Properties and regulation of a transiently assembled ERK2.Ets-1 signaling complex

ERK2 is a proline-directed protein kinase that displays a high specificity for a single threonine (Thr-38) on the substrate Ets-1, which lies within the consensus sequence 36phi-chi-Thr-Pro39 (where phi is typically a small hydrophobic residue and chi appears to be unrestricted). Thr-38 lies in a long flexible N-terminal tail (residues 1-52), which also contains a second potential phosphorylation site, Ser-26. How Ets-1 binds ERK2 to promote the phosphorylation of Thr-38 while simultaneously discriminating against the phosphorylation of Ser-26 is unclear. To delineate the details of the molecular recognition of Ets-1 by ERK2, the binding of various mutants and truncations of Ets-1 were analyzed by fluorescence anisotropy. The data that were obtained support the notion that the N-terminal tail contains a previously unrecognized docking site that promotes the phosphorylation of Thr-38. This new docking site helps assemble the complex of Ets-1 and ERK2 and makes a similar contribution to the stabilization of the complex as does the pointed domain of Ets-1. The in vitro activation of ERK2 by MKK1 induces a large conformational transition of the activation segment (DFG-APE), but neither induces self-association of ERK2 nor destabilizes the stability of the ERK2.Ets-1 complex. This latter observation suggests that interactions intrinsic to the active site are not important for complex assembly, a notion further supported by the observation that the substitution of a number of different amino acids for Pro-39 does not destabilize the complex. Mutagenesis of ERK2 within loop 13 suggests that Ets-1 binds the substrate-binding groove. These data suggest that ERK2 uses two weak docking interactions to specifically assemble the complex, perhaps in doing so denying Ser-26 access to the active site. Displacement of residues 1-138 of Ets-1 (EtsDelta138) from ERK2 by the peptide N-QKGKPRDLELPLSPSL-C, derived from Elk-1, suggests that Ets-1 engages the D-recruitment site (beta7-beta8 reverse turn and the alphaD-alphaE helix) of ERK2. Displacement of EtsDelta138 from ERK2 by the peptide N-AKLSFQFPS-C derived from Elk-1 shows that EtsDelta138 communicates with the F-recruitment site of ERK2 also.     

Examining docking interactions on ERK2 with modular peptide substrates

ERK2 primarily recognizes substrates through two recruitment sites, which lie outside the active site cleft of the kinase. These recruitment sites bind modular-docking sequences called docking sites and are potentially attractive sites for the development of non-ATP competitive inhibitors. The D-recruitment site (DRS) and the F-recruitment site (FRS) bind D-sites and F-sites, respectively. For example, peptides that target the FRS have been proposed to inhibit all ERK2 activity (Galanis, A., Yang, S. H., and Sharrocks, A. D. (2001) J. Biol. Chem. 276, 965-973); however, it has not been established whether this inhibition is steric or allosteric in origin. To facilitate inhibitor design and to examine potential coupling of recruitment sites to other ligand recognition sites within ERK2, energetic coupling within ERK2 was investigated using two new modular peptide substrates for ERK2. Modeling shows that one peptide (Sub-D) recognizes the DRS, while the other peptide (Sub-F) binds the FRS. A steady-state kinetic analysis reveals little evidence of thermodynamic linkage between the peptide substrate and ATP. Both peptides are phosphorylated through a random-order sequential mechanism with a k(cat)/K(m) comparable to Ets-1, a bona fide ERK2 substrate. Occupancy of the FRS with a peptide containing a modular docking sequence has no effect on the intrinsic ability of ERK2 to phosphorylate Sub-D. Occupancy of the DRS with a peptide containing a modular docking sequence has a slight effect (1.3 ± 0.1-fold increase in k(cat)) on the intrinsic ability of ERK2 to phosphorylate Sub-F. These data suggest that while docking interactions at the DRS and the FRS are energetically uncoupled, the DRS can exhibit weak communication to the active site. In addition, they suggest that peptides bound to the FRS inhibit the phosphorylation of protein substrates through a steric mechanism. The modeling and kinetic data suggest that the recruitment of ERK2 to cellular locations via its DRS may facilitate the formation of F-site selective ERK2 signaling complexes, while recruitment via the FRS will likely inhibit ERK2 through a steric mechanism of inhibition. Such recruitment may serve as an additional level of ERK2 regulation.     

Role of a cysteine residue in the active site of ERK and the MAPKK family

Kinases of mitogen-activated protein kinase (MAPK) cascades, including extracellular signal-regulated protein kinase (ERK), represent likely targets for pharmacological intervention in proliferative diseases. Here, we report that FR148083 inhibits ERK2 enzyme activity and TGFbeta-induced AP-1-dependent luciferase expression with respective IC50 values of 0.08 and 0.05 microM. FR265083 (1'-2' dihydro form) and FR263574 (1'-2' and 7'-8' tetrahydro form) exhibited 5.5-fold less and no activity, respectively, indicating that both the alpha,beta-unsaturated ketone and the conformation of the lactone ring contribute to this inhibitory activity. The X-ray crystal structure of the ERK2/FR148083 complex revealed that the compound binds to the ATP binding site of ERK2, involving a covalent bond to Sgamma of ERK2 Cys166, hydrogen bonds with the backbone NH of Met108, Nzeta of Lys114, backbone C=O of Ser153, Ndelta2 of Asn154, and hydrophobic interactions with the side chains of Ile31, Val39, Ala52, and Leu156. The covalent bond motif in the ERK2/FR148083 complex assures that the inhibitor has high activity for ERK2 and no activity for other MAPKs such as JNK1 and p38MAPKalpha/beta/gamma/delta which have leucine residues at the site corresponding to Cys166 in ERK2. On the other hand, MEK1 and MKK7, kinases of the MAPKK family which also can be inhibited by FR148083, contain a cysteine residue corresponding to Cys166 of ERK2. The covalent binding to the common cysteine residue in the ATP-binding site is therefore likely to play a crucial role in the inhibitory activity for these MAP kinases. These findings on the molecular recognition mechanisms of FR148083 for kinases with Cys166 should provide a novel strategy for the pharmacological intervention of MAPK cascades.     

The role of thiol groups in the structure and mechanism of action of arginine kinase

1. A detailed study of the reaction of iodoacetamide with arginine kinase has been carried out. 2. The enzyme contains five reactive thiol groups per 37000g. of protein, all of which can be alkylated. 3. Below pH8.5 loss of activity is substantially independent of pH and can be correlated with the alkylation of a single pH-independent thiol. 4. One catalytic site per enzyme molecule is inferred. 5. The progress curves of the alkylation reaction are polyphasic and reveal a pH-and time-dependent sequential release of thiols which is dependent upon the alkylation of the first pH-independent thiol. This is supported by electrophoretic investigations. 6. Comparison of alkylation rate and rate of loss of activity suggests that two thiol groups are not essential for catalytic activity. Variability in enzyme preparations with respect to alkylation rate appears to be associated with these two groups. 7. A complex protection pattern is revealed by the effects of various substrate combinations on rates of alkylation and of loss of activity. It is inferred that two thiol groups participate in conformational changes and nucleotide interactions. 8. Comparison with creatine kinase suggests a fundamentally similar catalytic mechanism, although for arginine kinase certain additional restrictions are necessary because of the protection observed with nucleotide substrates.     

A model of a MAPK•substrate complex in an active conformation: a computational and experimental approach

The mechanisms by which MAP kinases recognize and phosphorylate substrates are not completely understood. Efforts to understand the mechanisms have been compromised by the lack of MAPK-substrate structures. While MAPK-substrate docking is well established as a viable mechanism for bringing MAPKs and substrates into close proximity the molecular details of how such docking promotes phosphorylation is an unresolved issue. In the present study computer modeling approaches, with restraints derived from experimentally known interactions, were used to predict how the N-terminus of Ets-1 associates with ERK2. Interestingly, the N-terminus does not contain a consensus-docking site ((R/K)_2-3-X_2-6-Φ_A-X-Φ_B, where Φ is aliphatic hydrophobic) for ERK2. The modeling predicts that the N-terminus of Ets-1 makes important contributions to the stabilization of the complex, but remains largely disordered. The computer-generated model was used to guide mutagenesis experiments, which support the notion that Leu-11 and possibly Ile-13 and Ile-14 of Ets-1 1-138 (Ets) make contributions through binding to the hydrophobic groove of the ERK2 D-recruiting site (DRS). Based on the modeling, a consensus-docking site was introduced through the introduction of an arginine at residue 7, to give the consensus ⁷RK-X₂-Φ_A-X-Φ_B ¹³. This results in a 2-fold increase in k _cat/K _m for the phosphorylation of Ets by ERK2. Similarly, the substitution of the N-terminus for two different consensus docking sites derived from Elk-1 and MKK1 also improves k _cat/K _m by two-fold compared to Ets. Disruption of the N-terminal docking through deletion of residues 1-23 of Ets results in a 14-fold decrease in k _cat/K _m, with little apparent change in k _cat. A peptide that binds to the DRS of ERK2 affects K _m, but not k _cat. Our kinetic analysis suggests that the unstructured N-terminus provides 10-fold uniform stabilization of the ground state ERK2•Ets•MgATP complex and intermediates of the enzymatic reaction.     

Inactivation of GDP-mannose dehydrogenase from Pseudomonas aeruginosa by penicillic acid identifies a critical active site loop

The pathogenic bacterium Pseudomonas aeruginosa synthesizes alginate as one of a group of virulence factors that are produced during infections. The enzyme GDP-mannose dehydrogenase catalyzes the committed step in alginate biosynthesis. We show here that penicillic acid is an irreversible inactivator of GDP-mannose dehydrogenase. Inactivation occurs with a rate constant of 0.39+/-0.01 mM(-1) min(-1) at pH 8.0, and does not exhibit saturation behavior. Partial protection from inactivation is afforded by GDP-mannose, but not by the other substrate, NAD+. GMP and NAD+ together provide complete protection against inactivation. Analysis by mass spectrometry confirmed that the enzyme is alkylated at multiple cysteine residues by penicillic acid, including Cys 213, Cys 246, and the active site cysteine, Cys 268. However, the pH dependence of the inactivation rate suggested that alkylation of a single cysteine residue is sufficient to inactivate the enzyme. The C268A mutant protein was also susceptible to inactivation by penicillic acid. The presence of NAD+ and GMP provided partial protection of Cys 246 and Cys 268, and almost complete protection of Cys 213. Cys 213 is located on a helix that forms part of the binding pocket for GDP-mannose, and forms a hydrogen bond with Asn 252. Asn 252 is located on a loop that surrounds GDP-mannose. The C213A mutant enzyme exhibits a Vmax that is 1.8-fold greater than the wild-type enzyme, suggesting that the interaction between Cys 213 and Asn 252 helps to hold the loop in place during catalysis, and that opening the loop to release product is partially rate-limiting. Cys 246 is adjacent to the GDP-mannose binding loop, and its alkylation may also interfere with loop movement.     

Chemical modification of chalcone isomerase by mercurials and tetrathionate. Evidence for a single cysteine residue in the active site

Chalcone isomerase from soybean is inactivated by stoichiometric amounts of p-mercuribenzoate or HgCl2. Spectral titration of the enzyme with p-mercuribenzoate indicates that a single thiol group is modified. Treatment of modified enzyme with KCN or thiols results in a complete restoration of enzyme activity demonstrating that the inactivation is not due to irreversible protein denaturation. A product of the enzymatic reaction, naringenin, provides complete kinetic protection against inactivation by both mercurials. The binding constant (33 microM) for naringenin determined from the concentration dependence of the protection agrees with the inhibition constant (34 microM) for naringenin as a competitive inhibitor of the catalytic reaction. This agreement demonstrates that the observed kinetic protection results from the specific binding of naringenin to the active site. Incubation of native chalcone isomerase with sodium tetrathionate (0.1 M) results in a slow time-dependent loss of enzymatic activity. The inactivation of chalcone isomerase by tetrathionate and N-ethylmaleimide becomes very rapid in the presence of 6 M urea, indicating that the native tertiary structure is responsible for the low reactivity of the enzymatic thiol. The stoichiometric modification of reduced and denatured chalcone isomerase by [3H] N-ethylmaleimide indicates that the enzyme contains only a single cysteine residue and does not contain any disulfides. The evidence presented suggests that the only half-cystine residue in chalcone isomerase is located in the active site and thereby provides the first clue to the location of the active site in chalcone isomerase.     

Modification of the active site of isocitrate lyase from Pseudomonas indigofera

Kinetic analysis of inactivation of isocitrate lyase from Pseudomonas indigofera by 3-bromopyruvate established that enzyme binds this compound prior to alkylation and that substrate, D_s-isocitrate, competes for the same site on the enzyme. The rate of inactivation was increased by EDTA which is a promoter of catalysis in the presence of activated (reduced) enzyme and substrate. The combination of products, glyoxylate plus succinate, also protected against inactivation. Glyoxylate plus itaconate, phosphoenolpyruvate, or maleate also protected. However, each of the latter three compounds or glyoxylate or succinate alone provided little or no protection. Pyruvate, a competitive inhibitor with respect to glyoxylate in the condensation reaction, also failed to protect. However, two dicarboxylates, meso-tartrate and oxalate, that are also competitive inhibitors with respect to glyoxylate provide some protection against inactivation by BrP perhaps by bridging across cationic sites that facilitate glyoxylate and succinate binding. These and other results imply that alkylation by 3-bromopyruvate occurs at the succinate part of the active site. A mechanism which includes a catalytic role for the cysteine residue at the active site is presented and discussed.     

Active site directed inactivation of rat mammary gland fatty acid synthase by 3-chloropropionyl coenzyme A

3-Chloropropionyl coenzyme A (CoA) irreversibly inhibits rat mammary gland fatty acid synthase. Enzyme inactivation proceeds with first-order kinetics. NADPH (150 microM) as well as acetyl-CoA (500 microM) affords protection against inactivation, suggesting that the inhibitor is active site directed. In contrast, malonyl-CoA (500 microM) offers little protection. With chloro [1-14C]propionyl-CoA, stoichiometries of modification that approach one per enzyme protomer (240 kilodaltons) have been measured. When chloropropionyl-[3'-32P]CoA is used for inactivation, modification stoichiometries are less than 10% of the value observed in the 14C labeling experiments, suggesting that acylation of the enzyme occurs. Radioactivity remains associated with the 14C-labeled protein after performic acid oxidation, indicating that another linkage, in addition to the thio ester adduct, is formed during inactivation. Recovery of [( 14C]carboxyethyl)cysteine from digests of the inactivated enzyme indicates that alkylation of an active site cysteine occurs. The cysteamine sulfhydryl of the acyl carrier peptide is clearly not the site of modification. Loss of overall enzyme activity is tightly linked to decreases in the ketoacyl synthase partial reaction. This observation, coupled with the differential protection measured with acetyl-CoA and malonyl-CoA, suggests that the reagent modifies a residue at the active site involved in condensation. While inactivated enzyme shows good ketoacyl reductase activity when S-(acetoacetyl)-N-acetylcysteamine is used as a substrate, only poor activity for this partial reaction is measured when acetoacetyl-CoA is the substrate. This implies that the function of the acyl carrier peptide (ACP) is impaired during the inactivation process.(ABSTRACT TRUNCATED AT 250 WORDS)