epidermal-growth-factor and 5--methylthioadenosine

Fibroblast growth factor-2 (FGF2) activates the extracellular signal-regulated kinases 1 and 2 (ERK1/2) through its specific receptors. Interaction of FGF2 with cell-surface heparan sulfate proteoglycans has also been suggested to induce intracellular signals. Thus, we investigated whether FGF2 can stimulate ERK1/2 activation through heparan sulfate proteoglycans using mechanisms that do not depend on receptor activation in vascular smooth muscle cells. The activation of FGF receptors was inhibited by treating cells with 5'-deoxy-5'methyl-thioadenosine and by expressing truncated dominant-negative FGF receptors. In both cases, FGF2 was able to stimulate the phosphorylation of ERK1/2 despite the absence of detectable FGF receptor tyrosine kinase activity. The FGF2 activation of ERK1/2 in the absence of receptor activity was completely dependent on heparan sulfate, because this activity was abolished by heparinase III digestion of the cells. In contrast, heparinase III treatment of control cells, with functional FGF receptors, showed only slight changes in FGF2-mediated ERK1/2 activation kinetics. Thus, in addition to serving as coreceptors for FGF receptor activation, heparan sulfate proteoglycans might also function directly as receptors for FGF2-induced ERK1/2 activation. Activation of ERK1/2 via cell-surface proteoglycans could have significant biological consequences, potentially directing cell response toward growth, migration, or differentiation.

Topics: Animals; Cell Line; Cell Movement; CHO Cells; Cricetinae; Deoxyadenosines; DNA; Endothelial Cells; Enzyme Activation; Epidermal Growth Factor; Fibroblast Growth Factor 1; Fibroblast Growth Factor 10; Fibroblast Growth Factor 2; Fibroblast Growth Factors; Growth Substances; Heparan Sulfate Proteoglycans; Heparin-binding EGF-like Growth Factor; Heparitin Sulfate; Intercellular Signaling Peptides and Proteins; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Mutation; Polysaccharide-Lyases; Protein Binding; Receptor Protein-Tyrosine Kinases; Receptor, Fibroblast Growth Factor, Type 1; Receptors, Cell Surface; Receptors, Fibroblast Growth Factor; Thionucleosides

Stimulation of three human glioma cell lines with basic fibroblast growth factor (bFGF) led to the enhancement of cell growth and the rapid tyrosine phosphorylation of cellular proteins, including major substrates of 90 kD. A methyltransferase inhibitor, 5'-methylthioadenosine (MTA), inhibited dose dependently the bFGF-stimulated cell growth and protein tyrosine phosphorylation in glioma cells by blocking both receptor autophosphorylation and substrate phosphorylation, as shown by immunoblotting with antiphosphotyrosine antibodies and cross-linking bFGF to receptors. The antiproliferative activity of MTA correlated quantitatively with its potency as an inhibitor of bFGF-stimulated protein tyrosine kinase activity. The methyltransferase inhibitor MTA had no effect on either epidermal growth factor- or platelet-derived growth factor-stimulated protein tyrosine phosphorylation in glioma cells, but inhibited specifically bFGF-stimulated protein tyrosine kinase activity. The concentration of MTA required for inhibition of protein methylation correlated well with the concentration required for inhibition of bFGF-stimulated cell growth and protein tyrosine phosphorylation. Because MTA had no effect on numbers and dissociation constants of high- and low-affinity bFGF receptors, the inhibition of bFGF-stimulated bFGF receptor tyrosine kinase activity is not likely to be the result of a reduction in bFGF receptor and bFGF binding capacity. In fact, MTA delayed and reduced the internalization and nuclear translocation of bFGF, and the internalized bFGF was submitted to a limited proteolysis that converted it to lower molecular peptides whose presence remained for at least 22 hours. The effect of MTA on bFGF-stimulated tyrosine phosphorylation was immediate and readily reversible.

Topics: Antibodies; Cell Division; Cell Line; Cell Nucleus; Cross-Linking Reagents; Deoxyadenosines; Dose-Response Relationship, Drug; Epidermal Growth Factor; Fibroblast Growth Factor 2; Glioblastoma; Glioma; Humans; Immunoblotting; Iodine Radioisotopes; Methyltransferases; Neoplasm Proteins; Phosphorylation; Phosphotyrosine; Platelet-Derived Growth Factor; Precipitin Tests; Protein-Tyrosine Kinases; Receptors, Fibroblast Growth Factor; Thionucleosides; Tyrosine

Although epidermal growth factor (EGF) and nerve growth factor (NGF) have markedly different biological effects on PC-12 cells, many of the signaling events following ligand binding are similar. Both EGF and NGF result in the induction of the primary response gene egr-1/TIS8 and increased methylation of a variety of membrane-associated proteins as early as 5 min after EGF or NGF treatment using a methylation assay that detects methyl esters as well as methylated arginine residues. At 20 min after stimulation with these factors, the stimulation of methylation by NGF is greater than that of EGF, especially in the polypeptides of 36-42 and 20-22 kDa. To help dissect the pathways involved in these cellular responses, the protein kinase inhibitor K252a and the methyltransferase inhibitor 5'-methylthioadenosine (MTA) were used. Both K252a and MTA inhibit NGF-, but not EGF-mediated, primary response gene expression. In contrast, MTA, but not K252a, can block NGF-induced membrane associated protein methylation. These data suggest a role for differential protein methylation reactions in EGF and NGF signal transduction.

Topics: Animals; Carbazoles; Deoxyadenosines; DNA-Binding Proteins; Early Growth Response Protein 1; Epidermal Growth Factor; Gene Expression Regulation, Neoplastic; Immediate-Early Proteins; Indole Alkaloids; Membrane Proteins; Methylation; Neoplasm Proteins; Nerve Growth Factors; Nerve Tissue Proteins; PC12 Cells; Protein Kinase C; Protein Processing, Post-Translational; Rats; Signal Transduction; Thionucleosides; Transcription Factors

The effects of nerve growth factor (NGF) and epidermal growth factor (EGF) on the intracellular accumulation of inositol phosphates and on cytosolic free Ca2+ concentrations were studied in rat PC12 pheochromocytoma cells. Both NGF and EGF potentiate in these cells the increase in the accumulation of inositol phosphates that is elicited by bradykinin and carbachol. A corresponding potentiation was also found for the agonist-induced increase of cytosolic Ca2+ concentrations. The effect of NGF, but not that of EGF, is abolished when the cells are preincubated with 5'-deoxy-5'-methylthioadenosine, an inhibitor of S-adenosylhomocysteine hydrolase. These results suggest that an increased response to hormones, which act via phosphoinositide-derived second messengers, may be important in the mechanism of action of NGF and EGF.

Topics: Adenosine; Adrenal Gland Neoplasms; Animals; Bradykinin; Calcium; Cell Line; Deoxyadenosines; Dose-Response Relationship, Drug; Epidermal Growth Factor; Inositol Phosphates; Male; Mice; Nerve Growth Factors; Pheochromocytoma; Sugar Phosphates; Thionucleosides