Probing for drug-induced multiplex signal transduction pathways using high resolution two-dimensional gel electrophoresis: application to β-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 β-adrenoceptor (βAR)-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, NFκB/IκBα 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 βAR antagonist. In conclusion, an acute isoproterenol stimulus induced multiplex pathway modulation via the βAR 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.