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)

Type-2A protein phosphatase (PP2A) is a key regulator in many different cell signaling pathways and an important determinant in tumorigenesis. One of the signaling targets of PP2A is the mitogen-activated protein kinase (MAPK/ERK) cascade. In this study, we wanted to determine whether PP2A could be involved in regulation of death receptor activity through its capacity to regulate MAPK/ERK. To this end, we studied the effects of two different routes of protein phosphatase inhibition on death receptor-mediated apoptosis. We demonstrated that the apoptosis mediated by Fas, TNF-alpha, and TRAIL in U937 cells is suppressed by calyculin A, an inhibitor of type-1 and type-2A protein phosphatases. The inhibition of the protein phosphatase activity was shown to subsequently increase the MAPK activity in these cells, and the level of activation corresponded to the degree of suppression of cytokine-mediated apoptosis. A more physiological inhibitor, the intracellular PP2A inhibitor protein I2(PP2A), protected transfected HeLa cells in a similar way from Fas-mediated apoptosis and induced activation of MAPK in I2(PP2A) transfected cells. A corresponding inhibition could also be obtained by stable transfection with a constitutively active form of the MAPK kinase, MKK1 (also referred to as MEK1). The inhibitor-mediated protection was highly efficient in preventing early stages of apoptosis, as no caspase-8 cleavage occurred in these cells. The observed apoptosis suppression is likely to facilitate the tumor-promoting effect of a range of different type-2A protein phosphatase inhibitors, and could explain the reported tumor association of I2(PP2A).
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PMID:Type-2A protein phosphatase activity is required to maintain death receptor responsiveness. 1457 31

Angiotensin II (Ang II) stimulates protein synthesis in vascular smooth muscle cells (VSMCs), possibly secondary to regulatory changes at the initiation of mRNA translation. Mitogen-activated protein (MAP) kinase signal-integrating kinase-1 (Mnk1), a substrate of ERK and p38 MAP kinase, phosphorylates eukaryotic initiation factor 4E (eIF4E), an important factor in translation. The goal of the present study was to investigate the role of Mnk1 in Ang II-induced protein synthesis and to characterize the molecular mechanisms by which Mnk1 and eIF4E is activated in rat VSMCs. Ang II treatment resulted in increased Mnk1 activity and eIF4E phosphorylation. Expression of a dominant-negative Mnk1 mutant abolished Ang II-induced eIF4E phosphorylation. PD98059 or introduction of kinase-inactive MEK1/MKK1, but not SB202190 or kinase-inactive p38 MAP kinase, inhibited Ang II-induced Mnk1 activation and eIF4E phosphorylation, suggesting that ERK, but not p38 MAP kinase, is required for Ang II-induced Mnk1-eIF4E activation. Further, dominant-negative constructs for Ras, but not for Rho, Rac, or Cdc42, abolished Ang II-induced Mnk1 activation. Finally, treatment of VSMCs with CGP57380, a novel specific kinase inhibitor of Mnk1, resulted in dose-dependent decreases in Ang II-stimulated phosphorylation of eIF4E, protein synthesis, and VSMC hypertrophy. In summary, these data demonstrated that (1) Ang II-induced Mnk1 activation is mediated by the Ras-ERK cascade in VSMCs, and (2) Mnk1 is involved in Ang II-mediated protein synthesis and hypertrophy, presumably through the activation of translation-initiation. The Mnk1-eIF4E pathway may provide new insights into molecular mechanisms involved in vascular hypertrophy and other Ang II-mediated pathological states.
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PMID:Mnk1 is required for angiotensin II-induced protein synthesis in vascular smooth muscle cells. 1460 21

Phosphorylation-specific antibodies provide a powerful tool for analysing the regulation and activity of proteins in the MAP (mitogen-activated protein) kinase and other signalling pathways. Using synchronized cells, it was observed that phosphorylation-specific antibodies developed against the active form of MKK1/MKK2 (MAP kinase kinase-1 and -2) reacted with a protein that was approx. 35 kDa during G2/M-phase of the cell cycle. Failure of the 35 kDa protein to react with phosphorylation-independent MKK1/MKK2 antibodies suggested that this protein was not related to MKK1 or MKK2. Thus the 35 kDa protein was isolated by immunoprecipitation with the phospho-MKK1/MKK2 antibody and identified by MS. Peptide sequence analysis revealed matches with NPM (nucleophosmin/B23), a phosphoprotein involved in nucleolar assembly, centrosome duplication and ribosome assembly and transport. Biochemical and immunocytochemistry analyses verified that the phospho-MKK1/MKK2 antibodies cross-reacted with NPM that was phosphorylated at Thr234 and Thr237 during G2/M-phase, which are the same sites that are targeted by Cdc2 (cell division cycle protein-2) during mitosis. Using phosphorylation site mutants, we show that phosphorylation of Thr234 and Thr237 is required for NPM immunoreactivity with the phospho-MKK1/MKK2 antibody. Moreover, phosphorylation of Thr234 and Thr237 was demonstrated to regulate NPM localization to the centrosome after nuclear envelope breakdown in mitotic cells. These findings reveal a new insight into the role of phosphorylation in regulating NPM targeting during mitosis. However, caution should be used when using commercially available phospho-MKK1/MKK2 antibodies to examine the regulation of MKK1/MKK2 during mitotic transitions, owing to their cross-reactivity with phosphorylated NPM at this time of the cell cycle.
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PMID:Phosphorylation regulates nucleophosmin targeting to the centrosome during mitosis as detected by cross-reactive phosphorylation-specific MKK1/MKK2 antibodies. 1467 79

Cytochrome P450 2E1 (CYP2E1) is highly inducible in a subset of astrocytes in vivo following ischemic or mechanical injury and in vitro by lipopolysaccharide (LPS) or interleukin-1beta. We have studied the mechanism of induction, and found that transcriptional activation of CYP2E1 occurred within 3 h, and CYP2E1 dependent catalytic activity was induced more than 4-fold within 5 h. The induction was sensitive to several tyrosine kinase inhibitors, and was further modulated by inhibitors of p38 MAP kinase. MAP kinase kinase-3 (MKK3) was phosphorylated in response to LPS, and expression of constitutively active MKK3, but not the MAP kinase kinases MEKK1 or MKK1, activated CYP2E1. Transcriptional activation was mediated through a C/EBPbeta and -delta binding element situated at -486/-474, and appeared to involve activation of prebound factors as well as recruitment of newly synthesized C/EBPbeta and -delta. It is thus suggested that LPS induces MKK3 activation in astrocytes, which in turn stimulates a C/EBPbeta and -delta binding element to mediate transcriptional activation of CYP2E1.
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PMID:Lipopolysaccharide induces CYP2E1 in astrocytes through MAP kinase kinase-3 and C/EBPbeta and -delta. 1467 Sep 49

Uncoupling protein 3 (UCP3) is a mitochondrial protein with antioxidant properties and its regulation by factors promoting cell-survival may be important for protection of, for instance, neurons in states of oxidative stress. In the present study, we investigated regulatory pathways for UCP3 expression mediated by the neuroprotective hormone insulin-like growth factor type 1 (IGF-1) in human neuroblastoma SH-SY5Y cells. Northern blot analysis and RT-PCR showed that treatment with 10 nm IGF-1 increased the UCP3 mRNA levels 2.5-fold after 5 h. Co-incubation with the phosphatidylinositol 3 (PI3)-kinase inhibitor LY294002 prohibited IGF-1-mediated induction of both UCP3 mRNA and protein in a concentration-dependent manner, with a complete blockage at 1 microm, as shown by RT-PCR and western blot analyses. The mitogen-activated protein (MAP) kinase kinase 1 (MKK1 or MEK) inhibitor PD98059 also decreased the UCP3 mRNA expression at 10 microm, however, this concentration only partly inhibited the protein expression. We conclude that IGF-1 enhanced UCP3 expression at transcriptional level, primarily through the PI3-kinase-dependent pathway and partly through the MAP kinase pathway.
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PMID:Signalling pathways for insulin-like growth factor type 1-mediated expression of uncoupling protein 3. 1469 May 34

Nerve growth factor (NGF) increases expression of nitric oxide synthase (NOS) isozymes leading to enhanced production of nitric oxide (NO). NOS inhibitors attenuate NGF-mediated increases in cholinergic gene expression and neurite outgrowth. Mechanisms underlying this are unknown, but the mitogen-activated protein (MAP) kinase pathway plays an important role in NGF signaling. Like NGF, NO donors activate Ras leading to phosphorylation of MAP kinase. The present study investigated the role of NO in NGF-mediated activation of MAP kinase in PC12 cells. Cells were treated with 50 ng/mL NGF to establish the temporal pattern for rapid and sustained activation phases of MAP kinase kinase (MEK)-1/2 and p42/p44-MAP kinase. Subsequently, cells were pretreated with NOS inhibitors Nomega-nitro-L-arginine methylester and s-methylisothiourea and exposed to NGF for up to 24 h. NGF-induced activation of MEK-1/2 and p42/p44-MAP kinase was not dependent on NO, but sustained phosphorylation of MAP kinase was modulated by NO. This modulation did not occur at the level of Ras-Raf-MEK signaling or require activation of cGMP/PKG pathway. NOS inhibitors did not affect NGF-mediated phosphorylation of MEK. Expression of constitutively active-MEKK1 in cells led to phosphorylation of p42/p44-MAP kinase and robust neurite outgrowth; constitutively active-MKK1 also caused differentiation with neurite extension. NOS inhibitor treatment of cells expressing constitutively active kinases did not affect MAP kinase activation, but neurite outgrowth was attenuated. NOS inhibitors did not alter NGF-mediated nuclear translocation of phospho-MAP kinase, but phosphorylated kinases disappeared more rapidly from NOS inhibitor-treated cells suggesting greater phosphatase activity and termination of sustained activation of MAP kinase.
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PMID:Modulation of nerve growth factor-induced activation of MAP kinase in PC12 cells by inhibitors of nitric oxide synthase. 1471 89

Arctigenin, naturally occurring in Bardanae fructus, Saussurea medusa, Arctium lappa L., Torreya nucifera and Ipomea cairica, is a phenylpropanoid dibenzylbutyrolactone lignan with antioxidant and anti-inflammatory activities. Previously, we showed that arctigenin potently inhibited the induction of nitric oxide synthase (iNOS) by lipopolysaccharide (LPS), which involved suppression of NF-kappaB activation. In the present study, we examined the effects of arctigenin on mitogen-activated protein (MAP) kinase activation in Raw264.7 cells and MAP kinase kinase (MKK) activity. The effect of arctigenin on activator protein-1 (AP-1) activation was also studied in association with tumor necrosis factor-alpha (TNF-alpha) expression. Immunoblot analysis showed that arctigenin inhibited phosphorylation of MAP kinases ERK1/2, p38 kinase and JNK and their activities in Raw264.7 cells treated with LPS. Arctigenin potently inhibited the activity of MKK1 in vitro with the IC(50) value of 1 nM. Gel shift and reporter gene analyses revealed that arctigenin inhibited LPS-inducible AP-1 binding to the AP-1 consensus oligonucleotide and AP-1-mediated reporter gene expression. In view of the potential role of AP-1 in the induction of TNF-alpha, we next examined the inhibitory effects of arctigenin on the expression of TNF-alpha. Arctigenin blocked TNF-alpha production and decreased the level of TNF-alpha mRNA in the cells exposed to LPS. These results showed that arctigenin inhibited activation of MAP kinases including ERK1/2, p38 kinase and JNK through the inhibition of MKK activities, leading to AP-1 inactivation, which might, at least in part, contribute to the inhibition of TNF-alpha production.
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PMID:Arctigenin, a phenylpropanoid dibenzylbutyrolactone lignan, inhibits MAP kinases and AP-1 activation via potent MKK inhibition: the role in TNF-alpha inhibition. 1531 39

Normal pregnancy is associated with high angiotensin II (ANG II) concentrations in the maternal and fetal circulation. These high levels of ANG II may promote production vasodilators such as nitric oxide (NO). ANG II receptors are expressed in ovine fetoplacental artery endothelial (OFPAE) cells and mediate ANG II-stimulated OFPAE cell proliferation. Herein, we tested whether ANG II stimulated NO synthase 3 (NOS3, also known as eNOS) expression and total NO (NO(x)) production via activation of mitogen-activated protein kinase 3/1 (MAPK3/1, also known as ERK1/2) in OFPAE cells. ANG II elevated (P < 0.05) eNOS protein, but not mRNA levels with a maximum effect at 10 nM. ANG II also dose dependently increased (P < 0.05) NO(x) production with a maximal effect at doses of 1-100 nM. Activation of ERK1/2 by ANG II was determined by immunocytochemistry and Western blot analysis. ANG II rapidly induced positive staining for phosphorylated ERK1/2, appearing in cytosol after 1-5 min of ANG II treatment, accumulating in nuclei after 10 min, and disappearing at 15 min. ANG II increased (P < 0.05) phosphorylated ERK1/2 protein levels. Activation of ERK1/2 was confirmed by an immunocomplex kinase assay using ELK1 as a substrate. PD98059 significantly inhibited ANG II-induced ERK1/2 activation, and the ANG II-elevated eNOS protein levels but only partially reduced ANG II-increased NO(x) production. Thus, in OFPAE cells, the ANG II increased NO(x) production is associated with elevated eNOS protein expression, which is mediated at least in part via activation of the mitogen-activated protein kinase kinase1 and kinase2 (MAP2K1 and MAP2K2, known also as MEK1/2)/ERK1/2 cascade. Together with our previous observation that ANG II stimulates OFPAE cell proliferation, these data suggest that ANG II is a key regulator for both vasodilation and angiogenesis in the ovine fetoplacenta.
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PMID:Angiotensin II elevates nitric oxide synthase 3 expression and nitric oxide production via a mitogen-activated protein kinase cascade in ovine fetoplacental artery endothelial cells. 1572 93

Bovine type I collagen (BIC), which is widely used as a fibrous extracellular matrix component in cell culture models, inhibits the progression of melanoma cell cycle via p27 up-regulation. BIC also induces nitric oxide synthase in macrophages through JunB/AP-1 and NF-kappaB activation. Given the previous observations, this study investigates the effect of BIC on the cell cycle progression and regulatory function of Raw264.7 macrophage cells and the responsible signaling pathways. Cell cycle analysis revealed that BIC completely suppressed proliferation of Raw264.7 cells with inhibition of the percentage of cells in the S phase and the reciprocal decrease in the G0/G1 phase. DNA synthesis was also inhibited by BIC, as evidenced by a decrease in the cellular incorporation of [3H]thymidine. The G1/S arrest induced by BIC was reversed by chemical inhibition of phosphatidylinositol 3-kinase (PI3-kinase) or overexpression of the p85 subunit of PI3-kinase. Either PD98059 or stable transfection with mitogen-activated protein kinase kinase-1 [MKK1(-)] or c-Jun N-terminal kinase 1 [JNK1(-)] also released the cell cycle arrest. Immunoblot analyses revealed that the levels of cyclins D1, A and B1 were partly or completely down-regulated by BIC, but cyclin E, p21 and p27 were minimally changed. Chemical inhibition and dominant negative mutant overexpression experiments revealed that either PI3-kinase inhibition or JNK1(-) transfection prevented the decreases in cyclin D1, A and B1 by BIC, indicating that the PI3-kinase and JNK1 pathways were associated with disruption of the cyclins. The pathway involving MKK1-extracellular signal-regulated kinase-1/2 (ERK1/2) was responsible for the suppression of cyclin A and B1, but not that of cyclin D1. The present study showed that BIC inhibited proliferation of Raw264.7 cells and that the pathways involving PI3-kinase and mitogen-activated protein kinases regulate the cell cycle arrest.
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PMID:Bovine type I collagen inhibits Raw264.7 cell proliferation through phosphoinositide 3-kinase- and mitogen-activated protein kinase-dependent down-regulation of cyclins D1, A and B1. 1587 97

Glaucoma is the second leading cause of blindness in the world. Loss of vision in glaucomatous optic neuropathy is caused by the selective degeneration of retinal ganglion cells (RGCs). Ocular hypertension is a major risk factor in glaucoma, but visual field defects continue to progress in some patients despite the use of drugs that lower intraocular pressure. At present, there are no effective neuroprotective strategies for the treatment of this disease. The extracellular signal-regulated kinase (Erk) 1/2 pathway is an evolutionarily conserved mechanism used by several peptide factors to promote cell survival. Here we tested if selective activation of Erk1/2 protected RGCs in a rat model of experimental glaucoma. We used recombinant adeno-associated virus to transduce RGCs with genes encoding constitutively active or wild-type MEK1 (approved gene symbol MAP2K1), the upstream activator of Erk1/2. MEK1 gene transfer into RGCs markedly increased neuronal survival: 1366 +/- 70 RGCs/mm(2) (mean +/- SEM) were alive in the dorsal retina at 5 weeks after ocular hypertension surgery, a time when only 680 +/- 86 RGCs/mm(2) of these neurons remained in control eyes. We conclude that the Erk1/2 pathway plays a key role in the protection of RGCs from ocular hypertensive damage. This study identifies a novel gene therapy strategy in which selective activation of the Erk1/2 signaling pathway effectively slows cell death in glaucoma.
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PMID:Activation of the extracellular signal-regulated kinase 1/2 pathway by AAV gene transfer protects retinal ganglion cells in glaucoma. 1597 50


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