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 role of signal transducer and activator of transcription (STAT) signaling pathways in the interleukin-6 (IL-6)-induced morphological differentiation of PC12-E2 cells was assessed using wild type and dominant negative mutants of Stat1 and Stat3, containing Tyr --> Phe (YF), Ser --> Ala (SA), and the double mutations (DM), respectively. FS3-YF or FS3-DM markedly inhibited the IL-6-induced response, but overexpression of FS3-SA caused only a modest inhibition. Expression of all Stat3 mutants had no effect on NGF-induced neurite outgrowth. Overexpression of wild type Stat1 protein inhibited IL-6 activated DNA binding complexes containing Stat3 homodimers, which may explain the partial negative effect of Stat1 on IL-6-induced neurite outgrowth. Specificity of these STAT constructs was confirmed using luciferase reporter gene assays, which showed that IL-6-activated transcription was blocked by expression of FS3-YF and FS3-DM and that FS1 enhanced the interferon gamma-activated transcription. Thus, in PC12-E2 cells, Stat3 homodimers are preferentially activated by IL-6, indicating a role for Stat3 in the regulation of cellular differentiation. Furthermore, IL-6 induced robust neurite outgrowth in PC12-E2 cells expressing dominant negative forms of RAS or SHC or in cells pretreated with the mitogen-activated protein kinase mitogen-activated protein kinase kinase inhibitor, PD98059. Thus, activation of the Stat3 signaling pathway, but not RAS/ERK dependent pathways, is essential for differentiation of PC12-E2 cells by IL-6.
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PMID:Activation of the Stat3 signaling pathway is required for differentiation by interleukin-6 in PC12-E2 cells. 1063 20

The cellular responses to activated Ras vary depending on cell type. Normal cells are often induced into pathways that lead to cell growth arrest, senescence, and/or apoptosis in response to activated Ras expression. These are important protective anti-tumorigenic responses that restrict the propagation of cells bearing activated oncogenes. Here we show that induction of Ha-Ras(Val-12) in Rat-1 fibroblasts resulted in G(1) growth arrest and apoptosis with loss of viable cells that is accompanied by a marked decrease in cyclin D1 levels via increased ubiquitin-proteasome-dependent cyclin D1 turnover. This is in contrast with a rat intestinal epithelial cell line in which induction of Ha-Ras(Val-12) results in transformation associated with sustained proliferation and increased levels of cyclin D1, that is not accompanied by anoikis or apoptosis. Expression of the cyclin D1 mutant (T286A) that contains an alanine for threonine 286 substitution and is resistant to ubiquitin-proteasome degradation in the Ha-Ras(Val-12) expressing Rat-1 cells resulted in a sustained transformed phenotype with no accumulation of cells in G(1). Inhibition of mitogen-activated protein kinase (MEK1/2) pathway partially reversed the Ras-mediated decrease in cyclin D1. Induction of Ha-Ras(Val-12) resulted in activation of Akt kinase and inactivation of glycogen-synthase-3beta kinase that are associated with reduction of cyclin D1 protein. These results suggest that Ras-mediated cyclin D1 degradation in Rat-1 cells appears to be partially dependent on activation of mitogen-activated protein kinase pathway and independent of glycogen-synthase-3beta kinase pathway.
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PMID:Oncogenic Ras-mediated cell growth arrest and apoptosis are associated with increased ubiquitin-dependent cyclin D1 degradation. 1078 97

The functions of the extracellular domains of neural cell adhesion molecule (NCAM) have been studied extensively, whereas the roles of the cytoplasmic domains of the transmembrane forms of NCAM are less elucidated. We investigated the importance of the cytoplasmic domain of the 140-kDa NCAM isoform (cytNCAM-140) and of the 180-kDa NCAM isoform (cytNCAM-180) in NCAM-induced neurite extension by estimating NCAM-dependent neurite outgrowth from PC12-E2 cells grown in coculture with NCAM-negative or NCAM-positive fibroblasts. PC12-E2 cells were transiently transfected with expression plasmids encoding cytNCAM-140, cytNCAM-180, the constitutively active form of the mitogen-activated protein (MAP) kinase/extracellular signal-regulated kinase kinase (MEK2), and the enhanced variant of the green fluorescent protein (EGFP). EGFP expression was used for identification of transfected cells. We found that expression of cytNCAM-180 had no effect on NCAM-stimulated neuritogenesis, whereas expression of cytNCAM-140 strongly inhibited this process. However, if MEK2 was expressed concomitantly with cytNCAM-140, neurite outgrowth was rescued, indicating that cytNCAM-140 is involved in signaling via the Ras-MAP kinase pathway. PC12-E2 cells were subsequently transiently transfected with constructs encoding a series of fragments of cytNCAM-140 and various full-length cytNCAM-140 mutants, and the residues Thr-Glu-Val-Lys-Thr (839-843) were identified as essential in NCAM-stimulated neuritogenesis. The combined substitution of Glu(840) and Lys(842) with Ala abrogated the effect of the construct, assigning a critical role to these two residues.
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PMID:Identification of an amino acid sequence motif in the cytoplasmic domain of the NCAM-140 kDa isoform essential for its neuritogenic activity. 1093 11

Cisplatin activates multiple signal transduction pathways involved in coordinating cellular responses to stress. Here we demonstrate a requirement for extracellular signal-regulated protein kinase (ERK), a member of the mitogen-activated protein kinase family in mediating cisplatin-induced apoptosis of human cervical carcinoma HeLa cells. Cisplatin treatment resulted in dose- and time- dependent activation of ERK. That elevated ERK activity contributed to cell death by cisplatin was supported by several observations: 1) PD98059 and U0126, chemical inhibitors of the MEK/ERK signaling pathway, prevented apoptosis; 2) pretreatment of cells with TPA, an activator of the ERK pathway, enhanced their sensitivity to cisplatin; 3) suramin, a growth factor receptor antagonist that greatly suppressed ERK activation, likewise inhibited cisplatin-induced apoptosis; and, finally, 4) HeLa cell variants selected for cisplatin resistance showed reduced activation of ERK following cisplatin treatment. Cisplatin-induced apoptosis was associated with cytochrome c release and subsequent caspase-3 activation, both of which could be prevented by treatment with the MEK inhibitors. However, the caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone protected HeLa cells against apoptosis without affecting ERK activation. Taken together, our findings suggest that ERK activation plays an active role in mediating cisplatin-induced apoptosis of HeLa cells and functions upstream of caspase activation to initiate the apoptotic signal.
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PMID:Requirement for ERK activation in cisplatin-induced apoptosis. 1099 83

Galpha-interacting protein (GAIP) is a regulator of G protein signaling (RGS) that accelerates the rate of GTP hydrolysis by the alpha-subunit of the trimeric G(i3) protein. Both proteins are part of a signaling pathway that controls lysosomal-autophagic catabolism in human colon cancer HT-29 cells. Here we show that GAIP is phosphorylated by an extracellular signal-regulated (Erk1/2) MAP kinase-dependent pathway sensitive to amino acids, MEK1/2 (PD098059), and protein kinase C (GF109203X) inhibitors. An in vitro phosphorylation assay demonstrates that Erk2-dependent phosphorylation of GAIP stimulates its GTPase-activating protein activity toward the Galpha(i3) protein (k = 0.187 +/- 0.001 s(-)(1), EC(50) = 1.12 +/- 0.10 microm) when compared with unphosphorylated recombinant GAIP (k = 0.145 +/- 0.003 s(-)(1), EC(50) = 3.16 +/- 0. 12 microm) or to GAIP phosphorylated by other Ser/Thr protein kinases (protein kinase C, casein kinase II). This stimulation and the phosphorylation of GAIP by Erk2 were abrogated when serine at position 151 in the RGS domain was substituted by an alanine residue using site-directed mutagenesis. Furthermore, the lysosomal-autophagic pathway was not stimulated in S151A-GAIP mutant-expressing cells when compared with wild-type GAIP-expressing cells. These results demonstrate that the GTPase-activating protein activity of GAIP is stimulated by Erk2 phosphorylation. They also suggested that Erk1/2 and GAIP are engaged in the signaling control of a major catabolic pathway in intestinal derived cells.
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PMID:Erk1/2-dependent phosphorylation of Galpha-interacting protein stimulates its GTPase accelerating activity and autophagy in human colon cancer cells. 1099 92

A synthetic peptide, (D-Phe(6), beta-Ala(11), Phe(13), Nle(14))bombesin-(6-14) was used to investigate the signal transduction mechanisms of bombesin receptor subtype-3. Using NCI-1299#5 human lung cancer cells stably transfected with bombesin receptor subtype-3, 100 nM (D-Phe(6), beta-Ala(11), Phe(13), Nle(14))bombesin-(6-14) elevated the cytosolic Ca2+ from 150 to 250 nM within 10 s. Addition of (D-Phe(6), beta-Ala(11), Phe(13), Nle(14))bombesin-(6-14) caused phosphorylation of mitogen activated protein kinase in a time- and concentration-dependent manner. The mitogen activated protein kinase phosphorylation caused by (D-Phe(6), beta-Ala(11), Phe(13), Nle(14))bombesin-(6-14) was inhibited by 2'-amino-3'-methyoxyflavone (PD98059), a mitogen activated protein kinase kinase (MEK-1) inhibitor. Using a luciferase reporter gene construct, (D-Phe(6), beta-Ala(11), Phe(13), Nle(14))bombesin-(6-14) caused Elk-1 activation after 10 min and the increase in Elk-1 activation caused by (D-Phe(6), beta-Ala(11), Phe(13), Nle(14))bombesin-(6-14) was inhibited by PD98059 as well as a dominant-negative MEK-1. (D-Phe(6), beta-Ala(11), Phe(13), Nle(14))bombesin-(6-14) caused increased c-fos as well as c-jun mRNAs 1 h after addition to NCI-H1299#5 cells. The 47-fold increase in c-fos mRNA caused by 100 nM (D-Phe(6), beta-Ala(11), Phe(13), Nle(14))bombesin-(6-14) was inhibited by PD98059, a dominant-negative MEK-1 and a substance P antagonist but not (3-phenylpropanoyl-D-Ala(24), Pro(26), Psi(26,27), Phe(27))GRP-(20-27) (BW2258U89), a GRP receptor antagonist. These results indicate that (D-Phe(6), beta-Ala(11), Phe(13), Nle(14))bombesin-(6-14) caused increased nuclear oncogene expression and upstream events include mitogen activated protein kinase phosphorylation and Elk-1 activation.
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PMID:A bombesin receptor subtype-3 peptide increases nuclear oncogene expression in a MEK-1 dependent manner in human lung cancer cells. 1116 31

The NPXXY motif (X represents any amino acid) in the seventh transmembrane domain of the chemotactic formyl peptide receptor (FPR) is highly conserved among G protein-coupled receptors. Recent work suggested that this motif contributes to G protein-coupled receptor internalization and signal transduction; however, its role in FPR signaling remains unclear. In this study we replaced Asn(297) and Tyr(301) in the NPXXY motif of the human FPR with Ala (N297A) and Ala/Phe (Y301A/Y301F), respectively, and determined the effects of the substitutions on FPR functions in transfected rat basophilic leukemia cells. Whereas all the mutant receptors were expressed on the cell surface, the N297A receptor exhibited reduced binding affinity and was unable to mediate activation of phospholipase C-beta and the p42/44 mitogen-activated protein kinase (MAP kinase). The Y301F receptor displayed significantly decreased ligand-stimulated internalization and MAP kinase activation, suggesting that the hydrogen bonding at Tyr(301) is critical for these functions. The Y301F receptor showed a chemotactic response similar to that of wild-type FPR, indicating that cell chemotaxis does not require receptor internalization and hydrogen bonding at the Tyr(301) position. In contrast, the Y301A receptor displayed a left-shifted, but overall reduced, chemotaxis response that peaked at 0.1-1 nM. Finally, using a specific MAP kinase kinase inhibitor, we found that activation of MAP kinase is required for efficient FPR internalization, but is not essential for chemotaxis. These findings demonstrate that residues within the NPXXY motif differentially regulate the functions of FPR.
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PMID:Differential roles of the NPXXY motif in formyl peptide receptor signaling. 1123 59

Ca(2+)/calmodulin-dependent protein kinase phosphatase (CaMKPase) dephosphorylates and regulates multifunctional Ca(2+)/calmodulin-dependent protein kinases. In order to elucidate the mechanism of substrate recognition by CaMKPase, we chemically synthesized a variety of phosphopeptide analogs and carried out kinetic analysis using them as CaMKPase substrates. This is the first report using systematically synthesized phosphopeptides as substrates for kinetic studies on substrate specificities of protein Ser/Thr phosphatases. CaMKPase was shown to be a protein Ser/Thr phosphatase having a strong preference for a phospho-Thr residue. A Pro residue adjacent to the dephosphorylation site on the C-terminal side and acidic clusters around the dephosphorylation site had detrimental effects on dephosphorylation by CaMKPase. Deletion analysis of a model substrate peptide revealed that the minimal length of the substrate peptide was only 2 to 3 amino acid residues including the dephosphorylation site. The residues on the C-terminal side of the dephosphorylation site were not essential for dephosphorylation, whereas the residue adjacent to the dephosphorylation site on the N-terminal side was essential. Ala-scanning analysis suggested that CaMKPase did not recognize a specific motif around the dephosphorylation site. Myosin light chain phosphorylated by protein kinase C and Erk2 phosphorylated by MEK1 were poor substrates for CaMKPase, while a synthetic phosphopeptide corresponding to the sequence around the phosphorylation site of the former was not dephosphorylated by CaMKPase but that of the latter was fairly good substrate. These data suggest that substrate specificity of CaMKPase is determined by higher-order structure of the substrate protein rather than by the primary structure around its dephosphorylation site. Use of phosphopeptide substrates also revealed that poly-L-lysine, an activator for CaMKPase, activated the enzyme mainly through increase in the V(max) values.
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PMID:Substrate specificity of Ca(2+)/calmodulin-dependent protein kinase phosphatase: kinetic studies using synthetic phosphopeptides as model substrates. 1132 97

Docking between MEK1 and ERK2 is required for their stable interaction and efficient signal transmission. The MEK1 N terminus contains the ERK docking or D domain that consists of conserved hydrophobic and basic residues. We mutated the hydrophobic and basic residues individually and found that loss of either type reduced MEK1 phosphorylation of ERK2 in vitro and its ability to bind to ERK2 in vivo. Moreover, ERK2 was localized in both the cytoplasm and the nucleus when co-expressed with MEK1 that had mutations in either the hydrophobic or the basic residues. We then identified two conserved hydrophobic residues on ERK2 that play roles in docking with MEK1. Mutating these residues to alanine reduced the interaction of ERK2 with MEK1 in cells. These mutations also reduced the phosphorylation of MEK1 by ERK2 but had little effect on phosphorylation of MBP by ERK2. Finally, we generated docking site mutants in ERK2-MEK1 fusion proteins. Although the mutation of the MEK1 D domain significantly reduced ERK2-MEK1 activity, mutations of the putatively complementary acidic residues and hydrophobic residues on ERK2 did not change its activity. However, both types of mutations decreased the phosphorylation of Elk-1 caused by ERK2-MEK1 fusion proteins. These findings suggest complex interactions of MEK1 D domains with ERK2 that influence its activation and its effects on substrates.
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PMID:Hydrophobic as well as charged residues in both MEK1 and ERK2 are important for their proper docking. 1135 17

We previously described the identification of quail MafA, a novel transcription factor of the Maf bZIP (basic region leucine zipper) family, expressed in the differentiating neuroretina (NR). In the present study, we provide the first evidence that MafA is phosphorylated and that its biological properties strongly rely upon phosphorylation of serines 14 and 65, two residues located in the transcriptional activating domain within a consensus for phosphorylation by mitogen-activated protein kinases and which are conserved among Maf proteins. These residues are phosphorylated by ERK2 but not by p38, JNK, and ERK5 in vitro. However, the contribution of the MEK/ERK pathway to MafA phosphorylation in vivo appears to be moderate, implicating another kinase. The integrity of serine 14 and serine 65 residues is required for transcriptional activity, since their mutation into alanine severely impairs MafA capacity to activate transcription. Furthermore, we show that the MafA S14A/S65A mutant displays reduced capacity to induce expression of QR1, an NR-specific target of Maf proteins. Likewise, the integrity of serines 14 and 65 is essential for the MafA ability to stimulate expression of crystallin genes in NR cells and to induce NR-to-lens transdifferentiation. Thus, the MafA capacity to induce differentiation programs is dependent on its phosphorylation.
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PMID:Phosphorylation of MafA is essential for its transcriptional and biological properties. 1141 24


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