Design of a dual ERK5 kinase activation and autophosphorylation inhibitor to block cancer stem cell activity.

The importance of ERK5 kinase signaling in tumorigenicity, metastasis, and drug resistance of cancer stem cells (CSCs) has been recognized recently, and we report a unique dual inhibitor that blocks binding of the ERK5 activator and ERK5 autophosphorylation simultaneously. The conventional ATP-binding site inhibitors have not yet yielded expected level of anti-cancer effects, due to complexities in converting ERK5 activation into CSC biological effects. We designed the first ERK5-targeted anti-CSC dual active hetero-bivalent inhibitor that blocks the regulatory peptide interaction involved in ERK5 kinase activation and that simultaneously inhibits the conventional ATP-binding pocket as well. We utilized two assay systems to independently prove disruption of these two ERK5 activities via a single compound. We also showed that this compound inhibited CSC activities, such as colony formation, cell proliferation, and migration.

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.

Noncanonical function of MEKK2 and MEK5 PB1 domains for coordinated extracellular signal-regulated kinase 5 and c-Jun N-terminal kinase signaling.

MEKK2 and MEK5 encode Phox/Bem1p (PB1) domains that heterodimerize with one another. MEKK2, MEK5, and extracellular signal-related kinase 5 (ERK5) form a ternary complex through interactions involving the MEKK2 and MEK5 PB1 domains and a 34-amino-acid C-terminal extension of the MEK5 PB1 domain. This C-terminal extension encodes an ERK5 docking site required for MEK5 activation of ERK5. The PB1 domains bind in a front-to-back arrangement, with a cluster of basic amino acids in the front of the MEKK2 PB1 domain binding to the back-end acidic clusters of the MEK5 PB1 domain. The C-terminal moiety, including the acidic cluster of the MEKK2 PB1 domain, is not required for MEK5 binding and binds MKK7. Quiescent MEKK2 preferentially binds MEK5, and MEKK2 activation results in ERK5 activation. Activated MEKK2 binds and activates MKK7, leading to JNK activation. The findings define how the MEKK2 and MEK5 PB1 domains are uniquely used for differential binding of two mitogen-activated protein kinase kinases, MEK5 and MKK7, for the coordinated control of ERK5 and c-Jun N-terminal kinase activation.

Biochanin A protects against PM2.5-induced acute pulmonary cell injury by interacting with the target protein MEK5.

Epidemiological studies have shown that exposure to ambient fine particulate matter (PM2.5) is associated with an increased risk for cardiopulmonary diseases. The MEK5/ERK5 and NF-κB signaling pathways are closely related to the regulation of acute pulmonary cell injury (APCI) and may play an important role in the underlying pathophysiological mechanisms. Related studies have shown that Biochanin A (BCA) effectively interferes with APCI, but the underlying mechanism through which this occurs is not fully understood. Previously, based on proteomic and bioinformatic research, we found the indispensable role of MEK5 in mediating remission effects of BCA against PM2.5-induced lung toxicity. Therefore, using A549 adenocarcinoma human alveolar basal epithelial cells (A549 cells), we combined western blot and qRT-PCR to study the protective signaling pathways induced by BCA, indicating that MEK5/ERK5 and NF-κB are both involved in mediating APCI in response to PM2.5, and MEK5/ERK5 positively activated NF-κB and its downstream cellular regulatory factors. BCA significantly suppressed PM2.5-induced upregulation of MEK5/ERK5 expression and phosphorylation and activation of NF-κB. Furthermore, due to the specificity of the MEK5/ERK5 protein structure, the binding sites and binding patterns of BCA and MEK5 were analyzed using molecular docking correlation techniques, which showed that there are stable hydrogen bonds between BCA and the PB1 domain of MEK5 as well as its kinase domain. BCA forms a stable complex with MEK5, which has potential effects on MEKK2/3-MEK5-ERK5 ternary interactions, p62/αPKC-mediated NF-κB regulation, and inhibition of MEK5 target protein phosphorylation. Therefore, our study suggests that MEK5 is an important regulator of intracellular signaling of APCI in response to PM2.5 exposure. BCA may exert anti-APCI activity by targeting MEK5 to inhibit activation of the MEK5/ERK5/NF-κB signaling pathway.

Homogeneous time-resolved fluorescence resonance energy transfer assay for measurement of Phox/Bem1p (PB1) domain heterodimerization.

Twenty human proteins encode Phox/Bem1p (PB1) domains, which are involved in forming protein heterodimers. MEKK2, MEKK3, and MEK5 are 3 serine-threonine protein kinases that have PB1 domains. MEKK2, MEKK3, and MEK5 are the MAP3Ks and the MAP2K in the ERK5 mitogen-activated protein kinase (MAPK) signaling module. ERK5 is a critical MAPK for both development of the vasculature and vascular homeostasis in the adult, but no other MAPK has been shown to be critical in vascular maintenance in the adult animal. MEKK2 and MEKK3 are the only MAP3Ks shown to physically interact with and activate the MEK5-ERK5 signaling module. Interaction of MEKK2 or MEKK3 with MEK5 is mediated by heterodimerization of the MEKK2 (or MEKK3) PB1 and MEK5 PB1 domains. The authors have developed a homogeneous, time-resolved fluorescence resonance energy transfer (TR-FRET) assay to monitor PB1-PB1 domain heterodimerization. The assay uses a europium-chelate conjugated GST-MEK5 PB1 domain chimera, biotinylated MEKK2 PB1 domain, and streptavidin-Cy5. Interaction of the MEKK2 and MEK5 PB1 domains gives a robust FRET signal (Z' factor = 0.93), which is completely abrogated by mutation of 2 acidic residues (64D65E-->AA) within the MEK5 PB1 domain that causes loss of stable PB1-PB1 domain interaction. This assay can be used to study the specificity of PB1-PB1 domain interactions and to screen for molecules that can regulate MEKK2/MEKK3-MEK5 interactions. Disruption of PB1 domain interactions represents a novel approach for selectively regulating the ERK5 signaling pathway independent of kinase active site-directed adenosine triphosphate competitive inhibitors.

A novel mitogen-activated protein kinase docking site in the N terminus of MEK5alpha organizes the components of the extracellular signal-regulated kinase 5 signaling pathway.

The alternative splicing of the mek5 gene gives rise to two isoforms. MEK5beta lacks an extended N terminus present in MEK5alpha. Comparison of their activities led us to identify a novel mitogen-activated protein kinase (MAPK) docking site in the N terminus of MEK5alpha that is distinct from the consensus motif identified in the other MAPK kinases. It consists of a cluster of acidic residues at position 61 and positions 63 to 66. The formation of the MEK5/extracellular signal-regulated kinase 5 (ERK5) complex is critical for MEK5 to activate ERK5, to increase transcription via MEF2, and to enhance cellular survival in response to osmotic stress. Certain mutations in the ERK5 docking site that prevent MEK5/ERK5 interaction also abrogate the ability of MEKK2 to bind and activate MEK5. However, the identification of MEK5alpha mutants with selective binding defect demonstrates that the MEK5/ERK5 interaction does not rely on the binding of MEK5alpha to MEKK2 via their respective PB1 domains. Altogether these results establish that the N terminus of MEK5alpha is critical for the specific organization of the components of the ERK5 signaling pathway.

Insight into the binding properties of MEKK3 PB1 to MEK5 PB1 from its solution structure.

MEKK3 is a mitogen-activated protein kinase kinase kinase that participates in various signaling pathways. One of its functions is to activate the ERK5 signal pathway by phosphorylating and activating MEK5. MEKK3 and MEK5 each harbors a PB1 domain in the N-terminus, and they form a heterodimer via PB1-PB1 domain interaction that was reported to be indispensable to the activation of MEK5. Using NMR spectroscopy, we show here that a prolyl isomerization of the Gln38-Pro39 bond is present in MEKK3 PB1, which is the first case of structural heterogeneity within PB1 domains. We have solved the solution structures of both isomers and found a major difference between them in the Pro39 region. Residues Gly37-Leu40 form a type VIb beta-turn in the cis conformation, whereas no obvious character of beta-turn was observed in the trans conformation. Backbone dynamics studies have unraveled internal motions in the beta3/beta4-turn on a microsecond-millisecond time scale. Further investigation of its binding properties with MEK5 PB1 has demonstrated that MEKK3 PB1 binds MEK5 PB1 tightly with a Kd of about 10(-8) M. Mutagenesis analysis revealed that residues in the basic cluster of MEKK3 PB1 contributes differently to the PB1-PB1 interaction. Residues Lys 7 and Arg 5 play important roles in the interaction with MEK5 PB1. Taken together, this study provides new insights into structural details of MEKK3 PB1 and its binding properties with MEK5 PB1.