Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.11.25 (MEKK1)
 1,856 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Activation of the oncogenic potential of the MEK kinase TPL-2 (Cot) requires deletion of its C terminus. This mutation also weakens the interaction of TPL-2 with NF-kappaB1 p105 in vitro, although it is unclear whether this is important for the activation of TPL-2 oncogenicity. It is demonstrated here that TPL-2 stability in vivo relies on its high-affinity, stoichiometric association with NF-kappaB1 p105. Formation of this complex occurs as a result of two distinct interactions. The TPL-2 C terminus binds to a region encompassing residues 497 to 534 of p105, whereas the TPL-2 kinase domain interacts with the p105 death domain. Binding to the p105 death domain inhibits TPL-2 MEK kinase activity in vitro, and this inhibition is significantly augmented by concomitant interaction of the TPL-2 C terminus with p105. In cotransfected cells, both interactions are required for inhibition of TPL-2 MEK kinase activity and, consequently, the catalytic activity of a C-terminally truncated oncogenic mutant of TPL-2 is not affected by p105. Thus, in addition to its role as a precursor for p50 and cytoplasmic inhibitor of NF-kappaB, p105 is a negative regulator of TPL-2. Insensitivity of C-terminally truncated TPL-2 to this regulatory mechanism is likely to contribute to its ability to transform cells.
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PMID:NF-kappaB1 p105 negatively regulates TPL-2 MEK kinase activity. 1283 62

The tractive force generated by blood flow, called fluid shear stress, is an important regulator of endothelial cell gene expression. Several transcription factors are activated by shear stress, including members of the NF-kappaB/Rel family. The nature of the upstream-signaling components involved in the activation of NF-kappaB by flow has been studied in human endothelial cells. Flow rapidly increased endogenous IKK1/2 activity and transiently degraded IkappaBalpha and IkappaBbeta1, but not p105/p50. Nuclear translocation of the p65 subunit was induced by flow in wild-type (w/t) cells and in cells overexpressing w/t NIK, IKK1 or IKK2, but not in cells transiently transfected with kinase-inactive mutants of these enzymes. Nuclear translocation of p65 in response to flow was not affected by overexpressing a dominant-negative mutant of a MAPKKK related to NIK, called TPL2 kinase, nor by pretreating cells with the selective PKC inhibitor bisindoylmaleimide-1. Gel shift assays showed that the binding of p50/p65 heterodimer to radiolabeled oligonucleotide containing a shear-stress response element was increased by flow. The activity of a 3kappaB conA-luciferase reporter was also increased, confirming that NF-kappaB activated by flow was transcriptionally active. We conclude that shear stress induces gene transactivation by NF-kappaB (p50/p65) via the NIK-IKK1/2 pathway and proteosome-dependent degradation of IkappaB and that induction by flow does not involve TPL-2 kinase or PKC.
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PMID:Activation of NF-kappaB nuclear transcription factor by flow in human endothelial cells. 1297 91

NF-kappa B1 p105 forms a high-affinity, stoichiometric interaction with TPL-2, a MEK kinase essential for TLR4 activation of the ERK mitogen-activated protein kinase cascade in lipopolysaccharide (LPS)-stimulated macrophages. Interaction with p105 is required to maintain TPL-2 metabolic stability and also negatively regulates TPL-2 MEK kinase activity. Here, affinity purification identified A20-binding inhibitor of NF-kappa B 2 (ABIN-2) as a novel p105-associated protein. Cotransfection experiments demonstrated that ABIN-2 could interact with TPL-2 in addition to p105 but preferentially formed a ternary complex with both proteins. Consistently, in unstimulated bone marrow-derived macrophages (BMDMs), a substantial fraction of endogenous ABIN-2 was associated with both p105 and TPL-2. Although the majority of TPL-2 in these cells was complexed with ABIN-2, the pool of TPL-2 which could activate MEK after LPS stimulation was not, and LPS activation of TPL-2 was found to correlate with its release from ABIN-2. Depletion of ABIN-2 by RNA interference dramatically reduced steady-state levels of TPL-2 protein without affecting levels of TPL-2 mRNA or p105 protein. In addition, ABIN-2 increased the half-life of cotransfected TPL-2. Thus, optimal TPL-2 stability in vivo requires interaction with ABIN-2 as well as p105. Together, these data raise the possibility that ABIN-2 functions in the TLR4 signaling pathway which regulates TPL-2 activation.
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PMID:ABIN-2 forms a ternary complex with TPL-2 and NF-kappa B1 p105 and is essential for TPL-2 protein stability. 1516 88

The MEK kinase TPL-2 (also known as Cot) is required for lipopolysaccharide (LPS) activation of the extracellular signal-regulated kinase (ERK) mitogen-activated protein (MAP) kinase cascade in macrophages and consequent upregulation of genes involved in innate immune responses. In resting cells, TPL-2 forms a stoichiometric complex with NF-kappaB1 p105, which negatively regulates its MEK kinase activity. Here, it is shown that lipopolysaccharide (LPS) stimulation of primary macrophages causes the release of both long and short forms of TPL-2 from p105 and that TPL-2 MEK kinase activity is restricted to this p105-free pool. Activation of TPL-2, MEK, and ERK by LPS is also demonstrated to require proteasome-mediated proteolysis. p105 is known to be proteolysed by the proteasome following stimulus-induced phosphorylation of two serines in its PEST region by the IkappaB kinase (IKK) complex. Expression of a p105 point mutant, which is not susceptible to signal-induced proteolysis, in RAW264.7 macrophages impairs LPS-induced release of TPL-2 from p105 and its subsequent activation of MEK. Furthermore, expression of wild-type but not mutant p105 reconstitutes LPS stimulation of MEK and ERK phosphorylation in primary NF-kappaB1-deficient macrophages. Consistently, pharmacological blockade of IKK inhibits LPS-induced release of TPL-2 from p105 and TPL-2 activation. These data show that IKK-induced p105 proteolysis is essential for LPS activation of TPL-2, thus revealing a novel function of IKK in the regulation of the ERK MAP kinase cascade.
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PMID:Lipopolysaccharide activation of the TPL-2/MEK/extracellular signal-regulated kinase mitogen-activated protein kinase cascade is regulated by IkappaB kinase-induced proteolysis of NF-kappaB1 p105. 1548 31

To determine if nuclear factor-kappaB (NF-kB) plays a role in Mallory body (MB) formation, quantitative real-time RT-PCR assay was used to measure liver NF-kappaB1/p105 mRNA levels in 4 different groups of mice. Group 1: mice given IP saline for 15 weeks; group 2: mice fed diethyl 1,4-dihydro-2,4,6,-trimethyl-3,5-pyridinedicarboxylate (DDC) for 10 weeks when MBs were formed; group3: mice fed DDC 10 weeks, then withdrawn 5 weeks when MBs disappeared; group 4: mice fed DDC 10 weeks, withdrawn 4 weeks, then fed DDC+chlormethiazole (CMZ) for 1 week when MBs again formed. The mRNA for p105 NF-kappaB expression was significantly increased in the livers of mice treated with DDC (group 2) and DDC+CMZ (group 4) compared with the control livers (group 1) as well as the drug-withdrawal livers (group 3). Primary cultures of hepatocytes from drug-primed mice (the group 4 mice were withdrawn for another 4 weeks when the MBs had disappeared) were studied. The hepatocytes from drug-primed mice were MB free when isolated and used for primary culture. MBs began to form spontaneously within their cytoplasm after 2-3 days of culture. The NF-kappaB inhibitor (NF-kappaBi), a cell-permeable quinazoline compound that acts as a potent inhibitor of NF-kappaB transcriptional activation, was added to the medium 3 h after planting the cultures of liver cells. No MBs formed in the cells treated with 10 microM, 1 microM, and 0.1 microM NF-kappaBi for 6 days. MBs still formed in the cells treated with 10 nM NF-kappaBi for 6 days. Both DDC-primed and normal control liver cells began to enlarge and elongate after a few hours of culture. In contrast, the cells treated with NF-kappaBi stayed polyhedral in shape just as they appeared prior to culturing. The level of NF-kappaB1/p105 mRNA significantly increased in DDC-primed hepatocytes after 24 h of culture and in normal control hepatocytes after 48 h of culture. In DDC-primed hepatocytes, NF-kappaBi 0.1 muM treatment for 6 days significantly decreased mRNA expression of Src, p105/NF-kappaB1, ERK1, MEKK1, and JNK1/2. In normal control liver cells, NF-kappaBi treatment decreased mRNA expression of Src and JNK1 and stimulated the mRNA expression of p105/NF-kappaB1 and Junk2. NF-kappaBi treatment significantly decreased the total ERK1/2 protein and further decreased the phosphorylated (activated) form of ERK1/2 in the cultured hepatocytes. The results indicate that the p105 NF-kappaB pathway which putatively regulates ERK at both the transcriptional and post-translational levels regulates MB formation by way of changes in gene expression.
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PMID:The p105/50 NF-kappaB pathway is essential for Mallory body formation. 1592 71

Cot is one of the MAP kinase kinase kinases that regulates the ERK1/ERK2 pathway under physiological conditions. Cot is activated by LPS, by inducing its dissociation from the inactive p105 NFkappaB-Cot complex in macrophages. Here, we show that IL-1 promotes a 10-fold increase in endogenous Cot activity and that Cot is the only MAP kinase kinase kinase that activates ERK1/ERK2 in response to this cytokine. Moreover, in cells where the expression of Cot is blocked, IL-1 fails to induce an increase in IL-8 and MIP-1betamRNA levels. The activation of Cot-MKK1-ERK1/ERK2 signalling pathway by IL-1 is dependent on the activity of the transducer protein TRAF6. Most important, IL-1-induced ERK1/ERK2 activation is inhibited by PP1, a known inhibitor of Src tyrosine kinases, but this tyrosine kinase activity is not required for IL-1 to activate other MAP kinases such as p38 and JNK. This Src kinases inhibitor does not block the dissociation and subsequently degradation of Cot in response to IL-1, indicating that other events besides Cot dissociation are required to activate Cot. All these data highlight the specific requirements for activation of the Cot-MKK1-ERK1/ERK2 pathway and provide evidence that Cot controls the functions of IL-1 that are mediated by ERK1/ERK2.
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PMID:TRAF6 and Src kinase activity regulates Cot activation by IL-1. 1637 Dec 47

Toll-like receptors (TLRs) are a recently described receptor class involved in the regulation of innate and adaptive immunity. Here, we demonstrate that arrestin-2 and GRK5 (G protein-coupled receptor kinase 5), proteins that regulate G protein-coupled receptor signaling, play a negative role in TLR4 signaling in Raw264.7 macrophages. We find that lipopolysaccharide (LPS)-induced ERK1/2 phosphorylation is significantly enhanced in arrestin-2 and GRK5 knockdown cells. To elucidate the mechanisms involved, we tested the effect of arrestin-2 and GRK5 knockdown on LPS-stimulated signaling components that are upstream of ERK phosphorylation. Upon LPS stimulation, IkappaB kinase promotes phosphorylation and degradation of NFkappaB1 p105 (p105), which releases TPL2 (a MAP3K), which phosphorylates MEK1/2, which in turn phosphorylates ERK1/2. We demonstrate that knockdown of arrestin-2 leads to enhanced LPS-induced phosphorylation and degradation of p105, enhanced TPL2 release, and enhanced MEK1/2 phosphorylation. GRK5 knockdown also results in enhanced IkappaB kinase-mediated p105 phosphorylation and degradation, whereas GRK2 and GRK6 knockdown have no effect on this pathway. In vitro analysis demonstrates that arrestin-2 directly binds to the COOH-terminal domain of p105, whereas GRK5 binds to and phosphorylates p105. Taken together, these results suggest that p105 phosphorylation by GRK5 and binding of arrestin-2 negatively regulates LPS-stimulated ERK activation. These results reveal that arrestin-2 and GRK5 are important negative regulatory components in TLR4 signaling.
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PMID:Arrestin-2 and G protein-coupled receptor kinase 5 interact with NFkappaB1 p105 and negatively regulate lipopolysaccharide-stimulated ERK1/2 activation in macrophages. 1698 Mar 1

Tumor necrosis factor (TNF)-receptor-associated-factor-6 (TRAF6) is an adaptor protein involved in Toll-like receptor (TLR) signaling. Recent studies using macrophages from TRAF6 knockout mice have revealed that TRAF6 is required for TLR7 signaling. However, an essential role of TRAF6 in TLR4 signaling and cytokine production is slightly controversial. Using an RNAi approach to reduce the cellular levels of TRAF6, we tested the role of this adaptor protein on the sensitivity of the various components of the ERK pathway mediated by TLR4 and -7 in Raw264.7, a mouse macrophage cell line. ERK activation in macrophages by TLR4 and -7 is mediated via a MAP3K, called TPL2/COT, which under unstimulated conditions is associated with NF kappa B1 p105, a member of the I kappa B family of proteins. Upon stimulation with TLR ligands, p105 is phosphorylated by I kappa B kinase (IKK) complex and partially degraded, which releases TPL2. The free TPL2 is active and stimulates the ERK pathway via MEK1/2. The free TPL2, however, is also unstable and is targeted for degradation. We demonstrate here that reduced level of TRAF6 ( approximately 80% decrease) in macrophages does not significantly affect any of the components of the TLR4-stimulated ERK pathway, including p105 phosphorylation, TPL2 degradation and ERK1/2 phosphorylation. Surprisingly, however, TLR4-induced JNK1/2 phosphorylation is significantly blocked by TRAF6 knockdown, suggesting that ERK and JNK pathways are differentially sensitive to TRAF6 levels. Furthermore, although TLR4-mediated IKK-induced p105 phosphorylation is not sensitive to TRAF6 knockdown, I kappa B alpha phosphorylation (also, IKK-induced) is significantly blocked, suggesting that TLR4 activation results in a TRAF6-sensitive and -insensitive IKK activation in macrophages. In contrast to TLR4 signaling, TLR7 activation of ERK, JNK pathways and phosphorylation of p105 and I kappa B alpha are completely inhibited in TRAF6 knockdown cells. Compared to the signaling data, while TLR4-induced TNFalpha mRNA expression is not significantly inhibited by TRAF6 knockdown, TLR7-induced TNFalpha mRNA is significantly blocked. In contrast, both TLR4- and TLR7-induced IL6 mRNA are significantly blocked by TRAF6 knockdown. These results suggest that while TRAF6 is absolutely essential for TLR7 activation of ERK, JNK and NF kappa B pathways, TLR4-induced ERK, JNK pathways and IKK-mediated phosphorylation of I kappa B family members as well as cytokine expression are differentially sensitive to the cellular levels of TRAF6. These results have important implications in terms of therapeutic targeting of TRAF6 complexes in diseases where TLR4 and -7 are involved.
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PMID:Sensitivity of TLR4- and -7-induced NF kappa B1 p105-TPL2-ERK pathway to TNF-receptor-associated-factor-6 revealed by RNAi in mouse macrophages. 1750 94

Tumor progression locus 2 (TPL-2) kinase is essential for Toll-like receptor 4 activation of the mitogen-activated protein kinase extracellular signal-regulated kinase (ERK) and for upregulation of the inflammatory cytokine tumor necrosis factor (TNF) in lipopolysaccharide (LPS)-stimulated macrophages. LPS activation of ERK requires TPL-2 release from associated NF-kappaB1 p105, which blocks TPL-2 access to its substrate, the ERK kinase MEK. Here we demonstrate that TPL-2 activity is also regulated independently of p105, since LPS stimulation was still needed for TPL-2-dependent activation of ERK in Nfkb1(-/-) macrophages. In wild-type macrophages, LPS induced the rapid phosphorylation of serine (S) 400 in the TPL-2 C-terminal tail. Mutation of this conserved residue to alanine (A) blocked the ability of retrovirally expressed TPL-2 to induce the activation of ERK in LPS-stimulated Nfkb1(-/-) macrophages. TPL-2(S400A) expression also failed to reconstitute LPS activation of ERK and induction of TNF in Map3k8(-/-) macrophages, which lack endogenous TPL-2. Consistently, the S400A mutation was found to block LPS stimulation of TPL-2 MEK kinase activity. Thus, induction of TPL-2 MEK kinase activity by LPS stimulation of macrophages requires TPL-2 phosphorylation on S400, in addition to its release from NF-kappaB1 p105. Oncogenic C-terminal truncations of TPL-2 that remove S400 could promote its transforming potential by eliminating this critical control step.
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PMID:Phosphorylation of TPL-2 on serine 400 is essential for lipopolysaccharide activation of extracellular signal-regulated kinase in macrophages. 1770 78

Lithium is an anti-depressant drug that also possesses immunomodulatory functions. The anti-inflammatory effect of lithium is thought to involve activation of the transcription factor CREB, although the underlying mechanism is incompletely understood. We show here that in macrophages lithium stimulates Tpl2, a MAP kinase kinase kinase (MAP3K) known to mediate activation of extracellular signal regulated kinase (ERK) and the downstream target CREB. Lithium activates Tpl2 by inducing degradation of p105, an NF-kappaB precursor protein that functions as a physiological inhibitor of Tpl2. This novel function of lithium does not involve inhibition of a well-characterized lithium target, GSK3beta, since other known GSK3beta inhibitors do not induce p105 degradation or Tpl2 activation. Lithium also promotes the activation of Tpl2 and ERK by the TLR4 ligand LPS. On the other hand, prolonged incubation of macrophages with lithium results in dramatic loss of p105 and inhibition of LPS-stimulated NF-kappaB activation. Consequently, lithium both attenuates LPS-mediated pro-inflammatory gene induction and induces apoptosis in macrophages. These results provide novel insight into the anti-inflammatory function of lithium.
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PMID:Deregulation of Tpl2 and NF-kappaB signaling and induction of macrophage apoptosis by the anti-depressant drug lithium. 1915 80


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