Kinetic mechanism for p38 MAP kinase a A partial rapid-equilibrium random-order ternary-complex mechanism for the phosphorylation of a protein substrate Anna E Szafranska1 and Kevin N Dalby1,2 Division of Medicinal Chemistry, University of Texas at Austin, TX, USA Graduate Programs in Biochemistry and Molecular Biology and the Center for Molecular and Cellular Toxicology, University of Texas at Austin, TX, USA Keywords docking; inhibition; kinetic mechanism; MAP kinase; p38 MAPK Correspondence K N Dalby, Division of Medicinal Chemistry, College of Pharmacy, University of Texas at Austin, TX 78712, USA Fax: +1 512 232 2606 Tel: +1 512 471 9267 E-mail: Dalby@mail.utexas.edu (Received 28 February 2005, revised 18 May 2005, accepted 20 June 2005) doi:10.1111/j.1742-4658.2005.04827.x p38 Mitogen-activated protein kinase alpha (p38 MAPKa) is a member of the MAPK family It is activated by cellular stresses and has a number of cellular substrates whose coordinated regulation mediates inflammatory responses In addition, it is a useful anti-inflammatory drug target that has a high specificity for Ser-Pro or Thr-Pro motifs in proteins and contains a number of transcription factors as well as protein kinases in its catalog of known substrates Fundamental to signal transduction research is the understanding of the kinetic mechanisms of protein kinases and other protein modifying enzymes To achieve this end, because peptides often make only a subset of the full range of interactions made by proteins, protein substrates must be utilized to fully elucidate kinetic mechanisms We show using an untagged highly active form of p38 MAPKa, expressed and purified from Escherichia coli [Szafranska AE, Luo X & Dalby KN (2005) Anal Biochem 336, 1–10) that at pH 7.5, 10 mm Mg2+ and 27 °C p38 MAPKa phosphorylates ATF2D115 through a partial rapid-equilibrium randomorder ternary-complex mechanism This mechanism is supported by a combination of steady-state substrate and inhibition kinetics, as well as microcalorimetry and published structural studies The steady-state kinetic experiments suggest that magnesium adenosine triphosphate (MgATP), adenylyl (b,c-methylene) diphosphonic acid (MgAMP-PCP) and magnesium adenosine diphosphate (MgADP) bind p38 MAPKa with dissociation constants of KA ¼ 360 lm, KI ¼ 240 lm, and KI > 2000 lm, respectively Calorimetry experiments suggest that MgAMP-PCP and MgADP bind the p38 MAPKa–ATF2D115 binary complex slightly more tightly than they the free enzyme, with a dissociation constant of Kd  70 lm Interestingly, MgAMP-PCP exhibits a mixed inhibition pattern with respect to ATF2D115, whereas MgADP exhibits an uncompetitive-like pattern This discrepancy occurs because MgADP, unlike MgAMP-PCP, binds the free enzyme weakly Intriguingly, no inhibition by mm adenine or mm MgAMP was detected, suggesting that the presence of a b-phosphate is essential for significant binding of an ATP analog to the Abbreviations ATF2D115, glutathione S-transferase fusion protein of activating transcription factor residues 1–115; ERK, extracellular signal-regulated kinase; ITC, isothermal titration calorimetry; JNK, Jun N-terminal kinase; MAPK, mitogen-activated protein kinase; MgADP, magnesium adenosine diphosphate; MgAMP-PCP, adenylyl (beta,gamma-methylene) diphosphonic acid; MgATP, magnesium adenosine triphosphate; MKK3, MAP kinase kinase 3; MKK6, MAP kinase kinase 6; MKP3, MAP kinase phosphatase; NADH, nicotinamide adenine dinucleotide; p38 MAPKa, p38 mitogen-activated protein kinase alpha FEBS Journal 272 (2005) 4631–4645 ª 2005 FEBS 4631 Kinetic mechanism for p38 MAP kinase a A E Szafranska and K N Dalby enzyme Surprisingly, we found that inhibition by the well-known p38 MAPKa inhibitor SB 203580 does not follow classical linear inhibition kinetics at concentrations > 100 nm, as previously suggested, demonstrating that caution must be used when interpreting kinetic experiments using this inhibitor All organisms, from bacteria and yeasts to mammalian cells, respond to stimuli from the extracellular environment Incoming signals are sent via a cascade of proteins and enzymes from the surface of cells to their interior, causing alterations in gene expression and protein activity These, in turn, generate cellular responses, such as growth, differentiation, inflammation and apoptosis In eukaryotic cells, the mitogenactivated protein kinase (MAPK) module is a key element in the propagation, amplification and transport of extracellular signals to the nucleus [1] The MAPK superfamily includes the extracellular signalregulated kinases (ERKs), the Jun N-terminal kinases (JNKs) and the p38 MAP kinases, among others These enzymes are terminal components of three-tiered MAPK modules, each of which consists of a MAP kinase (MAPK), a MAPK kinase (MAPKK) and a MAPKK kinase (MAPKKK) MAPK modules operate in numerous biological settings where, through largely unknown mechanisms, multiple components impinge on a particular MAPKKK [1] In recent years there has been substantial interest in MAPKs due to their participation in numerous biological pathways and various human conditions and diseases One notable MAPK is p38 MAPKa whose activity has been associated with diseases such as cancer [2] or those with inflammatory components [3–5] p38 MAPKa is phosphorylated on Tyr180 and Thr182 by the upstream activators MAP kinase kinase (MKK3) and MAP kinase kinase (MKK6) Once activated, p38 MAPKa exerts its effect by directly phosphorylating transcription factors such as activating transcription factor (ATF2) and MEF2, or indirectly by activating downstream protein kinases such as MAPKAP-K2 and MAPKAP-K3, which in turn phosphorylate their own substrates [1] Despite a wealth of biological information, there are many unsolved issues concerning this and other MAPK signaling cascades Within the past decade, four isoforms of p38 MAPK termed a, b, c and d have been discovered, whose precise biological roles remain to be defined [1] Notably, the a and b isoforms are inhibited by the classic family of pyridinyl inhibitors related to SB 203580, whereas the c and d isoforms are not Thus, use of SB 203580, which has been the main 4632 pharmacological tool employed to date, is transparent to two of the p38 MAPK isoforms Although a number of structural studies have been reported, showing for example, inactive p38 MAPKa with and without inhibitors bound at the ATP site [6–12], the structure of an enzyme–substrate complex is notably lacking Although a number of mutagenesis studies have mapped sites of protein–protein interaction, the basis for and extent of the differences in specificity within the p38 MAPK family are still poorly understood Thus, we have no clear picture of how p38 MAPKs recognize protein substrates, or how this recognition is regulated in vivo Furthermore, we not know how cellular proteins such as scaffold proteins interact with p38 MAPK isoforms, how these interactions are regulated, how they interplay with catalysis, how they may be exploited therapeutically or how they differ within the family There is currently a lot of interest in understanding the molecular recognition events associated with MAPKs, because docking domains are thought to play a major role in determining the specificity of substrate–ligand and protein–ligand interactions [13–15] A growing number of enzymes are thought to utilize docking domains, which are substrate recognition elements lying outside the active site of the enzyme and which govern the formation of an enzyme–substrate complex [16–23] Several years ago, we showed that despite the presence of docking domains on p38 MAPKa, which could tether a protein substrate and facilitate multiple phosphorylations in one collision, p38 MAPKa phosphorylates ATF2D115 on Thr69 and Thr71 in a nonprocessive manner [24] Prior to this study, LoGrasso et al reported that p38 MAPKa phosphorylates ATF2D115 via a compulsory-order ternary-complex mechanism, in which the binding of ATF2D115 must precede that of magnesium ATP (MgATP) (Scheme 1B) [25] This possibility is intriguing because: (a) the proposed mechanism would appear to require novel communication between the enzyme and substrates to ensure that p38 MAPKa exclusively binds ATF2D115 before MgATP; and (b) such properties might be due to the employment of docking domains in substrate recognition However, the proposal of LoGrasso et al was challenged in a report that FEBS Journal 272 (2005) 4631–4645 ª 2005 FEBS A E Szafranska and K N Dalby Kinetic mechanism for p38 MAP kinase a mechanism We also show that nucleotides such as MgATP and particularly magnesium ADP (MgADP) bind preferentially to the binary p38 MAPKa– ATF2D115 complex, whereas no binding of magnesium AMP (MgAMP) or adenine was detected to any enzyme form This study provides the basis for the design of further structure ⁄ function and transient kinetic studies aimed at defining the kinetic mechanism and physical properties of p38 MAPKa in detail Results Steady-state kinetics Scheme (A) Random-order ternary-complex mechanism, (B) compulsory-order ternary-complex mechanism (ATF2D115 binds first, ATP second), (C) Compulsory-order ternary-complex mechanism (ATP binds first, peptide second) asserted that p38 MAPKa must bind MgATP before it binds a peptide substrate (Scheme 1C) [26] Recently, we established a new protocol for the preparation of recombinant murine p38 MAPKa [27], whose activity towards ATF2D115 is some 10-fold greater than previously reported [25] Given the availability of a highly active untagged form of p38 MAPKa, the potential novelty of its docking domaindependent substrate recognition, the uncertainty of it kinetic mechanism and the interest in the development of protein–protein interaction inhibitors, we decided to reinvestigate its kinetic mechanism using ATF2D115 as the substrate We describe a steady-state kinetic investigation of untagged p38 MAPKa and report that rather than following a compulsory-order ternarycomplex mechanism, as previously reported [25], p38 MAPKa phosphorylates ATF2D115 via a partial rapid-equilibrium random-order ternary-complex FEBS Journal 272 (2005) 4631–4645 ª 2005 FEBS Murine p38 MAPKa was expressed in Escherichia coli, purified and fully activated by constitutively active MKK6b according to the method of Szafranska and Dalby [27] (Fig 1) This preparation corresponds to the highest reported activity against ATF2D115 for this enzyme [26] To examine the propensity of p38 MAPKa to form a functional binary complex with MgATP, the ATPase activity of the enzyme was assessed In line with a previous report, p38 MAPKa displayed robust ATPase activity in the presence of 10 mm Mg2+ at pH 7.6 (kcat ¼ 0.3 s)1 and Km ¼ 353 lm) [26] The simplest mechanism accounting for the ATP hydrolysis is shown in Scheme 2A According to this mechanism, MgATP reversibly binds p38 MAPKa in the active site to form the binary complex MgATP (ka) This binding renders it susceptible to nucleophilic attack by hydroxyl nucleophiles, leading to the nucleophilic addition of a water molecule to the c-phosphoryl group of MgATP (kp), and the formation of MgADP and inorganic phosphate (Pi) These products then dissociate (kdiss) from the active site Given the slow turnover (kcat ¼ 0.3 s)1) for the hydrolysis reaction, and the relatively large Michaelis– Menten constant for MgATP, we assume a rapid-equilibrium mechanism where Km ¼ k–a ⁄ ka ¼ 353 lm A conservative estimate for the second-order rate constant of ka ẳ 104 m)1ặs)1 for the binding of MgATP to p38 MAPKa gives a rate constant for the dissociation of MgATP from the enzyme of k-a ¼ 3.5 s)1, if the dissociation constant KA ¼ 350 lm is used This value exceeds kcat by one order of magnitude, supporting the rapid-equilibrium assumption The ability of p38 MAPKa to bind MgATP and facilitate the nucleophilic attack of a water molecule with a turnover of 0.3 s)1, which is only fourfold lower than the turnover of ATF2D115 (see below), supports the notion that the MgATP complex is not a deadend complex with respect to the binding and phos4633 Kinetic mechanism for p38 MAP kinase a A E Szafranska and K N Dalby Fig Preparation of activated p38 MAPKa and ATF2D115 (A) 10% SDS ⁄ PAGE analysis showing activated, p38 MAPKa (lane 1) and its MS analysis (Mr 41 731 Da observed; 41 726 Da calculated) (B) 12% SDS ⁄ PAGE showing ATF2D115 (lane 1) and its MS analysis (Mr 39 658 Da observed; 39 650 Da calculated) Scheme (A) Mechanism of ATP hydrolysis by p38 MAPKa (B) Competitive inhibition of ATP hydrolysis with I dead-end complex phorylation of ATF2D115 Given the binding mode adopted by peptide substrates for a number of protein kinases, it is reasonable to assume that a protein substrate can bind productively to a preformed MgATP complex Thus, as pointed out by Chen et al [26], the robust ATPase activity exhibited by p38 MAPKa sheds some doubt on the compulsory-order ternarycomplex mechanism proposed by LoGrasso et al [25] We expressed and purified the glutathione S-transferase (GST) fusion protein of the N-terminal 115 residues of the transcription factor ATF2 (ATF2D115) essentially as described previously [25], with some minor modifications (Fig 1) [27] Having established 4634 the kinetic competence of the MgATP complex (with respect to nucleophilic attack by water), we conducted initial rate studies at various concentrations of ATF2D115 and MgATP Reciprocal plots of initial rate versus the concentration of ATF2D115 (Fig 2A) or ATP (Fig 2B) revealed an intersecting pattern of lines (> ⁄ v ¼ 0), indicative of a sequential kinetic mechanism, in which both substrates must bind to form a ternary complex before catalysis occurs Previously, we showed that ATF2D115 is phosphorylated twice by p38 MAPKa on Thr69 and Thr71 in a nonprocessive manner and that under initial rate conditions, only the mono-phosphorylated forms of ATF2D115 are produced at equal rates [24] Our results differ in two significant aspects from those previously reported for flag-tagged p38 MAPKa [25] First, in our case the double-reciprocal plots intersect above the x-axis (compared with below the x-axis for the flag-tagged enzyme) Second, the reported catalytic constant towards ATF2D115 is some 10-fold higher It is conceivable that these differences in activity reflect the presence of an N-terminal flag tag and ⁄ or the method by which the enzymes were overexpressed, activated and purified In our case a sensitive tryptic analysis indicates that the enzyme was fully activated [27] v Vmax ẳ AB aKA KB ỵ aKB A ỵ aKA B ỵ AB 1ị The rapid equilibrium assumption is a powerful approach used to simplify the analysis of enzyme mechanisms and for a ternary-complex mechanism it provides a good approximation to the reaction pathway when ligand-binding events are fast compared FEBS Journal 272 (2005) 4631–4645 ª 2005 FEBS FEBS Journal 272 (2005) 4631–4645 ª 2005 FEBS – 0.021 ± 0.001b – 0.021 ± 0.001b 241.5 ± 13 8.6 ± 0.5 – > 2000 UC C M C ND C C AMP-PCP 0.3 ± 0.01 – – a Lower estimate b KI, and bKI set equal – 353 ± 52 ATP ATPase SB 203580 AMP-PCP ATF2D115 ATP ATF2D115 ATP ATF2D115 ATP ATP ADP 1.1 ± 0.03 1.4 ± 0.1 38.6 ± 13.4 ± 15 ATF2D115 (B), ATP (A) Kinase 360 ± 17 Inhibitor kcat, (s ) aKB (lM) KB (lM) aKA (lM) KA (lM) 187.4 ± 37 9.7 ± 0.5 bKI (lM) a KI (lM) Inhibition constants Inhibition pattern Varied substrate Substrates with the interconversion of the central substrate and product complexes The lines in Fig represent the best fit of the experimental data to Eqn (1), which describes a rapid-equilibrium random-order ternarycomplex mechanism (Scheme 1A), according to the parameters shown in Table According to this fit, p38 MAPKa binds both substrates in the mid micromolar range [KB ¼ 39 lm (ATF2D115); KA ¼ 360 lm (MgATP)] to form the respective binary complexes We reasoned that with ligand binding to p38 MAPKa occurring in the micromolar range and a relatively low Activity Fig Two-substrate dependence kinetic analysis of p38 MAPKa (A) Double-reciprocal plot of ⁄ v versus ⁄ ATF2D115 at five fixed ATP concentrations (m, 12 lM; n, 25 lM; r, 50 lM; d, 100 lM; , 200 lM) (B) Double reciprocal plot of ⁄ v versus ⁄ ATP at five fixed ATF2D115 concentrations (., 2.5 lM; n, lM; r, 10 lM; m, 20 lM; d, 40 lM) Solid lines are the best fit through the experimental data to Eqn (1) )1 B Substrate dependence constants (lM) A Kinetic mechanism for p38 MAP kinase a Table Kinetic constants for p38 MAPKa obtained from two-substrate steady-state kinetics, and ATPase activity and inhibition studies C, competitive; UC, uncompetitive; M, mixed; ND, not determined A E Szafranska and K N Dalby 4635 Kinetic mechanism for p38 MAP kinase a catalytic constant of kcat ¼ 1.2 s)1 for the phosphorylation of ATF2D115, the rapid equilibrium assumption is likely to provide a reasonable description of the reaction mechanism and could be used to distinguish between several mechanistic possibilities For example, the rate-constant for the association of MgATP with a protein kinase is typically of the order of 105)106 m)1Ỉs)1, which, given a typical dissociation constant of 10 lm for MgATP, indicates a rate constant for MgATP dissociation of 1–10 s)1, which is at least as fast as the observed kcat Accordingly, we noted that the pattern of intersecting lines in Fig excludes a rapid-equilibrium compulsory-order ternary-complex mechanism where MgATP binds before ATF2D115 (Scheme 1C), because this mechanism requires that the lines in Fig intercept on the y-axis Thus, the observed ATPase activity, together with the substrate-dependence kinetics, appears to rule out possible compulsory-order ternary-complex mechanisms and support instead a rapid-equilibrium random-order ternary-complex mechanism Interestingly, the interaction coefficient of a ¼ 0.037 obtained from the fit would indicate that both substrates are held  27-fold more tightly in the ternary complex, compared with their respective binary complexes, if the mechanism was a full rapid-equilibrium mechanism More realistically however, the mechanism is likely to be a partial rapid-equilibrium mechanism, where the aKA represents a Michaelis–Menten constant and not a dissociation constant It should be noted that the values of Kd for MgATP obtained from both the single and bisubstrate kinetics are essentially identical (Table 1), which supports the mechanistic assignments Inhibitors AMP-PCP To examine the mechanism in more detail we examined the inhibition of p38 MAPKa by b,c-methylene ATP (AMP-PCP), a nonhydrolyzable analog of ATP Lineweaver–Burk plots at different concentrations of AMP-PCP show it to be a mixed inhibitor with respect to ATF2D115 (Fig 3A) and a competitive inhibitor with respect to MgATP (Fig 3B) Such patterns are consistent with a partial rapid-equilibrium randomorder ternary-complex mechanism (Scheme 3) [28] These lines represent the best fit of the experimental data to Eqn (2) and correspond to values of KI ¼ 187.4 lm and bKI ¼ 8.6 lm (Table 1), where KI, but not bKI is likely to be an equilibrium constant Not surprisingly, MgAMP-PCP and MgATP appear to display a similar degree of interaction with ATF2D115, suggesting that the bridging b,c oxygen does not 4636 A E Szafranska and K N Dalby contribute to MgATP binding In addition to the bisubstrate inhibition kinetics we also showed that AMP-PCP inhibits the ATPase activity of p38 MAPKa Analysis of the inhibition data (not shown), according to the mechanism in Scheme 2B, suggests that AMP-PCP binds the free enzyme with a dissociation constant of Ki ¼ 241 lm (Table 1), which is in fairly good agreement with KI ¼ 187.4 lm obtained from the bisubstrate kinetics v A   ẳ Vmax aKA ỵ KB ỵ IKB ỵ I ỵ A1 ỵ aKB B KI B B bKI rearranged v B     ¼ Vmax aKB ỵ KA ỵ IKA ỵ B ỵ aKA ỵ aKA I A KI A A bKI A ð2Þ MgADP We then examined the inhibitory effects of the product MgADP Lineweaver–Burk plots at different concentrations of MgADP and saturating MgATP (195 lm, ·7) suggest that MgADP is an uncompetitive-like inhibitor with respect to ATF2D115 (Fig 3C) and a competitive inhibitor with respect to MgATP (Fig 3D) Such patterns are not normally expected for a random-order ternary-complex mechanism, but can arise if the inhibitor displays selectivity towards certain enzyme forms We believe the uncompetitive pattern towards ATF2D115 (no slope effect) results because MgADP does not bind the free form of the enzyme to detectable levels under the conditions of the experiment The data in Fig 3C not rule out the possibility of a slight slope effect, however (and weak MgADP binding to the free enzyme), thus we conservatively assign a lower limit of KI > mm, the maximum concentration of MgADP used for the dissociation constant, which is in line with other reports [26] MgAMP and adenine We also tested whether adenine and MgAMP inhibit p38 MAPKa Surprisingly, neither compound inhibited the activity of p38 MAPKa, suggesting that the presence of the b-phosphate is essential for ATP analogs to bind SB 203580 The pyridinylimidazole inhibitor SB 203580 binds within the ATP-binding pocket of both active and FEBS Journal 272 (2005) 4631–4645 ª 2005 FEBS A E Szafranska and K N Dalby Kinetic mechanism for p38 MAP kinase a A B C D Fig Inhibition by MgAMP-PCP and MgADP (Upper) (A) Double-reciprocal plot of ⁄ v versus ⁄ ATF2D115 at five fixed MgAMP-PCP concentrations (., mM; n, 0.137 mM; r, 0.275 mM; , 0.55 mM; d, 1.1 mM) The ATP concentration was fixed at 226 lM (B) Double-reciprocal plot of ⁄ v versus ⁄ ATP at six fixed MgAMP-PCP concentrations (., lM; n, 32 lM; r, 64 lM; , 128 lM; d, 256 lM, m, 513 lM) The ATF2D115 concentration was fixed at 58 lM (C) Double-reciprocal plot of ⁄ v versus ⁄ ATF2D115 at six fixed MgADP concentrations (., mM; d, 0.125 mM; r, 0.25 mM; m, 0.5 mM; , 1.0 mM; n, 2.0 mM) The ATP concentration was fixed at 195 lM (D) Double-reciprocal plot of ⁄ v versus ⁄ ATP at five fixed MgADP concentrations (d, mM; , 0.25 mM; n, 0.5 mM; r, 1.0 mM; m, 2.0 mM) The ATF2D115 concentration was fixed at 52 lM Points represent experimentally determined initial velocities Solid lines are the best fit through the experimental data according to Eqn (2) inactive p38 MAPKa and has facilitated the dissection of several signaling pathways involving p38 MAPKa pathways [29,30] In the course of their studies, Lo-Grasso et al reported that SB 203580 is an uncompetitive inhibitor of p38 MAPKa with respect to ATF2D115 [25] Such a mechanism seemed to contradict the known predilection of the inhibitor for free p38 MAPKa, thus we decided to re-examine the mechanism of inhibition To so, we first fixed the concentration of ATF2D115 and varied SB 203580 over 0–80 nm A competitive inhibition pattern was obtained (not shown), as expected for an inhibitor that binds in the ATP-binding site The best fit to the kinetic data gave an approximate value for a competitive FEBS Journal 272 (2005) 4631–4645 ª 2005 FEBS inhibition constant that was in line with previous reports [31] Surprisingly, when we tried to extend our study to higher concentrations of the inhibitor we were not able to, because the mechanism of inhibition with respect to ATF2D115 at concentrations > 200 nm did not follow simple linear models of inhibition We tried two different preparations of SB 203580, a commercial source and a sample provided to us by Kevan Shokat’s laboratory The kinetic results were identical One possible reason for the poor fit is that SB 203580, which is fairly hydrophobic in character, aggregates at higher concentrations [32,33] The addition of 0.01% (v ⁄ v) Triton X-100, whose use is suggested to identify or reverse the action of aggregate-based inhibitors [34] 4637 Kinetic mechanism for p38 MAP kinase a Scheme Random-order substrate binding with I and IỈS dead end complexes proved inconclusive in our hands because the activity of the enzyme was also affected by the presence of Triton Isothermal titration calorimetry To lend further support to our conclusions, we examined the binding of ADP and AMP-PCP to p38 MAPKa in the presence and absence of ATF2D115 by isothermal titration calorimetry (ITC) ITC is the most direct method for the determination of macromolecular ligand dissociation constants (Kd), if it is feasible to conduct experiments in the appropriate range of protein and ligand concentrations [35] It is useful because it can be used to determine the binding stoichiometry, provided that the two interacting components are titrated at concentrations higher than the Kd When binding occurs, it can be readily observed from the change in shape of the binding isotherm The calorimetry experiments are significant for several reasons (The ITC experiments were designed so that the c-value, the factor characterizing the shape of titration curve, was not lower than 0.1 When c ¼ 0.1 the binding is very weak and yields a nearly horizontal isotherm with a poorly defined binding constant, Kd [39] The c-value in our experiments was in the range 0.5–0.6, which corresponds to 65–77 lm p38 MAPKa and represents a 13 000–15 400-fold increase in the enzyme concentration in comparison with the kinetic studies.) Notably, they show that the dissociation constants for ADP and AMP-PCP from the binary complex are fivefold higher than the values of bKi obtained 4638 A E Szafranska and K N Dalby kinetically, suggesting that, as suspected, ligand binding is not completely at equilibrium during turnover and that the mechanism is best described as a partial rapid-equilibrium mechanism For example, when ADP (0–223 lm) was titrated into a mixture of ATF2D115 (92 lm) and p38 MAPKa (68 lm), heat was evolved indicative of favorable nucleotide binding to the enzyme (Fig 4B) The best fit according to the two-component binding model provided a dissociation constant of Kd ¼ 62 ± lm, with n ¼ 0.52 ± 0.08 binding sites and the following thermodynamic parameters; DH ẳ )16 900 Jặmol)1, DS ẳ )37 0.32 JỈ mol)1ỈK)1 When MgAMP-PCP (0–183 lm) was titrated into a mixture of ATF2D115 (97 lm) and the enzyme (77 lm) (Fig 5B), a similar amount of heat was generated and the data analysis furnished the following values: Kd ¼ 69.6 ± lm, n ¼ 0.52 ± 0.08, DH ¼ )14 200 JỈmol)1, and DS ¼ )28.3 JỈmol)1ỈK)1 The calorimetry analysis supports the notion of synergy between nucleotides and ATF2D115 upon binding to p38 MAPKa For example, the binding of AMPPCP to the binary complex appears to be at least fivefold tighter than to the free enzyme When MgAMP-PCP (0–542 lm) was titrated into p38 MAPKa (194 lm), ~ 10-fold less heat was evolved compared with when MgAMP-PCP was added to the binary complex (Fig 5A) The heat generated was not sufficiently robust to enable an accurate titration, thus the best fit to the binding model gave values of Kd ¼ 300 ± 160 lm, n ¼ 1.4 ± 0.4, DH ẳ )1145 Jặmol)1, and DS ẳ +12.3 Jặmol)1ặK)1 This dissociation constant is in line with the value of K ¼ 184 lm obtained kinetically Interestingly, when ADP (0–550 lm) was titrated with p38 MAPKa (194 lm), no heat was detected This suggests like the inhibition data, that binding is probably weak (> 300 lm) and beyond the detection of the experiment Interestingly, the calorimetry experiments are consistent with only 0.5 binding sites per binary p38 MAPKATF2D115 complex, suggesting that the enzyme either has only one functional active site within the complex or that only 50% of the preparation is functional Discussion In recent years p38 MAPKa has emerged as a major practicable drug target, associated with several severe diseases of inflammation [3–5] The identification in 1994 of the pyridinyl class of p38 MAPKa inhibitors [29] fueled many studies aimed at exploiting the subtle differences between the active sites of protein kinases Despite these efforts, to date, only a handful of ATP competitive inhibitors have been developed that truly FEBS Journal 272 (2005) 4631–4645 ª 2005 FEBS A E Szafranska and K N Dalby Kinetic mechanism for p38 MAP kinase a Fig Isothermal titration calorimetry measurements of MgADP binding (A) (upper) Titration of MgADP (1.47 mM, 21 · lL) with activated p38 MAPKa (68 lM) at 27 °C; (lower) integrated enthalpy change for each injection (B) (upper) Titration of MgADP (1.47 mM, 21 · lL) with a mixture of activated p38 MAPKa (68 lM) and ATF2D115 (91.8 lM); (lower) integrated enthalpy change for each injection exhibit sufficient specificity to warrant development [36] Thus, there is a keen need to exploit other sites on protein kinases, such as cosubstrate or scaffoldbinding sites, which may offer alternative therapeutic avenues To this end, detailed kinetic and structure ⁄ function studies using protein substrates will help us to understand the full compliment of molecular interactions that govern the catalysis and regulation of these enzymes Kinetic mechanism Despite occupying an elevated position as a potentially important target of signal transduction therapy, little mechanistic work has been reported on p38 MAPKa or related family members, and as such there remains no clear model for their kinetic mechanisms ERK2 was originally proposed to phosphorylate myelin basic protein with rate-limiting (kcat ¼ 10 s)1) phosphorylation [37] However, with a physiologically relevant substrate (Ets1), ERK2 was shown to be activated by magnesium [38] and to follow a random-order ternaryFEBS Journal 272 (2005) 4631–4645 ª 2005 FEBS complex mechanism [39], with partially rate-limiting phosphorylation (k2 ¼ 109 s)1) and product release (k3 ¼ 56 s)1) [40] In this study, we focus on the steady-state kinetic mechanism of p38 MAPKa using the protein substrate ATF2D115 Previous studies on p38 MAPKa have been somewhat contradictory, suggesting that the enzyme follows compulsory-order ternary-complex mechanisms where the phosphoacceptor [25] or ATP [26] must bind first Compulsory-order mechanisms are ruled out in this study through: (a) their inconsistency with the substrate dependence and the dead end inhibitor kinetics (rules out the requirement that ATF2 must bind first); or (b) published structural studies and binding studies, which show that a peptides derived from a protein substrate can bind p38 MAPKa at docking domains outside of the active site in the absence of MgATP (rules out the requirement that MgATP must bind first) We show that, like the c-isoform [41], p38 MAPKa displays robust ATPase activity with a catalytic constant of kcat ¼ 0.3 s)1, which is very similar to 4639 Kinetic mechanism for p38 MAP kinase a A E Szafranska and K N Dalby Fig Isothermal titration calorimetry measurements of MgAMP-PCP binding (A) (upper) Titration of AMP-PCP (1.58 mM, 19 · lL) into activated p38 MAPKa (75 lM) at 27 °C; (lower) integrated enthalpy change for each injection (B) Titration of AMP-PCP into a mixture of activated p38 MAPKa (75 lM) and ATF2D115 (97 lM); (lower) integrated enthalpy change for each injection the catalytic constant for the phosphorylation of ATF2D115 that has a value of kcat ¼ 1.2 s)1 This and the work of Chen et al [26] are consistent with the notion that MgATP can bind the enzyme to form a functionally active complex There is substantial evidence to support the notion that p38 MAPKa binds protein substrates at its C-terminus in the absence of MgATP [42] Specifically, studies from the laboratories of both Goldsmith [43] and Ahn [44] showed that the inactive form of p38 MAPKa can bind to a peptide derived from the p38 MAPKa substrate MEF2A This peptide contains a consensus motif for docking of R ⁄ K-X4-FAX-FB (where X represents any amino acid and F represents a hydrophobic residue: Leu, Ile, or Val) and binds in a groove in the C-terminus of p38 MAPKa between the helices aD and aE and the reverse turn between strands b7 and b8 [43] As this consensus sequence is also present in ATF2, it is probable that p38 MAPKa binds ATF2 in the same groove as MEF2 Given that the unphosphorylated form of p38 MAPKa, whose active site is not prop4640 erly molded, still binds these pepides, it is extremely unlikely that the binding of MgATP must precede ATF2 Thus, taken together, these observations and our data support a partial rapid-equilibrium random-order ternary-complex mechanism (Scheme 1A), where both substrates (MgATP and ATF2D115) bind to p38 MAPKa with moderate affinities (MgATP, KA ¼ 360 lm; ATF2D115, KB ¼ 39 lm) (It is well known that steady-state kinetic studies not identify the extent to which binary complexes are actually functionally productive and that transient kinetic studies are required to determine this unequivocally However, protein kinases are considered to have extremely flexible active sites that can accommodate both substrates before they adopt a more closed conformation that facilitates catalysis [45] Therefore, it seems reasonable to assume that, for p38 MAPKa, which utilizes a docking domain, both binary complexes lie on the reaction pathway.) Jointly with calorimetry experiments, a fivefold synergy in substrate binding is indicated FEBS Journal 272 (2005) 4631–4645 ª 2005 FEBS A E Szafranska and K N Dalby Our conclusions and results differ on a number of counts to a previous report by LoGrasso et al who proposed a compulsory-order mechanism with ATF2D115 binding before MgATP [25] The major differences we found are as follows Firstly, MgAMPPCP exhibits a mixed inhibition pattern and not an uncompetitive pattern (Fig 3A) with respect to ATF2D115, which is consistent with a partial rapidequilibrium random-order ternary-complex mechanism (Scheme 1A) LoGrasso et al reported an uncompetitive pattern [25] Secondly, as with the previous study we found that MgADP exhibits an uncompetitive-like inhibition pattern with respect to ATF2D115 (Fig 3C) We interpret this as being due to its inability to bind free p38 MAPKa and not because MgATP, the substrate, binds free p38 MAPKa weakly That is, the observed pattern of inhibition reflects the selectivity of the product inhibitor ADP for the different enzyme forms and not the substrate ATP To corroborate this interpretation we showed that the binding of MgADP to p38 MAPKa could not be detected using microcalorimetry (Fig 4A), whereas robust binding of MgADP to the ATF2D115 binary complex (Fig 4B) was detected Thirdly, LoGrasso et al reported that SB 203580 displays an uncompetitive pattern with respect to ATF2D115 [25] This observation was curious because SB 203580 is known to bind p38 MAPKa very tightly [31] Therefore, the ability of SB 203580 to bind the same enzyme form as ATF2D115 predicts that there should be a significant ‘slope effect’ in a Lineweaver– Burk plot of ⁄ v against ⁄ ATF2D115 Driven by this curiosity, we examined the mechanism of inhibition by SB 203580 and found that it is not well described by any classical linear inhibition models, possibly because it aggregates in solution at concentrations above 200 nm, leading to nonspecific inhibition effects as seen for other protein kinase inhibitors [32–34] It is difficult to fully explain the differences between this study and LoGrasso’s While it is possible that the N-terminal FLAG tag utilized by LoGrasso et al for purification purposes [25] imparts an influence on substrate and nucleotide binding that could influence the inhibition patterns, other differences between our results cannot be explained by the tag It must be stated that the mechanism proposed by LoGrasso et al is unique for a protein kinase and neither followed by ERK2 [39] nor supported by the data presented here We propose, therefore, that the kinetic mechanism for p38 MAPKa should be reassigned as a partial rapidequilibrium random-order ternary-complex mechanism for the phosphorylation of ATF2D115 FEBS Journal 272 (2005) 4631–4645 ª 2005 FEBS Kinetic mechanism for p38 MAP kinase a In conclusion, activated p38 MAPKa has been prepared without a purification tag to the highest specific activity reported The enzyme phosphorylates ATF2D115 by a partial rapid-equilibrium randomorder ternary-complex mechanism and not through a compulsory-order mechanism as previously suggested [25] Interestingly, neither MgAMP nor adenine inhibited p38 MAPKa suggesting that the b-phosphate is necessary for MgATP binding It will be interesting to understand the basis for this selectivity Experimental procedures Buffers, reagents and plasmids Trizma base was purchased from EM Industries (Gibbstown, NJ, USA), Hepes from Sigma (St Louis, MO, USA) and ammonium carbonate from Fisher (Fair Lawn, NJ, USA) Glutathione (GSH) agarose and glutathione (reduced form) for GST-fusion protein purification were obtained from Sigma NiSO4 and Ni-NTA agarose for His6-p38 MAPKa purification were provided by Qiagen Inc (Santa Clarita, CA, USA) and Sigma, respectively The His6 tag was removed from p38 MAPKa using thrombin from Novagen (Madison, WI, USA) MgCl2 for p38 MAPKa activation and kinetic studies, as well as ATP analogs used in inhibition studies (adenine, AMP, AMPPCP, ADP) and SB 203580 were purchased from Sigma Kinase assays were conducted with Roche (Indianapolis, IN, USA) special quality ATP and [32P]ATP[cP] from ICN (Costa Mesa, CA, USA) Reagents for coupled enzyme assay, such as lactate dehydrogenase, pyruvate kinase, NADH and phosphoenol pyruvate, were supplied by Sigma All other buffer components and chemicals were obtained from Sigma The plasmid used to express His6p38 MAPKa [45] and GST-ATF2 (1–115) [27] have been reported previously The plasmid used to express GSTMKK6b (S207E T211E) was a gift from R Copeland (DuPont Merck, Delaware, USA) Proteins Expression and purification of tagless p38 MAPKa DNA sequences encoding p38 MAPKa cloned into pET14B (Novagen) was used to express p38 MAPKa as an N-terminal, hexa-histidine fusion protein in E coli BL21 (DE3) pLysS The enzyme was expressed and purified according to the method of Szafranska et al [27] Following removal of the hexa-histidine tag the enzyme preparation was dialyzed overnight at °C into storage buffer S1 [25 mm Hepes, mm dithiothreitol, 50 mm KCl, 5% (v ⁄ v) glycerol, pH 7.5], and stored at )80 °C at a concentration of approximately mgỈmL)1 until further use The concentration of p38 MAPKa was determined using the molar 4641 Kinetic mechanism for p38 MAP kinase a extinction coefficient (A280) of 52501 cm)1Ỉm)1, following the method of Gill and von Hippel [46] The homogeneity of the protein was routinely verified by 10% SDS ⁄ PAGE and by MS Expression and purification of GST-ATF2 (1–115) Residues 1–115 of the transcription factor ATF2 was expressed as a GST fusion protein from a pGEX-5T vector in E coli BL21 (DE3) pLysS and purified according to the previously reported method with two anion-exchange chromatography steps The protein substrate was stored at )80 °C in buffer S1 lacking glycerol The homogeneity of the protein was routinely verified by 12% SDS ⁄ PAGE and by MS The concentration of ATF2D115 was determined using the extinction coefficient (A280) of 48 490 cm)1Ỉm)1, following the method of Gill and von Hippel [46] Expression and purification of GST-MKK6b (S207E T211E) MKK6b (S207E T211E) was expressed as a GST fusion protein in E coli BL21 (DE3) pLysS The protein was purified by GSH-affinity chromatography according to standard protocols (Sigma Product Information for G4510) Pooled protein fractions were dialyzed at °C into buffer S1, concentrated and stored at )80 °C [27] Activation of p38 MAPKa by GST-MKK6b (S207E T211E) p38 MAPKa (10.7 lm, 14 mg) and GST-MKK6b (0.26 lm, 1.6 mg) were incubated for at 27 °C prior to the addition of ATP (4 mm) in 50 mL of activation buffer P1 (25 mm Hepes, mm dithiothrietol, 20 mm MgCl2, 0.5 mm EDTA, pH 7.5) and repurified, essentially according to the method of Szafranska et al [27] The activated, tagless p38 MAPKa was stored in buffer S1 at )80 °C until further use The homogeneity of the protein was routinely verified by 10% SDS ⁄ PAGE and by MS The concentration was assessed using calculated extinction coefficient (A280) of 52501 cm)1 m)1, according to the method of Gill and von Hippel [46] Electrospray mass spectrometry of proteins Protein samples were rid of excess salt by injection on a RPHPLC C18 column (Grace Vydac, 218TP54; Columbia, MD, USA) and elution over a standard gradient of 0–100%, (v ⁄ v) acetonitrile over 80 at a flow rate of 0.6 mLỈmin)1 Collected peaks were lyophilized, the residue re-suspended in 30 lL water: acetonitrile mixture (1 : 1) containing 0.1% (v ⁄ v) trifluoroacetic acid and analyzed by LC-MS with a Finnigan-MAT LCQ (Finnigan ⁄ Thermoquest, San Jose, 4642 A E Szafranska and K N Dalby CA) electrospray, ion trap mass spectrometer coupled with a Magic 2002 Microbore HPLC (Microchrom BioResource, Auburn, CA) The mass spectrometer was scanned over a 300–2000 m ⁄ z mass range using a : mixture of mobile phase A (acetonitrile ⁄ water ⁄ acetic acid ⁄ trifluoroacetic acid; : 98 : 0.1 : 0.02; v ⁄ v ⁄ v ⁄ v) and B (acetonitrile ⁄ water ⁄ acetic acid ⁄ trifluoroacetic acid; 10 : 90 : 0.009 : 0.02; v ⁄ v ⁄ v ⁄ v) at a flow rate of 10 lLỈmin)1 Steady-state kinetics p38 MAPKa assays were conducted at 27 °C in assay buffer A1 (20 mm Hepes, mm dithiothreitol, 100 mm KCl, 0.1 mm EDTA, 0.1 mm EGTA, 10 lgỈmL)1 bovine serum albumin, pH 7.6) containing 5–10 nm activated p38 MAPKa and 10 mm MgCl2 in a final volume of 100 lL At 10 mm magnesium the activity of the kinase is optimum The concentrations of substrates ranged as follows: ATF2D115 (2.5–52 lm), ATP (12.5–200 lm, 100–1000 c.p.m.Ỉpmol)1) The reaction mixture was incubated for before the addition of ATP Aliquots (10 lL) were taken at set time points and applied to a · cm P81 cellulose papers, which were allowed to air-dry, washed with 50 mm phosphoric acid (5 · 10 min), then in acetone (1 · 10 min) and dried The incorporation of radioactivity was determined by counting in 1.5 mL CytoScint on a Packard 1500 scintillation counter at a r-value of Each assay was performed at least twice Protein concentrations were determined at 280 nm using the following molar extinction coefficients: e ¼ 52 501 m)1Ỉcm)1 (p38 MAPKa), 48 490 m)1Ỉcm)1 (ATF2D115) ATP concentration was determined at 259 nm using e ¼ 15 400 m)1Ỉcm)1 The initial velocities were fitted using scientist for Windows v 2.0 (MicroMathÒ) Enzyme inhibition studies In general, enzyme inhibition studies were performed as described for two-substrate kinetics When ATF2D115 was the varied substrate (2.5–55 lm), ATP was fixed at 190–220 lm (100–1000 c.p.m.Ỉpmol)1) When ATP was the varied substrate (20–320 lm), ATF2D115 was fixed at 40–55 lm The concentrations of inhibitors ranged as follows: ADP (0.2–2 mm), SB 203580 (0.025–1.0 lm), AMP-PCP (0.18– 1.4 mm), AMP (0.5–2 mm) and adenine (0.21–1.7 mm) Where possible, the extinction coefficients were used to determine the accurate concentrations of inhibitors (e ¼ 15 400 m)1Ỉcm)1 for ADP, AMP-PCP and AMP, and 13 300 m)1Ỉcm)1 for adenine) Protein concentrations were determined using extinction coefficients as described above (Steady-state kinetics section) In each case inhibition reactions were performed twice The initial velocities data from the inhibition studies were fitted to the general velocity equations using scientist for Windows v 2.0 (MicroMathÒ) FEBS Journal 272 (2005) 4631–4645 ª 2005 FEBS A E Szafranska and K N Dalby Isothermal titration calorimetry Isothermal titration calorimetry was performed using an MCS isothermal titration calorimeter (Microcal Inc., Northampton, MA) connected to a water bath to maintain the constant temperature of 27 °C Ligand solutions were prepared in calorimetry buffer C1 (20 mm Hepes, pH 7.5–7.6, adjusted with m KOH, containing 100 mm KCl, mm 2-mercaptoethanol, 0.1 mm EDTA, 0.1 mm EGTA and 10 mm MgCl2) Protein solutions were dialyzed overnight at °C into the same buffer All solutions were centrifuged at 13 200 g for and degassed under vacuum for at least 30 prior to use After a stable baseline was achieved, ligand solutions were titrated into the stirred (410 r.p.m.) sample cell (1.325 mL) containing equilibrated (30 min) samples of the proteins at 27 °C The injection sequence consisted of an initial lL injection (not used in data fitting) followed by 26 injections of 8–10 lL, each at 180 s intervals until saturation was reached To correct for the heat of dilution and mixing, blank titrations of ligand into buffer were subtracted from the experimental titrations The reference offset was set at 20% Data evaluation, integration, analysis, and determination of binding parameters were performed using origin 5.0 software (Microcal, Inc.) Final protein and ligand concentrations were as follows: 65–77 lm activated p38 MAPKa, 91–97 lm ATF2D115, 223 lm ADP and 183 lm AMP-PCP To facilitate detection of binding between the enzyme and ATP analogs, additional ITC experiment was carried out where their concentrations were approximately doubled (194 lm p38 MAPKa, 549 lm ADP, 542 lm AMP-PCP) ATPase activity assay The ATPase activity of activated p38 MAPKa was characterized using the coupled assay method The decrease in NADH was monitored at 340 nm using a Cary 50 UV spectrophotometer The temperature was maintained at 27 °C using a VWR Refrigerated Circulator (Suwanee, GA, USA) The molar absorption coefficient (e) for NADH of 6.22 · 10)3 m)1Ỉcm)1 at 340 nm was used for calculations The coupled assay enzymes were purchased from Sigma as 3.2 m (pyruvate kinase) and 2.4 m (liver alcohol dehydrogenase) suspensions in (NH4)2SO4 Prior to the experiment they were buffer-exchanged with the assay buffer (4 · 400 lL for every 10 lL of enzyme suspension) using 0.5 mL Microcon centrifugal filter devices (13 200 g, °C) to rid of excess ammonium sulfate The resultant enzyme solutions were concentrated down to the approximate concentrations of 1.4 units (pyruvate kinase) and units (liver alcohol dehydrogenase) per 10 lL Both NADH and phosphoenolpyruvate solutions were prepared in 25 mm Hepes, pH 7.6 immediately prior to use The final assay volume of 200 lL contained: activated p38 MAPKa (0.4–0.45 lm), ATP (0.085–1.36 mm), PEP (1 mm), NADH (0.18– FEBS Journal 272 (2005) 4631–4645 ª 2005 FEBS Kinetic mechanism for p38 MAP kinase a 0.220 mm), pyruvate kinase (1.4 U) and liver alcohol dehydrogenase (3 U) in assay buffer Where applicable, AMP-PCP (0.25–2.2 mm) was added to the assay All assay components were preincubated at 27 °C for before addition of ATP and the data was acquired for the following under the same conditions Acknowledgements This research was supported in part by the Welch Foundation (F-1390) and the NIH 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MgATP complex is not a deadend complex with respect to the binding and phos4633 Kinetic mechanism for p38 MAP kinase a A E Szafranska and K N Dalby Fig Preparation of activated p38 MAPKa and ATF2D115... propose, therefore, that the kinetic mechanism for p38 MAPKa should be reassigned as a partial rapidequilibrium random-order ternary-complex mechanism for the phosphorylation of ATF2D115 FEBS Journal... Szafranska and K N Dalby Kinetic mechanism for p38 MAP kinase a mechanism We also show that nucleotides such as MgATP and particularly magnesium ADP (MgADP) bind preferentially to the binary p38 MAPKa–