D-recruitment Site Research Articles

Targeting ERK-MYD88 interaction leads to ERK dysregulation and immunogenic cancer cell death

The quest for targeted therapies is critical in the battle against cancer. The RAS/MAP kinase pathway is frequently implicated in neoplasia, with ERK playing a crucial role as the most distal kinase in the RAS signaling cascade. Our previous research demonstrated that the interaction between ERK and MYD88, an adaptor protein in innate immunity, is crucial for RAS-dependent transformation and cancer cell survival. In this study, we examine the biological consequences of disrupting the ERK-MYD88 interaction through the ERK D-recruitment site (DRS), while preserving ERK’s kinase activity. Our results indicate that EI-52, a small-molecule benzimidazole targeting ERK-MYD88 interaction induces an HRI-mediated integrated stress response (ISR), resulting in immunogenic apoptosis specific to cancer cells. Additionally, EI-52 exhibits anti-tumor efficacy in patient-derived tumors and induces an anti-tumor T cell response in mice in vivo. These findings suggest that inhibiting the ERK-MYD88 interaction may be a promising therapeutic approach in cancer treatment.

Hydrogen peroxide-dependent oxidation of ERK2 within its D-recruitment site alters its substrate selection

Extracellular signal-regulated kinases 1 and 2 (ERK1/2) are dysregulated in many pervasive diseases. Recently, we discovered that ERK1/2 is oxidized by signal-generated hydrogen peroxide in various cell types. Since the putative sites of oxidation lie within or near ERK1/2's ligand-binding surfaces, we investigated how oxidation of ERK2 regulates interactions with the model substrates Sub-D and Sub-F. These studies revealed that ERK2 undergoes sulfenylation at C159 on its D-recruitment site surface and that this modification modulates ERK2 activity differentially between substrates. Integrated biochemical, computational, and mutational analyses suggest a plausible mechanism for peroxide-dependent changes in ERK2-substrate interactions. Interestingly, oxidation decreased ERK2's affinity for some D-site ligands while increasing its affinity for others. Finally, oxidation by signal-generated peroxide enhanced ERK1/2's ability to phosphorylate ribosomal S6 kinase A1 (RSK1) in HeLa cells. Together, these studies lay the foundation for examining crosstalk between redox- and phosphorylation-dependent signaling at the level of kinase-substrate selection.

Identification and biochemical characterization of small molecule inhibitors of ERK2 that target the D-recruitment site.

Extracellular signal-regulated kinase (ERK) is the culmination of a mitogen-activated protein kinase cascade that regulates cellular processes like proliferation, migration, and survival. Consequently, abnormal ERK signaling often plays a role in the tumorigenesis and metastasis of numerous cancers. ERK inhibition is a sought-after treatment for cancers, especially since clinically approved drugs that target signaling upstream of ERK often induce acquired resistance. Furthermore, the ERK2 isoform may have a differential role in various cancers from the other canonical isoform, ERK1. We demonstrate that small molecules can inhibit ERK2 catalytic and noncatalytic functions by binding to the D-recruitment site (DRS), a protein-protein interaction site distal to the enzyme active site. Using a fluorescence anisotropy-based high-throughput screening, we identify compounds that bind to the DRS and exhibit dose-dependent inhibition of ERK2 activity and ERK2 phosphorylation. We characterize the dose-dependent potency of ERK2 inhibitors using fluorescence anisotropy-based binding assays, fluorescence-based ERK2 substrate phosphorylation assays, and in vitro ERK2 activation assays. In our example, the binding of a DRS inhibitor can be prevented by mutating the DRS residue Cys-159 to serine, indicating that this residue is essential for the interaction. Resulting inhibitors from this process can be assessed in cellular and in vivo experiments for inhibition of ERK signaling and can be evaluated as potential cancer drugs.

Modulating multi-functional ERK complexes by covalent targeting of a recruitment site in vivo

Recently, the targeting of ERK with ATP-competitive inhibitors has emerged as a potential clinical strategy to overcome acquired resistance to BRAF and MEK inhibitor combination therapies. In this study, we investigate an alternative strategy of targeting the D-recruitment site (DRS) of ERK. The DRS is a conserved region that lies distal to the active site and mediates ERK–protein interactions. We demonstrate that the small molecule BI-78D3 binds to the DRS of ERK2 and forms a covalent adduct with a conserved cysteine residue (C159) within the pocket and disrupts signaling in vivo. BI-78D3 does not covalently modify p38MAPK, JNK or ERK5. BI-78D3 promotes apoptosis in BRAF inhibitor-naive and resistant melanoma cells containing a BRAF V600E mutation. These studies provide the basis for designing modulators of protein–protein interactions involving ERK, with the potential to impact ERK signaling dynamics and to induce cell cycle arrest and apoptosis in ERK-dependent cancers.

Abstract 3872: Targeting multi-functional ERK-protein complexes in vivo

Abstract Mutations in pathways that enhance the activity of ERK1 and ERK2 are frequently present in human cancers, reflecting their important roles in tumor initiation, and progression. This notion is reinforced by observations in BRAF V600E mutant melanoma where the majority of the mechanisms of resistance to FDA-approved combination therapies targeting BRAF and MEK involve reactivation of ERK1 and ERK2. Recently, the direct targeting of the ERK enzymes using ATP-competitive inhibitors has emerged as an attractive strategy to overcome acquired resistance to current combination therapies. The ERK enzymes employ unique mechanisms of molecular recognition to engage protein components of the MAPK pathway. Here we report the potent targeting of an ERK-protein docking interaction by a small molecule thiotriazole, which abrogates ERK signaling in vivo. The thiotriazole binds covalently to a highly conserved cysteine residue within the D-recruitment site of ERK1/2 with more than a 100-fold discrimination over other MAPKs (e.g. JNK1/2, p38MAPKs and ERK5). Treatment of various BRAF-inhibitor naive or inhibitor-resistant melanoma cell lines expressing BRAF V600E with the thiotriazole for 2 hours induces dose-dependent inhibition of ERK activation and downstream signaling. Inhibition is maintained for up to 5 hours following thiotriazole washout and induces apoptosis and growth inhibition. Treatment of mice carrying a BRAFV600E A375 melanoma xenograft with the thiotriazole blocked tumor growth. Transient expression of a mutant form of ERK2, which is resistant to the thiotriazole, promotes survival of A375 and HEK 293 cells treated with thiotriazole. This study lays the foundation for developing a new modality for the treatment of solid tumors driven by excessive ERK signaling. Citation Format: Tamer S. Kaoud, William H. Johnson, Nancy D. Ebelt, Andrea Piserchio, Diana Zamora-Olivares, Sabrina V. Ravenstein, Jacey R. Pridgen, Ramakrishna Edupuganti, Rachel Sammons, Micael Cano, Mangalika Warthaka, Pengyu Ren, Eric V. Anslyn, Kenneth Y. Tsai, Ranajeet Ghose, Kevin N. Dalby. Targeting multi-functional ERK-protein complexes in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3872.

Targeting ERK beyond the boundaries of the kinase active site in melanoma

Extracellular signal-regulated kinase 1/2 (ERK1/2) constitute a point of convergence for complex signaling events that regulate essential cellular processes, including proliferation and survival. As such, dysregulation of the ERK signaling pathway is prevalent in many cancers. In the case of BRAF-V600E mutant melanoma, ERK inhibition has emerged as a viable clinical approach to abrogate signaling through the ERK pathway, even in cases where MEK and Raf inhibitor treatments fail to induce tumor regression due to resistance mechanisms. Several ERK inhibitors that target the active site of ERK have reached clinical trials, however, many critical ERK interactions occur at other potentially druggable sites on the protein. Here we discuss the role of ERK signaling in cell fate, in driving melanoma, and in resistance mechanisms to current BRAF-V600E melanoma treatments. We explore targeting ERK via a distinct site of protein-protein interaction, known as the D-recruitment site (DRS), as an alternative or supplementary mode of ERK pathway inhibition in BRAF-V600E melanoma. Targeting the DRS with inhibitors in melanoma has the potential to not only disrupt the catalytic apparatus of ERK but also its noncatalytic functions, which have significant impacts on spatiotemporal signaling dynamics and cell fate.

A Novel Class of Common Docking Domain Inhibitors That Prevent ERK2 Activation and Substrate Phosphorylation.

Extracellular signal-regulated kinases (ERK1/2) are mitogen-activated protein kinases (MAPKs) that play a pro-tumorigenic role in numerous cancers. ERK1/2 possess two protein-docking sites that are distinct from the active site: the D-recruitment site (DRS) and the F-recruitment site. These docking sites facilitate substrate recognition, intracellular localization, signaling specificity, and protein complex assembly. Targeting these sites on ERK in a therapeutic context may overcome many problems associated with traditional ATP-competitive inhibitors. Here, we identified a new class of inhibitors that target the ERK DRS by screening a synthetic combinatorial library of more than 30 million compounds. The screen detects the competitive displacement of a fluorescent peptide from the DRS of ERK2. The top molecular scaffold from the screen was optimized for structure-activity relationship by positional scanning of different functional groups. This resulted in 10 compounds with similar binding affinities and a shared core structure consisting of a tertiary amine hub with three functionalized cyclic guanidino branches. Compound 2507-1 inhibited ERK2 from phosphorylating a DRS-targeting substrate and prevented the phosphorylation of ERK2 by a constitutively active MEK1 (MAPK/ERK kinase 1) mutant. Interaction between an analogue, 2507-8, and the ERK2 DRS was confirmed by nuclear magnetic resonance and X-ray crystallography. 2507-8 forms critical interactions at the common docking domain residue Asp319 via an arginine-like moiety that is shared by all 10 hits, suggesting a common binding mode. The structural and biochemical insights reported here provide the basis for developing new ERK inhibitors that are not ATP-competitive but instead function by disrupting critical protein-protein interactions.

Allosteric Modulation of JNK Docking Site Interactions with ATP-Competitive Inhibitors.

The c-Jun N-terminal kinases (JNKs) play a wide variety of roles in cellular signaling processes, dictating important, and even divergent, cellular fates. These essential kinases possess docking surfaces distal to their active sites that interact with diverse binding partners, including upstream activators, downstream substrates, and protein scaffolds. Prior studies have suggested that the interactions of certain protein-binding partners with one such JNK docking surface, termed the D-recruitment site (DRS), can allosterically influence the conformational state of the ATP-binding pocket of JNKs. To further explore the allosteric relationship between the ATP-binding pockets and DRSs of JNKs, we investigated how the interactions of the scaffolding protein JIP1, as well as the upstream activators MKK4 and MKK7, are allosterically influenced by the ATP-binding site occupancy of the JNKs. We show that the affinity of the JNKs for JIP1 can be divergently modulated with ATP-competitive inhibitors, with a >50-fold difference in dissociation constant observed between the lowest- and highest-affinity JNK1-inhibitor complexes. Furthermore, we found that we could promote or attenuate phosphorylation of JNK1's activation loop by MKK4 and MKK7, by varying the ATP-binding site occupancy. Given that JIP1, MKK4, and MKK7 all interact with JNK DRSs, these results demonstrate that there is functional allostery between the ATP-binding sites and DRSs of these kinases. Furthermore, our studies suggest that ATP-competitive inhibitors can allosterically influence the intracellular binding partners of the JNKs.

Impact of Redox Modifications on ERK2 Substrate Phosphorylation

Extracellular regulated kinase 2 (ERK2), which is the terminal serine/threonine protein kinase in the Ras-Raf-MEK-ERK signaling axis, is involved in the regulation of many cellular processes, including cell proliferation, differentiation, survival, and cell cycle progression. Recently, ERK2 was shown to undergo reversible oxidation, inhibiting reactivity toward Elk1 substrate, following growth factor stimulation in a number of cell types (Keyes et al., FRBM, 2017). Interestingly, one of the residues exhibiting oxidation sensitivity (C159), is located within the D-recruitment site (DRS). Since the DRS is involved in binding a subset of ERK2 substrates, we compared the ability of wild-type ERK2 and ERK2(C159S) to phosphorylate a model DRS peptide substrate, Sub-D, following treatment with various concentrations of H2O2. Kinetic analysis demonstrated that, while both wild-type ERK2 and ERK2(C159S) exhibit similar apparent kcat and Km values for Sub-D in the absence of H2O2, treatment with H2O2 causes more significant changes in kcat,app and Km,app for Sub-D in wild-type compared with the C159S mutant of ERK2. Together, these data suggest that redox modification of ERK2 may alter its ability to phosphorylate substrates recognized by the DRS. This raised the intriguing possibility that oxidation could alter ERK2’s activity toward some, but not all, of its downstream substrates. To explore this possibility further, we investigated the impact of redox modification on ERK2’s global substrate selectivity using functional protein microarrays. Consistent with our hypothesis, ERK2-mediated phosphorylation of several substrates was unaffected by H2O2 treatment while others exhibited H2O2-dependent changes in their phosphorylation status, indicating that C159 oxidation controls ERK activity by modulating substrate specificity. Together, these studies offer important insights into redox regulation of ERK2 and provide potential points of signal integration between kinase- and redox-dependent signaling pathways.

Local destabilization, rigid body, and fuzzy docking facilitate the phosphorylation of the transcription factor Ets-1 by the mitogen-activated protein kinase ERK2

Mitogen-activated protein (MAP) kinase substrates are believed to require consensus docking motifs (D-site, F-site) to engage and facilitate efficient site-specific phosphorylation at specific serine/threonine-proline sequences by their cognate kinases. In contrast to other MAP kinase substrates, the transcription factor Ets-1 has no canonical docking motifs, yet it is efficiently phosphorylated by the MAP kinase ERK2 at a consensus threonine site (T38). Using NMR methodology, we demonstrate that this phosphorylation is enabled by a unique bipartite mode of ERK2 engagement by Ets-1 and involves two suboptimal noncanonical docking interactions instead of a single canonical docking motif. The N terminus of Ets-1 interacts with a part of the ERK2 D-recruitment site that normally accommodates the hydrophobic sidechains of a canonical D-site, retaining a significant degree of disorder in its ERK2-bound state. In contrast, the C-terminal region of Ets-1, including its Pointed (PNT) domain, engages in a largely rigid body interaction with a section of the ERK2 F-recruitment site through a binding mode that deviates significantly from that of a canonical F-site. This latter interaction is notable for the destabilization of a flexible helix that bridges the phospho-acceptor site to the rigid PNT domain. These two spatially distinct, individually weak docking interactions facilitate the highly specific recognition of ERK2 by Ets-1, and enable the optimal localization of its dynamic phospho-acceptor T38 at the kinase active site to enable efficient phosphorylation.

Abstract 3648: A high-throughput fluorescence anisotropy screening for discovery of inhibitors that target the D-recruitment site of ERK in vitro and in cells

Abstract Background: Docking sites on mitogen-activated protein kinases (MAPKs) direct protein binding interactions and generate signal specificity. Therapeutically targeting these sites in cancer offers a way to potentially overcome many issues associated with ATP-competitive MAPK inhibitor design, such as high cellular ATP concentrations and highly conserved active sites among MAPKs. Extracellular signal-regulated kinases 1/2 (ERK1/2) are MAPKs that have a pro-tumorigenic role in numerous cancers and possess two protein docking sites that are distinct from the active site: the D-recruitment site (DRS) and F-recruitment site (FRS). In this study, we have developed a high-throughput fluorescence anisotropy screening to identify small molecule inhibitors that target the DRS of ERK. Methods: DRS inhibitors were detected in the high-throughput screening by using fluorescence anisotropy to measure the ability of over 22,000 molecules to competitively displace a fluorescent peptide from the DRS of inactive ERK2. The top 100 compounds from this screening were validated with a secondary radioactive kinase assay. In this assay, the ability of each compound to prevent the phosphorylation of a DRS-specific peptide by active ERK2 was assessed using [γ-32P]-labeled ATP substrate to measure enzyme activity. The top nine compounds were characterized by in vitro dose-response assays against wild-type and C159S ERK2, a DRS mutant. Inhibition of ERK2 phosphorylation of an FRS substrate and JNK2 phosphorylation of c-Jun was evaluated in vitro to determine compound specificity. The potency and specificity of the compounds were also assessed in EGF-stimulated HEK293T cells for their ability to prevent ERK1/2, JNK1/2, and p38α phosphorylation using Western blot. The compounds were tested for irreversible inhibition in vitro through ERK activity tests after exposure to, and subsequent removal of the compounds. Results: Of the nine hit compounds found from the screening, four reversibly inhibited the DRS of ERK2 with in vitro IC50 and Ki values under 5 μM, and did not show significant inhibition of the FRS or C159S ERK2. Two of these compounds also reduced ERK activation to basal levels in MAPK pathway-stimulated HEK293T cells at concentrations of 5 μM. However, in specificity tests these inhibitors demonstrate ability to also target JNK2 and p38α. Conclusions: The fluorescence anisotropy screening developed here can be used to identify potent inhibitors that target the DRS of ERK. These inhibitors can be used to probe ERK signaling events that are mediated by the DRS and can be further optimized to yield highly selective ERK DRS inhibitors with potential therapeutic value. Citation Format: Rachel M. Sammons, Tamer S. Kaoud, Ashwini K. Devkota, Eun J. Cho, Kevin N. Dalby. A high-throughput fluorescence anisotropy screening for discovery of inhibitors that target the D-recruitment site of ERK in vitro and in cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3648. doi:10.1158/1538-7445.AM2015-3648

Elucidating binding modes of zuonin A enantiomers to JNK1 via in silico methods

Aberrant c-Jun N-terminal kinase (JNK) signaling is associated with a number of diseases, including neurological conditions and cancer. Enantiomers of the lignan zuonin A, (−)-zuonin A and (+)-zuonin A bind isoforms of JNK with similar affinity and disrupt protein-protein interactions at JNK's D-recruitment site. Thus, they are of interest as lead non-ATP competitive inhibitors of the JNKs. While (−)-zuonin A inhibits the activity of JNK toward c-Jun by 80% when saturating, (+)-zuonin A only inhibits by 15%. Molecular docking and molecular dynamics simulations were performed to gain a better understanding of how these inhibitors interact with JNK. The results of this study provide new insight into potential binding modes for (−)-zuonin A and suggest that (−)-zuonin A interacts with JNK via an induced fit mechanism near the highly conserved φA-X-φB recognition site. Binding of (+)-zuonin A to JNK displays no such dynamic feature. The different binding modes may help explain differences in the inhibitory properties of the enantiomers although further experimental work would be necessary to fully confirm this interpretation.

A fluorescence-based assay for p38α recruitment site binders: identification of rooperol as a novel p38α kinase inhibitor.

A new p38α inhibitor: Using a D-recruitment site (DRS) probe for p38α which exploits covalent interaction with Cys119 and alkyne-azide "click" chemistry to identify small molecules that recognize the p38α DRS, the anti-inflammatory natural product rooperol was identified as a novel p38α inhibitor.

Docking Interactions of Hematopoietic Tyrosine Phosphatase with MAP Kinases ERK2 and p38α

Hematopoietic tyrosine phosphatase (HePTP) regulates orthogonal MAP kinase signaling cascades by dephosphorylating both extracellular signal-regulated kinase (ERK) and p38. HePTP recognizes a docking site (D-recruitment site, DRS) on its targets using a conserved N-terminal sequence motif (D-motif). Using solution nuclear magnetic resonance spectroscopy and isothermal titration calorimetry, we compare, for the first time, the docking interactions of HePTP with ERK2 and p38α. Our results demonstrate that ERK2-HePTP interactions primarily involve the D-motif, while a contiguous region called the kinase specificity motif also plays a key role in p38α-HePTP interactions. D-Motif-DRS interactions for the two kinases, while similar overall, do show some specific differences.

Manipulating JNK Signaling with (−)-Zuonin A

Recently, in a virtual screening strategy to identify new compounds targeting the D-recruitment site (DRS) of the c-Jun N-terminal kinases (JNKs), we identified the natural product (-)-zuonin A. Here we report the asymmetric synthesis of (-)-zuonin A and its enantiomer (+)-zuonin A. A kinetic analysis for the inhibition of c-Jun phosphorylation by (-)-zuonin A revealed a mechanism of partial competitive inhibition. Its binding is proposed to weaken the interaction of c-Jun to JNK by approximately 5-fold, without affecting the efficiency of phosphorylation within the complex. (-)-Zuonin A inhibits the ability of both MKK4 and MKK7 to phosphorylate and activate JNK. The binding site of (-)-zuonin A is predicted by docking and molecular dynamics simulation to be located in the DRS of JNK. (+)-Zuonin A also binds JNK but barely impedes the binding of c-Jun. (-)-Zuonin A inhibits the activation of JNK, as well as the phosphorylation of c-Jun in anisomycin-treated HEK293 cells, with the inhibition of JNK activation being more pronounced. (-)-Zuonin A also inhibits events associated with constitutive JNK2 activity, including c-Jun phosphorylation, basal Akt activation, and MDA-MB-231 cell migration. Mutations in the predicted binding site for (-)-zuonin A can render it significantly more or less sensitive to inhibition than wild type JNK2, allowing for the design of potential chemical genetic experiments. These studies suggest that the biological activity reported for other lignans, such as saucerneol F and zuonin B, may be the result of their ability to impede protein-protein interactions within MAPK cascades.

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.

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 Anti-Apoptotic Protein PEA-15 Is a Tight Binding Inhibitor of ERK1 and ERK2, Which Blocks Docking Interactions at the D-Recruitment Site

PEA-15 is a small anti-apoptotic protein that is enriched in astrocytes, but expressed in a broad range of tissues. It sequesters the protein kinases ERK1 and 2 in the cytoplasm, thereby limiting their proximity to nuclear substrates. Using a fluorescence anisotropy approach, PEA-15 is shown to be a high-affinity ligand for both ERK1 and 2, exhibiting a dissociation constant in the range of Kd = 0.2-0.4 microM, regardless of their activation states. Neither the phosphorylation of PEA-15 (phospho Ser-104 and/or phospho Ser-116) nor the phosphorylation of ERK1/2 (by MKK1) significantly affects the stability of the ERK/PEA-15 interaction, and therefore it does not directly regulate the release of ERK2 to the nucleus. The extreme C-terminus of PEA-15 was previously shown by mutagenesis to be important for ERK2 binding; however, the site of binding was not established. Here it is demonstrated that the D-recruitment site (DRS) of ERK2 binds PEA-15, probably at the C-terminus, and renders PEA-15 an inhibitor of ERK2 docking interactions. Using fluorescence anisotropy competition assays it is shown that PEA-15 competes for binding to ERK1/2 with a peptide derived from the D-site of Elk-1, which binds the DRS of ERK1/2. Using modified ERK2 proteins containing single cysteine residues, PEA-15 was shown to protect single cysteines situated within the DRS from alkylation. The pattern and magnitude of protection were very similar to those induced by the binding of the peptide derived from the D-site of Elk-1. These and published data support the notion that PEA-15 binds two sites on ERK1/2 in a bidentate manner: the DRS and a site that includes the MAP kinase insert. Previous reports have suggested that PEA-15 is not an inhibitor of ERK2; however, it is shown here to potently inhibit the ability of ERK2 to phosphorylate two transcription factors, Elk-1 and Ets-1, which contain docking sites for the DRS of ERK2. Therefore, in addition to sequestering ERK1/2 in the cytoplasm, PEA-15 has the potential to modulate the activity of ERK2 in cells by competing directly with proteins that contain D-sites.

Expanding the Repertoire of an ERK2 Recruitment Site: Cysteine Footprinting Identifies the D-Recruitment Site as a Mediator of Ets-1 Binding

Many substrates of ERK2 contain a D-site, a sequence recognized by ERK2 that is used to promote catalysis. Despite lacking a canonical D-site, the substrate Ets-1 is displaced from ERK2 by peptides containing one. This suggests that Ets-1 may contain a novel or cryptic D-site. To investigate this possibility a protein footprinting strategy was developed to elucidate ERK2-ligand interactions. Using this approach, single cysteine reporters were placed in the D-recruitment site (DRS) of ERK2 and the resulting ERK2 proteins subjected to alkylation by iodoacetamide. The ability of residues 1-138 of Ets-1 to protect the cysteines from alkylation was determined. The pattern of protection observed is consistent with Ets-1 occupying a hydrophobic binding site within the DRS of ERK2. Significantly, a peptide derived from the D-site of Elk-1, which is known to bind the DRS, exhibits a similar pattern of cysteine protection. This analysis expands the repertoire of the DRS on ERK2 and suggests that other targeting sequences remain to be identified. Furthermore, cysteine-footprinting is presented as a useful way to interrogate protein-ligand interactions at the resolution of a single amino acid.

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.