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)

A new family of signaling intermediates for TGFbeta superfamily members and other growth factors has recently been identified and termed Smads. It has been suggested that the Smad1 subfamily is regulated primarily by the TGFbeta superfamily member bone morphogenetic protein (BMP). Here we demonstrate that TGFbeta induced phosphorylation of endogenous Smad1 in untransformed IECs and that the RI and RII TGFbeta receptors were detectable in Smad1 immunocomplexes. Expression of a dominant-negative mutant of Ras inhibited the ability of TGFbeta to phosphorylate endogenous Smad1. In a separate series of experiments, we have cloned a rat homologue of the drosophila mad gene (termed RSmad1) by screening an intestinal epithelial cell (IEC) cDNA library. By using an in vitro kinase assay with RSmad1 as the substrate, we demonstrate that the TGFbeta receptor complex can directly phosphorylate RSmad1. We show, further, that a dominant-negative mutant of MEK1 inhibited the ability of RSmad1 to induce the TGFbeta-responsive reporter p3TP-Lux in a human breast cancer cell line. Collectively, our data demonstrate that TGFbeta can regulate Smadl and that the Ras and MEK signaling components are partially required for the ability of TGFbeta to regulate Smad1.
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PMID:Cloning and expression of a rat Smad1: regulation by TGFbeta and modulation by the Ras/MEK pathway. 998 85

Smad proteins are essential components of the intracellular signaling pathways utilized by members of the transforming growth factor-beta (TGF-beta) superfamily of growth factors. Certain Smad proteins (e.g. Smad1, -2, and -3) can act as regulated transcriptional activators, a process that involves phosphorylation of these proteins by activated TGF-beta superfamily receptors. We demonstrate that the intracellular kinase mitogen-activated protein kinase kinase kinase-1 (MEKK-1), an upstream activator of the stress-activated protein kinase/c-Jun N-terminal kinase pathway, can participate in Smad2-dependent transcriptional events in cultured endothelial cells. A constitutively active form of MEKK-1 but not mitogen-activated protein kinase kinase-1 (MEK-1) or TGF-beta-activated kinase-1, two distinct intracellular kinases, can specifically activate a Gal4-Smad2 fusion protein, and this effect correlates with an increase in the phosphorylation state of the Smad2 protein. These effects do not require the presence of the C-terminal SSXS motif of Smad2 that is the site of TGF-beta type 1 receptor-mediated phosphorylation. Activation of Smad2 by active MEKK-1 results in enhanced Smad2-Smad4 interactions, nuclear localization of Smad2 and Smad4, and the stimulation of Smad protein-transcriptional coactivator interactions in endothelial cells. Overexpression of Smad7 can inhibit the MEKK-1-mediated stimulation of Smad2 transcriptional activity. A physiological level of fluid shear stress, a known activator of endogenous MEKK-1 activity in endothelial cells, can stimulate Smad2-mediated transcriptional activity. These data demonstrate a novel mechanism for activation of Smad protein-mediated signaling in endothelial cells and suggest that Smad2 may act as an integrator of diverse stimuli in these cells.
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PMID:MEKK-1, a component of the stress (stress-activated protein kinase/c-Jun N-terminal kinase) pathway, can selectively activate Smad2-mediated transcriptional activation in endothelial cells. 1008 21

Normal signaling by TGFbeta, in the absence of serum or exogenous factors, involves a rapid activation of Ras, Erks, and Sapks in proliferating cultures of TGFbeta-sensitive untransformed epithelial cells and human carcinoma cells. Expression of either RasN17 or dominant-negative (DN) MKK4, or addition of the MEK1 inhibitor PD98059, can block the ability of TGFbeta to induce AP-1 complex formation at the TGFbeta(1) promoter and to autoinduce its own production. The primary components present in this TGFbeta-stimulated AP-1 complex are JunD and Fra-2, although c-Jun, and possibly Fos B, may also be present. While there are two potential Smad binding elements (SBE's) in the TGFbeta(1) promoter, supershift assays suggest that at least one of these does not bind Smad4, and the other is unable to bind factors activated by TGFbeta. In contrast, TGFbeta autoinduction is Smad3-dependent, as DN Smad3 inhibits the ability of TGFbeta to stimulate TGFbeta(1) promoter activity. Our results indicate that TGFbeta can activate both the MKK4/Sapk and MEK/Erk pathways, through Ras and TGFbeta R(I) and R(II), to induce TGFbeta(1) production; Smad4 does not appear to be involved, and Smad3 appears to function independently of this Smad4. We also demonstrate that activation of the Ras/Mapk pathway by TGFbeta positively modulates Smad1-signaling-pathway activation by TGFbeta. In addition, Smad1 could enhance TGFbeta activation of the SBE reporter SBE-luc and this effect could be blocked by co-expression of a DN TGFbeta R(I) receptor or by the MEK1 inhibitor PD98059. This cross-talk between the MEK/Erk and Smad1 pathways was mediated through the four Erk consensus phosphorylation sites in the linker region of Smad1. Mutation of these sites resulted in a loss of the ligand-dependence of both Smad1-Smad4 interactions and nuclear accumulation of Smad1, as well as a loss of the ability of Smad1 to enhance TGFbeta-mediated SBE activation. Our results provide evidence that Erk-mediated phosphorylation of Smad1 in response to TGFbeta is critical for regulating Smad1 subcellular localization; this may be a key determinant in maintaining TGFbeta-dependent transcriptional activation.
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PMID:Role of Ras and Mapks in TGFbeta signaling. 1070 50

Bone morphogenetic protein-2 (BMP-2), a member of the transforming growth factor-beta (TGF-beta) family, regulates osteoblast differentiation and bone formation. Here we show a novel function of BMP-2 in human osteoblasts and identify a signaling pathway involved in this function. BMP-2 promotes apoptosis in primary human calvaria osteoblasts and in immortalized human neonatal calvaria osteoblasts, as shown by terminal deoxynucleotidyl transferase-mediated nick end labeling analysis. In contrast, TGF-beta 2 inhibits apoptosis in human osteoblasts. Studies of the mechanisms of action showed that BMP-2 increases the Bax/Bcl-2 ratio, whereas TG beta-2 has a negative effect. Moreover, BMP-2 increases the release of mitochondrial cytochrome c to the cytosol. Consistent with these results, BMP-2 increases caspase-9 and caspase-3, -6, and -7 activity, and an anti-caspase-9 agent suppresses BMP-2-induced apoptosis. Overexpression of dominant-negative Smad1 effectively blocks BMP-2-induced expression of the osteoblast transcription factor Runx2 but not the activation of caspases or apoptosis induced by BMP-2, indicating that the Smad1 signaling pathway is not involved in the BMP-2-induced apoptosis. The proapoptotic effect of BMP-2 is PKC-dependent, because BMP-2 increases PKC activity, and the selective PKC inhibitor calphostin C blocks the BMP-2-induced increased Bax/Bcl-2, caspase activity, and apoptosis. In contrast, the cAMP-dependent protein kinase A inhibitor H89, the p38 MAPK inhibitor SB203580, and the MEK inhibitor PD-98059 have no effect. The results show that BMP-2 uses a Smad-independent, PKC-dependent pathway to promote apoptosis via a Bax/Bcl-2 and cytochrome c-caspase-9-caspase-3, -6, -7 cascade in human osteoblasts.
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PMID:Bone morphogenetic protein-2 promotes osteoblast apoptosis through a Smad-independent, protein kinase C-dependent signaling pathway. 1139 80

Growth factors such as fibroblast growth factor-2 (FGF-2) and epidermal growth factor (EGF) that activate extracellular signal-regulated kinases (ERKs) through receptor tyrosine kinases (RTKs) stimulate proliferation but suppress differentiation of osteoblasts. To study the mechanism of this inhibitory action of these growth factors on osteoblastic differentiation, we evaluated Smad1 transactivity in MC3T3-E1 osteoblast-like cells by reporters of promoter activity of mouse Smad6, an early response gene to bone morphogenetic proteins (BMPs). FGF-2 and EGF inhibited alkaline phosphatase activity and Smad6 promoter activity stimulated by BMP-2. Overexpression of constitutively active MEK by adenovirus mimicked, but that of dominant negative Ras or treatment with a MEK1 inhibitor, PD098059, reversed, the inhibitory effects of these growth factors on both activities. These effects are mediated by BMP-responsive elements (BMPREs) on Smad6 promoter, because an artificial reporter driven by three tandem BMPREs gave similar results, and these effects were all abolished when the BMPREs were mutated. RTK-ERK activation inhibited the promoter activity even when BMP signal was mediated by a mutant Smad1, which lacks phosphorylation sites by ERKs, or by a Smad1 fused to Gal4 DNA binding domain, which constitutively localizes in the nucleus. These results show that the RTK-Ras-ERK pathway suppresses BMP signal by interfering with Smad1 transactivity. Because direct phosphorylation of Smad1 by ERKs is not required for the inhibition, other transcriptional factors that are phosphorylated by ERKs might be involved in the regulation of osteoblastic differentiation by ERKs.
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PMID:Receptor tyrosine kinases inhibit bone morphogenetic protein-Smad responsive promoter activity and differentiation of murine MC3T3-E1 osteoblast-like cells. 1273 21

Bone morphogenetic proteins (BMPs) are shown to potentiate NGF-induced neuronal differentiation in PC12 phaeochromocytoma cells grown on collagen under low-serum conditions. Whereas, cell bodies remained rounded in control medium or with only BMPs present, addition of BMP4 or BMP6 robustly increased the neuritogenic effect of NGF within 2 days. NGF-increased phosphorylation of p44(Erk1) and p42(Erk2) between 2 and 24h was unaffected by addition of BMP6. PC12 cells transfected with the SBE(4x)-luc reporter showed that BMP4 significantly increased receptor-activated Smad activity. Expression of constitutively active BMP receptor ALK2 activating Smad1 and Smad5 resulted in a strong increase in the SBE(4x)-luc reporter response. Adding the inhibitory Smad7 drastically reduced this signal. In contrast to wild-type (wt) Smad5, a Smad5 variant lacking five Erk phosphorylation sites in the linker region (designated Smad5/5SA) showed a strong background transcriptional activity. A fusion construct (Gal4-Smad5/5SA) was also highly transcriptionally active. Addition of the MEK inhibitor U0126 to PC12 cells expressing Gal4-Smad5/wt did not increase background transcriptional activity. However, upon activation by constitutively active ALK2 both Gal4-Smad5/wt and Gal4-Smad5/5SA strongly stimulated transcription. The data show that serine residues of the linker region of Smad5 reduce spontaneous transcriptional activity and that NGF-activated Erk does not antagonise BMP signalling at this site. Hence, NGF and BMP signals are likely to interact further downstream at the transcriptional level in neuronal differentiation of the PC12 cells.
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PMID:Bone morphogenetic protein signalling in NGF-stimulated PC12 cells. 1289 70

The role of TGF-beta/bone morphogenetic protein signaling in the chondrogenic differentiation of human synovial fibroblasts (SFs) was examined with the adenovirus vector-mediated gene transduction system. Expression of constitutively active activin receptor-like kinase 3 (ALK3CA) induced chondrocyte-specific gene expression in SFs cultured in pellets or in SF pellets transplanted into nude mice, in which both the Smad and p38 pathways are essential. To analyze downstream cascades of ALK3 signaling, we utilized adenovirus vectors carrying either Smad1 to stimulate Smad pathways or constitutively active MKK6 (MKK6CA) to activate p38 pathways. Smad1 expression had a synergistic effect on ALK3CA, while activation of p38 MAP kinase pathways alone by transduction of MKK6CA accelerated terminal chondrocytic differentiation, leading to type X collagen expression and enhanced mineralization. Overexpression of Smad1 prevented MKK6CA-induced type X collagen expression and maintained type II collagen expression. In a mouse model of osteoarthritis, activated p38 expression as well as type X collagen staining was detected in osteochondrophytes and marginal synovial cells. These results suggest that SFs can be differentiated into chondrocytes via ALK3 activation and that stimulating Smad pathways and controlling p38 activation at the proper level can be a good therapeutic strategy for maintaining the healthy joint homeostasis and treating degenerative joint disorders.
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PMID:Distinct roles of Smad pathways and p38 pathways in cartilage-specific gene expression in synovial fibroblasts. 1499 Oct 70

Bone morphogenetic proteins (BMPs) 4 and 6 as well as MEK inhibitors PD98059 and U0126 potentiate neurotrophin 3 (NT3)- and neurturin (NTN)-induced neurite outgrowth and survival of peripheral neurons from the E9 chicken embryo. Preexposure to BMP4 or PD98059 was sufficient to prime the potentiation of subsequently added NT3. Phosphorylation of Erk2, induced by NT3, was reduced by MEK inhibition but unaffected by BMP signaling. Real-time PCR showed that neither BMP stimulation nor MEK inhibition increased Trk receptor expression and that the BMP-induced genes Smad6 and Id1 were not upregulated by PD98059. In contrast, both MEK inhibition and BMP signaling suppressed transcription of the serum-response element (SRE)-driven Egr1 gene. A reporter assay using NGF-stimulated PC12 cells demonstrated that MEK/Erk/Elk-driven transcriptional activity was inhibited by Smad1/5 and by PD98059. Thus, suppression of SRE-controlled transcription represents a likely convergence point for pathways regulating neurotrophic responses.
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PMID:Blocked MAP kinase activity selectively enhances neurotrophic growth responses. 1501 50

NGF activates several signaling cascades in sympathetic neurons. We examined how activation of one of these cascades, the ERK/MAP (extracellular signal-regulated kinase/mitogen-activated protein) kinase pathway, affects dendritic growth in these cells. Dendritic growth was induced by exposure to NGF and BMP-7 (bone morphogenetic protein-7). Exposure to NGF increased phosphorylation of ERK1/2. Unexpectedly, two MEK (MAP kinase kinase) inhibitors (PD 98059 and U 0126) enhanced dendritic growth, and a ligand, basic FGF, that activates the ERK pathway inhibited the growth of these processes. The enhancement of dendritic growth by PD 98059 was associated with an increase in the number of axo-dendritic synapses, and it appeared to represent a specific morphogenic effect because neither axonal growth nor cell survival was affected. In addition, increased dendritic growth was not observed after exposure to inhibitors of other signaling pathways, including the phosphatidylinositol-3-kinase inhibitor LY 294002. Dendritic growth was also increased in cells transfected with dominant-negative mutants of MEK1 and ERK2 but not with dominant-negative mutants of MEK5 and ERK5, suggesting that ERK1/2 is the primary mediator of this effect. Exposure to BMP-7 induces nuclear translocation of Smad1 (Sma- and Mad-related protein 1), and PD 98059 treatment potentiated nuclear accumulation of Smad-1 induced by BMP-7 in sympathetic neurons, suggesting a direct enhancement of BMP signaling in cells treated with an MEK inhibitor. These observations indicate that one of the signaling cascades activated by NGF can act in an antagonistic manner in sympathetic neurons and reduce the dendritic growth induced by other NGF-sensitive pathways.
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PMID:Extracellular signal-regulated kinases regulate dendritic growth in rat sympathetic neurons. 1505 10

Tubulogenesis by epithelial cells regulates kidney, lung, and mammary development, whereas that by endothelial cells regulates vascular development. Although functionally dissimilar, the processes necessary for tubulation by epithelial and endothelial cells are very similar. We performed microarray analysis to further our understanding of tubulogenesis and observed a robust induction of regulator of G protein signaling 4 (RGS4) mRNA expression solely in tubulating cells, thereby implicating RGS4 as a potential regulator of tubulogenesis. Accordingly, RGS4 overexpression delayed and altered lung epithelial cell tubulation by selectively inhibiting G protein-mediated p38 MAPK activation, and, consequently, by reducing epithelial cell proliferation, migration, and expression of vascular endothelial growth factor (VEGF). The tubulogenic defects imparted by RGS4 in epithelial cells, including its reduction in VEGF expression, were rescued by overexpression of constitutively active MKK6, an activator of p38 MAPK. Similarly, RGS4 overexpression abrogated endothelial cell angiogenic sprouting by inhibiting their synthesis of DNA and invasion through synthetic basement membranes. We further show that RGS4 expression antagonized VEGF stimulation of DNA synthesis and extracellular signal-regulated kinase (ERK)1/ERK2 and p38 MAPK activation as well as ERK1/ERK2 activation stimulated by endothelin-1 and angiotensin II. RGS4 had no effect on the phosphorylation of Smad1 and Smad2 by bone morphogenic protein-7 and transforming growth factor-beta, respectively, indicating that RGS4 selectively inhibits G protein and VEGF signaling in endothelial cells. Finally, we found that RGS4 reduced endothelial cell response to VEGF by decreasing VEGF receptor-2 (KDR) expression. We therefore propose RGS4 as a novel antagonist of epithelial and endothelial cell tubulogenesis that selectively antagonizes intracellular signaling by G proteins and VEGF, thereby inhibiting cell proliferation, migration, and invasion, and VEGF and KDR expression.
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PMID:Identification and characterization of regulator of G protein signaling 4 (RGS4) as a novel inhibitor of tubulogenesis: RGS4 inhibits mitogen-activated protein kinases and vascular endothelial growth factor signaling. 1554


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