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)

Human GM-CSF (hGM-CSF) induces proliferation and sustains the viability of a mouse IL-3-dependent lymphoid cell line BA/F3 that expresses the functional hGM-CSF receptor (hGMR). To reveal an antiapoptotic mechanism of hGM-CSF, we analyzed various apoptotic markers of BA/F3 cells in various conditions. Within 24 hours of factor depletion, caspase 3-like, but not caspase 1-like, enzyme activity and DNA fragmentation were augmented. Analysis with the tyrosine kinase inhibitor (genistein) and an MEK1 inhibitor (PD98059) on antiapoptosis activity indicates that the activation of either the genistein-sensitive signaling pathway or the PD98059-sensitive signaling pathway of the betac subunit may be sufficient to suppress apoptosis through hGMR. Because hGMR mutants (which activate JAK2 but neither STAT5 nor the MAPK cascade) have antiapoptotic activity in BA/F3 cells, the involvement of JAK2, excluding the molecules mentioned earlier, for antiapoptosis activity seems likely. Because the JAK2 inhibitor AG-490 suppressed the antiapoptotic activity of hGM-CSF, the essential role for JAK2 activation to maintain the viability is considered. Interestingly, hGMR mutants, which lack MAPK cascade activation, require a higher dose of hGM-CSF than that for wild-type hGMR. Because the expression level and affinity to hGM-CSF among wild-type hGMR and mutant hGMR are the same, we speculated that biologic response is determined by a combination of strength of various signaling events.
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PMID:Analysis of antiapoptosis activity of human GM-CSF receptor. 1088 29

Colony-stimulating factor 1 (CSF-1) supports the proliferation, survival, and differentiation of bone marrow-derived cells of the monocytic lineage. In the myeloid progenitor 32D cell line expressing CSF-1 receptor (CSF-1R), CSF-1 activation of the extracellular signal-regulated kinase (ERK) pathway is both Ras and phosphatidylinositol 3-kinase (PI3-kinase) dependent. PI3-kinase inhibition did not influence events leading to Ras activation. Using the activity of the PI3-kinase effector, Akt, as readout, studies with dominant-negative and oncogenic Ras failed to place PI3-kinase downstream of Ras. Thus, PI3-kinase appears to act in parallel to Ras. PI3-kinase inhibitors enhanced CSF-1-stimulated A-Raf and c-Raf-1 activities, and dominant-negative A-Raf but not dominant-negative c-Raf-1 reduced CSF-1-provoked ERK activation, suggesting that A-Raf mediates a part of the stimulatory signal from Ras to MEK/ERK, acting in parallel to PI3-kinase. Unexpectedly, a CSF-1R lacking the PI3-kinase binding site (DeltaKI) remained capable of activating MEK/ERK in a PI3-kinase-dependent manner. To determine if Src family kinases (SFKs) are involved, we demonstrated that CSF-1 activated Fyn and Lyn in cells expressing wild-type (WT) or DeltaKI receptors. Moreover, CSF-1-induced Akt activity in cells expressing DeltaKI is SFK dependent since Akt activation was prevented by pharmacological or genetic inhibition of SFK activity. The docking protein Gab2 may link SFK to PI3-kinase. CSF-1 induced Gab2 tyrosyl phosphorylation and association with PI3-kinase in cells expressing WT or DeltaKI receptors. However, only in DeltaKI cells are these events prevented by PP1. Thus in myeloid progenitors, CSF-1 can activate the PI3-kinase/Akt pathway by at least two mechanisms, one involving direct receptor binding and one involving SFKs.
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PMID:Both src-dependent and -independent mechanisms mediate phosphatidylinositol 3-kinase regulation of colony-stimulating factor 1-activated mitogen-activated protein kinases in myeloid progenitors. 1095 75

1. The extent to which the p38 mitogen-activated protein (MAP) kinase and MAP kinase kinase (MKK)-1-signalling pathways regulate the expression of granulocyte/macrophage colony-stimulating factor (GM-CSF) from LPS-stimulated human monocytes has been investigated and compared to the well studied cytokine tumour necrosis factor-alpha (TNF alpha). 2. Lipopolysaccharide (LPS) evoked a concentration-dependent generation of GM-CSF from human monocytes. Temporally, this effect was preceded by an increase in GM-CSF mRNA transcripts and abolished by actinomycin D and cycloheximide. 3. LPS-induced GM-CSF release and mRNA expression were associated with a rapid and time-dependent activation of p38 MAP kinase, ERK-1 and ERK-2. 4. The respective MKK-1 and p38 MAP kinase inhibitors, PD 098059 and SB 203580, maximally suppressed LPS-induced GM-CSF generation by >90%, indicating that both of these signalling cascades co-operate in the generation of this cytokine. 5. Electrophoretic mobility shift assays demonstrated that LPS increased nuclear factor kappa B (NF-kappa B) : DNA binding. SN50, an inhibitor of NF-kappa B translocation, abolished LPS-induced NF-kappaB : DNA binding and the elaboration of TNFalpha, a cytokine known to be regulated by NF-kappaB in monocytes. In contrast, SN50 failed to affect the release of GM-CSF from the same monocyte cultures. 6. Collectively, these results suggest that the generation of GM-CSF by LPS-stimulated human monocytes is regulated in a co-operative fashion by p38 MAP kinase- and MKK-1-dependent signalling pathways independently of the activation of NF-kappa B.
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PMID:p38 MAP kinase and MKK-1 co-operate in the generation of GM-CSF from LPS-stimulated human monocytes by an NF-kappa B-independent mechanism. 1108 22

Prolonged eosinophil survival is an essential step in the late and chronic phases of allergic inflammation and is regulated by the eosinophil survival cytokines. Our work has demonstrated that tumour necrosis factor (TNF)-alpha enhances survival (Trypan blue exclusion test) of human peripheral blood eosinophils from mildly allergic patients in a dose-dependent manner. The survival activity of TNF-alpha was inhibited by anti-TNF-RI, anti-TNF-RII antagonist antibodies and anti-granulocyte-monocyte colony-stimulating factor (GM-CSF) neutralizing antibodies but not by anti-interleukin (IL)-3 or anti-IL-5 antibodies. Furthermore, TNF-alpha-induced GM-CSF release from eosinophils. Anti-TNF-alpha antibodies also inhibited GM-CSF release from eosinophils induced by rat mast cell sonicate, which enhances eosinophil survival. To define the signal transduction pathway involved in GM-CSF production, eosinophils were incubated either with various mitogen-activated protein kinases (MAPK) inhibitors (MEK, JNK, P38), or Cyclosporin A (calcineurin inhibitor), or MG-132 (proteasome inhibitor). Only the proteasome inhibitor significantly decreased both TNF-alpha-enhanced eosinophil survival (from 38.1+/-4.1% to 13.3+/-1.4%) and GM-CSF release (from 6.2+/-0.7 pg/ml to 0.3+/-0.1 pg/ml). TNF-alpha also induced nuclear factor-kappaB (NF-kappaB) translocation to the nucleus, an essential step in GM-CSF mRNA production. All these findings provide evidence that NF-kappaB is involved in TNF-alpha-enhanced eosinophil survival through the regulation of GM-CSF production by eosinophils.
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PMID:Mechanism of tumour necrosis factor alpha mediated eosinophil survival. 1150 5

Phosphoinositide 3-kinase (PI3-kinase)-dependent phosphorylation of the proapoptotic Bcl-2 family member Bad has been proposed as an important regulator of apoptotic cell death. To understand the importance of this pathway in nontransformed hematopoietic cells, we have examined the effect of survival cytokines on PI3-kinase activity and Bad expression and phosphorylation status in human neutrophils. Granulocyte macrophage-colony-stimulating factor (GM-CSF) and tumor necrosis factor-alpha (TNF-alpha) both reduced the rate of apoptosis in neutrophils cultured in vitro for 20 hours. Coincubation with the PI3-kinase inhibitor LY294002, which in parallel experiments abolished GM-CSF-primed, fMLP-stimulated superoxide anion production and GM-CSF-stimulated PtdIns(3,4,5)P(3) accumulation, inhibited the GM-CSF and TNF-alpha survival effect. In contrast, the MAP kinase kinase (MEK1/2) inhibitor PD98059 and the protein kinase A inhibitor H-89 had only a marginal effect on GM-CSF-mediated neutrophil survival. GM-CSF substantially increased Bad phosphorylation at Ser112 and Ser136 and increased the cytosolic accumulation of Bad. GM-CSF also regulated Bad at a transcription level with a marked decrease in mRNA levels at 4 hours. TNF-alpha caused a biphasic effect on the rate of morphologic apoptosis, which corresponded to an early increase, and a late inhibition, of Bad mRNA levels. LY294002 inhibited GM-CSF- and TNF-alpha-mediated changes in Bad phosphorylation and mRNA levels. These data suggest that the survival effect of GM-CSF and TNF-alpha in neutrophils is caused by a PI3-kinase-dependent phosphorylation and cytosolic translocation of Bad, together with an inhibition of Bad mRNA levels. This has important implications for the regulation of neutrophil apoptosis in vivo.
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PMID:Role of PI3-kinase-dependent Bad phosphorylation and altered transcription in cytokine-mediated neutrophil survival. 1223 75

Protein kinase C (PKC) proteins have been shown to be involved in diverse cellular responses of various cell types. In experiments to identify genes regulated during osteoclast differentiation by a cDNA microarray approach, we found that the gene expression of PKC-betaII was upregulated in differentiated cells. Reverse transcription-polymerase chain reaction and Western blotting analyses also showed an increase in PKC-betaI as well as PKC-betaII during osteoclast formation in mouse bone marrow cell cultures in the presence of macrophage-colony stimulating factor (M-CSF) and receptor activator of nuclear factor-kappaB ligand (RANKL). Use of an antisense oligonucleotide to PKC-betaII resulted in a reduction in the RANKL-driven osteoclastogenesis. Pharmacological intervention with PKC-beta activity by the specific inhibitor CG53353 suppressed cellular differentiation and fusion processes during osteoclastogenesis and inhibited bone-resorbing function of mature osteoclasts. PKC-beta inhibition abolished the ERK and MEK activation by macrophage-colony stimulating factor and RANKL in osteoclast precursor cells whereas the cytokine-induced NF-kappaB activation was not hampered by the PKC-beta inhibition. Our findings indicate that PKC-beta has a role in regulation of osteoclast formation and function potentially by participating in the ERK signaling pathway of M-CSF and RANKL.
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PMID:Participation of protein kinase C beta in osteoclast differentiation and function. 1266 49

We previously established two lung cancer cell lines, OKa-C-1 and MI-4, which constitutively produce abundant granulocyte-colony stimulating factor (G-CSF) and granulocyte macrophage-colony stimulating factor (GM-CSF). Inflammatory cytokines, tumor necrosis factor-alpha (TNF-alpha) and interleukin (IL)-1beta stimulated the expression of G-CSF, GM-CSF, and cyclooxygenase (COX)-2 in the two cell lines. It is known that increased COX-2 activity promotes tumor growth and induces G-CSF and GM-CSF expression in non-malignant cells, and that selective COX-2 inhibitors inhibit the growth of some types of malignant cells. Therefore, we hypothesized that inhibition of COX-2 activity might suppress constitutive production of G-CSF or GM-CSF in addition to reducing the growth of malignant cells. We confirmed that the selective COX-2 inhibitor, NS-398 suppressed the constitutive production of G-CSF and GM-CSF, and the cell growth in both OKa-C-1 and MI-4 cell lines. Prostaglandin E2 (PGE2) reversed the inhibitions of G-CSF and GM-CSF expression, as well as cell growth, by NS-398. This result confirms that the effects of NS-398 are based on the inhibition of COX activity. Some studies have indicated that nuclear factor kappa B (NF-kappaB) or MAPK (mitogen-activated protein kinase) activation is related to upregulation of G-CSF, GM-CSF or COX-2 expression in some types of cells. Therefore, we examined if the actions of NS-398 might be mediated by the MAP kinase pathway or NF-kappaB activity in OKa-C-1 and MI-4 cells. We found that NS-398 inhibits G-CSF and GM-CSF production and cell growth through an extracellular signal-regulated kinase kinase (MEK) signaling pathway in these cell lines. The prognosis of non-small cell lung cancer showing G-CSF gene expression is significantly worse. G-CSF overproduction by tumor cells is observed at an advanced clinical stage. Our findings imply that a COX-2 inhibitor might improve the prognosis of patients with lung cancer through the reduction of G-CSF or GM-CSF.
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PMID:Cyclooxygenase-2 inhibitor NS-398 suppresses cell growth and constitutive production of granulocyte-colony stimulating factor and granulocyte macrophage-colony stimulating factor in lung cancer cells. 1270 93

We report here for the first time the detection of the ribosomal p70S6 kinase (p70S6K) in a hematopoietic cell, the neutrophil, and the stimulation of its enzymatic activity by granulocyte macrophage colony-stimulating factor (GM-CSF). GM-CSF modified the Vmax of the enzyme (from 7.2 to 20.5 pmol/min/mg) and induced a time- and dose-dependent phosphorylation on p70S6K residues Thr389 and Thr421/Ser424. The immunosuppressant macrolide rapamycin caused either a decrease in intensity of phospho-Thr389 bands in Western blots, or as a downshift in the relative mobility of phospho-Thr421/Ser424 bands (consistent with the loss of phosphate), but not both simultaneously. The immunosuppressant FK506 failed to inhibit p70S6K activation, but was able to rescue the rapamycin-induced downshift, pointing to a role for the mammalian target of rapamycin (mTOR) kinase. Rapamycin also caused an inhibition (IC50 0.2 nm) of the in vitro enzymatic activity of p70S6K. However, the inhibition of activity was not complete, but only a 40-50%, indicating that neutrophil p70S6K activity has a rapamycin-resistant component. This component was totally inhibited by pre-incubating the cells with the mitogen-activated protein kinase (MAPK) kinase (MEK) inhibitor PD-98059 prior to treatment with rapamycin. This indicated that a kinase from the MEK/MAPK pathway also plays a role in p70S6K activation. Thus, GM-CSF causes the dual activation of a rapamycin-resistant, MAPK-related kinase, that targets Thr421/Ser424 S6K phosphorylation, and a rapamycin-sensitive, mTOR-related kinase, that targets Thr389, both of which are needed in cooperation to achieve full activation of neutrophil p70S6K.
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PMID:Mechanism of ribosomal p70S6 kinase activation by granulocyte macrophage colony-stimulating factor in neutrophils: cooperation of a MEK-related, THR421/SER424 kinase and a rapamycin-sensitive, m-TOR-related THR389 kinase. 1274 Mar 86

Granulocyte macrophage-colony-stimulating factor (GM-CSF), released from alveolar macrophages (AM), is an important regulator of eosinophil, T cell, and macrophage function and survival. We determined the mechanisms of GM-CSF regulation in AM from normal volunteers activated by lipopolysaccharide (LPS) by examining the role of nuclear factor-kappaB (NF-kappaB), and of p38 mitogen-activated protein (MAP) kinase and MAP kinase kinase (MKK-1). PD 098059 (10 microM), an inhibitor of upstream activator of MKK-1, inhibited GM-CSF expression, but the expression of GM-CSF was not inhibited by SB 203580 (10 microM), an inhibitor of p38-MAP kinase. Phosphorylation of extracellular signal-regulated kinase-1 (ERK-1), ERK-2, and p38 MAP kinase by LPS were demonstrated on Western blot analysis. LPS increased NF-kappaB:DNA binding as examined by electrophoretic mobility shift assay, but this was not suppressed by PD 098059 or by SB 203580. LPS induced an increase in NF-kappaB activation as examined by p50 translocation assay without suppression by PD 098059 or by SB 203580. SN50 (100 microM), an inhibitor of NF-kappaB translocation and the specific IKK-2-Inhibitor (AS602868; 10 microM), also prevented GM-CSF expression and release induced by LPS, indicating that GM-CSF release is NF-kappaB-dependent. PD 098059, but not SB 203580, inhibited LPS-induced histone acetyltransferase (HAT) activity, indicating chromatin modification. Furthermore, AS602868 and SN 50 suppressed LPS-induced HAT activity. TSA (10 ng/ml), an inhibitor of histone deacetylase (HDAC), reversed the inhibitory effect of PD 098059, SB 203580, SN 50 and AS602868 on GM-CSF release. GM-CSF expression and release in AM is controlled by NF-kappaB activation, and this is modulated by phosphorylation of MKK-1 and p38 MAP kinase acting on histone acetylation.
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PMID:Mitogen-activated protein kinase modulation of nuclear factor-kappaB-induced granulocyte macrophage-colony-stimulating factor release from human alveolar macrophages. 1287 51

In Alzheimer's disease (AD) brain the activity of protein phosphatase (PP)-2A is compromised and that of the extracellular signal-regulated protein kinase (ERK1/2) of the mitogen-activated protein kinase (MAPK) family, which can phosphorylate tau, is up-regulated. We investigated whether a decrease in PP-2A activity could underlie the activation of these kinases and the abnormal hyperphosphorylation of tau. Rat brain slices, 400-microm-thick, kept under metabolically active conditions in oxygenated (95% O(2), 5% CO(2)) artificial CSF were treated with 1.0 micromol/L okadaic acid (OA) for 1 hour at 33 degrees C. Under this condition, PP-2A activity was decreased to approximately 35% of the vehicle-treated control slices, and activities of PP-1 and PP-2B were not affected. In the OA-treated slices, we observed a dramatic increase in the phosphorylation/activation of ERK1/2, MEK1/2, and p70 S6 kinase both immunohistochemically and by Western blots using phosphorylation-dependent antibodies against these kinases. Treatment of 6-microm sections of the OA-treated slices with purified PP-2A reversed the phosphorylation/activation of these kinases. Hyperphosphorylation of tau at several abnormal hyperphosphorylation sites was also observed, as seen in AD brain. These results suggest 1) that PP-2A down-regulates ERK1/2, MEK1/2, and p70 S6 kinase activities through dephosphorylation at the serine/threonine residues of these kinases, and 2) that in AD brain the decrease in PP-2A activity could have caused the activation of ERK1/2, MEK1/2, and p70 S6 kinase, and the abnormal hyperphosphorylation of tau both via an increase in its phosphorylation and a decrease in its dephosphorylation.
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PMID:Okadaic-acid-induced inhibition of protein phosphatase 2A produces activation of mitogen-activated protein kinases ERK1/2, MEK1/2, and p70 S6, similar to that in Alzheimer's disease. 1293 26


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