Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.12.2 (MEK)
 18,161 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Recent evidence indicates that mutations in the gene encoding the WNK1 [with no K (lysine) protein kinase-1] results in an inherited hypertension syndrome called pseudohypoaldosteronism type II. The mechanisms by which WNK1 is regulated or the substrates it phosphorylates are currently unknown. We noticed that Thr-60 of WNK1, which lies N-terminal to the catalytic domain, is located within a PKB (protein kinase B) phosphorylation consensus sequence. We found that PKB phosphorylated WNK1 efficiently compared with known substrates, and both peptide map and mutational analysis revealed that the major PKB site of phosphorylation was Thr-60. Employing a phosphospecific Thr-60 WNK1 antibody, we demonstrated that IGF1 (insulin-like growth factor) stimulation of HEK-293 cells induced phosphorylation of endogenously expressed WNK1 at Thr-60. Consistent with PKB mediating this phosphorylation, inhibitors of PI 3-kinase (phosphoinositide 3-kinase; wortmannin and LY294002) but not inhibitors of mammalian target of rapamycin (rapamycin) or MEK1 (mitogen-activated protein kinase kinase-1) activation (PD184352), inhibited IGF1-induced phosphorylation of endogenous WNK1 at Thr-60. Moreover, IGF1-induced phosphorylation of endogenous WNK1 did not occur in PDK1-/- ES (embryonic stem) cells, in which PKB is not activated. In contrast, IGF1 still induced normal phosphorylation of WNK1 in PDK1(L155E/L155E) knock-in ES cells in which PKB, but not S6K (p70 ribosomal S6 kinase) or SGK1 (serum- and glucocorticoid-induced protein kinase 1), is activated. Our study provides strong pharmacological and genetic evidence that PKB mediates the phosphorylation of WNK1 at Thr-60 in vivo. We also performed experiments which suggest that the phosphorylation of WNK1 by PKB is not regulating its kinase activity or cellular localization directly. These results provide the first connection between the PI 3-kinase/PKB pathway and WNK1, suggesting a mechanism by which this pathway may influence blood pressure.
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PMID:WNK1, the kinase mutated in an inherited high-blood-pressure syndrome, is a novel PKB (protein kinase B)/Akt substrate. 1461 43

HER2, a member of the human epidermal growth factor (EGF) receptor family, not only plays important roles in the progression of breast cancer tumorigenesis and metastasis, but may protect cancer cells from conventional cytotoxic therapies as well. In the current study, we evaluated the effect of targeting HER2 on radiosensitization of human breast cancer cells. Using six breast cancer cell lines with various levels of HER2 (BT474, SKBR3, MDA453, MCF7, ZR75B, and MDA468), we found that trastuzumab (Herceptin), a humanized monoclonal antibody that may inhibit breast cancer cell proliferation but does not induce apoptosis when used alone, enhanced radiation-induced apoptosis of the cells in a HER2 level-dependent manner. We furthered this study in MCF7 cells transfected for high levels of HER2 (MCF7HER2). Compared with parental or control vector-transfected MCF7 cells, MCF7HER2 cells showed increased phosphorylation of at least two important HER2 downstream molecules, protein kinase B/Akt and mitogen-activated protein kinase (MAPK), and increased resistance to radiotherapy, as shown by reduced induction of apoptosis and increased cell clonogenic survival after radiation. Exposure of the cells to trastuzumab down-regulated the levels of HER2 and reduced phosphorylation levels of Akt and MAPK in MCF7HER2 cells, and sensitized these cells to radiotherapy. When specific inhibitors of the phosphatidylinositol 3-kinase (PI3-K) and MAPK kinase (MEK) pathways were used, we found that exposure of MCF7HER2 cells to the PI3-K inhibitor LY294002 inhibited Akt phosphorylation and radiosensitized the cells, whereas the radiosensitization effect by the MEK inhibitor PD98059 was relatively weaker, albeit the phosphorylation of MAPK was reduced by PD98059 treatment. Our results indicate that the PI3-K pathway might be the major pathway for trastuzumab-mediated radiosensitization of breast cancer cells.
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PMID:Sensitization of breast cancer cells to radiation by trastuzumab. 1461 84

Tumor necrosis factor alpha (TNF-alpha) modulates various events through several different pathways. Many tumor cells are resistant to this cytokine. Pretreatment of these cells with actinomycin D enhances TNF-alpha-induced apoptosis. In the present study, we investigated the mechanism of this enhancement and whether or not the apoptosis of TNF-alpha-resistant cancer cells can be induced by the inhibition of Protein kinase C (PKC). When TNF-alpha was added after inhibition of PKC by H7, apoptosis was observed, and companied with the activation of nuclear factor kappa B (NF-kappaB). After the inhibition of protein kinase B (Akt) by LY294002 or p38 mitogen-activated protein kinase (p38MAPK) by SB203580, the addition of TNF-alpha did not cause apoptosis. However, after the inhibition of MAPK/extracellular signal-regulated kinase kinase 1/2 (MEK1/2) with U0126, apoptosis was observed when TNF-alpha was added. In the Western blotting analysis, phosphorylation of MEK1/2 occurred at 60 minutes after the addition of TNF-alpha. However, it was noted that after pretreatment with H7, a significant decrease in phosphorylated MEK1/2 was observed. The present findings suggest that MEK1/2 plays an important role in TNF-alpha-resistance in TNF-alpha-resistant B16 melanoma BL6 cells. Furthermore, it was found that MEK1/2 is more important than NF-kappaB, Akt, and p38MAPK in anti-apoptotic PKC signaling and that TNF-alpha-resistance can be overcome by inhibiting MEK1/2. These results suggest the possibility of development of a new anticancer drug treatment.
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PMID:Pretreatment with PKC inhibitor triggers TNF-alpha induced apoptosis in TNF-alpha-resistant B16 melanoma BL6 cells. 1465 70

Akt (protein kinase B, PKB) is one of the major downstream pathways of neurotrophin signaling and plays important roles in the cell survival and synaptic plasticity of the central nervous system. Electroconvulsive shock (ECS) has neurotrophic effect and it affects the synaptic plasticity. It can activate another major pathway of neurotrophin signaling, i.e., Ras-Raf-MEK-Erk cascade. In this paper, the authors investigated whether ECS can activate Akt signaling in the rat hippocampus. After a single ECS, the phosphorylation of Akt was increased, as were the signals detected by phospho-PDK1 substrate antibody, which suggests the activation of PDK1, an upstream molecule of Akt. The phosphorylation of downstream molecules of Akt, forkhead transcription factors (FKHR), endothelial nitric oxide synthase (eNOS), and glycogen synthase kinase-3beta (GSK-3beta) was also increased. The increased phosphorylation of Akt appeared within 5 min of ECS and its time frame paralleled that of the phosphorylation of Erks. Taken together, these results suggest that ECS activates Akt signaling over a similar time scale to that of Erks in the rat hippocampus.
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PMID:Activation of protein kinase B (Akt) signaling after electroconvulsive shock in the rat hippocampus. 1468 55

1. Platelet-activating factor (PAF) is known to stimulate a variety of neutrophil activities, including chemotaxis, phagocytosis, degranulation, reactive oxygen species production and intracellular pH increase. The purpose of this study was to investigate the effect of PAF on pH((i)), specifically if these changes in pH are the result of phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) pathway activation in bovine neutrophils. 2. PAF caused intracellular alkalinization in 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester-loaded bovine neutrophils. This phenomenon seems to be mediated by amiloride-sensitive Na(+)/H(+) exchange, and is inhibited by WEB2086 (a selective PAF receptor antagonist), genistein (a tyrosine kinase inhibitor), wortmannin and LY294002 (PI3K inhibitors), and PD98059 and UO126 (MEK inhibitors). 3. PAF 100 nm induced an increase in tyrosine phosphorylation of proteins 62, 44 and 21 kDa with a maximum response at 2 min of incubation. 4. Unlike human neutrophils, bovine neutrophils are strongly stimulated by PAF via phosphorylation of ERK1/2 (extracellular-signal-regulated protein kinase) with an EC(50) of 30 and 13 nm, respectively. 5. PAF MAPK activation was also inhibited by WEB2086, pertussis toxin (PTX), genistein, wortmannin, LY294002, PD98059 and UO126 in bovine neutrophils. The ERK1/2 activation is dependent on PI3K pathway, because protein kinase B was phosphorylated by PAF and inhibited by wortmannin and LY294002, but not by U0126. 6. Our results suggest that PAF induces intracellular alkalinization via PI3K-MAPK activation. This effect is upstream regulated by PAF receptor, PTX-sensitive G protein, tyrosine kinase, PI3K and MEK1/2 in bovine neutrophils.
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PMID:Platelet-activating factor increases pH(i) in bovine neutrophils through the PI3K-ERK1/2 pathway. 1469 Oct 48

Reactive oxygen species including H(2)O(2) activate an array of intracellular signalling cascades that are closely associated with cell death and cell survival pathways. The human neuroblastoma SH-SY5Y cell line is widely used as model cell system for studying neuronal cell death induced by oxidative stress. However, at present very little is known about the signalling pathways activated by H(2)O(2) in SH-SY5Y cells. Therefore, in this study we have investigated the effect of H(2)O(2) on extracellular signal-regulated kinase 1/2 (ERK1/2), c-Jun N-terminal kinase (JNK), p38 mitogen-activated protein kinase (p38 MAPK) and protein kinase B (PKB) activation in undifferentiated and differentiated SH-SY5Y cells. H(2)O(2) stimulated time and concentration increases in ERK1/2, JNK and PKB phosphorylation in undifferentiated and differentiated SH-SY5Y cells. No increases in p38 MAPK phosphorylation were observed following H(2)O(2) treatment. The phosphatidylinositol 3-kinase (PI-3K) inhibitors wortmannin and LY 294002 ((2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one) inhibited H(2)O(2)-induced increases in ERK1/2 and PKB phosphorylation. Furthermore, H(2)O(2)-mediated increases in ERK1/2 activation were sensitive to the MAPK kinase 1 (MEK1) inhibitor PD 98059 (2'-amino-3'-methoxyflavone), whereas JNK responses were blocked by the JNK inhibitor SP 600125 (anthra[1-9-cd]pyrazol-6(2H)-one). Treatment of SH-SY5Y cells with H(2)O(2) (1 mM; 16 h) significantly increased the release of lactate dehydrogenase (LDH) into the culture medium indicative of a decrease in cell viability. Pre-treatment with wortmannin, SP 600125 or SB 203580 (4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole; p38 MAPK inhibitor) had no effect on H(2)O(2)-induced LDH release from undifferentiated or differentiated SH-SY5Y cells. In contrast, PD 98059 and LY 294002 significantly decreased H(2)O(2)-induced cell death in both undifferentiated and differentiated SH-SY5Y cells. In conclusion, we have shown that H(2)O(2) stimulates robust increases in ERK1/2, JNK and PKB in undifferentiated and differentiated SH-SY5Y cells. Furthermore, the data presented clearly suggest that inhibition of the ERK1/2 pathway protects SH-SY5Y cells from H(2)O(2)-induced cell death.
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PMID:Activation of ERK1/2, JNK and PKB by hydrogen peroxide in human SH-SY5Y neuroblastoma cells: role of ERK1/2 in H2O2-induced cell death. 1472 4

Undersulfation of the basement membrane matrix of alveolar type II (AT2) cells compared with that of neighboring type I cells is believed to account for some of the known morphological and functional differences between these pneumocytes. Heparin, a model for sulfated components of basement membrane matrices, is known to inhibit fibroblast growth factor (FGF)-2-stimulated DNA synthesis as well as gene expression of FGF-2 and its receptor in AT2 cells. To determine whether these end points result from specific effects of heparin on FGF-related signaling pathways, isolated rat AT2 cells were treated with 100 ng/ml FGF-1 or FGF-2 in the presence of up to 500 microg/ml heparin. In addition, experiments were done on cells grown in the presence of 20 mM sodium chlorate (sulfation inhibitor). High-dose heparin reduced FGF-1- or FGF-2-stimulated phosphorylation of mitogen-activated protein kinase kinases (MEK1/2), p44/42 mitogen-activated protein kinases (MAPK/ERK1/2), stress-activated protein kinase/c-Jun NH(2)-terminal kinase, Akt/protein kinase B, and p90(RSK). FGF-2-stimulated signaling was more sensitive to heparin's effects than was signaling stimulated by FGF-1. Heparin had an additive effect on the reduced [(3)H]thymidine incorporation in FGF-2-treated AT2 cells caused by inhibition of the MEK/ERK pathway by the MEK inhibitor PD-98059. The data suggest that heparin's known capacity to alter DNA synthesis and, possibly, other biological end points is realized via cross talk between multiple signaling pathways.
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PMID:Heparin affects signaling pathways stimulated by fibroblast growth factor-1 and -2 in type II cells. 1496 81

The matrix metalloproteinase (MMP)-2 has been recognized as a major mediator of basement membrane degradation, angiogenesis, tumor invasion, and metastasis. The factors that regulate its expression have not, however, been fully elucidated. We previously identified the type I insulin-like growth factor (IGF-I) receptor as a regulator of MMP-2 synthesis. The objective of the present study was to investigate the signal transduction pathway(s) mediating this regulation. We show here that in Lewis lung carcinoma subline H-59 cells treated with IGF-I (10 ng/ml), the PI 3-kinase (phosphatidylinositol 3'-kinase) /protein kinase B (Akt) and C-Raf/ERK pathways were activated, and MMP-2 promoter activity, mRNA, and protein synthesis were induced. MMP-2 induction was blocked by the PI 3-kinase inhibitors LY294002 and wortmannin, by overexpression of a dominant-negative Akt or wild-type PTEN (phosphatase and tensin homologue deleted on chromosome 10), and by rapamycin. In contrast, a MEK inhibitor PD98059 failed to reduce MMP-2 promoter activation and actually increased MMP-2 mRNA and protein synthesis by up to 30%. Interestingly, suppression of PI 3-kinase signaling by a dominant-negative Akt enhanced ERK activity in cells stimulated with 10 ng/ml but not with 100 ng/ml IGF-I. Furthermore, at the higher (100 ng/ml) IGF-I concentration, C-Raf and ERK, but not PI 3-kinase activation, was enhanced, and this resulted in down-regulation of MMP-2 synthesis. This effect was reversed in cells expressing a dominant-negative ERK mutant. The results suggest that IGF-I can up-regulate MMP-2 synthesis via PI 3-kinase/Akt/mTOR (the mammalian target of rapamycin) signaling while concomitantly transmitting a negative regulatory signal via the Raf/ERK pathway. The outcome of IGF-IR (the receptor for IGF-I) activation may ultimately depend on factors, such as ligand bioavailability, that can shift the balance preferentially toward one pathway or the other.
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PMID:Dual regulation of MMP-2 expression by the type 1 insulin-like growth factor receptor: the phosphatidylinositol 3-kinase/Akt and Raf/ERK pathways transmit opposing signals. 1499 22

To determine whether Insulin-like growth factor (IGF-I) treatment represents a potential means of enhancing the survival of cardiac muscle cells from adriamycin (ADR)-induced cell death, the present study examined the ability of IGF-I to prevent cell death. The study was performed utilising the embryonic, rat, cardiac muscle cell line, H9C2. Incubating cardiac muscle cells in the presence of adriamycin increased cell death, as determined by MTT assay and annexin V-positive cell number. The addition of 100 ng/mL IGF-I, in the presence of adriamycin, decreased apoptosis. The effect of IGF-I on phosphorylation of PI, a substrate of phosphatidylinositol 3-kinase (PI 3-kinase) or protein kinase B (AKT), was also examined in H9C2 cardiac muscle cells. IGF-I increased the phosphorylation of ERK 1 and 2 and PKC zeta kinase. The use of inhibitors of PI 3-kinase (LY 294002), in the cell death assay, demonstrated partial abrogation of the protective effect of IGF-I. The MEK1 inhibitor-PD098059 and the PKC inhibitor-chelerythrine exhibited no effect on IGF-1-induced cell protection. In the regulatory subunit of PI3K-p85- dominant, negative plasmid-transfected cells, the IGF-1-induced protective effect was reversed. This data demonstrates that IGF-I protects cardiac muscle cells from ADR-induced cell death. Although IGF-I activates several signaling pathways that contribute to its protective effect in other cell types, only activation of PI 3-kinase contributes to this effect in H9C2 cardiac muscle cells.
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PMID:Signal transduction of the protective effect of insulin like growth factor-1 on adriamycin-induced apoptosis in cardiac muscle cells. 1508 39

Several forms of human dwarfism are due to activating mutations in FGFR3 highlighting the role of FGF signaling in the growth attenuation of cartilage. Here, we studied the effects of FGF2 on RCS chondrocytes. Treatment with FGF2 induced growth arrest in the G1 phase of the cell cycle and partial de-differentiation of cells manifested by changes in cell morphology, loss of the cartilage-like extracellular matrix, and down-regulation of aggrecan expression. FGF2 activated phospholipase Cgamma, protein kinase B, and Erk and p38 MAP kinases. Chemical inhibition of FGFR3 and MEK1/2 antagonized FGF2-mediated growth arrest. Expression of a dominant-negative Ras mutant resulted in a partial reversal of growth inhibition while expression of constitutively activated Ras led to Erk-dependent growth arrest, further demonstrating the role of the Ras/Erk pathway in this phenotype. At the molecular level, FGF2-induced growth arrest was initiated by disintegration of cyclin D3-cdk6 complex followed by increased association of p21(WAF1) and p27(Kip1) with the cyclin-cdk2 and cyclin-cdk4 complexes leading to inhibition of their kinase activities and ultimately to underphosphorylation of the p107 and p130 pocket proteins. Both p21(WAF1) and p27(Kip1) accumulated upon FGF2 treatment, but this accumulation occurred at the protein level at least partially due to interaction with transcriptionally induced cyclin D1.
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PMID:FGF2 inhibits proliferation and alters the cartilage-like phenotype of RCS cells. 1519 33


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