Docking motif interactions in MAP kinases revealed by hydrogen exchange mass spectrometry

Protein interactions between MAP kinases and substrates, activators, and scaffolding proteins are regulated by docking site motifs, one containing basic residues proximal to Leu-X-Leu (DEJL) and a second containing Phe-X-Phe (DEF). Hydrogen exchange mass spectrometry was used to identify regions in MAP kinases protected from solvent by docking motif interactions. Protection by DEJL peptide binding was observed in loops spanning beta7-beta8 and alphaD-alphaE in p38alpha and ERK2. In contrast, protection by DEF binding to ERK2 revealed a distinct hydrophobic pocket for Phe-X-Phe binding formed between the P+1 site, alphaF helix, and the MAP kinase insert. In inactive ERK2, this pocket is occluded by intramolecular interactions with residues in the activation lip. In vitro assays confirm the dependence of Elk1 and nucleoporin binding on ERK2 phosphorylation, and provide a structural basis for preferential involvement of active ERK in substrate binding and nuclear pore protein interactions.     

Crystal structures of MAP kinase p38 complexed to the docking sites on its nuclear substrate MEF2A and activator MKK3b

The structures of the MAP kinase p38 in complex with docking site peptides containing a phi(A)-X-phi(B) motif, derived from substrate MEF2A and activating enzyme MKK3b, have been solved. The peptides bind to the same site in the C-terminal domain of the kinase, which is both outside the active site and distinct from the "CD" domain previously implicated in docking site interactions. Mutational analysis on the interaction of p38 with the docking sites supports the crystallographic models and has uncovered two novel residues on the docking groove that are critical for binding. The two peptides induce similar large conformational changes local to the peptide binding groove. The peptides also induce unexpected and different conformational changes in the active site, as well as structural disorder in the phosphorylation lip.     

Solution NMR insights into docking interactions involving inactive ERK2

The mitogen-activated protein (MAP) kinase ERK2 contains recruitment sites that engage canonical and noncanonical motifs found in a variety of upstream kinases, regulating phosphatases and downstream targets. Interactions involving two of these sites, the D-recruitment site (DRS) and the F-recruitment site (FRS), have been shown to play a key role in signal transduction by ERK/MAP kinases. The dynamic nature of these recruitment events makes NMR uniquely suited to provide significant insight into these interactions. While NMR studies of kinases in general have been greatly hindered by their large size and complex dynamic behavior leading to the suboptimal performance of standard methodologies, we have overcome these difficulties for inactive full-length ERK2 and obtained an acceptable level of backbone resonance assignments. This allowed a detailed investigation of the structural perturbations that accompany interactions involving both canonical and noncanonical recruitment events. No crystallographic information exists for the latter. We found that the chemical shift perturbations in inactive ERK2, indicative of structural changes in the presence of canonical and noncanonical motifs, are not restricted to the recruitment sites but also involve the linker that connects the N- and C-lobes and, in most cases, a gatekeeper residue that is thought to exert allosteric control over catalytic activity. We also found that, even though the canonical motifs interact with the DRS utilizing both charge-charge and hydrophobic interactions, the noncanonical interactions primarily involve the latter. These results demonstrate the feasibility of solution NMR techniques for a comprehensive analysis of docking interactions in a full-length ERK/MAP kinase.     

The third conformation of p38α MAP kinase observed in phosphorylated p38α and in solution

MAPKs engage substrates, MAP2Ks, and phosphatases via a docking groove in the C-terminal domain of the kinase. Prior crystallographic studies on the unphosphorylated MAPKs p38α and ERK2 defined the docking groove and revealed long-range conformational changes affecting the activation loop and active site of the kinase induced by peptide. Solution NMR data presented here for unphosphorylated p38α with a MEK3b-derived peptide (p38α/pepMEK3b) validate these findings. Crystallograhic data from doubly phosphorylated active p38α (p38α/T?GY?/pepMEK3b) reveal a structure similar to unphosphorylated p38α/MEK3b, and distinct from phosphorylated p38γ (p38γ/T?GY?) and ERK2 (ERK2/T?EY?). The structure supports the idea that MAP kinases adopt three distinct conformations: unphosphorylated, phosphorylated, and a docking peptide-induced form.     

ERK2 shows a restrictive and locally selective mechanism of recognition by its tyrosine phosphatase inactivators not shared by its activator MEK1

The two regulatory residues that control the enzymatic activity of the mitogen-activated protein (MAP) kinase ERK2 are phosphorylated by the unique MAP kinase kinases MEK1/2 and dephosphorylated by several tyrosine-specific and dual specificity protein phosphatases. Selective docking interactions facilitate these phosphorylation and dephosphorylation events, controlling the specificity and duration of the MAP kinase activation-inactivation cycles. We have analyzed the contribution of specific residues of ERK2 in the physical and functional interaction with the ERK2 phosphatase inactivators PTP-SL and MKP-3 and with its activator MEK1. Single mutations in ERK2 that abrogated the dephosphorylation by endogenous tyrosine phosphatases from HEK293 cells still allowed efficient phosphorylation by endogenous MEK1/2. Discrete ERK2 mutations at the ERK2 docking groove differentially affected binding and inactivation by PTP-SL and MKP-3. Remarkably, the cytosolic retention of ERK2 by its activator MEK1 was not affected by any of the analyzed ERK2 single amino acid substitutions. A chimeric MEK1 protein, containing the kinase interaction motif of PTP-SL, bound tightly to ERK2 through its docking groove and behaved as a gain-of-function MAP kinase kinase that hyperactivated ERK2. Our results provide evidence that the ERK2 docking groove is more restrictive and selective for its tyrosine phosphatase inactivators than for MEK1/2 and indicate that distinct ERK2 residues modulate the docking interactions with activating and inactivating effectors.     

Substantial Conformational Change Mediated by Charge-Triad Residues of the Death Effector Domain in Protein-Protein Interactions

Protein conformational changes are commonly associated with the formation of protein complexes. The non-catalytic death effector domains (DEDs) mediate protein-protein interactions in a variety of cellular processes, including apoptosis, proliferation and migration, and glucose metabolism. Here, using NMR residual dipolar coupling (RDC) data, we report a conformational change in the DED of the phosphoprotein enriched in astrocytes, 15 kDa (PEA-15) protein in the complex with a mitogen-activated protein (MAP) kinase, extracellular regulated kinase 2 (ERK2), which is essential in regulating ERK2 cellular distribution and function in cell proliferation and migration. The most significant conformational change in PEA-15 happens at helices α2, α3, and α4, which also possess the highest flexibility among the six-helix bundle of the DED. This crucial conformational change is modulated by the D/E-RxDL charge-triad motif, one of the prominent structural features of DEDs, together with a number of other electrostatic and hydrogen bonding interactions on the protein surface. Charge-triad motif promotes the optimal orientation of key residues and expands the binding interface to accommodate protein-protein interactions. However, the charge-triad residues are not directly involved in the binding interface between PEA-15 and ERK2.     

A MAP kinase docking site is required for phosphorylation and activation of p90(rsk)/MAPKAP kinase-1

Activation of the various mitogen-activated protein (MAP) kinase pathways converts many different extracellular stimuli into specific cellular responses by inducing the phosphorylation of particular groups of substrates. One important determinant for substrate specificity is likely to be the amino-acid sequence surrounding the phosphorylation site; however, these sites overlap significantly between different MAP kinase family members. The idea is now emerging that specific docking sites for protein kinases are involved in the efficient binding and phosphorylation of some substrates [1] [2] [3] [4]. The MAP kinase-activated protein (MAPKAP) kinase p90 rsk contains two kinase domains [5]: the amino-terminal domain (D1) is required for the phosphorylation of exogenous substrates whereas the carboxy-terminal domain (D2) is involved in autophosphorylation. Association between the extracellular signal-regulated kinase (Erk) MAP kinases and p90(rsk) family members has been detected in various cell types including Xenopus oocytes [6] [7] [8], where inactive p90(rsk) is bound to the inactive form of the Erk2- like MAP kinase p42(mpk1). Here, we identify a new MAP kinase docking site located at the carboxyl terminus of p90(rsk). This docking site was required for the efficient phosphorylation and activation of p90(rsk) in vitro and in vivo and was also both necessary and sufficient for the stable and specific association with p42(mpk1). The sequence of the docking site was conserved in other MAPKAP kinases, suggesting that it might represent a new class of interaction motif that facilitates efficient and specific signal transduction by MAP kinases.     

Effect of the DEF motif on phosphorylation of peptide substrates by ERK

MAP kinase ERK maintains specificity by binding to docking sites such as the DEF domain or D domain. It was previously shown that appending peptides derived from D domains to a substrate peptide increased apparent efficiency of peptide phosphorylation while preserving its apparent specificity for ERK. Here we determine the effect of the DEF motif on efficiency and specificity of peptide phosphorylation by ERK. The DEF motif modulated the apparent affinity of the peptide for ERK while the substrate motif dominated the apparent catalytic rate. Attachment of the DEF sequence improved apparent phosphorylation efficiency by 60-fold. Addition of peptides possessing both the DEF and D motif to a substrate sequence did not yield additive effects on the K_M of the substrate for ERK. Further, the DEF motif diminished the apparent specificity for ERK and increased the apparent efficiencies of phosphorylation of the substrate peptide by p38α kinase and JNK1.     

Quantitative analysis of ERK2 interactions with substrate proteins: roles for kinase docking domains and activity in determining binding affinity

Extracellular signal-regulated kinase-1 and -2 (ERK1/2) proteins regulate a variety of cellular functions, including cell proliferation and differentiation, by interacting with and phosphorylating substrate proteins. Two docking sites, common docking (CD/ED) domain and F-site recruitment site (FRS), on ERK proteins have been identified. Specific interactions with the CD/ED domain and the FRS occur with substrates containing a docking site for ERK and JNK, LXL (DEJL) motif (D-domain) and a docking site for ERK, FXF (DEF) motif (F-site), respectively. However, the relative contributions of the ERK docking sites in mediating substrate interactions that allow efficient phosphate transfer are largely unknown. In these studies, we provide a quantitative analysis of ERK2 interactions with substrates using surface plasmon resonance to measure real time protein-protein interactions. ERK2 interacted with ELK-1 (DEF and DEJL motifs), RSK-1 (DEJL motif), and c-Fos (DEF motif) with K(D) values of 0.25, 0.15, and 0.97 μM, respectively. CD/ED domain mutations inhibited interactions with ELK-1 and RSK-1 by 6-fold but had no effect on interactions with c-Fos. Select mutations in FRS residues differentially inhibited ELK-1 or c-Fos interactions with ERK2 but had little effect on RSK-1 interactions. Mutations in both the ED and FRS docking sites completely inhibited ELK-1 interactions but had no effect on interactions with stathmin, an ERK substrate whose docking site is unknown. The phosphorylation status of ERK2 did not affect interactions with RSK-1 or c-Fos but did inhibit interactions with ELK-1 and stathmin. These studies provide a quantitative evaluation of specific docking domains involved in mediating interactions between ERK2 and protein substrates and define the contributions of these interactions to phosphate transfer.     

Docking of PRAK/MK5 to the Atypical MAPKs ERK3 and ERK4 Defines a Novel MAPK Interaction Motif

ERK3 and ERK4 are atypical MAPKs in which the canonical TXY motif within the activation loop of the classical MAPKs is replaced by SEG. Both ERK3 and ERK4 bind, translocate, and activate the MAPK-activated protein kinase (MK) 5. The classical MAPKs ERK1/2 and p38 interact with downstream MKs (RSK1–3 and MK2–3, respectively) through conserved clusters of acidic amino acids, which constitute the common docking (CD) domain. In contrast to the classical MAPKs, the interaction between ERK3/4 and MK5 is strictly dependent on phosphorylation of the SEG motif of these kinases. Here we report that the conserved CD domain is dispensable for the interaction of ERK3 and ERK4 with MK5. Using peptide overlay assays, we have defined a novel MK5 interaction motif (FRIEDE) within both ERK4 and ERK3 that is essential for binding to the C-terminal region of MK5. This motif is located within the L16 extension lying C-terminal to the CD domain in ERK3 and ERK4 and a single isoleucine to lysine substitution in FRIEDE totally abrogates binding, activation, and translocation of MK5 by both ERK3 and ERK4. These findings are the first to demonstrate binding of a physiological substrate via this region of the L16 loop in a MAPK. Furthermore, the link between activation loop phosphorylation and accessibility of the FRIEDE interaction motif suggests a switch mechanism for these atypical MAPKs in which the phosphorylation status of the activation loop regulates the ability of both ERK3 and ERK4 to bind to a downstream effector.Mitogen-activated protein kinase (MAPK)² phosphorylation cascades play important roles in the regulation of diverse cellular functions such as cell proliferation, differentiation, migration, and apoptosis (1, 2). A characteristic and conserved feature of this family of signaling pathways is their organization into modules comprising a sequential three-tiered kinase cascade. This contains a MAPK kinase kinase, a MEK, and the MAPK itself. Four such MAPK signaling modules have been described in mammals: ERK1 and ERK2, the c-Jun N-terminal kinases 1–3, the p38 kinases (p38α/β/γ/δ), and ERK5 (3–7). The MAPK kinase kinases phosphorylate and activate the MEKs, which in turn activate the MAPKs by dual phosphorylation on both the threonine and the tyrosine residue of a highly conserved TXY motif in the kinase activation loop. MAPKs are Ser/Thr kinases, which phosphorylate a wide range of substrates with the minimal consensus sequence (S/T)P (2).ERK4 and its close relative ERK3 are regarded as atypical members of the MAPK family. In contrast to the classical MAPKs, ERK3 and ERK4 harbor an SEG motif in the activation loop and thus lack a second phosphoacceptor site. In addition, protein kinases all possess a conserved APE motif located just C-terminal to the phosphoacceptor sites within subdomain VIII, in which the conserved glutamate is important for maintaining the stability of the kinase domain. In both ERK3 and ERK4, this motif is substituted by SPR, and ERK3 and ERK4 are the only two protein kinases in the human genome with an arginine residue in this position (8). Although they display significant sequence homology (44% identity) with ERK1 and ERK2 within their kinase domains, both ERK3 and ERK4 have unique C-terminal extensions, which account for the large differences in size observed between ERK1/2 (∼360 amino acids) and ERK3/ERK4 (721/587 amino acids). Whereas classical MAPKs have been highly conserved throughout evolution, with examples found in both unicellular and multicellular organisms, ERK3 and ERK4 are only present in vertebrates. Finally, in contrast to many of the classical MAPKs, the regulation, substrate specificity, and physiological functions of ERK3 and ERK4 are poorly understood. Although ERK3 and ERK4 are very similar to each other, there are significant differences between them. For instance, whereas ERK4, like most classical MAPKs, is a stable protein, ERK3 is highly unstable and subject to rapid proteosomal degradation. Thus, ERK3 activity may be regulated at the level of cellular abundance, and taken together these features indicate that ERK3 and ERK4 may perform specialized functions and enjoy different modes of regulation when compared with classical MAPKs (9–11).Despite the striking differences between ERK3 and ERK4 and the classical MAPKs, they do share one property with the ERK1/2, p38, and ERK5, namely the ability to interact with a group of downstream Ser/Thr protein kinases, termed MAPK-activated protein kinases (MAPKAPKs or MKs) (12, 13). In the case of ERK3 and ERK4, both proteins interact with, translocate, and activate the MK5 protein kinase. Several studies have drawn attention to the role of specific docking interactions that contribute to both substrate selectivity and regulation in MAPK pathways (14–17). These interactions involve docking domains, which specifically recognize small peptide docking motifs (D motifs) located on functional MAPK partner proteins including downstream substrates, scaffold proteins, as well as positive and negative regulators. The docking domains, although located within the kinase domains, are distinct from the active site. Similarly the D motifs, which these docking domains recognize, are also distinct from the phosphoacceptor sites within protein substrates (18). There are several classes of D motifs. The motifs found in MAPKAP kinases including MK5 have the consensus sequence LX_1–2(K/R)_2–5 where X is any amino acid (12). The corresponding docking domains within the MAPKs have also been characterized (16, 19, 20). The common docking (CD) domain is a cluster of negatively charged amino acids located in the L16 extension directly C-terminal to the kinase domain in the MAPK primary structure. A second domain termed ED (Glu-Asp) also contributes to binding specificity. This latter site is located near the CD domain in the MAPK tertiary structure. Whereas the CD domain is considered commonly important for all docking interactions, the ED site is thought to be important for the determination of specificity (16). Other residues and regions distinct from the ED and CD domains have also been shown to be important for docking.(21–25).This work has so far been largely confined to analysis of the classical MAPKs, and much less is known about the nature of substrate or regulatory docking interactions for the atypical MAPKs. We and others (9, 11, 26) have recently reported that the region encompassing residues 326–340 within both ERK3 and ERK4 is required for their ability to interact with and activate MK5. Furthermore, a truncated mutant of MK5, which lacks the 50 C-terminal residues (MK5 1–423), was unable to bind to ERK4 despite the fact that it retains its D domain. Finally, in contrast to conventional MAPKs, the interaction between ERK3 and ERK4 and MK5 requires activation loop phosphorylation of ERK3 and ERK4 (27, 28). Taken together these observations suggest that the mechanism by which the atypical MAPKs recognize and bind to at least one important class of effector kinases may be distinct to that found in the classical MAPKs such as ERK1/2 and p38.Here we demonstrate that two separate C-terminal regions, encompassing residues 383–393 and 460–465, respectively, are necessary for MK5 to interact with both ERK3 and ERK4. These regions are distinct from the D motif previously identified within MK5, suggesting that binding to ERK3 and ERK4 may be mediated by a different mechanism to that seen in the classical MAPKs. In support of this, the conserved CD domains within ERK3 and ERK4 are shown to be completely dispensable for MK5 interaction. Using peptide overlay assays, we have defined a minimal MK5 interaction motif FRIEDE in ERK4. Furthermore, we demonstrate that a single point mutation (ERK3 I334K or ERK4 I330K) within this FRIEDE motif is sufficient to disrupt the binding of both ERK3 and ERK4 to MK5 and consequently their ability to both translocate and activate MK5. The FRIEDE motif is located within the L16 extension C-terminal to the CD domain in both ERK3 and ERK4. Interestingly, molecular modeling of the corresponding region in ERK2 suggests that it undergoes a significant conformational change as a result of activation loop phosphorylation, making this part of the L16 extension more accessible (29). We propose that the FRIEDE motif represents a novel MAPK interaction motif, the function of which is linked to activation loop phosphorylation and MAPK activation.     

Docking interactions in protein kinase and phosphatase networks

To achieve high biological specificity, protein kinases and phosphatases often recognize their targets through interactions that occur outside of the active site. Although the role of modular protein-protein interaction domains in kinase and phosphatase signaling has been well characterized, it is becoming clear that many kinases and phosphatases utilize docking interactions - recognition of a short peptide motif in target partners by a groove on the catalytic domain that is separate from the active site. Docking is particularly prevalent in serine/threonine kinases and phosphatases, and is a versatile organizational tool for building complex signaling networks; it confers a high degree of specificity and, in some cases, allosteric regulation.     

Two clusters of residues at the docking groove of mitogen-activated protein kinases differentially mediate their functional interaction with the tyrosine phosphatases PTP-SL and STEP.

Regulated function of mitogen-activated protein (MAP) kinases involves their selective association through docking sites with both activating MAP kinase kinases and inactivating phosphatases, including dual specificity and protein-tyrosine phosphatases (PTP). Site-directed mutagenesis on the mammalian MAP kinases ERK2 and p38alpha identified within their C-terminal docking grooves two clusters of residues important for association with their regulatory PTPs, PTP-SL and STEP. ERK2 and p38alpha mutations that resembled the sevenmaker gain-of-function mutation in the Rolled D. melanogaster ERK2 homologue failed to associate with PTP-SL, were not retained in the cytosol, and were poorly inactivated by this PTP. Additional ERK2 mutations at the docking groove showed deficient association and dephosphorylation by PTP-SL, although their cytosolic retention was unaffected. Other ERK2 mutations, resembling gain-of-function mutations in the FUS3 yeast ERK2 homologue, associated to PTP-SL and were inactivated normally by this PTP. Our results demonstrate that mutations at distinct regions of the docking groove of ERK2 and p38alpha differentially affect their association and regulation by the PTP-SL and STEP PTPs.     

The PHD domain of MEKK1 acts as an E3 ubiquitin ligase and mediates ubiquitination and degradation of ERK1/2

ERK1/2 MAP kinases are important regulators in cellular signaling, whose activity is normally reversibly regulated by threonine-tyrosine phosphorylation. In contrast, we have found that stress-induced ERK1/2 activity is downregulated by ubiquitin/proteasome-mediated degradation of ERK1/2. The PHD domain of MEKK1, a RING finger-like structure, exhibited E3 ubiquitin ligase activity toward ERK2 in vitro and in vivo. Moreover, both MEKK1 kinase activity and the docking motif on ERK1/2 were involved in ERK1/2 ubiquitination. Significantly, cells expressing ERK2 with the docking motif mutation were resistant to sorbitol-induced apoptosis. Therefore, MEKK1 functions not only as an upstream activator of the ERK and JNK through its kinase domain, but also as an E3 ligase through its PHD domain, providing a negative regulatory mechanism for decreasing ERK1/2 activity.     

Docking sites on substrate proteins direct extracellular signal-regulated kinase to phosphorylate specific residues

Mitogen-activated protein (MAP) kinases such as extracellular signal-regulated kinase (ERK) are important signaling proteins that phosphorylate (S/T)P sites in many different protein substrates. ERK binding to substrate proteins is mediated by docking sites including the FXFP motif and the D-domain. We characterized the sequence of amino acids that can constitute the FXFP motif using peptide and protein substrates. Substitutions of the phenylalanines at positions 1 and 3 had significant effects, indicating that these phenylalanines provide substantial binding affinity, whereas substitutions of the residues at positions 2 and 4 had less effect. The FXFP and D-domain docking sites were analyzed in a variety of positions and arrangements in the proteins ELK-1 and KSR-1. Our results indicate that the FXFP and D-domain docking sites form a flexible, modular system that has two functions. First, the affinity of a substrate for ERK can be regulated by the number, type, position, and arrangement of docking sites. Second, in substrates with multiple potential phosphorylation sites, docking sites can direct phosphorylation of specific (S/T)P residues. In particular, the FQFP motif of ELK-1 is necessary and sufficient to direct phosphorylation of serine 383, whereas the D-domain directs phosphorylation of other (S/T)P sites in ELK-1.     

Identification of a docking groove on ERK and p38 MAP kinases that regulates the specificity of docking interactions

MAP kinases (MAPKs) form a complex with MAPK kinases (MAPKKs), MAPK-specific phosphatases (MKPs) and various targets including MAPKAPKs. These docking interactions contribute to regulation of the specificity and efficiency of the enzymatic reactions. We have previously identified a docking site on MAPKs, termed the CD (common docking) domain, which is utilized commonly for docking interactions with MAPKKs, MKPs and MAPKAPKs. However, the CD domain alone does not determine the docking specificity. Here we have identified a novel site on p38 and ERK2 MAPKs that regulates the docking specificity towards MAPKAPKs. Remarkably, exchange of two amino acids in this site of ERK2 for corresponding residues of p38 converted the docking specificity for MAPKAPK-3/3pk, which is a dominant target of p38, from the ERK2 type to the p38 type, and vice versa. Furthermore, our detailed analyses with a number of MAPKAPKs and MKPs suggest that a groove in the steric structure of MAPKs, which comprises the CD domain and the site identified here, serves as a common docking region for various MAPK-interacting molecules.     

Crystal structure of PTP-SL/PTPBR7 catalytic domain: implications for MAP kinase regulation

Protein tyrosine phosphatases PTP-SL and PTPBR7 are isoforms belonging to cytosolic membrane-associated and to receptor-like PTPs (RPTPs), respectively. They represent a new family of PTPs with a major role in activation and translocation of MAP kinases. Specifically, the complex formation between PTP-SL and ERK2 involves an unusual interaction leading to the phosphorylation of PTP-SL by ERK2 at Thr253 and the inactivating dephosphorylation of ERK2 by PTP-SL. This interaction is strictly dependent upon a kinase interaction motif (KIM) (residues 224-239) situated at the N terminus of the PTP-SL catalytic domain. We report the first crystal structure of the catalytic domain for a member of this family (PTP-SL, residues 254-549, identical with residues 361-656 of PTPBR7), providing an example of an RPTP with single cytoplasmic domain, which is monomeric, having an unhindered catalytic site. In addition to the characteristic PTP-core structure, PTP-SL has an N-terminal helix, possibly orienting the KIM motif upon interaction with the target ERK2. An unusual residue in the catalytically important WPD loop promotes formation of a hydrophobically and electrostatically stabilised clamp. This could induce increased rigidity to the WPD loop and therefore reduced catalytic activity, in agreement with our kinetic measurements. A docking model based on the PTP-SL structure suggests that, in the complex with ERK2, the phosphorylation of PTP-SL should be accomplished first. The subsequent dephosphorylation of ERK2 seems to be possible only if a conformational rearrangement of the two interacting partners takes place.     

The activity of the extracellular signal-regulated kinase 2 is regulated by differential phosphorylation in the activation loop

The mitogen-activated protein kinases (MAP kinases) play a central role in signaling pathways initiated by extracellular stimuli such as growth factors, cytokines, and various forms of environmental stress. Full activation of the MAP kinases requires dual phosphorylation of the Thr and Tyr residues in the TXY motif of the activation loop by MAP kinase kinases. Interestingly, down-regulation of MAP kinase activity can be initiated by multiple Ser/Thr phosphatases, Tyr-specific phosphatases, and dual-specificity phosphatases. This would inevitable lead to the formation of monophosphorylated MAP kinases. However, in much of the literature investigating MAP kinase signaling, there has been the implicit assumption that the monophosphorylated forms are inactive. Thus, the significance for the need of multiple phosphatases in regulating MAP kinase activity is not clear, and the biological functions of these monophosphorylated MAP kinases are currently unknown. We have prepared extracellular signal-regulated protein kinase 2 (ERK2) in all phosphorylated forms and kinetically characterized them using two proteins (the myelin basic protein and Elk-1) and ATP as substrates. Our results revealed that a single phosphorylation in the activation loop of ERK2 produces an intermediate activity state. Thus, the catalytic efficiencies of the monophosphorylated ERK2/pY and ERK2/pT (ERK2 phosphorylated on Tyr-185 and Thr-183, respectively) are approximately 2-3 orders of magnitude higher than that of the unphosphorylated ERK2 and are only 1-2 orders of magnitude lower than that of the fully active bisphosphorylated ERK2/pTpY. This raises the possibility that the monophosphorylated ERK2s may have distinct biological roles in vivo. Different phosphorylation states in the activation loop could be linked to graded effects on a single ERK2 function. Alternatively, they could be linked to distinct ERK2 functions. Although less active than the bisphosphorylated species, the monophosphorylated ERK2s may differentially phosphorylate pathway components.     

The structure of phosphorylated p38gamma is monomeric and reveals a conserved activation-loop conformation.

BACKGROUND: Mitogen-activated protein (MAP) kinases mediate the cellular response to stimuli such as pro-inflammatory cytokines and environmental stress. P38gamma is a new member of the MAP kinase family, and is expressed at its highest levels in skeletal muscle. P38gamma is 63% identical in sequence to P38alpha. The structure of P38alpha MAP kinase has been determined in the apo, unphosphorylated, inactive form. The structures of apo unphosphorylated ERK2, a related MAP kinase, and apo phosphorylated ERK2 have also been determined. RESULTS: We have determined the structure of doubly phosphorylated P38gamma in complex with an ATP analog by X-ray crystallography. This is the first report of a structure of an activated kinase in the P38 subfamily, and the first bound to a nucleotide. P38gamma residue phosphoryl-Thr183 forms hydrogen bonds with five basic amino acids, and these interactions induce an interdomain rotation. The conformation of the activation loop of P38gamma is almost identical to that observed in the structure of activated ERK2. However, unlike ERK2, the crystal structure and solution studies indicate that activated P38gamma exists as a monomer. CONCLUSIONS: Interactions mediated by phosphoryl-Thr183 induce structural changes that direct the domains and active-site residues of P38gamma into a conformation consistent with catalytic activity. The conformation of the phosphorylation loop is likely to be similar in all activated MAP kinases, but not all activated MAP kinases form dimers.     

Regulation of the inducible nuclear dual-specificity phosphatase DUSP5 by ERK MAPK

DUSP5 is an inducible, nuclear, dual-specificity phosphatase, which specifically interacts with and inactivates the ERK1/2 MAP kinases in mammalian cells. In addition, expression of DUSP5 causes nuclear translocation of ERK2 indicating that it may act as a nuclear anchor for the inactive kinase. Here we show that induction of DUSP5 mRNA and protein in response to growth factors is dependent on ERK1/2 activation and that the accumulation of DUSP5 protein is regulated by rapid proteasomal degradation. DUSP5 is phosphorylated by ERK1/2 both in vitro and in vivo on three sites (Thr321, Ser346 and Ser376) within its C-terminal domain. DUSP5 phosphorylation is absolutely dependent on the conserved kinase interaction motif (KIM) within the amino-terminal domain of DUSP5, indicating that the same protein–protein contacts are required for both the inactivation of ERK2 by DUSP5 and for DUSP5 to act as a substrate for this MAPK. Using a combination of pharmacological inhibitors and phospho-site mutants we can find no evidence that phosphorylation of DUSP5 by ERK2 significantly affects either the half-life of the DUSP5 protein or its ability to bind to, inactivate or anchor ERK2 in the nucleus. However, co-expression of ERK2 results in significant stabilisation of DUSP5, which is accompanied by reduced levels of DUSP5 ubiquitination. These changes are independent of ERK2 kinase activity but absolutely depend on the ability of ERK2 to bind to DUSP5. We conclude that DUSP5 is stabilised by complex formation with its physiological substrate and that this may reinforce its activity as both a phosphatase and nuclear anchor for ERK2.