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 nerve growth factor (NGF)-mediated activation of the mitogen-activated protein (MAP) kinase cascade is an obligatory step in the morphological differentiation of PC12 cells. Signal transduction through the MAP kinase cascade is dependent upon activation of p21(ras) which binds directly to Raf family protein kinases, mediating their association with the membrane and activation. PC12 cells express two Raf isoforms, c-Raf and B-Raf. The activation of the MAP kinase cascade in response to NGF is due principally to the action of B-Raf. NGF treatment of PC12 cells resulted in the enhanced phosphorylation of B-Raf and c-Raf, and both exhibit reduced electrophoretic mobilities following stimulation of the cells. The NGF-stimulated phosphorylation of B-Raf was correlated with its enzymatic activation as measured by the phosphorylation of its substrate MEK. However, c-Raf does not exhibit significant levels of activity. B-Raf was present as a component of a high molecular mass complex, which included the molecular chaperone, heat shock protein 90 (HSP90). Importantly, c-Raf did not participate in the formation of such complexes. The B-Raf containing HSP90 complexes were normally present in PC12 cells, and their assembly was not dependent upon NGF stimulation. These data suggest that the ability of B-Raf to activate the MAP kinase cascade is due to its association with a large signaling complex, which is likely to impart signaling pathway specificity.
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PMID:Nerve growth factor-mediated activation of the mitogen-activated protein (MAP) kinase cascade involves a signaling complex containing B-Raf and HSP90. 879 78

Confocal laser scanning microscopy was used to identify the cells within organotypic slice cultures of the developing mouse cerebral cortex that respond to estradiol treatment by phosphorylation of ERK1 and ERK2. Estrogen-responsive cells resembled neurons morphologically and expressed the neuronal marker microtubule-associated protein 2B. The intracellular distribution of the phospho-ERK signal was both cytoplasmic and nuclear, but inhibition of protein synthesis abolished the appearance of the nuclear signal. ERK1and ERK2 also coimmunoprecipitated with heat shock protein 90 (Hsp90) in the cerebral cortical explants. Geldanamycin effectively disrupted this association and prevented ERK phosphorylation. Surprisingly, MEK2 but not MEK1 was the principal mediator of estradiol-induced activation of ERK. Our data demonstrate the requirement for Hsp90 in estrogen-induced activation of ERK1 and ERK2 by MEK2 in the developing mouse cerebral cortex and also provide insight into alternative mechanisms by which estradiol may influence cytoplasmic and nuclear events in responsive neurons via the MAP kinase cascade.
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PMID:Estradiol-induced phosphorylation of ERK1/2 in explants of the mouse cerebral cortex: the roles of heat shock protein 90 (Hsp90) and MEK2. 1174 28

A requirement for cyclin D2 in G(1)-to-S phase progression has been definitively established in mature B cells stimulated via the B cell antigen receptor (BCR). However, the identity of constituents of the BCR signaling cascade that leads to cyclin D2 accumulation remains incomplete. We report that inhibition of mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK)-1/2 blocked BCR-induced activation of extracellular signal-regulated kinase (ERK). Inhibition of the MEK1/2-ERK pathway was sufficient to abrogate BCR-induced cyclin D2 expression at the mRNA and protein levels. Disruption of endogenous heat shock protein 90 (hsp90) function with geldanamycin abrogated BCR-induced cyclin D2 expression and proliferation. Geldanamycin effects were attributed to a selective depletion of cellular Raf-1 that interrupted BCR-coupled activation of MEK1/2 and ERK. By contrast, signaling through the phosphatidylinositol 3-kinase and protein kinase C pathways was not affected, suggesting that disruption of hsp90 function did not cause a general impairment of BCR signaling. These results suggest that the MEK1/2-ERK pathway is essential for BCR signaling to cyclin D2 accumulation in ex vivo splenic B lymphocytes. Furthermore, these findings imply that hsp90 function is required for BCR signaling through the Raf-1-MEK1/2-ERK pathway but not through the phosphatidylinositol 3-kinase- or protein kinase C-dependent pathways.
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PMID:Requirement for a hsp90 chaperone-dependent MEK1/2-ERK pathway for B cell antigen receptor-induced cyclin D2 expression in mature B lymphocytes. 1182 72

Interactions between the protein kinase C (PKC) and Chk1 inhibitor UCN-01 and the heat shock protein 90 (Hsp90) antagonist 17-AAG have been examined in human leukemia cells in relation to effects on signal transduction pathways and apoptosis. Simultaneous exposure (30 hours) of U937 monocytic leukemia cells to minimally toxic concentrations of 17-AAG (eg, 400 nM) and UCN-01 (eg, 75 nM) triggered a pronounced increase in mitochondrial injury (ie, loss of mitochondrial membrane potential [Deltapsim]; cytosolic release of cytochrome c), caspase activation, and apoptosis. Synergistic induction of apoptosis was also observed in other human leukemia cell types (eg, Jurkat, NB4). Coexposure of human leukemia cells to 17-AAG and the PKC inhibitor bisindolylmaleimide (GFX) did not result in enhanced lethality, arguing against the possibility that the PKC inhibitory actions of UCN-01 are responsible for synergistic interactions. The enhanced cytotoxicity of this combination was associated with diminished Akt activation and marked down-regulation of Raf-1, MEK1/2, and mitogen-activated protein kinase (MAPK). Coadministration of 17-AAG and UCN-01 did not modify expression of Hsp90, Hsp27, phospho-JNK, or phospho-p38 MAPK, but was associated with further p34cdc2 dephosphorylation and diminished expression of Bcl-2, Mcl-1, and XIAP. In addition, inducible expression of both a constitutively active MEK1/2 or myristolated Akt construct, which overcame inhibition of ERK and Akt activation, respectively, significantly attenuated 17-AAG/UCN-01-mediated lethality. Together, these findings indicate that the Hsp90 antagonist 17-AAG potentiates UCN-01 cytotoxicity in a variety of human leukemia cell types and suggest that interference with both the Akt and Raf-1/MEK/MAP kinase cytoprotective signaling pathways contribute to this phenomenon.
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PMID:Synergistic antileukemic interactions between 17-AAG and UCN-01 involve interruption of RAF/MEK- and AKT-related pathways. 1273 74

Hypoxia inducible factor 1 (HIF-1), a heterodimeric transcription factor composed of HIF-1alpha and HIF-1beta subunits, serves as a key regulator of metabolic adaptation to hypoxia. The amount of HIF-1alpha protein is regulated either by attenuating von Hippel-Lindau protein (pVHL)-dependent ubiquitination and subsequent 26 S proteasomal degradation or by enhancing cap-dependent mRNA translation, presumably involving a phosphatidyinositol 3-kinase (PI3K)/Akt-regulated pathway. In addition, it became apparent that Hsp90 protects HIF-1alpha from oxygen-independent degradation. Here we present evidence that PI3K/Akt is required for heat shock proteins to stabilize HIF-1alpha. In pVHL-deficient renal cell carcinoma cells, PI3K inhibition by LY294002 and wortmannin or transfection of either a dominant negative PI3K or a kinase-dead Akt mutant substantially lowered constitutively expressed HIF-1alpha without altering HIF-1alpha mRNA. Inhibitors of mitogen-activated protein kinase kinase (MAPKK) such as PD98059 or the p38 MAPK inhibitor SB203580 showed no interference. Considering that PI3K inhibitors down-regulated heat shock protein 90 (Hsp90) as well as Hsp70 expression and moreover attenuated heat- or hypoxia-induced Hsp70 as well as hypoxia-induced Hsp90 up-regulation we conclude that PI3K inhibition promoted degradation of HIF-1alpha indirectly by reducing steady state concentrations of Hsp90 and/or Hsp70. HIF-1alpha co-immunoprecipitated with Hsp90/Hsp70 and direct binding of Hsp70 to the oxygen-dependent degradation domain (ODD) of HIF-1alpha was proven by a pull-down assay and a peptide array. PI3K-mediated degradation of HIF-1alpha was confirmed in HEK 293 cells under hypoxia, suggesting that heat shock proteins constitute an integral component for HIF-1alpha accumulation. We conclude that PI3K/Akt contributes to HIF-1alpha stabilization by provoking expression of heat shock proteins.
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PMID:PI3K/Akt is required for heat shock proteins to protect hypoxia-inducible factor 1alpha from pVHL-independent degradation. 1472 29

Human telomerase activity is induced by Ag receptor ligation in T and B cells. However, it is unknown whether telomerase activity is increased in association with activation and proliferation of NK cells. We found that telomerase activity in a human NK cell line (NK-92), which requires IL-2 for proliferation, was increased within 24 h after stimulation with IL-2. Levels of human telomerase reverse transcriptase (hTERT) mRNA and protein correlated with telomerase activity. ERK1/2 and Akt kinase (Akt) were activated by IL-2 stimulation. LY294002, an inhibitor of PI3K, abolished expression of hTERT mRNA and protein expression and abolished hTERT activity, whereas PD98059, which inhibits MEK1/2 and thus ERK1/2, had no effect. In addition, radicicol, an inhibitor of heat shock protein 90 (Hsp90), and rapamycin, an inhibitor of the mammalian target of rapamycin (mTOR), blocked IL-2-induced hTERT activity and nuclear translocation of hTERT but not hTERT mRNA expression. hTERT was coimmunoprecipitated with Akt, Hsp90, mTOR, and p70 S6 kinase (S6K), suggesting that these molecules form a physical complex. Immunoprecipitates of Akt, Hsp90, mTOR, and S6K from IL-2-stimulated NK-92 cells contained telomerase activity. Furthermore, the findings that Hsp90 and mTOR immunoprecipitates from primary samples contained telomerase activity are consistent with the results from NK-92 cells. These results indicate that IL-2 stimulation induces hTERT activation and that the mechanism of IL-2-induced hTERT activation involves transcriptional or posttranslational regulation through the pathway including PI3K/Akt, Hsp90, mTOR, and S6K in NK cells.
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PMID:IL-2 increases human telomerase reverse transcriptase activity transcriptionally and posttranslationally through phosphatidylinositol 3'-kinase/Akt, heat shock protein 90, and mammalian target of rapamycin in transformed NK cells. 1584 22

The multidrug resistance gene 1 (MDR1) product, P-glycoprotein (P-gp), pumps out a variety of anticancer agents from the cell, including anthracyclines, Vinca alkaloids, and taxanes. The expression of P-gp therefore confers resistance to these anticancer agents. In our present study, we found that FTI-277 (a farnesyltransferase inhibitor), U0126 [an inhibitor of mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) kinase (MEK)], and 17-allylamino-17-demethoxygeldanamycin (an inhibitor of heat shock protein 90) reduced the endogenous expression levels of P-gp in the human colorectal cancer cells, HCT-15 and SW620-14. In contrast, inhibitors of phosphatidylinositol 3-OH kinase, mammalian target of rapamycin, p38 mitogen-activated protein kinase, and c-Jun NH(2)-terminal kinase did not affect P-gp expression in these cells. We further found that U0126 down-regulated exogenous P-gp expression in the MDR1-transduced human breast cancer cells, MCF-7/MDR and MDA-MB-231/MDR. However, the MDR1 mRNA levels in these cells were unaffected by this treatment. PD98059 (a MEK inhibitor), ERK small interfering RNA, and p90 ribosomal S6 kinase (RSK) small interfering RNA also suppressed P-gp expression. Conversely, epidermal growth factor and basic fibroblast growth factor enhanced P-gp expression, but the MDR1 mRNA levels were unchanged in epidermal growth factor-stimulated cells. Pulse-chase analysis revealed that U0126 promoted P-gp degradation but did not affect the biosynthesis of this gene product. The pretreatment of cells with U0126 enhanced the paclitaxel-induced cleavage of poly(ADP-ribose) polymerase and paclitaxel sensitivity. Furthermore, U0126-treated cells showed high levels of rhodamine123 uptake. Hence, our present data show that inhibition of the MEK-ERK-RSK pathway down-regulates P-gp expression levels and diminishes the cellular multidrug resistance.
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PMID:Inhibition of the mitogen-activated protein kinase pathway results in the down-regulation of P-glycoprotein. 1762 Apr 38

This study explored the effect of MS-275, a novel histone deacetylase inhibitor (HDACI), against a variety of human leukemia cells with defined genetic alterations. MS-275 profoundly induced growth arrest of acute myelogenous leukemia (AML) MOLM13 and biphenotypic leukemia MV4-11 cells, which possess internal tandem duplication mutation in the fms-like tyrosine kinase 3 (FLT3) gene (FLT3-ITD), with IC50s less than 1 microM, as measured by 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay on day two of culture. Exposure of these cells to MS-275 decreased levels of total, as well as, phosphorylated forms of FLT3, resulting in inactivation of its downstream signal pathways, including Akt, ERK, and STAT5. Further studies found that MS-275 induced acetylation of heat shock protein 90 (HSP90) in conjunction with ubiquitination of FLT3, leading to degradation of FLT3 proteins in these cells. This was blunted by treatment with the proteasome inhibitor bortezomib, confirming that FLT was degraded via ubiquitin/proteasome pathway. Moreover, we found that further inhibition of MEK/ERK signaling potentiated the action of MS-275 in leukemia cells. Taken together, MS-275 may be useful for treatment of individuals with leukemia possessing activating mutation of FLT3 gene.
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PMID:MS-275, a novel histone deacetylase inhibitor with selectivity against HDAC1, induces degradation of FLT3 via inhibition of chaperone function of heat shock protein 90 in AML cells. 1839 2

Increased Akt phosphorylation was reported in cancer cell lines and tumor tissues of patients exposed to rapamycin, a response likely contributing to the attenuated antitumor activity of rapamycin. It is, therefore, necessary to develop and validate combination strategies to reverse rapamycin-induced Akt signaling. We now report that Akt activation in response to rapamycin is abrogated by 17-allylamino-17-demethoxygeldanamycin (17-AAG), a heat shock protein 90 (HSP90) inhibitor. Rapamycin/17-AAG combination results in an enhanced antiproliferative activity in both MCF-7 and MDA-MB-231 breast cancer cells. In combination 17-AAG confers potent suppression of Raf-MEK-extracellular signal-regulated kinase signaling, a pathway that is otherwise not inhibited by rapamycin individually. Importantly, 17-AAG cooperates with rapamycin to block the phosphorylation of the mammalian target of rapamycin at Ser2448, as well as its downstream effectors ribosomal p70 S6 kinase and eukaryotic initiation factor 4E binding protein 1, which is accompanied by a substantial reduction in cyclins D1 and E. The potency of rapamycin/17-AAG combination is not affected by the activation of insulin-like growth factor 1 receptor signaling, which has been previously shown to diminish the antiproliferative activity of rapamycin. Rapamycin/17-AAG combination alleviates the induction of HSP90 protein, a heat shock response frequently associated with 17-AAG monotherapy. Our findings establish a mechanistic rationale for a combination approach using rapamycin and 17-AAG in the treatment of breast cancer.
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PMID:Combination of rapamycin and 17-allylamino-17-demethoxygeldanamycin abrogates Akt activation and potentiates mTOR blockade in breast cancer cells. 1859 9

The geldanamycin derivatives 17-allylamino-17-demethoxygeldanamycin (17-AAG) and 17-dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG) are promising chemotherapeutic drugs that inhibit heat shock protein 90 (HSP90) function. Previous studies have shown that 17-AAG/DMAG treatment induces the degradation of mutant BRAF (V600E) and inhibits the activation of mitogen-activated protein/extracellular signal-regulated kinase 1/2 (MEK1/2). We have found, however, that HSP90 inhibition alone is not sufficient for efficient BRAF(V600E) degradation in some cells. HSP90 inhibitors structurally unrelated to geldanamycin, radicicol and novobiocin, while inducing the degradation of the HSP90 client protein RAF-1 fail to induce BRAF(V600E) degradation or inhibit MEK1/2 activation in HT29 human colon cancer cells. Moreover, after treatment with 17-DMAG, the kinase activity of residual, undegraded BRAF(V600E) was also lost. Incubation of cells with a reactive oxygen species (ROS) scavenger, N-acetyl cysteine, partially restored kinase activity and also partially prevented BRAF(V600E) degradation due to 17-DMAG treatment. Conversely, treatment with the ROS producing drug menadione clearly inhibited MEK1/2 and reduced BRAF(V600E). These results suggest that in addition to direct inhibition of HSP90, the antitumor effect of geldanamycin and its derivatives is also mediated though the production of ROS, which may directly inactivate tumorigenic mutant BRAF(V600E).
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PMID:Oxidative stress plays a critical role in inactivating mutant BRAF by geldanamycin derivatives. 1867 57


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