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.     

The docking interaction of caspase-9 with ERK2 provides a mechanism for the selective inhibitory phosphorylation of caspase-9 at threonine 125

Caspase-9 plays a critical role in the initiation of apoptosis by the mitochondrial pathway. Activation of caspase-9 is inhibited by phosphorylation at Thr(125) by ERK1/2 MAPKs in response to growth factors. Here, we show that phosphorylation of this site is specific for these classical MAPKs and is not strongly induced when JNK and p38alpha/beta MAPKs are activated by anisomycin. By deletion and mutagenic analysis, we identify domains in caspase-9 and ERK2 that mediate their interaction. Binding of ERK2 to caspase-9 and subsequent phosphorylation of caspase-9 requires a basic docking domain (D domain) in the N-terminal prodomain of the caspase. Mutational analysis of ERK2 reveals a (157)TTCD(160) motif required for recognition of caspase-9 that acts independently of the putative common docking domain. Molecular modeling supports the conclusion that Arg(10) in the D domain of caspase-9 interacts with Asp(160) in the TTCD motif of ERK2. Differences in the TTCD motif in other MAPK family members could account for the selective recognition of caspase-9 by ERK1/2. This selectivity may be important for the antiapoptotic role of classical MAPKs in contrast to the proapoptotic roles of stress-activated MAPKs.     

Activation of ERK induces phosphorylation of MAPK phosphatase-7, a JNK specific phosphatase,at Ser-446

We previously showed that MKP-7 suppresses MAPK activation in COS-7 cells in the order of selectivity, JNK > p38 > ERK, but interacts with ERK as well as JNK and p38. In this study we found that, when expressed in COS-7 cells with HA-ERK2, the mobility of FLAG-MKP-7 was decreased on SDS-PAGE gels depending on several stimuli, including phorbol 12-myristate 13-acetate, fetal bovine serum, epidermal growth factor, H2O2, and ionomycin. By using U0126, a MEK inhibitor, and introducing several point mutations, we demonstrated that this upward mobility shift is because of phosphorylation and identified Ser-446 of MKP-7 as the phosphorylation site targeted by ERK activation. To determine how MKP-7 interacts with MAPKs, we identified three domains in MKP-7 required for interaction with MAPKs, namely, putative MAP kinase docking domains (D-domain) I and II and a long COOH-terminal stretch unique to MKP-7. The D-domain I is required for interaction with ERK and p38, whereas the D-domain II is required for interaction with JNK and p38, which is likely to be important for MKP-7 to suppress JNK and p38 activations. The COOH-terminal stretch of MKP-7 was shown to determine JNK preference for MKP-7 by masking MKP-7 activity toward p38 and is a domain bound by ERK. These data strongly suggested that Ser-446 of MKP-7 is phosphorylated by ERK.     

Substrate discrimination among mitogen-activated protein kinases through distinct docking sequence motifs

Mitogen-activated protein kinases (MAPKs) mediate cellular responses to a wide variety of extracellular stimuli. MAPK signal transduction cascades are tightly regulated, and individual MAPKs display exquisite specificity in recognition of their target substrates. All MAPK family members share a common phosphorylation site motif, raising questions as to how substrate specificity is achieved. Here we describe a peptide library screen to identify sequence requirements of the DEF site (docking site for ERK FXF), a docking motif separate from the phosphorylation site. We show that MAPK isoforms recognize DEF sites with unique sequences and identify two key residues on the MAPK that largely dictate sequence specificity. Based on these observations and computational docking studies, we propose a revised model for MAPK interaction with substrates containing DEF sites. Variations in DEF site sequence requirements provide one possible mechanism for encoding complex target specificity among MAPK isoforms.     

Sequence Alignment Reveals Possible MAPK Docking Motifs on HIV Proteins

Over the course of HIV infection, virus replication is facilitated by the phosphorylation of HIV proteins by human ERK1 and ERK2 mitogen-activated protein kinases (MAPKs). MAPKs are known to phosphorylate their substrates by first binding with them at a docking site. Docking site interactions could be viable drug targets because the sequences guiding them are more specific than phosphorylation consensus sites. In this study we use multiple bioinformatics tools to discover candidate MAPK docking site motifs on HIV proteins known to be phosphorylated by MAPKs, and we discuss the possibility of targeting docking sites with drugs. Using sequence alignments of HIV proteins of different subtypes, we show that MAPK docking patterns previously described for human proteins appear on the HIV matrix, Tat, and Vif proteins in a strain dependent manner, but are absent from HIV Rev and appear on all HIV Nef strains. We revise the regular expressions of previously annotated MAPK docking patterns in order to provide a subtype independent motif that annotates all HIV proteins. One revision is based on a documented human variant of one of the substrate docking motifs, and the other reduces the number of required basic amino acids in the standard docking motifs from two to one. The proposed patterns are shown to be consistent with in silico docking between ERK1 and the HIV matrix protein. The motif usage on HIV proteins is sufficiently different from human proteins in amino acid sequence similarity to allow for HIV specific targeting using small-molecule drugs.     

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.     

Differential phosphorylation of c-Jun and JunD in response to the epidermal growth factor is determined by the structure of MAPK targeting sequences

MAPK phosphorylation of various substrates is mediated by the presence of docking sites, including the D domain and the DEF motif. Depending on the number and sequences of these domains, substrates are phosphorylated by specific subsets of MAPKs. For example, a D domain targets JNK to c-Jun, whereas a DEF motif is required for ERK phosphorylation of c-Fos. JunD, in contrast, contains both D and DEF domains. Here we show that these motifs mediate JunD phosphorylation in response to either ERK or JNK activation. An intact D domain is required for phosphorylation and activation of JunD by both subtypes of MAPK. The DEF motif acts together with the D domain to elicit efficient phosphorylation of JunD in response to the epidermal growth factor (EGF) but has no function on JunD phosphorylation and activation by JNK signaling. Furthermore, we show that conversion of a c-Jun sequence to a canonical DEF domain, as it is present in JunD, elicits c-Jun activation in response to EGF. Our results suggest that evolution of a particular modular system of MAPK targeting sequences has determined a differential response of JunD and c-Jun to ERK activation.     

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.     

Molecular determinants that mediate selective activation of p38 MAP kinase isoforms

The p38 mitogen-activated protein kinase (MAPK) group is represented by four isoforms in mammals (p38alpha, p38beta2, p38gamma and p38delta). These p38 MAPK isoforms appear to mediate distinct functions in vivo due, in part, to differences in substrate phosphorylation by individual p38 MAPKs and also to selective activation by MAPK kinases (MAPKKs). Here we report the identification of two factors that contribute to the specificity of p38 MAPK activation. One mechanism of specificity is the selective formation of functional complexes between MAPKK and different p38 MAPKs. The formation of these complexes requires the presence of a MAPK docking site in the N-terminus of the MAPKK. The second mechanism that confers signaling specificity is the selective recognition of the activation loop (T-loop) of p38 MAPK isoforms. Together, these processes provide a mechanism that enables the selective activation of p38 MAPK in response to activated MAPKK.     

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 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.     

Substrate recognition domains within extracellular signal-regulated kinase mediate binding and catalytic activation of mitogen-activated protein kinase phosphatase-3

Mitogen-activated protein (MAP) kinase phosphatase-3 (MKP-3) is a dual specificity phosphatase that inactivates extracellular signal-regulated kinase (ERK) MAP kinases. This reflects tight and specific binding between ERK and the MKP-3 amino terminus with consequent phosphatase activation and dephosphorylation of the bound MAP kinase. We have used a series of p38/ERK chimeric molecules to identify domains within ERK necessary for binding and catalytic activation of MKP-3. These studies demonstrate that ERK kinase subdomains V-XI are necessary and sufficient for binding and catalytic activation of MKP-3. These domains constitute the major COOH-terminal structural lobe of ERK. p38/ERK chimeras possessing these regions display increased sensitivity to inactivation by MKP-3. These data also reveal an overlap between ERK domains interacting with MKP-3 and those known to confer substrate specificity on the ERK MAP kinase. Consistent with this, we show that peptides representing docking sites within the target substrates Elk-1 and p90(rsk) inhibit ERK-dependent activation of MKP-3. In addition, abolition of ERK-dependent phosphatase activation following mutation of a putative kinase interaction motif (KIM) within the MKP-3 NH(2) terminus suggests that key sites of contact for the ERK COOH-terminal structural lobe include residues localized between the Cdc25 homology domains (CH2) found conserved between members of the DSP gene family.     

Hydrophobic as well as charged residues in both MEK1 and ERK2 are important for their proper docking

Docking between MEK1 and ERK2 is required for their stable interaction and efficient signal transmission. The MEK1 N terminus contains the ERK docking or D domain that consists of conserved hydrophobic and basic residues. We mutated the hydrophobic and basic residues individually and found that loss of either type reduced MEK1 phosphorylation of ERK2 in vitro and its ability to bind to ERK2 in vivo. Moreover, ERK2 was localized in both the cytoplasm and the nucleus when co-expressed with MEK1 that had mutations in either the hydrophobic or the basic residues. We then identified two conserved hydrophobic residues on ERK2 that play roles in docking with MEK1. Mutating these residues to alanine reduced the interaction of ERK2 with MEK1 in cells. These mutations also reduced the phosphorylation of MEK1 by ERK2 but had little effect on phosphorylation of MBP by ERK2. Finally, we generated docking site mutants in ERK2-MEK1 fusion proteins. Although the mutation of the MEK1 D domain significantly reduced ERK2-MEK1 activity, mutations of the putatively complementary acidic residues and hydrophobic residues on ERK2 did not change its activity. However, both types of mutations decreased the phosphorylation of Elk-1 caused by ERK2-MEK1 fusion proteins. These findings suggest complex interactions of MEK1 D domains with ERK2 that influence its activation and its effects on substrates.