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)

There is emerging evidence indicating that smooth muscle contraction and Ca2+ influx through voltage-dependent L-type Ca2+ channels are regulated by tyrosine kinases; however, the specific kinases involved are largely unknown. In rabbit colonic muscularis mucosae cells, tyrosine-phosphorylated proteins of approximately 60 and 125 kDa were observed in immunoblots using an anti-phosphotyrosine antibody and were identified as c-Src and focal adhesion kinase (FAK) by immunoblotting with specific antibodies. FAK co-immunoprecipitated with c-Src, and the phosphorylation of the c-Src.FAK complex was markedly enhanced by platelet-derived growth factor (PDGF) BB. The presence of activated c-Src in unstimulated cells was identified in cell lysates by immunoblotting with an antibody recognizing the autophosphorylated site (P416Y). In whole-cell patch-clamp studies, intracellular dialysis of a Src substrate peptide and anti-c-Src and anti-FAK antibodies suppressed Ca2+ currents by 60, 62, and 43%, respectively. In contrast, intracellular dialysis of an anti-mouse IgG or anti-Kv1.5 antibody did not inhibit Ca2+ currents. Co-dialysis of anti-c-Src and anti-FAK antibodies inhibited Ca2+ currents (63%) equivalent to dialysis with the anti-c-Src antibody alone. PDGF-BB enhanced Ca2+ currents by 43%, which was abolished by the anti-c-Src and anti-FAK antibodies. Neither the MEK inhibitor PD 098059 nor an anti-Ras antibody inhibited basal Ca2+ currents or PDGF-stimulated Ca2+ currents. The alpha1C subunit of the L-type Ca2+ channel co-immunoprecipitated with anti-c-Src and anti-phosphotyrosine antibodies, indicating direct association of c-Src kinase with the Ca2+ channel. These data suggest that c-Src and FAK, but not the Ras/mitogen-activated protein kinase cascade, modulate basal Ca2+ channel activity and mediate the PDGF-induced enhancement of L-type Ca2+ currents in differentiated smooth muscle cells.
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PMID:Modulation of voltage-dependent Ca2+ channels in rabbit colonic smooth muscle cells by c-Src and focal adhesion kinase. 947 93

We have identified two RHAMM (receptor for hyaluronan-mediated motility) isoforms that encode an alternatively spliced exon 4 (Hall, C. L., Yang, B., Yang, X., Zhang, S., Turley, M., Samuel, S., Lange, L. A., Wang, C., Curpen, G. D., Savani, R. C., Greenberg, A. H., and Turley, E. A. (1995) Cell 82, 19-26 and Wang, C., Entwistle, J., Hou, G., Li, Q., and Turley, E. A. (1996) Gene 174, 299-306). One of these, RHAMM variant 4 (RHAMMv4), is transforming when overexpressed and regulates Ras signaling (Hall et al.). Here we note using flow cytometry and confocal analysis that RHAMM isoforms encoding exon 4 occur both on the cell surface and in the cytoplasm. Epitope-tagging experiments indicate that RHAMMv4 occurs only in the cytoplasm. Several observations suggest that both cell surface RHAMM isoforms and RHAMMv4 are involved in regulating extracellular-regulated kinase (ERK) activity. Affinity-purified anti-RHAMM exon 4 antibodies block the ability of platelet-derived growth factor to activate ERK, and these reagents modify the protein tyrosine phosphorylation profile of proteins resulting from treatment with platelet-derived growth factor. A dominant negative form of RHAMMv4 inhibits mutant active Ras activation of ERK and coimmunoprecipitates with both mitogen-activated protein kinase kinase and ERK, suggesting that the intracellular RHAMMv4 acts downstream of Ras, possibly at the level of mitogen-activated protein kinase kinase-ERK interactions. Consistent with this, overexpression of RHAMMv4 constitutively activates ERK. These results identify a novel mechanism for the regulation of the Ras-ERK signaling pathway and suggest that RHAMM plays multiple roles in this regulation.
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PMID:The hyaluronan receptor RHAMM regulates extracellular-regulated kinase. 955 28

Cells grown in 3-dimensional collagen gels adopt a nonproliferative, contractile phenotype which is more characteristic of cells in vivo than cells grown in 2-dimensional culture. The floating collagen gel contraction assay is a well-defined system used to study cell-extracellular matrix interactions grown in 3-dimensional culture. Although the cell biology of this system is well defined, the cell signaling associated with gel contraction has not been well characterized. In this study we demonstrate that fetal bovine (FBS) and platelet-derived growth factor (PDGF)-induced mesangial cell-collagen gel contraction is associated with increased tyrosine phosphorylation of a number of proteins including focal adhesion kinase (FAK) and the 42-kDa isoform of MAPK (ERK2). FBS-induced gel contraction is not affected by the presence of the MEK inhibitor PD098059. Low concentrations of PDGF-BB (10 ng/ml) induce gel contraction; however, at higher PDGF-BB concentrations (80 ng/ml) gel contraction is not observed. PDGF-BB-induced gel contraction as well as tyrosine phosphorylation of FAK are inhibited in the presence of the PI-3 kinase inhibitor wortmanin. Minimal autophosphorylation of the PDGF-beta receptor is observed under 3-dimensional culture conditions following PDGF-BB stimulation; however, when mesangial cells grown in 2-dimensional culture are exposed to PDGF-BB, the PDGF-beta receptor was prominently phosphorylated. We conclude that induction of collagen gel contraction by FBS and PDGF-BB is associated with tyrosine kinase phosphorylation and that these responses differ substantially from what occurs in 2-dimensional cultures in the presence of the same agonists.
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PMID:Tyrosine kinase cell signaling pathways of rat mesangial cells in 3-dimensional cultures: response to fetal bovine serum and platelet-derived growth factor-BB. 957 Sep 28

Proteins comprising the mitogen-activated protein (MAP) kinase signaling cascade are activated by a variety of growth factors, but the precise role of this series of kinase reactions, especially Raf kinase and MAP kinase kinase (MEK), in vascular smooth muscle (VSM) cell mitogenesis is not known. In this study, a specific and selective inhibitor of MEK, PD-98059, was used to examine the role of MEK in DNA synthesis and Raf-1 activity in VSM cells stimulated with serum as well as with growth factors encompassing both tyrosine kinase and G protein-coupled receptor classes. Although significant increases in DNA synthesis are seen after stimulation of VSM cells with either 10% serum,platelet-derived growth factor (PDGF)-BB, or alpha-thrombin, preincubation of the cells with 50 microM PD-98059 for 1 h inhibits stimulation by PDGF and thrombin, but not by serum. There is a dose-dependent inhibition of the mitogenic effect by PD-98059 in all cases; these results are not affected when PD-98059 is added at times ranging from 4 h before to 2 h after growth factor addition (times at which PD-98059 exerts its inhibitory effect). In the presence of PD-98059, stimulated MAP kinase activity is attenuated when growth factors are added between 10 min and 4 h, times which correspond to both early and sustained phases of MAP kinase activity. In addition, Raf-1 activity is markedly increased by incubation of the cells with PD-98059,despite attenuation of hyperphosphorylation of this kinase. Thus growth factors coupled to both tyrosine kinase and G protein receptors require components of the MAP kinase cascade (MEK and/or MAP kinase) for VSM cell mitogenesis, whereas serum is capable of stimulatory effects in the absence of active MEK and MAP kinase. Furthermore, there exists a functional feedback stimulatory effect of inhibited MEK on its upstream activator Raf-1 in the case of serum as well as growth factors coupled to tyrosine kinase and G protein receptors.
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PMID:MEK inhibition augments Raf activity, but has variable effects on mitogenesis, in vascular smooth muscle cells. 969 94

In fibroblasts transforming growth factor-beta1 (TGF-beta1) regulates cell proliferation and turnover of macromolecular components of the extracellular matrix. Here, intracellular signaling events in growth-inhibited embryonic rat lung fibroblasts (RFL-6) upon stimulation with TGF-beta1 were investigated. TGF-beta1 rapidly induced the activation of c-Raf-1, MEK-1, and MAPK p42 and p44. The activation of this pathway by TGF-beta1 did not depend on autocrine platelet-derived growth factor (PDGF) or basic fibroblast growth factor (bFGF). Inhibition of the binding of growth factors to their tyrosine kinase receptors did not affect MAPK activation by TGF-beta1. Ras activation by TGF-beta1 was significantly lower compared to the activation by PDGF or bFGF. The intracellular transduction of the TGF-beta1 signal was completely suppressed by depletion or inhibition of protein kinase C (PKC). It is shown that calcium-dependent isoforms of PKC are required for MAPK activation by TGF-beta1.
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PMID:Transforming growth factor-beta1-induced activation of the Raf-MEK-MAPK signaling pathway in rat lung fibroblasts via a PKC-dependent mechanism. 971 18

Phosphatidylinositol 3-kinase (PI3K) activation is necessary for insulin-responsive glucose transporter (GLUT4) translocation and glucose transport. Insulin and platelet-derived growth factor (PDGF) stimulate PI3K activity in 3T3-L1 adipocytes, but only insulin is capable of stimulating GLUT4 translocation and glucose transport. We found that PDGF causes serine/threonine phosphorylation of insulin receptor substrate 1 (IRS-1) in 3T3-L1 cells, measured by altered mobility on SDS-polyacrylamide gel, and this leads to a decrease in insulin-stimulated tyrosine phosphorylation of IRS-1. The PI3K inhibitors wortmannin and LY294002 inhibit the PDGF-induced phosphorylation of IRS-1, whereas the MEK inhibitor PD98059 was without a major effect. PDGF pretreatment for 60-90 min led to a marked 80-90% reduction in insulin stimulatable phosphotyrosine and IRS-1-associated PI3K activity. We examined the functional consequences of this decrease in IRS-1-associated PI3K activity. Interestingly, insulin stimulation of GLUT4 translocation and glucose transport was unaffected by 60-90 min of PDGF preincubation. Furthermore, insulin activation of Akt and p70(s6kinase), kinases downstream of PI3K, was unaffected by PDGF pretreatment. Wortmannin was capable of blocking these insulin actions following PDGF pretreatment, suggesting that PI3K was still necessary for these effects. In conclusion, 1) PDGF causes serine/threonine phosphorylation of IRS-1, and PI3K, or a kinase downstream of PI3K, mediates this phosphorylation. 2) This PDGF-induced phosphorylation of IRS-1 leads to a significant decrease in insulin-stimulated PI3K activity. 3) PDGF has no effect on insulin stimulation of Akt, p70(s6kinase), GLUT4 translocation, or glucose transport. 4) This suggests the existence of an IRS-1-independent pathway leading to the activation of PI3K, Akt, and p70(s6kinase); GLUT4 translocation; and glucose transport.
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PMID:Platelet-derived growth factor inhibits insulin stimulation of insulin receptor substrate-1-associated phosphatidylinositol 3-kinase in 3T3-L1 adipocytes without affecting glucose transport. 973 73

Mitogen-activated protein (MAP) kinase cascades are major signaling systems by which cells transduce extracellular cues into intracellular responses. In general, MAP kinases are activated by phosphorylation on tyrosine and threonine residues and inactivated by dephosphorylation. Therefore, MAP kinase phosphatase-1 (MKP-1), a dual-specificity protein tyrosine phosphatase that exhibits catalytic activity toward both regulatory sites on MAP kinases, is suggested to be responsible for the downregulation of extracellular signal-regulated kinase (ERK), stress-activated protein kinase (SAPK), and p38 MAP kinase. In the present study, we examined the role of these MAP kinases in the induction of MKP-1 in vascular smooth muscle cells (VSMCs). Extracellular stimuli such as platelet-derived growth factor (PDGF), 12-O-tetradecanoylphorbol 13-acetate (TPA), and angiotensin II, which activated ERK but not SAPK/p38 MAP kinase, induced a transient induction of MKP-1 mRNA and its intracellular protein. In addition, PD 098059, an antagonist of MEK (MAP kinase/ERK kinase), the upstream kinase of ERK, significantly reduced the PDGF-induced activation of ERK and potently inhibited the expression of MKP-1 after stimulation with PDGF, thereby demonstrating the induction of MKP-1 in response to activation of the ERK signaling cascade. Furthermore, anisomycin, a potent stimulus of SAPK and p38 MAP kinase, also induced MKP-1 mRNA expression. This effect of anisomycin was significantly inhibited in the presence of the p38 MAP kinase antagonist SB 203580. These data suggest the induction of MKP-1, not only after stimulation of the cell growth promoting ERK pathway but also in response to activation of stress-responsive MAP kinase signaling cascades. We suggest that this pattern of MKP-1 induction may be a negative feedback mechanism in the control of MAP kinase activity in VSMCs.
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PMID:Regulation of mitogen-activated protein kinase phosphatase-1 in vascular smooth muscle cells. 977 60

Fibronectin seems to play an important role in promoting the characteristic changes of vascular smooth muscle cells in diabetes mellitus including overexpression of the platelet-derived growth factor beta-receptor. To determine the regulatory mechanism of the beta-receptor by fibronectin, we have analyzed the effect of fibronectin on the expression of the beta-receptor in cultured rat aortic smooth muscle cells using the beta-receptor promoter/luciferase expression vector system. Fibronectin was found to stimulate the expression of the beta-receptor at the transcriptional level. Both a MEK1 inhibitor PD98059 and a tyrosine kinase inhibitor herbimycin A significantly inhibited the fibronectin-stimulated receptor transcription. Herbimycin A also completely inhibited the fibronectin-stimulated increase in tyrosine phosphorylation of focal adhesion kinase. These data suggest the involvement of the integrin-mediated mitogen-activated protein kinase pathway downstream of fibronectin stimulation in the activation process of the beta-receptor promoter.
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PMID:Fibronectin stimulates transcription of the platelet-derived growth factor beta-receptor in cultured rat aortic smooth muscle cells. 979 Sep 68

The tumor suppressor PTEN dephosphorylates focal adhesion kinase (FAK) and inhibits integrin-mediated cell spreading and cell migration. We demonstrate here that expression of PTEN selectively inhibits activation of the extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) pathway. PTEN expression in glioblastoma cells lacking the protein resulted in inhibition of integrin-mediated MAP kinase activation. Epidermal growth factor (EGF) and platelet-derived growth factor (PDGF)- induced MAPK activation were also blocked. To determine the specific point of inhibition in the Ras/Raf/ MEK/ERK pathway, we examined these components after stimulation by fibronectin or growth factors. Shc phosphorylation and Ras activity were inhibited by expression of PTEN, whereas EGF receptor autophosphorylation was unaffected. The ability of cells to spread at normal rates was partially rescued by coexpression of constitutively activated MEK1, a downstream component of the pathway. In addition, focal contact formation was enhanced as indicated by paxillin staining. The phosphatase domain of PTEN was essential for all of these functions, because PTEN with an inactive phosphatase domain did not suppress MAP kinase or Ras activity. In contrast to its effects on ERK, PTEN expression did not affect c-Jun NH2-terminal kinase (JNK) or PDGF-stimulated Akt. Our data suggest that a general function of PTEN is to down-regulate FAK and Shc phosphorylation, Ras activity, downstream MAP kinase activation, and associated focal contact formation and cell spreading.
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PMID:Tumor suppressor PTEN inhibits integrin- and growth factor-mediated mitogen-activated protein (MAP) kinase signaling pathways. 983 64

Flt3 ligand (FL) is an early-acting potent co-stimulatory cytokine that regulates proliferation and differentiation of a number of blood cell lineages. Its receptor Flt3/Flk2 belongs to class III receptor tyrosine kinases that also include the receptors for colony-stimulating factor 1, Steel factor, and platelet-derived growth factor. Using CSF-1 receptor/Flt3 chimeras, two groups have characterized some of the post-receptor signaling events and substrate specificity of murine Flt3 receptor. However, there are few studies on the signaling pathway through human Flt3. We examined human Flt3 signaling pathways in a murine IL-3-dependent hematopoietic cell line Baf3, which stably expresses full-length human Flt3 receptor. This subline proliferates in response to human FL. Like the chimeric murine Flt3, human Flt3 undergoes autophosphorylation, associates with Grb2, and leads to tyrosine phosphorylation of Shc on ligand binding. We found that SHP-2, but not SHP-1, is tyrosine-phosphorylated by FL stimulation. SHP-2 does not associate with Flt3, but binds directly to Grb2. SHIP is also tyrosine-phosphorylated and associates with Shc after FL simulation. We further examined the downstream signaling pathway. FL transiently activates MAP kinase. This activation could be blocked by PD98059, a specific MEK inhibitor. PD98059 also blocked cell proliferation in response to FL. These results demonstrate that SHP-2 and SHIP are important components in the human Flt3 signaling pathway and suggest that SHP-2 and SHIP, by forming complexes with adapter proteins Grb2 and Shc, may modulate MAP kinase activation, which may be necessary for the mitogenic signaling of Flt3.
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PMID:Flt3 signaling involves tyrosyl-phosphorylation of SHP-2 and SHIP and their association with Grb2 and Shc in Baf3/Flt3 cells. 1008 May 42


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