Mitogen-activated Protein Kinase Binding Research Articles

Design, Synthesis, and Molecular Docking of Novel Miscellaneous Chalcones as p38α Mitogen-Activated Protein Kinase Inhibitors.

New chalcones were synthesized and evaluated to serve as p38-α type of mitogen-activated protein kinase (MAPK) inhibitors. According to the National Cancer Institute, the findings indicated that at a 10 μM dosage, compounds 3a and 6 were the most active among all the compounds examined, with mean growth inhibition% of 94.83 and 58.49, respectively. In 5-dose testing, they showed anticancer activity in the micro-molar range with GI50 in the range of 1.41-46.1 and 2.07-31.3 μM, respectively. Besides, powerful activity, especially against the leukaemia cell lines and good selectivity to cancer cells compared to normal PCS-800-017 with a selectivity index=12.41 and 23.77, respectively. Compounds 3a and 6 inhibited p38α MAPK with IC50 values of 0.1462±0.0063 and 0.4356±0.0189 μM, correspondingly. 3a showed good inhibition for HL-60(TB) cells and induced cell cycle arrest in HL-60(TB) cells at the G2/M phase. Besides, it elevated the total apoptosis by 14.68-fold and increased the caspase-3 level by 3.52-fold compared with doxorubicin, which raised it by 4.30-fold, inducing apoptosis by acting as caspase-dependent inducers. These results suggest that 3a is a promising antiproliferative and p38α MAPK inhibitor, confirmed by molecular docking with high compatibility 3a with the p38α MAPK binding site.

A novel site on dual-specificity phosphatase MKP7/DUSP16 is required for catalysis and MAPK binding

The dual-specificity phosphatases responsible for the inactivation of the mitogen-activated protein kinases (MAPKs) are designated as the MAPK phosphatases (MKPs). We demonstrated previously that MKP5 is regulated through a novel allosteric site suggesting additional regulatory mechanisms of catalysis exist amongst the MKPs. Here, we sought to determine whether the equivalent site within the phosphatase domain of a highly similar MKP family member, MKP7, is also important for phosphatase function. We found that mutation of tyrosine 271 (Y271) in MKP7, which represents the comparable Y435 within the MKP5 allosteric pocket, inhibited MKP7 catalytic activity. Consistent with this, when MKP7 Y271 mutants were overexpressed in cells, the substrates of MKP7, p38 MAPK or JNK, failed to undergo dephosphorylation. The binding efficiency of MKP7 to p38 MAPK and JNK1/2 was also reduced when MKP7 Y271 is mutated. Consistent with reduced MAPK binding, we observed a greater accumulation of nuclear p38 MAPK and JNK when the MKP7 Y271 mutants are expressed in cells as compared with WT MKP7, which sequesters p38 MAPK/JNK in the cytoplasm. Therefore, we propose that Y271 is critical for effective MAPK dephosphorylation through a mechanism whereby binding to this residue precedes engagement of the catalytic site and upon overexpression, MKP7 allosteric site mutants potentiate MAPK signaling. These results provide insight into the regulatory mechanisms of MKP7 catalysis and interactions with the MAPKs. Furthermore, these data support the generality of the MKP allosteric site and provide a basis for small molecule targeting of MKP7.

Co-regulation of the transcription controlling ATF2 phosphoswitch by JNK and p38

Transcription factor phosphorylation at specific sites often activates gene expression, but how environmental cues quantitatively control transcription is not well-understood. Activating protein 1 transcription factors are phosphorylated by mitogen-activated protein kinases (MAPK) in their transactivation domains (TAD) at so-called phosphoswitches, which are a hallmark in response to growth factors, cytokines or stress. We show that the ATF2 TAD is controlled by functionally distinct signaling pathways (JNK and p38) through structurally different MAPK binding sites. Moreover, JNK mediated phosphorylation at an evolutionarily more recent site diminishes p38 binding and made the phosphoswitch differently sensitive to JNK and p38 in vertebrates. Structures of MAPK-TAD complexes and mechanistic modeling of ATF2 TAD phosphorylation in cells suggest that kinase binding motifs and phosphorylation sites line up to maximize MAPK based co-regulation. This study shows how the activity of an ancient transcription controlling phosphoswitch became dependent on the relative flux of upstream signals.

An allosteric site on MKP5 reveals a strategy for small-molecule inhibition.

The mitogen-activated protein kinase (MAPK) phosphatases (MKPs) have been considered "undruggable," but their position as regulators of the MAPKs makes them promising therapeutic targets. MKP5 has been suggested as a potential target for the treatment of dystrophic muscle disease. Here, we identified an inhibitor of MKP5 using a p38α MAPK-derived, phosphopeptide-based small-molecule screen. We solved the structure of MKP5 in complex with this inhibitor, which revealed a previously undescribed allosteric binding pocket. Binding of the inhibitor to this pocket collapsed the MKP5 active site and was predicted to limit MAPK binding. Treatment with the inhibitor recapitulated the phenotype of MKP5 deficiency, resulting in activation of p38 MAPK and JNK. We demonstrated that MKP5 was required for TGF-β1 signaling in muscle and that the inhibitor blocked TGF-β1-mediated Smad2 phosphorylation. TGF-β1 pathway antagonism has been proposed for the treatment of dystrophic muscle disease. Thus, allosteric inhibition of MKP5 represents a therapeutic strategy against dystrophic muscle disease.

MAP Kinases Mediate Regulation of eNOS through Phosphorylation of Different Sites

Mitogen activated protein kinases (MAPK) p38 and ERK, have both been reported to bind endothelial nitric oxide synthase (eNOS) with submicromolar affinity via hypothesized interactions with a pentabasic non‐canonical MAPK binding sequence in the autoinhibitory insertion of eNOS. The neuronal isoform, which lacks the pentabasic motif, did not bind either MAPK. In the present study, eNOS phosphorylation by MAPKs (P38, ERK, JNK) was examined using in vitro kinase assays and immunoblotting. JNK phosphorylation at pS116 contrasts with ERK, which phosphorylated pS602, and p38, which phosphorylated both sites. Literature reports that JNK1/2 activation does not alter eNOS activity while prior work in our lab shows that ERK phosphorylation negativity impacts NO output. We will present in vitro results comparing the direct impact of each kinase on eNOS activity using the oxyhemoglobin (Oxy‐Hb) assay. In HMEC‐1 cells and in vitro we observe that phosphorylation at these MAPK sites happens concurrently with activating sites (pS1179), creating antagonistically phosphorylated eNOS. These results underscore the importance of MAPK interactions with eNOS showing that each MAPK creates a unique phosphorylation pattern and NO production outcome. These findings strengthen the emerging paradigm of eNOS as a junction of multiple signaling pathways.Support or Funding InformationR15GM110634‐01A1 supported this work.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Combinatorial Phosphorylation-Dependent Synergy Between a G Protein Subunit and the Ste5 Scaffold Regulates Differential MAP-Kinase Activation in Yeast

Heterotrimeric G proteins (Gαβγ) play a critical role as transducers in G protein signaling systems responsible for activating a wide range of physiological processes including neurotransmitter and hormone signaling. In yeast, heterotrimeric G protein signaling results in the distinctly different activation of two mitogen-activated protein kinases (MAPKs) - Fus3 and Kss1 - a phenomenon that requires conformational switching of the scaffold protein Ste5. Here we show that rapid receptor-activated phosphorylation of the intrinsically disordered N-terminal tail of the yeast Gγ subunit (Ste18-Nt) has co-evolved to synergize with MAPK binding on Ste5 to control the kinetics and amplitude of Fus3 and Kss1 signaling. By modulating this synergistic component genetically, we show that the activation kinetics and amplitude of either kinase can be controlled - leading to preferential hyperactivation of Fus3 or Kss1. These results further resolve the mechanism by which scaffolds control pathway activation and provide compelling evidence that the intrinsically disordered N-terminal tails of Gγ subunits can serve as regulators of G protein signaling.

C‐JUN N‐terminal kinase (JNKα1) binds eNOS and phosphorylates at S116, differentiating it from p38 and ERK phosphorylation

We have previously shown that the mitogen activated protein kinases (MAPK), p38 and ERK bind endothelial nitric oxide synthase (eNOS) with submicromolar affinity via interactions with a pentabasic non‐canonical MAPK binding sequence in the autoinhibitory insertion of eNOS. The neuronal isoform, which lacks the pentabasic motif, did not bind MAPK. In the present study, eNOS binding by c‐Jun N‐terminal kinase (JNKα1) was examined using optical biosensing. Similar to p38 and ERK, JNK evinced binding to eNOS (31 nM KD) but not nNOS as measured by fit to a one‐state global model. Rate constants were determined (kon = 4125 M−1s−1 and koff = 1.3 × 10−4 s−1). In vitro kinase assays and immunoblotting identified phosphorylation by JNK at S116, contrasting with ERK, which phosphorylated S602, and p38, which phosphorylated both sites. Phosphorylation by JNK at S116 did not alter the conformational manifold, where phosphorylation at S602 by ERK and p38 does. In HMEC1 cells we observe that phosphorylation at these MAPK sites happens concurrently with activating sites (S1177), creating antagonistically phosphorylated eNOS. Further, we examine how phosphorylation of eNOS alters the cellular interactions of the MAP kinases. These results underscore the importance of MAPK interactions with eNOS showing that each MAPK creates a unique phosphorylation pattern. These findings strengthen the emerging paradigm of eNOS as a junction of multiple signaling pathways.Support or Funding InformationNIH 1 R15 GM110634‐01A1 | R25GM111565This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Exploration of Protein Regions Involved in Map Kinase Mediated Signaling

Many signaling relevant intracellular protein-protein interactions rely on binding events between dedicated surfaces of signaling enzymes and linear binding motifs found in their partner proteins. We have been studying the role of linear binding motifs capable of binding to human mitogen activated protein kinases (MAPK). These enzymes are mostly known for their ubiquitous role in signaling networks through which cells respond to extracellular stimuli. How abundant these linear binding motifs are and how they mechanistically contribute to MAPK based cellular signaling? After exploring MAPK binding motifs in the disordered human proteome we are focusing on the exploration of functional sites through which phosphorylation may exert a functional change on the binding partner (e.g. by changing activity, turnover, trafficking or protein-protein interaction capacity). These studies give insights into the molecular level strategies that MAPKs use to control cellular physiology. We suggest that MAPKs mostly exploit the possibilities that evolutionarily fast changing and intrinsically disordered regions of various proteomes provide for the phosphorylation based regulatory control of protein function.

Structural assembly of the signaling competent ERK2–RSK1 heterodimeric protein kinase complex

Mitogen-activated protein kinases (MAPKs) bind and activate their downstream kinase substrates, MAPK-activated protein kinases (MAPKAPKs). Notably, extracellular signal regulated kinase 2 (ERK2) phosphorylates ribosomal S6 kinase 1 (RSK1), which promotes cellular growth. Here, we determined the crystal structure of an RSK1 construct in complex with its activator kinase. The structure captures the kinase-kinase complex in a precatalytic state where the activation loop of the downstream kinase (RSK1) faces the enzyme's (ERK2) catalytic site. Molecular dynamics simulation was used to show how this heterodimer could shift into a signaling-competent state. This structural analysis combined with biochemical and cellular studies on MAPK→MAPKAPK signaling showed that the interaction between the MAPK binding linear motif (residing in a disordered kinase domain extension) and the ERK2 "docking" groove plays the major role in making an encounter complex. This interaction holds kinase domains proximal as they "readjust," whereas generic kinase domain surface contacts bring them into a catalytically competent state.

Structural Basis for the Regulation of the Mitogen-activated Protein (MAP) Kinase p38α by the Dual Specificity Phosphatase 16 MAP Kinase Binding Domain in Solution

Mitogen-activated protein kinases (MAPKs) fulfill essential biological functions and are key pharmaceutical targets. Regulation of MAPKs is achieved via a plethora of regulatory proteins including activating MAPKKs and an abundance of deactivating phosphatases. Although all regulatory proteins use an identical interaction site on MAPKs, the common docking and hydrophobic pocket, they use distinct kinase interaction motif (KIM or D-motif) sequences that are present in linear, peptide-like, or well folded protein domains. It has been recently shown that a KIM-containing MAPK-specific dual specificity phosphatase DUSP10 uses a unique binding mode to interact with p38α. Here we describe the interaction of the MAPK binding domain of DUSP16 with p38α and show that despite belonging to the same dual specificity phosphatase (DUSP) family, its interaction mode differs from that of DUSP10. Indeed, the DUSP16 MAPK binding domain uses an additional helix, α-helix 4, to further engage p38α. This leads to an additional interaction surface on p38α. Together, these structural and energetic differences in p38α engagement highlight the fine-tuning necessary to achieve MAPK specificity and regulation among multiple regulatory proteins.

Structural Mechanism for the Specific Assembly and Activation of the Extracellular Signal Regulated Kinase 5 (ERK5) Module

Mitogen-activated protein kinase (MAPK) activation depends on a linear binding motif found in all MAPK kinases (MKK). In addition, the PB1 (Phox and Bem1) domain of MKK5 is required for extracellular signal regulated kinase 5 (ERK5) activation. We present the crystal structure of ERK5 in complex with an MKK5 construct comprised of the PB1 domain and the linear binding motif. We show that ERK5 has distinct protein-protein interaction surfaces compared with ERK2, which is the closest ERK5 paralog. The two MAPKs have characteristically different physiological functions and their distinct protein-protein interaction surface topography enables them to bind different sets of activators and substrates. Structural and biochemical characterization revealed that the MKK5 PB1 domain cooperates with the MAPK binding linear motif to achieve substrate specific binding, and it also enables co-recruitment of the upstream activating enzyme and the downstream substrate into one signaling competent complex. Studies on present day MAPKs and MKKs hint on the way protein kinase networks may evolve. In particular, they suggest how paralogous enzymes with similar catalytic properties could acquire novel signaling roles by merely changing the way they make physical links to other proteins.

Label Transfer Reagents to Probe p38 MAPK Binding Partners

Protein kinases are essential enzymes for cellular signaling, and are often regulated by participation in protein complexes. The mitogen-activated protein kinase (MAPK) p38 is involved in multiple pathways, and its regulation depends on its interactions with other signaling proteins. However, the identification of p38-interacting proteins is challenging. For this reason, we have developed label transfer reagents (LTRs) that allow labeling of p38 signaling complexes. These LTRs leverage the potency and selectivity of known p38 inhibitors to place a photo-crosslinker and tag in the vicinity of p38 and its binding partners. Upon UV irradiation, proteins that are in close proximity to p38 are covalently crosslinked, and labeled proteins are detected and/or purified with an orthogonal chemical handle. Here we demonstrate that p38-selective LTRs selectively label a diversity of p38 binding partners, including substrates, activators, and inactivators. Furthermore, these LTRs can be used in immunoprecipitations to provide low-resolution structural information on p38-containing complexes.

Control of Sty1 MAPK activity through stabilisation of the Pyp2 MAPK phosphatase

In all eukaryotes tight control of mitogen-activated protein kinase (MAPK) activity plays an important role in modulating intracellular signalling in response to changing environments. The fission yeast MAPK Sty1 (also known as Spc1 or Phh1) is highly activated in response to a variety of external stresses. To avoid segregation of damaged organelles or chromosomes, strong Sty1 activation transiently blocks mitosis and cell division until such stresses have been dealt with. MAPK phosphatases dephosphorylate Sty1 to reduce kinase activity. Therefore, tight control of MAPK phosphatases is central for stress adaptation and for cell division to resume. In contrast to Pyp1, the fission yeast Pyp2 MAPK phosphatase is under environmental control. Pyp2 has a unique sequence (the linker region) between the catalytic domain and the N-terminal MAPK-binding site. Here we show that the Pyp2 linker region is a destabilisation domain. Furthermore, the linker region is highly phosphorylated to increase Pyp2 protein stability and this phosphorylation is Sty1 dependent. Our data suggests that Sty1 activation promotes Pyp2 phosphorylation to increase the stability of the phosphatase. This MAPK-dependent Pyp2 stabilisation allows cells to attenuate MAPK signalling and resume cell division, once stresses have been dealt with.

Specificity of linear motifs that bind to a common mitogen-activated protein kinase docking groove.

Mitogen-activated protein kinases (MAPKs) have a docking groove that interacts with linear "docking" motifs in binding partners. To determine the structural basis of binding specificity between MAPKs and docking motifs, we quantitatively analyzed the ability of 15 docking motifs from diverse MAPK partners to bind to c-Jun amino-terminal kinase 1 (JNK1), p38α, and extracellular signal-regulated kinase 2 (ERK2). Classical docking motifs mediated highly specific binding only to JNK1, and only those motifs with a sequence pattern distinct from the classical MAPK binding docking motif consensus differentiated between the topographically similar docking grooves of ERK and p38α. Crystal structures of four complexes of MAPKs with docking peptides, representing JNK-specific, ERK-specific, or ERK- and p38-selective binding modes, revealed that the regions located between consensus positions in the docking motifs showed conformational diversity. Although the consensus positions in the docking motifs served as anchor points that bound to common MAPK surface features and mostly contributed to docking in a nondiscriminatory fashion, the conformation of the intervening region between the anchor points mostly determined specificity. We designed peptides with tailored MAPK binding profiles by rationally changing the length and amino acid composition of intervening regions located between anchor points. These results suggest a coherent structural model for MAPK docking specificity that reveals how short linear motifs binding to a common kinase docking groove can mediate diverse interaction patterns and contribute to correct MAPK partner selection in signaling networks.

A chromatin-modifying function of JNK during stem cell differentiation

Signaling mediates cellular responses to extracellular stimuli. The c-Jun NH(2)-terminal kinase (JNK) pathway exemplifies one subgroup of the mitogen-activated protein (MAP) kinases, which, besides having established functions in stress response, also contribute to development by an unknown mechanism. We show by genome-wide location analysis that JNK binds to a large set of active promoters during the differentiation of stem cells into neurons. JNK-bound promoters are enriched with binding motifs for the transcription factor NF-Y but not for AP-1. NF-Y occupies these predicted sites, and overexpression of dominant-negative NF-YA reduces the JNK presence on chromatin. We find that histone H3 Ser10 (H3S10) is a substrate for JNK, and JNK-bound promoters are enriched for H3S10 phosphorylation. Inhibition of JNK signaling in post-mitotic neurons reduces phosphorylation at H3S10 and the expression of target genes. These results establish MAP kinase binding and function on chromatin at a novel class of target genes during stem cell differentiation.

FGF-receptor substrate 2 functions as a molecular sensor integrating external regulatory signals into the FGF pathway

Fibroblast growth factor (FGF) receptor substrate 2alpha (FRS2alpha) is the main mediator of signaling in the FGF pathway. Recent studies have shown that mitogen-activated protein kinase (MAPK) phosphorylates serine and threonine residues in FRS2, negatively affecting FGF-induced tyrosine phosphorylation (PY) of FRS2. Several kinds of stimuli can induce serine/threonine phosphorylation (PS/T) of FRS2, indicating that FRS2 may be useful for studying crosstalk between growth factor signaling pathways. Here, we report that FGF-induced PY of FRS2 can be attenuated by EGF co-stimulation in PC12 cells; this inhibitory effect could be completely reversed by U0126, an inhibitor of MEK. We further identified the ERK1/2-binding motif in FRS2 and generated FRS2-3KL, a mutant lacking MAPK binding and PT upon FGF and/or EGF stimulation. Unlike wild-type (WT) FRS2, FGF-induced PY of FRS2-3KL could not be inhibited by EGF co-stimulation, and FRS2-3KL-expressing PC12 cells exhibited more differentiating potential than FRS2-WT-expressing cells in response to FGF treatment. These results suggest that PS/T of FRS2 mediated by the FRS2-MAPK negative regulatory loop may function as a molecular switch integrating negative regulatory signals from other pathways into FGFR-generated signal transduction.

The Structure of the MAP2K MEK6 Reveals an Autoinhibitory Dimer

MAP2Ks are dual-specificity protein kinases functioning at the center of three-tiered MAP kinase modules. The structure of the kinase domain of the MAP2K MEK6 with phosphorylation site mimetic aspartic acid mutations (MEK6/DeltaN/DD) has been solved at 2.3 angstroms resolution. The structure reveals an autoinhibited elongated ellipsoidal dimer. The enzyme adopts an inactive conformation, based upon structural queues, despite the phosphomimetic mutations. Gel filtration and small-angle X-ray scattering analysis confirm that the crystallographically observed ellipsoidal dimer is a feature of MEK6/DeltaN/DD and full-length unphosphorylated wild-type MEK6 in solution. The interface includes the phosphate binding ribbon of each subunit, part of the activation loop, and a rare "arginine stack" between symmetry-related arginine residues in the N-terminal lobe. The autoinhibited structure likely confers specificity on active MAP2Ks. The dimer may also serve the function in unphosphorylated MEK6 of preventing activation loop phosphorylation by inappropriate kinases.

Plant Glycoprotein Modulates the Expression of Interleukin-1βviaInhibition of MAP Kinase in HMC-1 Cells

Dioscorea batatas Decne (DBD) is used to heal various disorders of the kidney and lungs as an herbal agent in Korea. The purpose of the present study was to determine whether the DBD glycoprotein regulates the inflammatory reaction stimulated by phorbol-12-myristate 13-acetate plus calcium ionophore A23187 (PMACI) in human mast cells (HMC-1). The results indicate that DBD glycoprotein decreased gene expression of interleukin (IL)-1beta and cyclooxygenase (COX)-2 in PMACI-stimulated HMC-1 cells through blocking of phosphorylation of p44/42 mitogen-activated protein kinase (MAPK) and p38 MAPK and DNA binding activities of nuclear factor (NF)-kappaB and activator protein (AP)-1. The production of intracellular reactive oxygen species (ROS) and nitric oxide (NO) is gradually reduced by concentration-dependent DBD glycoprotein treatment in PMACI-stimulated HMC-1 cells. Hence, we propose the hypothesis that DBD glycoprotein can serve as a potent anti-inflammatory agent in the treatment of inflammatory allergic diseases through inhibition of inflammation-related signal transduction in mast cell activation.

17β-Estradiol attenuates cardiac dysfunction and decreases NF-κB binding activity in mechanically stretched rat hearts

This study was to examine the effect of estrogen on mechanical stretching-induced cardiac dysfunction in an isolated heart model. The isolated rat hearts were perfused via the Langendorff system and exposed to left ventricular stretching. One group hearts ( n = 6) were perfused with 17β-estradiol (100 nM) and the other group hearts ( n = 6) were perfused with estrogen plus its receptor antagonist ICI182,780 (1 μM) before myocardial stretching was performed. Control hearts ( n = 6) were perfused with perfusion buffer. Cardiac functions were recorded. At the end of perfusion, the hearts were harvested and the levels of tumor necrosis factor-alpha (TNF-alpha), phospho-p38 mitogen-activated protein kinase (MAPK) and nuclear factor-κB (NF-κB) binding activity were examined. Acute ventricular stretching resulted in significantly decrease in left ventricular developed pressure (LVDP) by 42.7%, maximal positive and negative values of the first derivative of pressure (+d P/d t and −d P/d t) by 43.2%, and 43.5%, respectively. The levels of TNF-alpha, phospho-p38 MAPK and NF-κB DNA binding activity were significantly increased following myocardial stretching. In 17β-estradiol treated hearts, the myocardial functions were significantly improved. The levels of TNF-alpha, phospho-p38 MAPK, and NF-κB binding activity in myocardium were also significantly reduced by 35.7%, 56.9%, and 50%, respectively, compared with untreated stretched hearts. The beneficial effects of 17β-estradiol on the stretched hearts were abolished by ICI182,780. The results suggest that pharmacological dose of 17β-estradiol will attenuate stretching-induced cardiac dysfunction in an isolated heart model. The mechanisms could involve in blunting p38 MAPK and NF-κB signaling.

Functional assignment of MAPK phosphatase domains

Mitogen-activated protein kinase (MAPK) pathways are well conserved in most organisms, from yeast to humans. The principal components of these pathways are MAP kinases whose activity is regulated by phosphorylation, implicating various MAPK protein effectors-in particular, protein phosphatases that inactivate MAPKs by dephosphorylation. The molecular basis of binding specificity of such regulatory phosphatases to MAPKs is poorly understood. To try to pinpoint potential functional regions within the sequences and to help identify new family members, we have applied a multimotif pattern-recognition approach to characterize two MAPK phosphatase subfamilies (tyrosine-specific and dual specificity) that are crucial in the regulation of MAPKs. We built "fingerprints" for these two subfamilies that are unique to, and highly discriminatory for, each group of proteins. The fingerprints were used in a genome-wide screen, identifying more than 80 MAPK phosphatase domains, several of which were in partial sequences or unclassified proteins. We confirmed experimentally that one predicted MAPK phosphatase orthologue in Xenopus binds to ERK1/2, suggesting a role in MAPK signaling and thus supporting our functional predictions. Further analysis, mapping the fingerprints on the three-dimensional structure of MAPK phosphatases, revealed that some of the fingerprint motifs reside in the N-terminal noncatalytic regions coinciding with reported MAPK binding sites, while others lie within the catalytic phosphatase domain. These results also suggest the presence of putative allosteric sites in the catalytic region for modulation of protein-protein interactions, and provide a framework for future experimental validation.