Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.12.2 (MEK)
18,161 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Earlier studies have implicated the significance of transforming growth factor-beta3 (TGFbeta3) in the regulation of Sertoli cell tight junction (TJ) dynamics, possibly via its inhibitory effects on the expression of occludin, claudin-11, and zonula occludens-1 (ZO-1). Yet the mechanism by which TGFbeta3 regulates the Sertoli cell TJ-permeability barrier is not known. Using techniques of semiquantitative reverse transcription-PCR (RT-PCR), immunoblotting, immunohistochemistry, and inhibitors against different kinases coupled with physiological techniques to assess the Sertoli cell TJ barrier function, it was shown that this TGFbeta3-induced effect on Sertoli cell TJ dynamics is mediated via the p38 mitogen-activated protein (MAP) kinase pathway. First, the assembly of the Sertoli cell-TJ barrier was shown to be associated with a transient but significant decline in both the TGFbeta3 production and expression by Sertoli cells. Furthermore, addition of TGFbeta3 to Sertoli cell cultures during TJ assembly indeed perturbed the TJ barrier with an IC50 at approximately 9 pM. Second, the TGFbeta3-induced disruption of the TJ barrier was associated with a transient induction in MEKK2 but not the other upstream signaling molecules that mediate TGFbeta3 action, such as Smad2, Cdc42, Rac2, and N-Ras, suggesting this effect might be mediated via the p38 MAP kinase pathway. This postulate was confirmed by the observation that TGFbeta3 also induced the protein level of the activated and phosphorylated form of p38 MAP kinase at the time the TJ barrier was perturbed. Third, and perhaps the most important of all, this TGFbeta3-mediated inhibitory effect on the TJ barrier and the TGFbeta3-induced p-p38 MAP kinase production could be blocked by SB202190, a specific p38 MAP kinase inhibitor, but not U0126, a specific MEK1/2 kinase inhibitor. These results thus unequivocally demonstrate that TGFbeta3 utilizes the p38 MAP kinase pathway to regulate Sertoli cell TJ dynamics.
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PMID:Transforming growth factor beta3 regulates the dynamics of Sertoli cell tight junctions via the p38 mitogen-activated protein kinase pathway. 1260 50

Protein kinase C-associated kinase (PKK, also known as RIP4/DIK) activates NFkappaB when overexpressed in cell lines and is required for keratinocyte differentiation in vivo. However, very little is understood about the factors upstream of PKK or how PKK activates NFkappaB. Here we show that certain catalytically inactive mutants of PKK can activate NFkappaB, although to a lesser degree than wild type PKK. The deletion of specific domains of wild type PKK diminishes the ability of this enzyme to activate NFkappaB; the same deletions made on a catalytically inactive PKK background completely ablate NFkappaB activation. PKK may be phosphorylated by two specific mitogen-activated protein kinase kinase kinases, MEKK2 and MEKK3, and this interaction may in part be mediated through a critical activation loop residue, Thr184. Catalytically inactive PKK mutants that block phorbol ester-induced NFkappaB activation do not interfere with, but unexpectedly enhance, the activation of NFkappaB by these two mitogen-activated protein kinase kinase kinases. Taken together, these data indicate that PKK may function in both a kinase-dependent as well as a kinase-independent manner to activate NFkappaB.
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PMID:Protein kinase C-associated kinase can activate NFkappaB in both a kinase-dependent and a kinase-independent manner. 1267 34

The mitogen-activated protein kinase (MAPK) c-Jun N-terminal kinase (JNK) is a critical regulator of collagenase-1 production in rheumatoid arthritis (RA). The MAPKs are regulated by upstream kinases, including MAPK kinases (MAPKKs) and MAPK kinase kinases (MAP3Ks). The present study was designed to evaluate the expression and regulation of the JNK pathway by MAP3K in arthritis. RT-PCR studies of MAP3K gene expression in RA and osteoarthritis synovial tissue demonstrated mitogen-activated protein kinase/ERK kinase kinase (MEKK) 1, MEKK2, apoptosis-signal regulating kinase-1, TGF-beta activated kinase 1 (TAK1) gene expression while only trace amounts of MEKK3, MEKK4, and MLK3 mRNA were detected. Western blot analysis demonstrated immunoreactive MEKK2, TAK1, and trace amounts of MEKK3 but not MEKK1 or apoptosis-signal regulating kinase-1. Analysis of MAP3K mRNA in cultured fibroblast-like synoviocytes (FLS) showed that all of the MAP3Ks examined were expressed. Western blot analysis of FLS demonstrated that MEKK1, MEKK2, and TAK1 were readily detectable and were subsequently the focus of functional studies. In vitro kinase assays using MEKK2 immunoprecipitates demonstrated that IL-1 increased MEKK2-mediated phosphorylation of the key MAPKKs that activate JNK (MAPK kinase (MKK)4 and MKK7). Furthermore, MEKK2 immunoprecipitates activated c-Jun in an IL-1 dependent manner and this activity was inhibited by the selective JNK inhibitor SP600125. Of interest, MEKK1 immunoprecipitates from IL-1-stimulated FLS appeared to activate c-Jun through the JNK pathway and TAK1 activation of c-Jun was dependent on JNK, ERK, and p38. These data indicate that MEKK2 is a potent activator of the JNK pathway in FLS and that signal complexes including MEKK2, MKK4, MKK7, and/or JNK are potential therapeutic targets in RA.
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PMID:Regulation of c-Jun N-terminal kinase by MEKK-2 and mitogen-activated protein kinase kinase kinases in rheumatoid arthritis. 1473 42

The ERK5 signaling cascade acts through sequential activation of MEKK2/3, MEK5 and ERK5 and transmits signals to a variety of stress and mitogenic related targets. In this study we examined the subcellular localization of the components of the ERK5 cascade and found that in resting, as well as in EGF-stimulated HeLa and Rat-1 cells, endogenous ERK5 is localized mainly in the nucleus. This location is different from the previously described location of exogenous ERK5, in the cytosol of resting cells, which is confirmed in this study. The reason for the different localization could be a saturation of anchoring moieties by the endogenous ERK5. Indeed, in situ detergent extraction analysis using Nonidet P-40, revealed that ERK5 is bound to detergent resistant moieties in the nucleus, while the exogenous protein fails to interact with those anchors. The upstream activator MEK5 is also localized in the nucleus both before and after EGF stimulation and is resistant to NP-40 extraction in resting cells. ERK5 remains bound to these nuclear moieties even after stimulation, while MEK5 is detached from the anchors but remains localized in the nucleus. Unlike ERK5 and MEK5, their upstream activator MEKK2 is localized mainly in the cytosol of resting cells, and translocates into the nucleus upon EGF stimulation, allowing transmission of signals to the nuclear MEK5. The nuclear localization of MEK5 and ERK5 is different from that of ERK1/2 and MEK1/2 in resting cells, indicating that each MAPK cascade uses distinct mechanisms to transmit extracellular signals to their nuclear targets.
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PMID:MEK5 and ERK5 are localized in the nuclei of resting as well as stimulated cells, while MEKK2 translocates from the cytosol to the nucleus upon stimulation. 1507 38

MEKK2, MEK5, and extracellular signal-regulated kinase 5 (ERK5) are members of a three-kinase cascade for the activation of ERK5. MEK5 is the only MAP2K to express a PB1 domain, and we have shown that it heterodimerizes with the PB1 domain of MEKK2. Here we demonstrate the MEK5 PB1 domain is a scaffold that also binds ERK5, functionally forming a MEKK2-MEK5-ERK5 complex. Reconstitution assays and CFP/YFP imaging (fluorescence resonance energy transfer [FRET]) measuring YFP-MEKK2/CFP-MEK5 and CFP-MEK5/YFP-ERK5 interactions define distinct MEK5 PB1 domain binding sites for MEKK2 and ERK5, with a C-terminal extension of the PB1 domain contributing to ERK5 binding. Stimulus-dependent CFP/YFP FRET in combination with mutational analysis was used to define MEK5 PB1 domain residues critical for the interaction of MEKK2/MEK5 and MEK5/ERK5 required for activation of the ERK5 pathway in living cells. Fusion of the MEK5 PB1 domain to the N terminus of MEK1 confers ERK5 regulation by a MAP2K normally regulating only ERK1/2. The MEK5 PB1 domain confers stringent MAP3K regulation of ERK5 relative to more promiscuous MAP3K control of ERK1/2, JNK, and p38.
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PMID:PB1 domain-dependent signaling complex is required for extracellular signal-regulated kinase 5 activation. 1650 87

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.
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PMID:Noncanonical function of MEKK2 and MEK5 PB1 domains for coordinated extracellular signal-regulated kinase 5 and c-Jun N-terminal kinase signaling. 1745 62

c-Jun N-terminal kinase (JNK) contributes to metalloproteinase (MMP) gene expression and joint destruction in inflammatory arthritis. It is phosphorylated by at least two upstream kinases, the mitogen-activated protein kinase kinases (MEK) MKK4 and MKK7, which are, in turn, phosphorylated by MEK kinases (MEKKs). However, the MEKKs that are most relevant to JNK activation in synoviocytes have not been determined. These studies were designed to assess the hierarchy of upstream MEKKs, MEKK1, MEKK2, MEKK3, and transforming growth factor-beta activated kinase (TAK)1, in rheumatoid arthritis (RA). Using either small interfering RNA (siRNA) knockdown or knockout fibroblast-like synoviocytes (FLSs), MEKK1, MEKK2, or MEKK3 deficiency (either alone or in combination) had no effect on IL-1beta-stimulated phospho-JNK (P-JNK) induction or MMP expression. However, TAK1 deficiency significantly decreased P-JNK, P-MKK4 and P-MKK7 induction compared with scrambled control. TAK1 knockdown did not affect p38 activation. Kinase assays showed that TAK1 siRNA significantly suppressed JNK kinase function. In addition, MKK4 and MKK7 kinase activity were significantly decreased in TAK1 deficient FLSs. Electrophoretic mobility shift assays demonstrated a significant decrease in IL-1beta induced AP-1 activation due to TAK1 knockdown. Quantitative PCR showed that TAK1 deficiency significantly decreased IL-1beta-induced MMP3 gene expression and IL-6 protein expression. These results show that TAK1 is a critical pathway for IL-1beta-induced activation of JNK and JNK-regulated gene expression in FLSs. In contrast to other cell lineages, MEKK1, MEKK2, and MEKK3 did not contribute to JNK phosphorylation in FLSs. The data identify TAK1 as a pivotal upstream kinase and potential therapeutic target to modulate synoviocyte activation in RA.
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PMID:Regulation of the JNK pathway by TGF-beta activated kinase 1 in rheumatoid arthritis synoviocytes. 1755 74

Proteins containing the PB1 domain, a protein interaction module conserved in animals, fungi, amoebas, and plants, participate in diverse biological processes. The PB1 domains adopt a ubiquitin-like beta-grasp fold, containing two alpha helices and a mixed five-stranded beta sheet, and are classified into groups harboring an acidic OPCA motif (type I), the invariant lysine residue on the first beta strand (type II), or both (type I/II). The OPCA motif of a type I PB1 domain forms salt bridges with basic residues, especially the conserved lysine, of a type II PB1 domain, thereby mediating a specific PB1-PB1 heterodimerization, whereas additional contacts contribute to high affinity and specificity of the modular interaction. The canonical PB1 dimerization is required for the formation of complexes between p40(phox) and p67(phox) (for activation of the NADPH oxidase crucial for mammalian host defense), between the scaffold Bem1 and the guanine nucleotide exchange factor Cdc24 (for polarity establishment in yeasts), and between the polarity protein Par6 and atypical protein kinase C (for cell polarization in animal cells), as well as for the interaction between the mitogen-activated protein kinase kinase kinases MEKK2 or MEKK3 and the downstream target mitogen-activated protein kinase kinase MEK5 (for early cardiovascular development in mammals). PB1 domains can also mediate interactions with other protein domains. For example, an intramolecular interaction between the PB1 and PX domains of p40(phox) regulates phagosomal targeting of the microbicidal NADPH oxidase; the PB1 domain of MEK5 is likely responsible for binding to the downstream kinase ERK5, which lacks a PB1 domain; and the scaffold protein Nbr1 associates through a PB1-containing region with titin, a sarcomere protein without a PB1 domain. This Review describes various aspects of PB1 domains at the molecular and cellular levels.
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PMID:Structure and function of the PB1 domain, a protein interaction module conserved in animals, fungi, amoebas, and plants. 1772 78

The therapeutic effects of alpha-lipoic acid (alpha-LA) via NF-kappa B down regulation were demonstrated on joint inflammation and erosion in an animal model. In this study, we investigated how alpha-LA inhibits the pathway of NF-kappa B activation by TNF-alpha via the mitogen-activated protein kinase (MAPK) pathway in rheumatoid arthritis (RA) fibroblast-like synovial cells (FLS). FLS were stimulated with TNF-alpha following pre-treatment with or without alpha-LA. Electrophoretic mobility shift assays (EMSA) revealed that TNF-alpha activates NF-kappa B in FLS. This was inhibited by alpha-LA at concentrations of 1 mM. TNF-alpha induced IKK mediated phosphorylation of GST-I kappa B and pre-treatment with alpha-LA inhibited this pathway. FLS constitutively express MEKK1, MEKK2, MEKK3, and TAK1 and that their levels are unaffected by TNF-alpha or alpha-LA. Immunoprecipitation using anti-MEKK1 antibody phosphorylated GST-I kappa B and pre-treating the cells with alpha-LA could abolish the reaction. FLS were immunoprecipitated using an antibody to MEKK1, and MKK4 was coprecipitated with MEKK1. In addition, immune complexes precipitated with anti-MKK4 antibody phosphorylated GST-I kappa B, and pre-treatment with alpha-LA inhibited the phosphorylation. Immunoprecipitation assay showed that MEKK1, MKK4, IKK-alpha, IKK-beta, I kappa B, and NF-kappa B comprised immunocomplex. It can be concluded that TNF-alpha activates NF-kappa B in FLS through MEKK1-MKK4-IKK signaling complex, and alpha-LA inhibits this signaling at the level of or upstream of IKK-alpha and IKK-beta.
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PMID:Alpha-lipoic acid inhibits TNF-alpha induced NF-kappa B activation through blocking of MEKK1-MKK4-IKK signaling cascades. 1818 52

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.
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PMID:Homogeneous time-resolved fluorescence resonance energy transfer assay for measurement of Phox/Bem1p (PB1) domain heterodimerization. 1848 Apr 72


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