Probing for drug-induced multiplex signal transduction pathways using high resolution two-dimensional gel electrophoresis: application to beta-adrenoceptor stimulation in the rat C6 glioma cell.
Whole-cell [(32)P]-protein phosphorylation assays and two-dimensional gel electrophoresis (2-DGE) were applied to the analysis of the beta-adrenoceptor (betaAR)-linked signal transduction pathway. Rat C6 glioma cells were stimulated with isoproterenol and the protein lysates were resolved by 2-DGE. Two dimensional [(32)P]-phosphoprotein 'maps' were generated depicting the modulation of intracellular proteins after isoproterenol stimulation versus unstimulated cells. A total of 274 distinct phosphoprotein spots were detected, of which 200 were up-regulated, 69 were down-regulated, and 5 remained unchanged. An evaluation of isoproterenol's activity across several kinase pathways was performed using a computer-generated 2-DGE template incorporating the location and identification of individual signaling phosphoprotein intermediaries. The template served as a 'reference map' for drug treatment comparisons. We observed a significant increase in the phosphorylation states of several nuclear transcription factors, notably CREB-1, ATF-1, NFkappaB/IkappaBalpha and ELK-1, but not c-Jun. A parallel series of radioimmunoprecipitation studies confirmed our 2-DGE findings. Moreover, isoproterenol increased the phosphorylation state of PKC and of several MAPK-dependent pathway kinases which correlated with a significant increase in their endogenous kinase activity. Isoproterenol's effects on PKA, PKC and ERK-dependent activities were blocked by propranolol, a betaAR antagonist. In conclusion, an acute isoproterenol stimulus induced multiplex pathway modulation via the betaAR in the C6 glioma cell indicating that signaling pathway cross-talk is an essential feature for the regulation of cellular function. Moreover, the immediate advantages of the 2-DGE analytical approach were apparent, and further development of the protein database will provide a valuable tool to screen for broad-based drug-mediated signaling activities.
CNS Disorders, Wyeth-Ayerst Research, CN 8000, Princeton, NJ 08543-8000, USA.
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D000818:Animals; D001932:Brain Neoplasms; D000242:Cyclic AMP / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods; D005910:Glioma; D007545:Isoproterenol / Q000494:pharmacology*; D007700:Kinetics; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D009363:Neoplasm Proteins / Q000302:isolation & purification / Q000378:metabolism; D010750:Phosphoproteins / Q000302:isolation & purification / Q000378:metabolism*; D010766:Phosphorylation; D011433:Propranolol / Q000494:pharmacology; D011493:Protein Kinase C / Q000378:metabolism*; D051381:Rats; D011943:Receptors, Adrenergic, beta / Q000187:drug effects / Q000502:physiology*; D015398:Signal Transduction / Q000187:drug effects*; D014157:Transcription Factors / Q000302:isolation & purification / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D009363:Neoplasm Proteins; D010750:Phosphoproteins; D011943:Receptors, Adrenergic, beta; D014157:Transcription Factors; D011433:Propranolol; D000242:Cyclic AMP; D011493:Protein Kinase C; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D007545:Isoproterenol