Title: Phosphorylation of MAP Kinases by MAP/ERK Involves Multiple Regions of MAP Kinases
Abstract: Mitogen-activated protein (MAP) kinases are activated with great specificity by MAP/ERK kinases (MEKs). The basis for the specific activation is not understood. In this study chimeras composed of two MAP kinases, extracellular signal-regulated protein kinase 2 and p38, were assayed in vitro for phosphorylation and activation by different MEK isoforms to probe the requirements for productive interaction of MAP kinases with MEKs. Experimental results and modeling support the conclusion that the specificity of MEK/MAP kinase phosphorylation results from multiple contacts, including surfaces in both the N- and C-terminal domains. Mitogen-activated protein (MAP) kinases are activated with great specificity by MAP/ERK kinases (MEKs). The basis for the specific activation is not understood. In this study chimeras composed of two MAP kinases, extracellular signal-regulated protein kinase 2 and p38, were assayed in vitro for phosphorylation and activation by different MEK isoforms to probe the requirements for productive interaction of MAP kinases with MEKs. Experimental results and modeling support the conclusion that the specificity of MEK/MAP kinase phosphorylation results from multiple contacts, including surfaces in both the N- and C-terminal domains. Mitogen-activated protein (MAP) 1The abbreviations used are: MAP, mitogen-activated protein; ERK, extracellular signal-regulated protein kinase; MEK, MAP/ERK kinase; MEKK, MEK kinase; PTP1, protein-tyrosine phosphatase, type 1 1The abbreviations used are: MAP, mitogen-activated protein; ERK, extracellular signal-regulated protein kinase; MEK, MAP/ERK kinase; MEKK, MEK kinase; PTP1, protein-tyrosine phosphatase, type 1 kinase or extracellular signal-regulated protein kinase (ERK) cascades are present in all eukaryotes and are utilized in almost all signal transduction pathways originating from receptors at the cell surface (1Lewis T.S. Shapiro P.S. Ahn N.G. Cancer Res. 1998; 74: 49-139Crossref Google Scholar, 2Robinson M.J. Cobb M.H. Curr. Opin. Cell Biol. 1997; 9: 180-186Crossref PubMed Scopus (2274) Google Scholar). A plethora of different stimuli, including growth factors, cytokines, heat shock, and ultraviolet light, can initiate signaling through these cascades. Each cascade consists of a three kinase module: a MAP kinase, a MAP kinase/ERK kinase (MEK) that activates the MAP kinase, and a MEK kinase (MEKK) that activates the MEK (3Neiman A.M. Stevenson B.J. Xu H.-P. Sprague Jr., G.F. Herskowitz I. Wigler M. Marcus S. Mol. Biol. Cell. 1993; 4: 107-120Crossref PubMed Scopus (125) Google Scholar). The MAP kinase in each cascade preferentially phosphorylates substrates with a serine or threonine followed by a proline. There are three mammalian MAP kinase modules that have been extensively studied. These include the ERK1/2 module, the c-Jun N-terminal protein kinase/stress-activated protein kinase module, and the p38 module. ERK3, ERK4, and ERK5 and other p38 isoforms have also been identified, but the cascades leading to activation of these kinases are not well characterized (4Boulton T.G. Cobb M.H. Cell Regul. 1991; 2: 357-371Crossref PubMed Scopus (279) Google Scholar, 5Boulton T.G. Nye S.H. Robbins D.J. Ip N.Y. Radziejewska E. Morgenbesser S.D. DePinho R.A. Panayotatos N. Cobb M.H. Yancopoulos G.D. Cell. 1991; 65: 663-675Abstract Full Text PDF PubMed Scopus (1476) Google Scholar, 6Gonzalez F.A. Raden D.L. Rigby M.R. Davis R.J. FEBS Lett. 1992; 304: 170-178Crossref PubMed Scopus (110) Google Scholar, 7Zhou G. Bao Z.Q. Dixon J.E. J. Biol. Chem. 1995; 270: 12665-12669Abstract Full Text Full Text PDF PubMed Scopus (535) Google Scholar, 8Lee J.-D. Ulevitch R.J. Han J. Biochem. Biophys. Res. Commun. 1995; 213: 715-724Crossref PubMed Scopus (285) Google Scholar, 9Jiang Y. Gram H. Zhao M. New L. Gu J. Feng L. Di Padova F. Ulevitch R.J. Han J. J. Biol. Chem. 1997; 272: 30122-30128Abstract Full Text Full Text PDF PubMed Scopus (430) Google Scholar, 10Li Z. Jiang Y. Ulevitch R.J. Han J. Biochem. Biophys. Res. Commun. 1996; 228: 334-340Crossref PubMed Scopus (350) Google Scholar, 11Lechner C. Zahalka M.A. Giot J.F. Moller N.P. Ullrich A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 4355-4359Crossref PubMed Scopus (274) Google Scholar).Like other protein kinases, the MAP kinases are folded into two domains (12Goldsmith E. Cobb M.H. Curr. Opin. Struct. Biol. 1994; 4: 833-840Crossref PubMed Scopus (63) Google Scholar). The smaller N-terminal domain is composed mostly of β strands, whereas the C-terminal domain is made up of α helices. ATP binds between the two domains, and protein substrate is believed to bind on the surface of the C-terminal domain. Alignment of the amino acid sequences of many protein kinases reveals a common core catalytic domain of 250–300 residues encoding the two-domain structure (13Hanks S.K. Quinn A.M. Hunter T. Science. 1988; 241: 42-52Crossref PubMed Scopus (3782) Google Scholar). Protein kinases possess 12 conserved stretches of amino acids within their catalytic domains known as subdomains (13Hanks S.K. Quinn A.M. Hunter T. Science. 1988; 241: 42-52Crossref PubMed Scopus (3782) Google Scholar, 14Knighton D.R. Zheng J. Ten Eyck L.F. Ashford V.A. Xuong N.-H. Taylor S.S. Sowadski J.M. Science. 1991; 253: 407-413Crossref PubMed Scopus (1437) Google Scholar, 15Hanks S.K. Hunter T. FASEB J. 1995; 9: 576-596Crossref PubMed Scopus (2259) Google Scholar). These conserved elements as well as unique structures contribute to catalysis by and regulation of protein kinases. The functions of several of the conserved and unique structural motifs are known. A glycine-rich loop in the N-terminal domain, termed the phosphate anchor ribbon, has a role in binding ATP. Also in the N-terminal domain, subdomain III encodes the C helix, which contains an invariant Glu involved in binding MgATP (16Knighton D.R. Zheng J. Ten Eyck L.F. Xuong N.-H. Taylor S.S. Sowadski J.M. Science. 1991; 253: 414-429Crossref PubMed Scopus (803) Google Scholar, 17Taylor S.S. Knighton D.R. Zheng J. Sowadski J.M. Gibbs C.S. Zoller M.J. Trends Biochem. Sci. 1993; 18: 84-89Abstract Full Text PDF PubMed Scopus (186) Google Scholar). This helix is important for maintaining an open domain conformation in unphosphorylated ERK2 (18Zhang F. Strand A. Robbins D. Cobb M.H. Goldsmith E.J. Nature. 1994; 367: 704-710Crossref PubMed Scopus (532) Google Scholar), aligning catalytic residues in Src (19Xu W. Harrison S.C. Eck M.J. Nature. 1997; 385: 595-602Crossref PubMed Scopus (1242) Google Scholar) and Cdk2 (20DeBondt H.L. Rosenblatt J. Jancarik J. Jones H.D. Morgan D.O. Kim S.-H. Nature. 1993; 363: 595-602Crossref PubMed Scopus (827) Google Scholar), and binding to the cyclin regulatory subunit in Cdk2 (21Jeffrey P.D. Russo A.A. Polyak K. Gibbs E. Hurwitz J. Massague J. Pavletich N.P. Nature. 1995; 376: 313-320Crossref PubMed Scopus (1208) Google Scholar). The activation loop, known as the phosphorylation lip in MAP kinases, is a poorly conserved element in the C-terminal domain and contains the two MAP kinase phosphorylation site residues within a TXY sequence. Also in the C-terminal domain is the MAP kinase insert. It is composed of 32 residues and is only found in the MAP kinases and the Cdks. The MAP kinase insert and a loop consisting of residues 199–205 of ERK2 interact with the phosphorylation lip in the unphosphorylated form of ERK2. Finally, a loop at the C terminus of the MAP kinases, L16 or the C-terminal tail, wraps around the back of the structure and interacts with the N-terminal domain (18Zhang F. Strand A. Robbins D. Cobb M.H. Goldsmith E.J. Nature. 1994; 367: 704-710Crossref PubMed Scopus (532) Google Scholar).The crystal structures of unphosphorylated and phosphorylated ERK2 and unphosphorylated p38 have been solved (18Zhang F. Strand A. Robbins D. Cobb M.H. Goldsmith E.J. Nature. 1994; 367: 704-710Crossref PubMed Scopus (532) Google Scholar, 22Wang Z. Harkins P.C. Ulevitch R.J. Han J. Cobb M.H. Goldsmith E.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2327-2332Crossref PubMed Scopus (244) Google Scholar, 23Canagarajah B.J. Khokhlatchev A. Cobb M.H. Goldsmith E. Cell. 1997; 90: 859-869Abstract Full Text Full Text PDF PubMed Scopus (616) Google Scholar, 24Wilson K.P. Fitzgibbon M.J. Caron P.R. Griffith J.P. Chen W. McCaffrey P.G. Chambers S.P. Su M.S.S. J. Biol. Chem. 1996; 271: 27696-27700Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar). Several important differences exist between the structures of the unphosphorylated forms of ERK2 and p38. There is a wider domain separation in p38 than in ERK2. The phosphorylation lip in p38 is six residues shorter and has a different conformation. In p38 the lip is folded up between the two domains, and the C-terminal portion of the lip forms a turn of helix that blocks the P+1 specificity pocket. This pocket directs the proline specificity of MAP kinases. The axis of the C helix is rotated, and the N terminus of this helix is shifted by 6 Å relative to the helix in ERK2. The helix at the end of L16 is extended by 7 Å, and there is an increase in the hydrophilicity of residues that form contacts between L16 and the N-terminal domain. This results in a less intimate interaction between L16 and the N-terminal domain in p38 (22Wang Z. Harkins P.C. Ulevitch R.J. Han J. Cobb M.H. Goldsmith E.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2327-2332Crossref PubMed Scopus (244) Google Scholar, 24Wilson K.P. Fitzgibbon M.J. Caron P.R. Griffith J.P. Chen W. McCaffrey P.G. Chambers S.P. Su M.S.S. J. Biol. Chem. 1996; 271: 27696-27700Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar).Within MAP kinase cascades, the MEKs are the most specific enzymes. These dual specificity kinases activate their respective MAP kinase substrates by phosphorylating the threonine and tyrosine of the specific TXY sequence located in the phosphorylation lip. The only known substrates of MEK1 and MEK2 are ERK1 and ERK2 (25Seger R. Ahn N.G. Posada J. Munar E.S. Jensen A.M. Cooper J.A. Cobb M.H. Krebs E.G. J. Biol. Chem. 1992; 267: 14373-14381Abstract Full Text PDF PubMed Google Scholar). Other MEKs also phosphorylate only a small subset of the MAP kinase family. For example, MEK3 and MEK6 will only phosphorylate p38 isoforms, whereas MEK5 will only phosphorylate ERK5 (26Dérijard B. Raingeaud J. Barrett T. Wu I.-H. Han J. Ulevitch R.J. Davis R.J. Science. 1995; 267: 682-685Crossref PubMed Scopus (1404) Google Scholar, 27Stein B. Brady H. Yang M.X. Young D.B. Barbosa M.S. J. Biol. Chem. 1996; 271: 11427-11433Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar, 28Kato Y. Kravchecnko V.V. Tapping R.I. Han J. Ulevitch R.J. Lee J.D. EMBO J. 1997; 16: 7054-7066Crossref PubMed Scopus (491) Google Scholar). In addition, the MEKs require native MAP kinases as substrates; they will not phosphorylate denatured proteins or peptides derived from the phosphorylation lip (25Seger R. Ahn N.G. Posada J. Munar E.S. Jensen A.M. Cooper J.A. Cobb M.H. Krebs E.G. J. Biol. Chem. 1992; 267: 14373-14381Abstract Full Text PDF PubMed Google Scholar). Little is known about the structural basis of the specificity of interactions between MEKs and MAP kinases despite information from the crystal structures of ERK2 and p38.Previous in vitro studies concluded that neither the phosphorylation lip length nor the residue between the phosphorylation sites is a critical element for directing MEK specificity (29Robinson M.J. Cheng M. Khokhlatchev A. Ebert D. Ahn N. Guan K.-L. Stein B. Goldsmith E. Cobb M.H. J. Biol. Chem. 1996; 271: 29734-29739Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 30Jiang Y. Li Z. Schwarz E.M. Lin A. Guan K. Ulevitch R.J. Han J. J. Biol. Chem. 1998; 272: 11096-11102Abstract Full Text Full Text PDF Scopus (74) Google Scholar). Brunet and Pouysségur (31Brunet A. Pouysségur J. Science. 1996; 272: 1653-1655Crossref Scopus (118) Google Scholar) measured the in vivoactivities of chimeras of p38 and ERK1 after treatment of transfected cells with stimuli that activated either p38 or ERK1. Based on these findings they proposed that a 40-residue stretch of amino acids within subdomains III and IV of p38 was important for directing the specificity of activation. In addition, they suggested that the C helix within subdomain III was the key element for recognition of MAP kinases by MEKs.To define structural elements of the MAP kinases that are required to direct specific recognition by MEKs, chimeras of ERK2 and p38 were tested for phosphorylation and activation by five different MEKsin vitro. Compilation of the data reveals multiple spatially segregated contacts in the MEK/MAP kinase interface.RESULTSMEKs recognize the native MAP kinase structure with considerable selectivity. The primary sequence surrounding the TXY phosphorylation sites of the ERKs is not sufficient to determine MEK recognition. Neither peptides derived from the double phosphorylation site nor denatured proteins are phosphorylated by MEKs (25Seger R. Ahn N.G. Posada J. Munar E.S. Jensen A.M. Cooper J.A. Cobb M.H. Krebs E.G. J. Biol. Chem. 1992; 267: 14373-14381Abstract Full Text PDF PubMed Google Scholar). These findings support the conclusion that MEKs require specific conformations or secondary determinants for productive interaction with their MAP kinase substrates. We wished to identify the structural features of MAP kinases that allow MEKs to distinguish among them as substrates. To do this we created chimeras between ERK2 and p38 that contained selected intact structural elements from each enzyme (Fig.1 A). The chimeras were composed of ERK2 and p38 because a related set of chimeras had been tested in transfected cells (31Brunet A. Pouysségur J. Science. 1996; 272: 1653-1655Crossref Scopus (118) Google Scholar), and the structures of both of these MAP kinases were available for analysis (18Zhang F. Strand A. Robbins D. Cobb M.H. Goldsmith E.J. Nature. 1994; 367: 704-710Crossref PubMed Scopus (532) Google Scholar, 22Wang Z. Harkins P.C. Ulevitch R.J. Han J. Cobb M.H. Goldsmith E.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2327-2332Crossref PubMed Scopus (244) Google Scholar, 24Wilson K.P. Fitzgibbon M.J. Caron P.R. Griffith J.P. Chen W. McCaffrey P.G. Chambers S.P. Su M.S.S. J. Biol. Chem. 1996; 271: 27696-27700Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar). The chimeras were expressed and purified and then tested in vitro for activation by MEK family members, MEKs 1, 2, 3, 4, and 6, which phosphorylate ERK2 or p38 in vitro. Measurements included rate and extent of phosphorylation, phosphoamino acids, recognition by antibodies selective for phosphorylated ERK2, and activated activity relative to the wild type proteins. ERK2 and p38 proteins containing inserted restriction sites were also purified and tested as controls.The MAP kinase catalytic core contains two domains. The N-terminal domain is composed of N-terminal sequence (white andred in Fig. 1 B) and the C-terminal tail, L16 (yellow in Fig. 1 B), and is primarily involved in binding ATP. In the MAP kinases L16 lies on the surface of the N-terminal domain. The C-terminal domain (blue in Fig.1 B) binds the protein substrate. To identify important structural features, we first created a chimera, PIVECTP, which contained one of its two major structural domains from each kinase (Fig. 1). This chimera contained the larger C-terminal structural domain from ERK2 and the smaller N-terminal domain from p38. The p38 elements making up the N-terminal domain are the N terminus through subdomain IV and L16 (shown in white, red, andyellow in Fig. 1 B).We examined phosphorylation of this chimera by MEKs, the amino acids phosphorylated, and its activity after phosphorylation. This chimera was phosphorylated by MEK1 and MEK2 and by MEK3, MEK4, and MEK6, although the stoichiometry of phosphorylation by MEK3/4/6 was lower (Fig. 2). The rates of phosphorylation of the chimera by MEK2 and MEK1 were similar to the rates of phosphorylation of wild type ERK2 by these MEKs (see Figs.5 A and 7 A). Likewise, the rates of phosphorylation of this chimera by MEK6 and MEK3 were similar to their rates of phosphorylation of p38 (see Figs. 6 A and7 B). In each case, both tyrosine and threonine were phosphorylated (see Fig. 5 C and 6 C). These results indicated that interaction determinants were present in both domains. All five MEKs activated PIVECTP, and in each case the activity was proportional to the extent of phosphorylation, with MEK4 being the least effective (Figs. 2 and 3). The activity of this unphosphorylated chimera was similar to that of wild type ERK2. Maximal activity was about 15% of the activity of wild type ERK2 (Fig. 3). This may be due to imperfect folding of the chimera.Figure 2Phosphorylation of chimeras by MEK isoforms. Relative stoichiometry of phosphate incorporation into ERK2, PIVECTP, PIVE, PIIE, EIIPIVE, EIIP, and p38 by the indicated MEKs. Data for each MEK are expressed relative to stoichiometry of phosphorylation of ERK2 by MEK1 and MEK2 and of p38 by MEK3, MEK4, and MEK6. Data are the means ± S.E. of three to five independent experiments.View Large Image Figure ViewerDownload (PPT)Figure 7Time course of phosphorylation and activation of chimeras by MEK1, MEK3, and MEK4. A, phosphate incorporation into ERK2 (●), PIIE (▴), PIVE (▪), PIVECTP (♦), and EIIPIVE (▾) after incubation with MEK1 for 5, 10, 15, 20, 30, 60, 90, 120, 180, and 240 min. B, phosphate incorporation into p38 (●) and PIVECTP (▴) after incubation with MEK3 for the times listed in A. C, phosphate incorporation into p38 (●) and EIIP (▴) after incubation with MEK4 for the times listed inA.View Large Image Figure ViewerDownload (PPT)Figure 3Kinase activity of chimeras after phosphorylation by MEK isoforms. Relative myelin basic protein kinase activity of ERK2, PIVECTP, PIVE, PIIE, EIIPIVE, and EIIP after phosphorylation by the indicated MEKs. Data are expressed relative to specific activity of ERK2 after phosphorylation by MEK2, set to 1. Data are the means ± S.E. of three to five independent experiments.View Large Image Figure ViewerDownload (PPT)Inclusion of residues from p38 in the N-terminal domain of the chimera allowed phosphorylation by MEK3/6 in addition to MEK1/2. We next wanted to determine which structural motifs within the N-terminal domain were responsible for this expanded interaction. Therefore, three additional chimeras containing less of the N-terminal domain of p38 were constructed and analyzed as described next.To determine the contribution of L16 (yellow in Fig.1 B) to phosphorylation by MEK, the PIVE chimera, which had all of the N-terminal domain except L16 from p38, was generated. It contained p38 residues from the N terminus through subdomain IV and ERK2 residues from subdomain V through the C terminus (Fig.1 A). Its activity in the unphosphorylated state was similar to wild type ERK2. This chimera was phosphorylated by MEK1, MEK2, and MEK6 but not by MEK3 or MEK4 (Fig. 2). All three MEKs that phosphorylated PIVE activated it equally (Fig. 3). It had stimulated protein kinase activity about 20% of that of ERK2. The rates of phosphorylation of PIVE by MEK1, MEK2, and MEK6 were similar to the rates of phosphorylation of the wild type proteins (see Figs.5 A, 6 A, and 7 A). These results suggested that the C-terminal tail within the N-terminal domain contributed to activation by MEK3.EIIPIVE had only p38 subdomains III and IV, which contained the C helix and β strands 4 and 5 (red in Fig. 1 B). Without phosphorylation by MEK, this chimera had slightly higher activity than wild type ERK2. EIIPIVE was phosphorylated and activated by MEK1 and MEK2 (Figs. 2 and 3). When phosphorylated, this chimera demonstrated decreased activity relative to wild type ERK2 (Fig. 3). MEK3, MEK4, and MEK6 did not phosphorylate it. Therefore, the C helix, β4, and β5 of MAP kinases were not sufficient for phosphorylation of a MAP kinase by these MEKs. EIIPIVE also was phosphorylated by MEK1 and MEK2 at rates similar to the rates of phosphorylation of ERK2 (see Figs.5 A and 7 A).To determine the role of the first two subdomains (white in Fig. 1 B) in phosphorylation by MEKs, PIIE was tested. This chimera contained p38 residues in the N terminus through subdomain II and ERK2 residues in subdomain III through the C terminus (Fig.1 A). This chimera had activity comparable with that of unphosphorylated ERK2. Once again, only MEK1 and MEK2 phosphorylated and activated this chimera (Figs. 2 and 3). The rates of phosphorylation of PIIE by MEK1 and MEK2 were similar to the rates of phosphorylation of wild type ERK2 by MEK1 and MEK2 (see Fig.5 A and 7 A). Results from these four chimeras indicated that many p38 residues must be present in the N-terminal domain of each chimera for phosphorylation by the p38-specific MEKs.We wanted to determine whether any differences existed in the pattern of phosphorylation and activation by MEKs if the other MAP kinase was at the N terminus of the chimera. Thus, we expressed a reciprocal chimera, EIIP. EIIP contained ERK2 residues from the N terminus through subdomain II (white in Fig. 1 B) and p38 residues from subdomain III through the C terminus. This chimera was phosphorylated by MEK4 and MEK6 but not by MEK1, MEK2, or MEK3 (Fig.2). The time courses of phosphorylation were slightly more rapid (see Fig. 7 C). This chimera had very low basal activity, less than 1% of wild type ERK2, suggesting that it was poorly folded. After phosphorylation, this chimera was inactive (Fig. 3). Analysis of its phosphoamino acids after phosphorylation by MEK4 revealed that it was inactive in part because there was little or no threonine phosphorylated (Fig. 4). Domains I and II of ERK2 alter the position of the C helix in the chimera, which may account for the inability of MEKs to phosphorylate threonine.Figure 4Phosphoamino acid analysis. Phosphoamino acid analysis of p38 and p38II after incubation with MEK3 and EIIP after incubation with MEK4. The positions of the three phosphoamino acid standards are indicated.View Large Image Figure ViewerDownload (PPT)The two control proteins were also tested in the in vitrokinase assays to determine whether any residues changed by the insertion of the restriction sites caused the changes in interaction with MEKs. The ERK2 control, ERK2II→CT was phosphorylated and activated only by MEK1 and MEK2 (data not shown). Therefore, residues that were changed by the insertion of restriction sites did not affect the productive interaction of ERK2 chimeras with MEKs. The p38 control, p38II, was phosphorylated by MEK3, MEK4, and MEK6 (data not shown). p38II was activated normally by MEK4/6 but less well by MEK3 (data not shown). The phosphoamino acid analysis (Fig. 4) indicates that p38II is not fully activated by MEK3 because it is poorly phosphorylated on threonine.The maximum stoichiometries of phosphorylation of the chimeras by MEKs 1–4 and 6 were evaluated (Table I) and were consistent with the extents of activation of the chimeras. With the exception of EIIP, the time courses of phosphorylation of the chimeras by individual MEK family members were similar to their rates with their normal substrates, ERK2 or p38 (Figs.Figure 5, Figure 6, Figure 7).Table IMaximum stoichiometry of phosphorylation by MEKsERK2p38PIIEPIVEPIVECTPEIIPEIIPIVEAlone0.040.010.020.130.020.000.17MEK11.350.030.612.120.470.000.75MEK22.450.060.892.690.910.021.16MEK30.051.360.020.110.180.030.12MEK40.052.680.040.150.081.380.15MEK60.061.450.070.880.280.460.08Maximum stoichiometry of phosphorylation of ERK2, p38, and each chimera is listed as mol PO4/mol MAP kinase. Reactions were performed at 30 °C for 1 h. There was no significant increase in the stoichiometry of phosphorylation at longer times. Data are representative of four or five experiments for each MAP kinase. Open table in a new tab To determine whether phosphorylation occurred on the predicted residues (Thr183 and Tyr185), chimeras phosphorylated by MEKs for different times were immunoblotted with antibodies that selectively recognize the doubly phosphorylated ERK2 epitope. These antibodies do not detect unphosphorylated or singly phosphorylated ERK2 at equivalent protein concentrations. The immunoblots reveal an excellent parallel between increasing phosphorylation and appearance of the phospho epitope (Figs. 5 and 6). Phosphorylation of the chimeras saturated at the same times as did blotting intensity. This is consistent with the conclusion that phosphorylation occurred largely on Thr183 and Tyr185 and not on previously unidentified residues.These immunoblotting findings agree with phosphoamino acid analysis, which showed the majority of phosphate on tyrosine and threonine. However, PIVECTP, for example, apparently contained less phosphotyrosine in these analyses than did wild type ERK2. To determine whether this was due to prior autophosphorylation of the chimera, we compared the stoichiometry of phosphorylation of wild type ERK2 and three of the chimeras before and after dephosphorylation with the tyrosine phosphatase PTP1 (Fig.8 A). The stoichiometry of phosphorylation of ERK2 was unchanged by pretreatment with phosphatase; on the other hand, phosphorylation of two of the chimeras increased by 20–30%. The increase was primarily due to increased tyrosine phosphorylation as was apparent from the increase in phosphotyrosine recovered in the phosphoamino acid analysis (Fig. 8, B andC). This is consistent with the idea that the chimeras have enhanced abilities to autophosphorylate on Tyr185 prior to isolation from bacteria. Because this tyrosine is already partly phosphorylated when the proteins are purified, less phosphate can be transferred to tyrosine by MEKs.Figure 8Autophosphorylation accounts for decreased tyrosine phosphorylation of PIVE, PIVECTP, and EIIPIVE by MEK2. A, relative stoichiometry of phosphate incorporation by MEK2 into ERK2, PIVE, PIVECTP, and EIIPIVE without or with pretreatment with PTP1. Data are expressed as the means ± S.E. for three independent experiments. B, phosphoamino acid analysis of ERK2, PIVE, PIVECTP, and EIIPIVE not dephosphorylated with PTP1.C, phosphoamino acid analysis of ERK2, PIVE, PIVECTP, and ERK2IIPIVE first dephosphorylated with PTP1. For B andC, the positions of the three phosphoamino acid standards are indicated.View Large Image Figure ViewerDownload (PPT)DISCUSSIONMEK isoforms recognize members of the MAP kinase family with selectivity. The residues or structural motifs that are required for this selective recognition have not been defined. ERK1/2 and p38 isoforms share about 40–45% sequence identity (5Boulton T.G. Nye S.H. Robbins D.J. Ip N.Y. Radziejewska E. Morgenbesser S.D. DePinho R.A. Panayotatos N. Cobb M.H. Yancopoulos G.D. Cell. 1991; 65: 663-675Abstract Full Text PDF PubMed Scopus (1476) Google Scholar, 36Han J. Lee J.-D. Bibbs L. Ulevitch R.J. Science. 1994; 265: 808-811Crossref PubMed Scopus (2401) Google Scholar, 37Zheng C.F. Ohmichi M. Saltiel A.R. Guan K.-L. Biochemistry. 1994; 33: 5595-5599Crossref PubMed Scopus (41) Google Scholar), and the structures of the unphosphorylated forms of ERK2 and p38 are similar (18Zhang F. Strand A. Robbins D. Cobb M.H. Goldsmith E.J. Nature. 1994; 367: 704-710Crossref PubMed Scopus (532) Google Scholar, 22Wang Z. Harkins P.C. Ulevitch R.J. Han J. Cobb M.H. Goldsmith E.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2327-2332Crossref PubMed Scopus (244) Google Scholar). The goal of this project was to use chimeras containing swapped structural elements of ERK2 and p38 to identify regions of the MAP kinases that direct interactions with particular MEKs.Our findings using ERK2/p38 chimeras indicate that no single MAP kinase structural motif forms a sufficient interaction surface with MEK. The N-terminal structural domain likely forms part of the interface. However, even the entire domain is not sufficient to restrict phosphorylation by MEKs, because the chimera containing the N-terminal domain from p38 was phosphorylated by MEK1 and MEK2 in addition to MEK3/4/6. This demonstrates that there are interaction determinants in the C-terminal domain. In addition, a fairly large surface area within the N-terminal domain must be present for the interaction of the chimeras with MEK3/4/6. Swapping only subdomains I and II or subdomains III and IV was not enough to allow MEK3/4/6 to phosphorylate the chimeras. Based on these data, it appears that MEKs require multiple interacting sites within both domains to specifically phosphorylate different MAP kinases.Our results with ERK2/p38 chimeras are in part consistent with work from Brunet and Pouysségur (31Brunet A. Pouysségur J. Science. 1996; 272: 1653-1655Crossref Scopus (118) Google Scholar), who expressed p38/ERK1 chimeras in mammalian cells. A p38/ERK1 chimera equivalent to PIVE was activated by both growth factors and cell stress, whereas a chimera equivalent to PIIE was only activated by growth factors. We have shown with further subdivision of the swapped regions that in contrast to the suggestion from the p38/ERK1 studies, the C helix alone is not the key element determining which MEKs will phosphorylate a particular MAP kinasein vitro.Additional findings suggest that the MAP kinase insert is required for phosphorylation by MEKs. To address the function of this insert, it was deleted from wild type ERK2. 2J. L. Wilsbacher and M. H. Cobb, unpublished results. Examination of the crys