzymes are postulated to exist in a protein kinase-mediated signal transduction cascade in which MEK activates MAPK(1Robbins D.J. Zhen E. Cheng M. Xu S. Ebert D. Cobb M.H. Adv. Cancer Res. 1994; 63: 93-116Crossref PubMed Google Scholar, 2Sturgill T.W. Wu J. Biochim. Biophys. Acta. 1991; 1092: 350-357Crossref PubMed Scopus (328) Google Scholar, 3Davis R.J. J. Biol. Chem. 1993; 268: 14553-14556Abstract Full Text PDF PubMed Google Scholar, 4Crews C.M. Erikson R.L. Cell. 1993; 74: 215-217Abstract Full Text PDF PubMed Scopus (296) Google Scholar). Indeed, many laboratories have observed a potent and rapid activation of both MEK and MAPK in several cell types in response to various agonists(1Robbins D.J. Zhen E. Cheng M. Xu S. Ebert D. Cobb M.H. Adv. Cancer Res. 1994; 63: 93-116Crossref PubMed Google Scholar, 2Sturgill T.W. Wu J. Biochim. Biophys. Acta. 1991; 1092: 350-357Crossref PubMed Scopus (328) Google Scholar, 3Davis R.J. J. Biol. Chem. 1993; 268: 14553-14556Abstract Full Text PDF PubMed Google Scholar, 4Crews C.M. Erikson R.L. Cell. 1993; 74: 215-217Abstract Full Text PDF PubMed Scopus (296) Google Scholar). To investigate the hypothesis that other factors may be required to bring about a more potent phosphorylation and activation of MAPK, we utilized the recombinant constitutively active MEK mutant S218D/S222D MEK1 (30Yan M. Templeton D.J. J. Biol. Chem. 1994; 269: 19067-19073Abstract Full Text PDF PubMed Google Scholar, 31Alessi D.R. Saito Y. Campbell D.G. Cohen P. Sitanandam G. Rapp U. Ashworth A. Marshall C.J. Cowley S. EMBO J. 1994; 13: 1610-1619Crossref PubMed Scopus (464) Google Scholar, 32Pages G. Brunet A. Allemain G.L. Pouyssegur J. EMBO J. 1994; 13: 3003-3010Crossref PubMed Scopus (114) Google Scholar, 33Huang W. Erickson R.L. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8960-8963Crossref PubMed Scopus (131) Google Scholar, 34Mansour S.J. Matten W.T. Hermann A.S. Candia J.M. Rong S. Fukasawa K. Vande Woude G.F. Ahn N.G. Science. 1994; 265: 967-970Crossref Scopus (1254) Google Scholar) to search for factors in rabbit skeletal muscle extracts that would enhance the ability of MEK to phosphorylate and activate MAPK. Using the S218D/S222D MEK1 mutant, rather than native MEK, excluded the possibility of detecting MEK kinases in our assays(23Dent P. Haser W. Haystead T.A.J. Vincent L.A. Roberts T.M. Sturgill T.W. Science. 1992; 257: 1404-1407Crossref PubMed Scopus (496) Google Scholar, 29Reuter C.W. Cattling A.D. 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