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)

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

CYLD is a tumor suppressor that is mutated in familial cylindromatosis, an autosomal dominant predisposition to multiple tumors of the skin appendages. Recent studies suggest that transfected CYLD has deubiquitinating enzyme activity and inhibits the activation of transcription factor NF-kappaB. However, the role of endogenous CYLD in regulating cell signaling remains poorly defined. Here we report a critical role for CYLD in negatively regulating the c-Jun NH(2)-terminal kinase (JNK). CYLD knockdown by RNA interference results in hyper-activation of JNK by diverse immune stimuli, including tumor necrosis factor-alpha, interleukin-1, lipopolysaccharide, and an agonistic anti-CD40 antibody. The JNK-inhibitory function of CYLD appears to be specific for immune receptors because the CYLD knockdown has no significant effect on stress-induced JNK activation. Consistently, CYLD negatively regulates the activation of MKK7, an upstream kinase known to mediate JNK activation by immune stimuli. We further demonstrate that CYLD also negatively regulates IkappaB kinase, although this function of CYLD is seen in a receptor-dependent manner. These findings identify the JNK signaling pathway as a major downstream target of CYLD and suggest a receptor-dependent role of CYLD in regulating the IkappaB kinase pathway.
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PMID:Negative regulation of JNK signaling by the tumor suppressor CYLD. 1549

Development of drug resistance in cancer is one of the main challenges in chemotherapy, and many mechanisms are still unknown. In this study, we show that tumor necrosis factor alpha (TNFalpha) increases postdrug survival from 5-fluoro-2'-deoxyuridine (FdUrd) in two human colon tumor cell lines. This resulted in the development of drug-resistant cells in a TNFalpha-dependent manner. Interestingly, although the drug-resistant cells were selected using FdUrd, they are also resistant to a number of other antimetabolites in the DNA synthesis pathway in a TNFalpha-dependent manner. Only in the drug-resistant cells (p35-colo201) TNFalpha treatment resulted in G(0)-G(1) arrest but not in the parental colo201 and other cell types. Blocking TNFalpha-induced cell cycle arrest sensitized drug-resistant cells to FdUrd. TNFalpha-induced cell cycle arrest required IKK. IKK inhibition by a small molecule inhibitor or by the knockdown of IKKalpha, IKKbeta, or RelA/p65 using siRNA, but not the inhibition of JNK, MEK, p38, or caspase-8 pathways, blocked TNFalpha-induced G(0)-G(1) arrest and restored sensitivity to FdUrd of drug-resistant cells. TNFalpha reduced the transcripts and protein levels of phosphorylated retinoblastoma protein (Rb), Rb, E2F1, and Cdk4 only in drug-resistant p35-colo201 cells. This effect of TNFalpha was reversed by IKK inhibitor, suggesting that TNFalpha-induced cell cycle arrest is probably due to the reduction of Rb, E2F1, and Cdk4. Taken together, this study shows that, in vitro, TNFalpha-induced cell cycle arrest through IKK can provide a mechanism for the development of drug resistance to anti-cancer drugs, purine and pyrimidine analogues.
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PMID:Tumor necrosis factor alpha-dependent drug resistance to purine and pyrimidine analogues in human colon tumor cells mediated through IKK. 1561 Oct 81

Lysophosphatidic acid (LPA) enhances urokinase plasminogen activator (uPA) expression in ovarian cancer cells; however, the molecular mechanisms responsible for this event have not been investigated. In this study, we used the invasive ovarian cancer SK-OV-3 cell line to explore the signaling molecules and pathways essential for LPA-induced uPA up-regulation. With the aid of specific inhibitors and dominant negative forms of signaling molecules, we determined that the G(i)-associated pathway mediates this LPA-induced event. Moreover, constitutively active H-Ras and Raf-1-activating H-Ras mutant enhance uPA expression, whereas dominant negative H-Ras and Raf-1 block LPA-induced uPA up-regulation, suggesting that the Ras-Raf pathway works downstream of G(i) to mediate this LPA-induced process. Surprisingly, dominant negative MEK1 or Erk2 displays only marginal inhibitory effect on LPA-induced uPA up-regulation, suggesting that a signaling pathway distinct from Raf-MEK1/2-Erk is the prominent pathway responsible for this process. In this report, we demonstrate that LPA activates NF-kappaB in a Ras-Raf-dependent manner and that blocking NF-kappaB activation with either non-phosphorylable IkappaB or dominant negative IkappaB kinase abolished LPA-induced uPA up-regulation and uPA promoter activation. Furthermore, introducing mutations to knock out the NF-kappaB binding site of the uPA promoter results in over 80% reduction in LPA-induced uPA promoter activation, whereas this activity is largely intact with the promoter containing mutations in the AP1 binding sites. Thus these results suggest that the G(i)-Ras-Raf-NF-kappaB signaling cascade is responsible for LPA-induced uPA up-regulation in ovarian cancer cells.
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PMID:Signaling mechanisms responsible for lysophosphatidic acid-induced urokinase plasminogen activator expression in ovarian cancer cells. 1565 92

Overexpression of inducible nitric oxide synthase (iNOS) has been reported in several human cancers, including esophageal squamous cell carcinoma (SCC). Benzo[a]pyrene (B[a]P), a polycyclic hydrocarbon carcinogen found in tobacco smoke and in the environment, induces cancer in multiple organ sites in animals and may be a causative agent for certain human cancers, such as esophageal cancer. In the present study, the effects of B[a]P on the induction of iNOS and the signaling pathways that lead to the induction were investigated in cultured rat esophageal epithelial (RE-149) cells. Treatment of RE-149 cells with B[a]P led to a marked increase in the expression of iNOS. The induction of iNOS by B[a]P was found to occur through an extracellular signal-regulated protein kinases (ERKs)-dependent pathway, since inhibition of ERKs by either pretreatment of RE-149 cells with PD98059, an inhibitor of ERKs upstream kinase MEK1/2, or overexpression of DN-ERK2, blocked the induction of iNOS by B[a]P. Furthermore, impairing nuclear factor-kappaB (NFkappaB) activation by either NEMO-BDBP, an NFkappaB specific inhibitor, or overexpression of DN-IkappaBalpha or IKK-KM markedly inhibited the expression of B[a]P-induced iNOS, suggesting that the NFkappaB pathway is also required for the induction of iNOS by B[a]P. In addition, treatment of RE-149 cells with either SB202190, a p38 kinase inhibitor, or c-JunN-terminal kinase inhibitor II, resulted in an increased induction of iNOS. Pretreatment of RE-149 cells with wortmannin, a PI-3K inhibitor, or with rapamycin, an mTOR/p70S6K pathway inhibitor, had no effect on the expression of iNOS. These results suggest that B[a]P initiates the signaling pathways leading to the induction of iNOS in cultured rat esophageal epithelial cells. In view of the potential role of iNOS in the development of esophageal SCC in humans, we speculate that the induction of iNOS by B[a]P may be one mechanism by which B[a]P could produce carcinogenic effects in the human esophagus.
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PMID:Differential requirement of signal pathways for benzo[a]pyrene (B[a]P)-induced nitric oxide synthase (iNOS) in rat esophageal epithelial cells. 1571 51

We examined the role of the IkappaB kinase complex in nerve growth factor (NGF)-induced neuronal differentiation of PC12 cells. We showed that neurite outgrowth is accompanied by an activation of the IKK complex and a delayed elevation of NF-kappaB-dependent transcription. Ectopic expression of a constitutively active form of IKK2 but not of IKK1 promoted neurite outgrowth in the absence of NGF. In addition, increased expression of Bcl-2 and Bcl-xL and resistance to apoptosis upon serum withdrawal were found. The IKK2-driven neurite outgrowth was not blocked by MEK1/2 and PI3K inhibitors but was repressed by the SN50 peptide suggesting that NF-kappaB activation is critical for this differentiation process. Transdominant mutants of IkappaBalpha (32/36-SS/AA) and IKK1 only marginally reduced NGF-driven neuritogenesis. However, a dominant negative mutant of IKK2 or an IkappaBalpha protein lacking the complete N-terminus was able to repress neuritogenesis. We also detected tyrosine phosphorylation of IkappaBalpha during differentiation. Consequently, PC12 cells expressing mutant IkappaBalpha (Y42F) show an impaired neuritogenesis. Furthermore, PC12 cells ectopically expressing p65 show almost no signs of neurite outgrowth which is, however, found to some extent in c-Rel-expressing cells. Our data suggest that NGF-induced PC12 differentiation includes activation of IKK2 which may promote the release of c-Rel-containing dimers.
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PMID:Activation of the IkappaB kinase complex is sufficient for neuronal differentiation of PC12 cells. 1593 65

The transcription factor nuclear factor-kappa B (NF-kappaB) subunit p65 is phosphorylated by IkappaB kinase (IKK) at S536 in transactivation domain (TAD) 1. In this study, we investigate the presence of IKK sites in TAD2 of p65. Recombinant IKKbeta, but not IKKalpha, phosphorylated a GST-p65 substrate in which TAD1 was deleted. Mutational analysis revealed S468 as the only IKK site in TAD2. S468 phosphorylation occurred rapidly after TNF-alpha and IL-1beta in T cell, B cell, cervix carcinoma, hepatoma, breast cancer, and astrocytoma lines and in primary hepatic stellate cells as well as peripheral blood mononuclear cells. S468-phosphorylated p65 coimmunoprecipitated with IkappaBalpha, indicating that p65 is phosphorylated while bound to IkappaBalpha. Dominant negative IKKbeta or pharmacological IKK inhibition blocked S468 phosphorylation after TNF-alpha or IL-1beta, whereas dominant negative IKKalpha or inhibitors of MEK, p38, JNK, PI-3 kinase, or GSK-3 had no effect. p65S468A-reconstituted p65-/- mouse embryonic fibroblasts (MEFs) showed a small, but significant, elevation of NF-kappaB-driven luciferase activity and RANTES mRNA levels after TNF-alpha and IL-1beta in comparison to wtp65-reconstituted MEFs. p65 nuclear translocation was not altered in p65S468A-expressing MEFs. In conclusion, our results indicate that 1) IKKbeta phosphorylates multiple p65 sites, 2) IKKbeta phosphorylates p65 in an IkappaB-p65 complex, and 3) S468 phosphorylation slightly reduces TNF-alpha- and IL-1beta-induced NF-kappaB activation.
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PMID:IKKbeta phosphorylates p65 at S468 in transactivaton domain 2. 1604 71

Unveiling of endothelial nuclear factor-kappaB (NF-kappaB) activation is pivotal for understanding the inflammatory reaction and the pathogenesis of inflammatory vascular diseases. We here report the novel function of extracellular signal-related kinase (ERK) in controlling endothelial NF-kappaB activation and inflammatory responses. In human endothelial cells, vascular endothelial growth factor (VEGF) induced NF-kappaB-dependent transcription of cell adhesion molecules (CAMs) and monocyte adhesion. These effects were prominently enhanced by either pretreatment with the MEK inhibitors, PD98059 and U0126 or overexpression of a dominant negative form of MEK, but blocked by a wild type ERK. Consistently, inhibition of ERK significantly increased IkappaB kinase (IKK) activity, IkappaBalpha phosphorylation, and nuclear translocation of NF-kappaB induced by VEGF, whereas overexpression of ERK resulted in the loss of these responses to VEGF. Using two PKC inhibitors has demonstrated that VEGF concomitantly stimulates IKK and its negative regulatory signal ERK through PKC that lies downstream of KDR/Flk-1. Strikingly, elevation of ERK in endothelial cells markedly inhibited CAM expression and NF-kappaB activation as well as monocyte adhesion induced by IL-1beta and TNF-alpha. The data collectively suggest that ERK serves as an anti-inflammatory signal that suppresses expression of NF-kappaB-dependent inflammatory genes by inhibiting IKK activity in endothelial cells. Measuring the existence of ERK activity in vascular endothelial cells may be useful for predicting the feasibility and potency of inflammatory reactions in the vasculature.
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PMID:ERK is an anti-inflammatory signal that suppresses expression of NF-kappaB-dependent inflammatory genes by inhibiting IKK activity in endothelial cells. 1624 16

The chemokine stromal-derived factor-1alpha (SDF-1alpha/CXCL-12) and its receptor, CXCR4, play a crucial role in adhesion and transendothelium migration (TEM) of prostate cancer cells. We tested the hypothesis that enhanced expression of CXCR4 in prostate cancer cells is dependent upon SDF-1alpha-mediated activation of nuclear factor-kappaB (NF-kappaB). SDF-1alpha increased the CXCR4 mRNA and protein expression in PC-3 cells but not in LNCaP cells. Similarly, SDF-1alpha enhanced the NF-kappaB-dependent transcriptional activity in PC-3 cells but not in LNCaP cells. SDF-1alpha increased PC-3 cell adhesion to the human umbilical vein endothelial cell monolayer and enhanced TEM, which was abrogated with anti-CXCR4 monoclonal antibody (mAb). Suppression of NF-kappaB activity in PC-3 cells by a mutant IkappaBalpha super-repressor adenoviral vector decreased the CXCR4 mRNA expression and inhibited adhesion and TEM. Transient overexpression of p65 subunit of NF-kappaB in PC-3 cells up-regulated CXCR4 receptor expression and increased the adhesion and TEM of these cells in response to SDF-1alpha gradient. Treatment of PC-3 cells with SDF-1alpha leads to nuclear translocation of NF-kappaB protein within 15 to 30 minutes, which correlated with IkappaBalpha phosphorylation. A p42/44 mitogen-activated protein kinase [MAPK, extracellular signal regulated kinase-1/2 (ERK-1/2)] biphasic activation pattern was observed in these cells at 15 minutes and 3 hours after SDF-1alpha treatment. Phosphorylation of IkappaB kinase alpha was observed within 30 minutes, which was blocked by PD98059 [MAPK kinase (MEK) inhibitor]. PD98059 cotreatment significantly inhibited SDF-1alpha-induced NF-kappaB reporter activity and CXCR4 receptor expression as shown by flow cytometry. These data suggest that SDF-1alpha-induced expression of CXCR4 in PC-3 cells is dependent on MEK/ERK signaling cascade and NF-kappaB activation.
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PMID:Up-regulation of CXCR4 expression in PC-3 cells by stromal-derived factor-1alpha (CXCL12) increases endothelial adhesion and transendothelial migration: role of MEK/ERK signaling pathway-dependent NF-kappaB activation. 1626 13

Cytosolic phospholipase A2 (cPLA2) plays a pivotal role in mediating agonist-induced arachidonic acid (AA) release for prostaglandins (PG) synthesis induced by bacterial lipopolysaccharide (LPS) and cytokines. However, the intracellular signaling pathways mediating LPS-induced cPLA2 expression and PGE2 synthesis in canine tracheal smooth muscle cells (TSMCs) remains unknown. LPS-induced expression of cPLA2 and release of PGE2 was attenuated by inhibitors of tyrosine kinase (genistein), phosphatidylcholine-phospholipase C (D609), phosphatidylinositol-phospholipase C (U73122), PKC (GF109203X and staurosporine), removal of Ca2+ by BAPTA/AM plus EDTA, MEK1/2 (PD98059), p38 (SB202190), JNK (SP600125), and phosphatidylinositol 3-kinase (PI3-K; LY294002 and wortmannin). The involvement of MPAKs in LPS-induced responses was further confirmed by transfection of TSMCs with dominant negative mutants of ERK2 and p38. LPS-induced cPLA2 expression and PGE2 synthesis was inhibited by a selective NF-kappaB inhibitor (helenalin) and transfection with dominant negative mutants of NF-kappaB inducing kinase (NIK), IkappaB kinase (IKK)-alpha, and IKK-beta, consistent with that LPS-stimulated both IkappaB-alpha degradation and NF-kappaB translocation into nucleus in these cells. LPS-stimulated cPLA2 phosphorylation was inhibited by PD98059, GF109203X, and staurosporine, indicating the regulation by p42/p44 MAPK and PKC. Moreover, LPS-induced up-regulation of cPLA2 and COX-2 linked to PGE2 synthesis was inhibited by AACOCF3 (a selective cPLA2 inhibitor), implying the involvement of cPLA2 in these responses. These findings suggest that phosphorylation and expression of cPLA2 correlates with the release of PGE2 from LPS-challenged TSMCs, at least in part, mediated through MAPKs and NF-kappaB signaling pathways. LPS-mediated responses were modulated by PLC, Ca2+, PKC, tyrosine kinase, and PI3-K in TSMCs.
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PMID:Induction of cytosolic phospholipase A2 by lipopolysaccharide in canine tracheal smooth muscle cells: involvement of MAPKs and NF-kappaB pathways. 1627 65


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