scholarly journals Differential regulation of Raf-1 and B-Raf and Ras-dependent activation of mitogen-activated protein kinase by cyclic AMP in PC12 cells.

1995 ◽  
Vol 15 (10) ◽  
pp. 5524-5530 ◽  
Author(s):  
P Erhardt ◽  
J Troppmair ◽  
U R Rapp ◽  
G M Cooper
Keyword(s):  
Growth Factor ◽  
Cyclic Amp ◽  
Map Kinase ◽  
Pc12 Cells ◽  
Dominant Negative ◽  
Mitogen Activated Protein Kinase ◽  
Mitogen Activated Protein ◽  
Map Kinase Pathway ◽  
Kinase Pathway ◽  
Stimulation Of

Growth factor stimulation of the mitogen-activated protein (MAP) kinase pathway in fibroblasts is inhibited by cyclic AMP (cAMP) as a result of inhibition of Raf-1. In contrast, cAMP inhibits neither nerve growth factor-induced MAP kinase activation nor differentiation in PC12 pheochromocytoma cells. Instead, in PC12 cells cAMP activates MAP kinase. Since one of the major differences between the Ras/Raf/MAP kinase cascades of these cell types is the expression of B-Raf in PC12 cells, we compared the effects of cAMP on Raf-1 and B-Raf. In PC12 cells maintained in serum-containing medium, B-Raf was refractory to inhibition by cAMP, whereas Raf-1 was effectively inhibited. In contrast, both B-Raf and Raf-1 were inhibited by cAMP in serum-starved PC12 cells. The effect of cAMP is thus dependent upon growth conditions, with B-Raf being resistant to cAMP inhibition in the presence of serum. These results were extended by studies of Rat-1 fibroblasts into which B-Raf had been introduced by transfection. As in PC12 cells, B-Raf was resistant to inhibition by cAMP in the presence of serum, whereas Raf-1 was effectively inhibited. In addition, the expression of B-Raf rendered Rat-1 cells resistant to the inhibitory effects of cAMP on both growth factor-induced activation of MAP kinase and mitogenesis. These results indicate that Raf-1 and B-Raf are differentially sensitive to inhibition by cAMP and that B-Raf expression can contribute to cell type-specific differences in the regulation of the MAP kinase pathway. In contrast to the situation in PC12 cells, cAMP by itself did not stimulate MAP kinase in B-Raf-expressing Rat-1 cells. The activation of MAP kinase by cAMP in PC12 cells was inhibited by the expression of a dominant negative Ras mutant, indicating that cAMP acts on a target upstream of Ras. Thus, it appears that a signaling component upstream of Ras is also require for cAMP stimulation of MAP kinase in PC12 cells.

scholarly journals Mitogen-activated protein kinase activation is insufficient for growth factor receptor-mediated PC12 cell differentiation.

1995 ◽  
Vol 15 (7) ◽  
pp. 3644-3653 ◽  
Author(s):  
R R Vaillancourt ◽  
L E Heasley ◽  
J Zamarripa ◽  
B Storey ◽  
M Valius ◽  
...  
Keyword(s):  
Cell Differentiation ◽  
Growth Factor ◽  
Map Kinase ◽  
Pc12 Cells ◽  
Pc12 Cell ◽  
Growth Factor Receptor ◽  
Receptor Activation ◽  
Mitogen Activated Protein ◽  
Map Kinase Pathway ◽  
Kinase Pathway

When expressed in PC12 cells, the platelet-derived growth factor beta receptor (beta PDGF-R) mediates cell differentiation. Mutational analysis of the beta PDGF-R indicated that persistent receptor stimulation of the Ras/Raf/mitogen-activated protein (MAP) kinase pathway alone was insufficient to sustain PC12 cell differentiation. PDGF receptor activation of signal pathways involving p60c-src or the persistent regulation of phospholipase C gamma was required for PC12 cell differentiation. beta PDGF-R regulation of phosphatidylinositol 3-kinase, the GTPase-activating protein of Ras, and the tyrosine phosphatase, Syp, was not required for PC12 cell differentiation. In contrast to overexpression of oncoproteins involved in regulating the MAP kinase pathway, growth factor receptor-mediated differentiation of PC12 cells requires the integration of other signals with the Ras/Raf/MAP kinase pathway.

Dibucaine and Tetracaine Inhibit the Activation of Mitogen-activated Protein Kinase Mediated by L-type Calcium Channels in PC12 Cells 

1999 ◽  
Vol 91 (6) ◽  
pp. 1798-1798 ◽  
Author(s):  
Masumi Kansha ◽  
Taro Nagata ◽  
Kazuo Irita ◽  
Shosuke Takahashi
Keyword(s):  
Map Kinase ◽  
Pc12 Cells ◽  
Local Anesthetics ◽  
Mitogen Activated Protein ◽  
Ic50 Values ◽  
Map Kinase Pathway ◽  
Kinase Signaling Pathway ◽  
Kinase Pathway ◽  
Map Kinase Signaling Pathway ◽  
Ca2 Channels

Background An elevation of the intracellular calcium level, which is mediated by N-methyl-D-aspartate receptors and L-type Ca2+ channels both, activates the mitogen-activated protein (MAP) kinase signaling pathway involved in synaptic modification. It has recently been suggested that MAP kinase plays a role in coupling the synaptic excitation to gene expression in the nucleus of postsynaptic neurons. Because the effects of local anesthetics on cellular signal transduction in neuronal cells are not well-known, the authors investigated whether they affect the MAP kinase signaling pathway using PC12 cells. Methods The cells were stimulated with either 50 mM KCl or 1 microM ionomycin, and activated MAP kinase was thus immunoprecipitated. The immunocomplexes were then subjected to an Elk1 phosphorylation assay. Both the phosphorylation of MAP kinase and the induction of c-Fos were detected by immunoblotting. Results Pretreatment of the cells with 1 mM (ethylenedioxy)-diethyl-enedinitrilotetraacetic acid or 5 micron nifedipine blocked the MAP kinase activation induced by 50 mM KCl, whereas pretreatment with 2 microM omega-conotoxin GIVA did not. The expression of c-Fos induced by potassium chloride was also suppressed by dibucaine, tetracaine (concentrations that inhibited 50% of the activity of positive control [IC50s] were 16.2+/-0.2 and 73.2+/-0.7 microM, respectively), and PD 98059, a mitogen-activated/extracellular receptor-regulated kinase inhibitor. Higher concentrations of dibucaine and tetracaine were needed to suppress the activation of MAP kinase induced by ionomycin (the IC50 values of dibucaine and tetracaine were 62.5+/-2.2 and 330.5+/-32.8 microM, respectively) compared with potassium chloride (the IC50 values of dibucaine and tetracaine were 17.7+/-1.0 and 70.2+/-1.2 microM, respectively). Although probable targets of these local anesthetics might be L-type Ca2+ channels or components between Ca2+ and Ras in MAP kinase pathway, the possibility that they directly affect MAP kinase still remains. Conclusions Dibucaine and tetracaine at clinical concentrations were found to inhibit the activation of MAP kinase and the expression of c-Fos mediated by L-type Ca2+ channels in PC12 cells. The suppression of MAP kinase pathway may thus be a potential target site for the actions of dibucaine and tetracaine, including the modification of the synaptic functions.

scholarly journals The K5 Capsule of Escherichia coli Strain Nissle 1917 Is Important in Stimulating Expression of Toll-Like Receptor 5, CD14, MyD88, and TRIF Together with the Induction of Interleukin-8 Expression via the Mitogen-Activated Protein Kinase Pathway in Epithelial Cells

2010 ◽  
Vol 78 (5) ◽  
pp. 2153-2162 ◽  
Author(s):  
Mohamed Hafez ◽  
Kelly Hayes ◽  
Marie Goldrick ◽  
Richard K. Grencis ◽  
Ian S. Roberts
Keyword(s):  
Escherichia Coli ◽  
Epithelial Cells ◽  
Map Kinase ◽  
Interleukin 8 ◽  
Mitogen Activated Protein Kinase ◽  
Probiotic Properties ◽  
Toll Like Receptor ◽  
Mitogen Activated Protein ◽  
Map Kinase Pathway ◽  
Kinase Pathway

ABSTRACT Escherichia coli strain Nissle 1917, which has been widely used as a probiotic for the treatment of inflammatory bowel disorders, expresses a K5 capsule, the expression of which is often associated with extraintestinal and urinary tract isolates of E. coli. Previously, it had been shown that the expression of a K5 capsule by Nissle 1917 was important in mediating interactions with epithelial cells and the extent of chemokine expression. In this paper, we show that infection with Nissle 1917 induces expression of Toll-like receptor 4 (TLR4) and TLR5 in Caco-2 cells and that maximal induction of TLR5 required the K5 capsule. In addition, purified K5 polysaccharide was capable of inducing expression of TLR5 and mCD14 and potentiated the activity of both TLR4 and TLR5 agonists to increase the proinflammatory response. Infection with Nissle 1917 also increased the expression of the adaptor molecules MyD88 and TRIF, which was K5 capsule dependent. By Western blot analysis, it was possible to show that induction of interleukin-8 by Nissle 1917 was predominantly through the mitogen-activated protein (MAP) kinase pathway and that expression of the K5 capsule was important for activation of the MAP kinase pathway. This paper provides new information on the function of the K5 capsule in mediating interactions between Nissle 1917 and epithelial cells and the mechanisms that underlie the probiotic properties of Nissle 1917.

scholarly journals Nitric Oxide Synthase Inhibitors Modulate Nerve-Growth-Factor Mediated Activation of Akt

2012 ◽  
Vol 2012 ◽  
pp. 1-11 ◽  
Author(s):  
Cheryl L. Cragg ◽  
Janet C. MacKinnon ◽  
Bettina E. Kalisch
Keyword(s):  
Nitric Oxide ◽  
Nerve Growth Factor ◽  
Growth Factor ◽  
Map Kinase ◽  
Pc12 Cells ◽  
Cell Lines ◽  
Nerve Growth ◽  
Akt Phosphorylation ◽  
Map Kinase Pathway ◽  
Kinase Pathway

Nitric oxide (NO) modulates nerve-growth-factor- (NGF-) mediated signaling and gene expression. In the present paper, the role of NO in NGF-mediated Akt activation in PC12 and IMR32 cells was investigated. Cells were treated with NGF (50 ng/mL) in the presence or absence of NO synthase (NOS) inhibitors and Akt phosphorylation assessed by western blot analysis. In both cell lines, Akt was phosphorylated within 15 min of NGF treatment. In PC12 cells, this level of phosphorylation was sustained for 60 min, while in IMR32 cells, the activation decreased after 30 min of NGF treatment. The nonselective NOS inhibitor Nω-nitro-L-arginine methylester (L-NAME; 20 mM) had no effect on NGF-mediated Akt phosphorylation in PC12 cells but in combination with NGF, the iNOS selective inhibitor s-methylisothiourea (S-MIU; 2.0 mM) maintained Akt phosphorylation up to 2 h. In IMR32 cells, both L-NAME and S-MIU prolonged the activation of Akt. Pretreatment with 50 μM U0126, a MAP kinase pathway inhibitor, also increased the activation of Akt in both cell lines. These data suggest that NO modulates the duration of phosphorylation of Akt in response to NGF and that this effect may, in part, be mediated by the effects of NO on the Ras-MAP kinase pathway.

The mitogen-activated protein kinase cascade is activated by B-Raf in response to nerve growth factor through interaction with p21ras

1994 ◽  
Vol 14 (10) ◽  
pp. 6944-6953
Author(s):  
R K Jaiswal ◽  
S A Moodie ◽  
A Wolfman ◽  
G E Landreth
Keyword(s):  
Nerve Growth Factor ◽  
Growth Factor ◽  
Map Kinase ◽  
Pc12 Cells ◽  
Mitogen Activated Protein Kinase ◽  
Stable Complex ◽  
Nerve Growth ◽  
Map Kinase Cascade ◽  
Mitogen Activated Protein ◽  
Kinase Cascade

Nerve growth factor (NGF) activates the mitogen-activated protein (MAP) kinase cascade through a p21ras-dependent signal transduction pathway in PC12 cells. The linkage between p21ras and MEK1 was investigated to identify those elements which participate in the regulation of MEK1 activity. We have screened for MEK activators using a coupled assay in which the MAP kinase cascade has been reconstituted in vitro. We report that we have detected a single NGF-stimulated MEK-activating activity which has been identified as B-Raf. PC12 cells express both B-Raf and c-Raf1; however, the MEK-activating activity was found only in fractions containing B-Raf. c-Raf1-containing fractions did not exhibit a MEK-activating activity. Gel filtration analysis revealed that the B-Raf eluted with an apparent M(r) of 250,000 to 300,000, indicating that it is present within a stable complex with other unidentified proteins. Immunoprecipitation with B-Raf-specific antisera quantitatively precipitated all MEK activator activity from these fractions. We also demonstrate that B-Raf, as well as c-Raf1, directly interacted with activated p21ras immobilized on silica beads. NGF treatment of the cells had no effect on the ability of B-Raf or c-Raf1 to bind to activated p21ras. These data indicate that this interaction was not dependent upon the activation state of these enzymes; however, MEK kinase activity was found to be associated with p21ras following incubation with NGF-treated samples at levels higher than those obtained from unstimulated cells. These data provide direct evidence that NGF-stimulated B-Raf is responsible for the activation of the MAP kinase cascade in PC12 cells, whereas c-Raf1 activity was not found to function within this pathway.

scholarly journals ras2 Controls Morphogenesis, Pheromone Response, and Pathogenicity in the Fungal Pathogen Ustilago maydis

2002 ◽  
Vol 1 (6) ◽  
pp. 954-966 ◽  
Author(s):  
Nancy Lee ◽  
James W. Kronstad
Keyword(s):  
Protein Kinase ◽  
Map Kinase ◽  
Ustilago Maydis ◽  
Mitogen Activated Protein Kinase ◽  
Gene Encoding ◽  
Mitogen Activated Protein ◽  
Kinase Cascade ◽  
Map Kinase Pathway ◽  
Altered Cell ◽  
Kinase Pathway

ABSTRACT Ustilago maydis, a pathogen of maize, is a useful model for the analysis of mating, pathogenicity, and the morphological transition between budding and filamentous growth in fungi. As in other fungi, these processes are regulated by conserved signaling mechanisms, including the cyclic AMP (cAMP)/protein kinase A (PKA) pathway and at least one mitogen-activated protein kinase (MAP kinase) pathway. A current challenge is to identify additional factors that lie downstream of the cAMP pathway and that influence morphogenesis in U. maydis. In this study, we identified suppressor mutations that restored budding growth to a constitutively filamentous mutant with a defect in the gene encoding a catalytic subunit of PKA. Complementation of one suppressor mutation unexpectedly identified the ras2 gene, which is predicted to encode a member of the well-conserved ras family of small GTP-binding proteins. Deletion of the ras2 gene in haploid cells altered cell morphology, eliminated pathogenicity on maize seedlings, and revealed a role in the production of aerial hyphae during mating. We also used an activated ras2 allele to demonstrate that Ras2 promotes pseudohyphal growth via a MAP kinase cascade involving the MAP kinase kinase Fuz7 and the MAP kinase Ubc3. Overall, our results reveal an additional level of crosstalk between the cAMP signaling pathway and a MAP kinase pathway influenced by Ras2.

Bradykinin-stimulated protein synthesis by myocytes is dependent on the MAP kinase pathway and p70S6K

1999 ◽  
Vol 276 (4) ◽  
pp. H1393-H1398 ◽  
Author(s):  
Rebecca H. Ritchie ◽  
James D. Marsh ◽  
Rick J. Schiebinger
Keyword(s):  
Protein Synthesis ◽  
Map Kinase ◽  
Mrna Translation ◽  
S6 Kinase ◽  
Mitogen Activated Protein ◽  
Map Kinase Pathway ◽  
Ribosomal S6 Kinase ◽  
Pathway Inhibition ◽  
Kinase Pathway ◽  
Stimulation Of

Bradykinin (BK) has a direct hypertrophic effect on rat ventricular cardiomyocytes (VCM) as defined by an increase in protein synthesis and an increase in atrial natriuretic peptide mRNA and secretion. In the current study, we have examined the dependence of BK-induced protein synthesis on activation of 90-kDa ribosomal S6 kinase (p90rsk) and 70-kDa S6 kinase (p70S6K). Both of these kinases possess the ability to phosphorylate the ribosomal protein S6, which plays an important role in initiating mRNA translation. Stimulation of adult VCM with 10 μM BK increased p90rsk activity by 2.5 ± 0.3-fold and increased p70S6Kactivity by 2.0 ± 0.3-fold. p90rsk is a terminal kinase in the mitogen-activated protein (MAP) kinase pathway. Inhibition of MAP kinase kinase activation by Raf in the MAP kinase pathway with PD-098059 (25 μM) blocked BK-stimulated activation of p90rsk by 70% and unexpectedly blocked p70S6K by 72%. Rapamycin inhibited BK-stimulated p70S6Kactivity by 93% but had no effect on p90rsk activation by BK. Inhibition of the MAP kinase pathway and p70S6K with PD-098059 was paralleled by changes in protein synthesis. BK (10 μM) increased [3H]phenylalanine incorporation by 27 ± 3 and 39 ± 6% in cultured adult and neonatal VCM, respectively. Treatment with PD-098059 or rapamycin abolished the increase in protein synthesis stimulated by BK. These results suggest that 1) BK activates p70S6K and p90rsk; 2) although both p70S6K and p90rsk have the potential to phosphorylate the ribosomal S6 protein, p70S6K and not p90rsk is the predominant kinase involved in increasing protein synthesis by BK; and 3) p70S6K activation is dependent on stimulation of the MAP kinase pathway at a point distal to Raf.

scholarly journals A Mitogen-Activated Protein Kinase That Senses Nitrogen Regulates Conidial Germination and Growth in Aspergillus fumigatus

2004 ◽  
Vol 3 (2) ◽  
pp. 557-560 ◽  
Author(s):  
Tao Xue ◽  
C. Kim Nguyen ◽  
Angela Romans ◽  
Gregory S. May
Keyword(s):  
Protein Kinase ◽  
Aspergillus Fumigatus ◽  
Map Kinase ◽  
Conidial Germination ◽  
Mitogen Activated Protein Kinase ◽  
Protein Map ◽  
Mitogen Activated Protein ◽  
Map Kinase Pathway ◽  
Germination And Growth ◽  
Kinase Pathway

ABSTRACT We show that the mitogen-activated protein (MAP) kinase pathway that responds to osmotic stress in Aspergillus fumigatus is also involved in nutritional sensing. This MAP kinase regulates conidial germination in response to the nitrogen source and is activated upon starvation for either carbon or nitrogen during vegetative growth.

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