Architecture of the MKK6-p38α complex defines the basis of MAPK specificity and activation.

The mitogen-activated protein kinase (MAPK) p38α is a central component of signaling in inflammation and the immune response and is, therefore, an important drug target. Little is known about the molecular mechanism of its activation by double phosphorylation from MAPK kinases (MAP2Ks), because of the challenge of trapping a transient and dynamic heterokinase complex. We applied a multidisciplinary approach to generate a structural model of p38α in complex with its MAP2K, MKK6, and to understand the activation mechanism. Integrating cryo-electron microscopy with molecular dynamics simulations, hydrogen-deuterium exchange mass spectrometry, and experiments in cells, we demonstrate a dynamic, multistep phosphorylation mechanism, identify catalytically relevant interactions, and show that MAP2K-disordered amino termini determine pathway specificity. Our work captures a fundamental step of cell signaling: a kinase phosphorylating its downstream target kinase.

Dissection of Kinase Isoforms through Orthogonal and Chemical Inducible Signaling Cascades.

Interference from endogenous signaling enzymes represents a major hurdle for building orthogonal signaling cascades inside cells, particularly among closely related isoforms within an enzyme family. Here, we employed a genetically encoded chemical decaging strategy to build orthogonally activated kinase isoforms, with the endogenous counterparts temporally disabled by an extracellularly delivered bacterial effector. This approach eliminated any potential interference from other kinase isoforms as well as endogenous kinases, which allowed the specific, gain-of-function report of mitogen-activated protein kinase kinase 1 (MEK1) activity as opposed to MEK2 with high temporal resolution. Our study dissected the distinct enzymatic activity, feedback regulation and signal outputs between these closely related kinase isoforms.

BRAF-inhibitor Associated MEK Mutations Increase RAF-Dependent and -Independent Enzymatic Activity.

Alterations in MEK1/2 occur in cancers, both in the treatment-naïve state and following targeted therapies, most notably BRAF and MEK inhibitors in BRAF-V600E-mutant melanoma and colorectal cancer. Efforts were undertaken to understand the effects of these mutations, based upon protein structural location, and MEK1/2 activity. Two categories of MEK1/2 alterations were evaluated, those associated with either the allosteric pocket or helix-A. Clinically, MEK1/2 alterations of the allosteric pocket are rare and we demonstrate that they confer resistance to MEK inhibitors, while retaining sensitivity to BRAF inhibition. Most mutations described in patients fall within, or are associated with, helix-A. Mutations in this region reduce sensitivity to both BRAF and MEK inhibition and display elevated phospho-ERK1/2 levels, independent from increases in phospho-MEK1/2. Biochemical experiments with a representative helix-A variant, MEK1-Q56P, reveal both increased catalytic efficiency of the activated enzyme, and phosphorylation-independent activity relative to wild-type MEK1. Consistent with these findings, MEK1/2 alterations in helix A retain sensitivity to downstream antagonism via pharmacologic inhibition of ERK1/2. This work highlights the importance of classifying mutations based on structural and phenotypic consequences, both in terms of pathway signaling output and response to pharmacologic inhibition.Implications: This study suggests that alternate modes of target inhibition, such as ERK inhibition, will be required to effectively treat tumors harboring these MEK1/2-resistant alleles. Mol Cancer Res; 15(10); 1431-44. ©2017 AACR.

The WW domain of the scaffolding protein IQGAP1 is neither necessary nor sufficient for binding to the MAPKs ERK1 and ERK2.

Mitogen-activated protein kinase (MAPK) scaffold proteins, such as IQ motif containing GTPase activating protein 1 (IQGAP1), are promising targets for novel therapies against cancer and other diseases. Such approaches require accurate information about which domains on the scaffold protein bind to the kinases in the MAPK cascade. Results from previous studies have suggested that the WW domain of IQGAP1 binds to the cancer-associated MAPKs ERK1 and ERK2, and that this domain might thus offer a new tool to selectively inhibit MAPK activation in cancer cells. The goal of this work was therefore to critically evaluate which IQGAP1 domains bind to ERK1/2. Here, using quantitative in vitro binding assays, we show that the IQ domain of IQGAP1 is both necessary and sufficient for binding to ERK1 and ERK2, as well as to the MAPK kinases MEK1 and MEK2. Furthermore, we show that the WW domain is not required for ERK-IQGAP1 binding, and contributes little or no binding energy to this interaction, challenging previous models of how WW-based peptides might inhibit tumorigenesis. Finally, we show that the ERK2-IQGAP1 interaction does not require ERK2 phosphorylation or catalytic activity and does not involve known docking recruitment sites on ERK2, and we obtain an estimate of the dissociation constant (Kd ) for this interaction of 8 µm These results prompt a re-evaluation of published findings and a refined model of IQGAP scaffolding.

Functional redundancy of the kinases MEK1 and MEK2: Rescue of the Mek1 mutant phenotype by Mek2 knock-in reveals a protein threshold effect.

The mammalian genome contains two mitogen-activated protein kinase (MAPK) kinase (MEK)-encoding genes, Mek1 and Mek2. MEKs phosphorylate and activate the two extracellular signal-regulated kinase (ERK) isoforms ERK1 and ERK2. Mek1(-/-) embryos die due to placental defects, whereas Mek2(-/-) mice survive with a normal life span and fertility, suggesting that MEK1 has functions not shared by MEK2. However, most Mek1(+/-)Mek2(+/-) embryos also die from placental defects, indicating that both Mek genes contribute to placental development. To assess the functional specificity of the Mek1 and Mek2 genes, we produced a Mek1 knock-in allele in which the Mek2 coding sequences were placed under the control of Mek1 regulatory sequences (Mek1(2) allele). Mek1(2/2) mice were viable with no apparent phenotype, indicating rescue by MEK2 and functional redundancy between the two MEK proteins. However, Mek1(2/-) embryos with Mek2 in only one of the Mek1 alleles and the other Mek1 allele null died from abnormal placenta, suggesting a dosage effect. Analysis of mice from a Mek1 Mek2 allelic series revealed that the occurrence of the placenta phenotype correlated with the amount of MEK protein independently of which MEK isoform was produced. Thus, although MEK1 and MEK2 can substitute for each other, a minimum amount of MEK is critical for placenta development and embryo survival.

In silico investigation of new binding pocket for mitogen activated kinase kinase (MEK): Development of new promising inhibitors.

It has been previously shown that the inhibition of mitogen activated protein kinase kinase (MEK) contributes to apoptosis and suppression of different cancer cells. Correspondingly, a number of MEK1/2 inhibitors have been designed and evaluated since 2001. However, they did not satisfy essential pharmacokinetic (PK) and pharmacodynamic (PD) properties thus, almost most of them were terminated in pre-clinical or clinical studies. This study aims to design new specific MEK1/2 inhibitors with improved PK/PD profiles to be used as alternative cancer medications. In first part of this study, a comprehensive screening, for the first time, was done on well-known MEK1/2 inhibitors using a number of computational programs such as AutoDock Tools 4.2 (ADT) and AutoDock Vina. Therefore a valuable training dataset as well as a reliable pharmacophore model were provided which were then used to design new inhibitors. According to the results of training dataset, Trametinib was determined as the best inhibitor provided, so far. So, Trametinib was used as the lead structure to design new inhibitors in this study. In second part of this investigation, a set of new allosteric MEK1/2 inhibitors were designed significantly improving the binding energy as well as the ADMET properties, suggesting more specific and stable ligand-receptor complexes. Consequently, the structures 14 and 15 of our inhibitors, as the most potent structures, are great substituents for Trametinib to be used and evaluated in clinical trials as alternative cancer drugs.

MEK1/2 Inhibitors: Molecular Activity and Resistance Mechanisms.

Aberrant activation of the three-layered protein kinase cascade, Raf/MEK/ERK, is often detected in human cancer, which is mainly attributed to the oncogenic alterations of RAF, or its upstream activators RAS or cell surface receptor tyrosine kinases. Deregulated activity of the Raf/MEK/ERK pathway drives uncontrolled tumor cell proliferation and survival, thus providing a rational therapeutic target for the treatment of many cancers. While Raf, MEK1/2, and ERK1/2 are equally important targets for the design of therapeutic small molecular weight inhibitors, the effort to develop MEK1/2-specific inhibitors has been greatly successful. Particularly, MEK1/2 have been relatively advantageous for the design of highly selective adenosine triphosphate (ATP)-noncompetitive inhibitors. Indeed, a plethora of highly selective and potent MEK1/2 inhibitors are now available and many of those inhibitors have been evaluated for their therapeutic potential. Herein, we review different MEK1/2 inhibitors that have been studied for their inhibitory mechanisms and therapeutic potential in cancer. Some of the key structural features of MEK1/2 that are important for the efficacy of these inhibitors are also discussed. In addition, we discuss current challenges and future prospective in using these advanced MEK1/2 inhibitors for cancer therapy.

Discovery and characterization of MAPK-activated protein kinase-2 prevention of activation inhibitors.

Two structurally distinct series of novel, MAPK-activated kinase-2 prevention of activation inhibitors have been discovered by high throughput screening. Preliminary structure-activity relationship (SAR) studies revealed substructural features that influence the selective inhibition of the activation by p38α of the downstream kinase MK2 in preference to an alternative substrate, MSK1. Enzyme kinetics, surface plasmon resonance (SPR), 2D protein NMR, and X-ray crystallography were used to determine the binding mode and the molecular mechanism of action. The compounds bind competitively to the ATP binding site of p38α but unexpectedly with higher affinity in the p38α-MK2 complex compared with p38α alone. This observation is hypothesized to be the origin of the substrate selectivity. The two lead series identified are suitable for further investigation for their potential to treat chronic inflammatory diseases with improved tolerability over previously studied p38α inhibitors.

Towards the development of chromone-based MEK1/2 modulators.

Inhibition or allosteric modulation of mitogen-activated protein kinase kinases MEK1 and MEK2 (MEK1/2) represent a promising strategy for the discovery of new specific anticancer agents. In this paper, structure-based design, beginning from the lead compound PD98059, was used to study potential structural modifications on the chromone structure in order to obtain highly potent derivatives that target the allosteric pocket in MEK1. Subsequently, a small series of PD98059 analogs were synthesized to provide a first generation of chromone-based derivatives that inhibit the activation of MEK1 with IC50 values as low as 30 nM in vitro. Complementary cellular studies also showed that two of the compounds in the series inhibit the activity of MEK1/2 with IC50 values in the nanomolar range (73-97 nM). In addition, compounds in this series were found to inhibit the proliferation of a small panel of human cancer cell lines.

Concurrent MEK2 mutation and BRAF amplification confer resistance to BRAF and MEK inhibitors in melanoma.

Although BRAF and MEK inhibitors have proven clinical benefits in melanoma, most patients develop resistance. We report a de novo MEK2-Q60P mutation and BRAF gain in a melanoma from a patient who progressed on the MEK inhibitor trametinib and did not respond to the BRAF inhibitor dabrafenib. We also identified the same MEK2-Q60P mutation along with BRAF amplification in a xenograft tumor derived from a second melanoma patient resistant to the combination of dabrafenib and trametinib. Melanoma cells chronically exposed to trametinib acquired concurrent MEK2-Q60P mutation and BRAF-V600E amplification, which conferred resistance to MEK and BRAF inhibitors. The resistant cells had sustained MAPK activation and persistent phosphorylation of S6K. A triple combination of dabrafenib, trametinib, and the PI3K/mTOR inhibitor GSK2126458 led to sustained tumor growth inhibition. Hence, concurrent genetic events that sustain MAPK signaling can underlie resistance to both BRAF and MEK inhibitors, requiring novel therapeutic strategies to overcome it.

Direct targeting of MEK1/2 and RSK2 by silybin induces cell-cycle arrest and inhibits melanoma cell growth.

Abnormal functioning of multiple gene products underlies the neoplastic transformation of cells. Thus, chemopreventive and/or chemotherapeutic agents with multigene targets hold promise in the development of effective anticancer drugs. Silybin, a component of milk thistle, is a natural anticancer agent. In the present study, we investigated the effect of silybin on melanoma cell growth and elucidated its molecular targets. Our study revealed that silybin attenuated the growth of melanoma xenograft tumors in nude mice. Silybin inhibited the kinase activity of mitogen-activated protein kinase (MEK)-1/2 and ribosomal S6 kinase (RSK)-2 in melanoma cells. The direct binding of silybin with MEK1/2 and RSK2 was explored using a computational docking model. Treatment of melanoma cells with silybin attenuated the phosphorylation of extracellular signal-regulated kinase (ERK)-1/2 and RSK2, which are regulated by the upstream kinases MEK1/2. The blockade of MEK1/2-ERK1/2-RSK2 signaling by silybin resulted in a reduced activation of NF-κB, activator protein-1, and STAT3, which are transcriptional regulators of a variety of proliferative genes in melanomas. Silybin, by blocking the activation of these transcription factors, induced cell-cycle arrest at the G1 phase and inhibited melanoma cell growth in vitro and in vivo. Taken together, silybin suppresses melanoma growth by directly targeting MEK- and RSK-mediated signaling pathways.

MEK1/2 dual-specificity protein kinases: structure and regulation.

MEK1 and MEK2 are related protein kinases that participate in the RAS-RAF-MEK-ERK signal transduction cascade. This cascade participates in the regulation of a large variety of processes including apoptosis, cell cycle progression, cell migration, differentiation, metabolism, and proliferation. Moreover, oncogenic mutations in RAS or B-RAF are responsible for a large proportion of human cancers. MEK1 is activated by phosphorylation of S218 and S222 in its activation segment as catalyzed by RAF kinases in an intricate process that involves a KSR scaffold. Besides functioning as a scaffold, the kinase activity of KSR is also required for MEK activation. MEK1 regulation is unusual in that S212 phosphorylation in its activation segment is inhibitory. Moreover, active ERK catalyzes a feedback inhibitory phosphorylation of MEK1 T292 that serves to downregulate the pathway.

Cell intrinsic roles of apoptosis-associated speck-like protein in regulating innate and adaptive immune responses.

The role of apoptosis-associated speck-Like protein (ASC) in the assembly of the inflammasome complex within macrophages has been elucidated in several studies. In this particular role, ASC functions as an adaptor protein by linking nod-like receptors (NLRs) and procaspase-1, thereby leading to the activation of caspase-1 to cleave inflammatory cytokines IL-1ß and IL-18 and inducing pyroptosis. It has been noted that ASC maintains inflammasome-independent roles, including but not limited to controlling the expression of Dock2 and mitogen-activated protein kinases (MAPK/ERK2) and regulating the NF-κB pathway. This paper will emphasize the major roles of ASC during pathogen infection, the mechanisms by which it mediates inflammation, and discuss its more recently discovered functions.

The MEK2-binding tumor suppressor hDlg is recruited by E-cadherin to the midbody ring.

BACKGROUND: The human homologue of the Drosophila Discs-large tumor suppressor protein, hDlg, is a multi-domain cytoplasmic protein that localizes to the membrane at intercellular junction sites. At both synaptic junctions and epithelia cell-cell junctions, hDlg is known to recruit several signaling proteins into macromolecular complexes. hDlg is also found at the midbody, a small microtubule-rich structure bridging the two daughter cells during cytokinesis, but its function at this site is not clear. RESULTS: Here we describe the interaction of hDlg with the activated form of MEK2 of the canonical RAF/MEK/ERK pathway, a protein that is found at the midbody during cytokinesis. We show that both proteins localize to a sub-structure of the midbody, the midbody ring, and that the interaction between the PDZ domains of hDlg and the C-terminal portion of MEK2 is dependent on the phosphorylation of MEK2. Finally, we found that E-cadherin also localizes to the midbody and that its expression is required for the isoform-specific recruitment of hDlg, but not activated MEK2, to that structure. CONCLUSION: Our results suggest that like at other cell-cell junction sites, hDlg is part of a macromolecular complex of structural and signaling proteins at the midbody.

A full-length 3D structure for MAPK/ERK kinase 2 (MEK2).

As a pivotal signal pathway, the Ras/Raf/MEK/ERK cascade can be activated by multiple extracellular stimuli and can transmit signals to diverse substrates. It remains to be elucidated how so many different signals can be variously transferred by only two MEK molecules (MEK1 and MEK2). Because of technological limitations the complete structures of the MEKs are still unavailable. Here, we report the full-length structure of MEK2 obtained by homology modeling and molecular dynamics simulations. The simulations show that the N-terminal part of MEK2 is highly flexible and this flexibility may enable MEK2 to interact with ERKs and other ligands in diverse manners that correspond to various upstream signals and downstream consequences.

4-anilino-5-carboxamido-2-pyridone derivatives as noncompetitive inhibitors of mitogen-activated protein kinase kinase.

A new series of MEK1 inhibitors, the 4-anilino-5-carboxamido-2-pyridones, were designed and synthesized using a combination of medicinal chemistry, computational chemistry, and structural elucidation. The effect of variation in the carboxamide side chain, substitution on the pyridone nitrogen, and replacement of the 4'-iodide were all investigated. This study afforded several compounds which were either equipotent or more potent than the clinical candidate CI-1040 (1) in an isolated enzyme assay, as well as murine colon carcinoma (C26) cells, as measured by suppression of phosphorylated ERK substrate. Most notably, pyridone 27 was found to be more potent than 1 in vitro and produced a 100% response rate at a lower dose than 1, when tested for in vivo efficacy in animals bearing C26 tumors.

Retrophosphorylation of Mkk1 and Mkk2 MAPKKs by the Slt2 MAPK in the yeast cell integrity pathway.

In Saccharomyces cerevisiae, a variety of stresses and aggressions to the cell wall stimulate the activation of the cell wall integrity MAPK pathway, which triggers the expression of a series of genes important for the maintenance of cell wall homeostasis. This MAPK module lies downstream of the Rho1 small GTPase and protein kinase C Pkc1 and consists of MAPKKK Bck1, MAPKKs Mkk1 and Mkk2, and the Slt2 MAPK. In agreement with previous reports suggesting that Mkk1 and Mkk2 were functionally redundant, we show here that both Mkk1 and Mkk2 alone or even chimerical proteins constructed by interchanging their catalytic and regulatory domains are able to efficiently maintain signal transduction through the pathway. Both Mkk1 and Mkk2 are phosphorylated in vivo concomitant to activation of the cell integrity pathway. Interestingly, hyperphosphorylation of the MEKs required not only the upstream components of the pathway, but also a catalytically competent Slt2 MAPK downstream. Active Slt2 purified from yeast extracts was able to phosphorylate Mkk1 and Mkk2 in vitro. We have mapped Ser(50) as a direct phosphorylation target for Slt2 in Mkk2. However, substitution of all (Ser/Thr)-Pro canonical MAPK target sites with alanine did not totally abrogate Slt2-dependent Mkk2 phosphorylation. Mutation or deletion of a conserved MAPK-docking site at the N-terminal extension of Mkk2 precluded its interaction with Slt2 and negatively affected retrophosphorylation. Our data show that the cell wall integrity MAPKKs are targets for their downstream MAPK, suggesting the existence of complex feedback regulatory mechanisms at this level.

The expression of the homologue of the Caenorhabditis elegans lin-45 raf is regulated in the motile stages of the plant parasitic nematode Meloidogyne artiellia.

The Ras-MAPK signal transduction pathway controls multiple developmental events and is involved in the processing of olfactory information in the free living nematode Caenorhabditis elegans. We have studied the Ras-MAPK pathway in the plant parasitic nematode Meloidogyne artiellia. The genes Mt-let-60, Mt-lin-45, Mt-mek-2 and Mt-mpk-1 have been isolated and sequenced. Each of them shows a high level of sequence similarity to its presumed ortholog in C. elegans and key functional domains are structurally conserved. Furthermore, we show that the M. artiellia recombinant MEK-2 protein can phosphorylate and activate the M. artiellia recombinant MPK-1 and the recombinant MEK-2 itself can be phosphorylated and activated by immunoprecipitated mammalian Raf. Surprisingly, the Mt-lin-45 message is not detectable in freshly emerged juveniles or in male specimens, suggesting that it may be quickly degraded in these life stages.

Phosphorylation of HSF1 by MAPK-activated protein kinase 2 on serine 121, inhibits transcriptional activity and promotes HSP90 binding.

Heat shock transcription factor 1 (HSF1) monitors the structural integrity of intracellular proteins and its regulation is essential for the health and longevity of eukaryotic organisms. HSF1 also plays a role in the acute inflammatory response in the negative regulation of cytokine gene transcription. Here we show, for the first time, that HSF1 is regulated by the proinflammatory protein kinase MAPKAP kinase 2 (MK2). We have shown that MK2 directly phosphorylates HSF1 and inhibits activity by decreasing its ability to bind the heat shock elements (HSE) found in the promoters of target genes encoding the HSP molecular chaperones and cytokine genes. We show that activation of HSF1 to bind HSE in hsp promoters is inhibited through the phosphorylation of a specific residue, serine 121 by MK2. A potential mechanism for MK2-induced HSF1 inactivation is suggested by the findings that phosphorylation of serine 121 enhances HSF1 binding to HSP90, a major repressor of HSF1. Dephosphorylation of serine 121 in cells exposed to non-steroidal anti-inflammatory drugs leads to HSP90 dissociation from HSF1, which then forms active DNA binding trimers. These experiments indicate a novel mechanism for the regulation of HSF1 by proinflammatory signaling and may permit HSF1 to respond rapidly to extracellular events, permitting optimal physiological regulation.

Structures of human MAP kinase kinase 1 (MEK1) and MEK2 describe novel noncompetitive kinase inhibition.

MEK1 and MEK2 are closely related, dual-specificity tyrosine/threonine protein kinases found in the Ras/Raf/MEK/ERK mitogen-activated protein kinase (MAPK) signaling pathway. Approximately 30% of all human cancers have a constitutively activated MAPK pathway, and constitutive activation of MEK1 results in cellular transformation. Here we present the X-ray structures of human MEK1 and MEK2, each determined as a ternary complex with MgATP and an inhibitor to a resolution of 2.4 A and 3.2 A, respectively. The structures reveal that MEK1 and MEK2 each have a unique inhibitor-binding pocket adjacent to the MgATP-binding site. The presence of the potent inhibitor induces several conformational changes in the unphosphorylated MEK1 and MEK2 enzymes that lock them into a closed but catalytically inactive species. Thus, the structures reported here reveal a novel, noncompetitive mechanism for protein kinase inhibition.