(134f) Manipulation and Kinetic Analysis of Crosstalk between Pi 3-Kinase/Akt and Ras/Erk Signal Transduction Pathways

The analysis of signal transduction networks, which control cell behavior in the proper context of external stimuli, is confounded by crosstalk interactions among the so-called “pathways” that represent the presumably dominant routes of information flow within the cell. In previous studies, we characterized the mechanism of crosstalk inherent in the direct interaction between Ras-GTP and phosphoinositide (PI) 3-kinase, signaling enzymes that play a decisive role in cell proliferation, survival, and migration. That analysis was performed using cells expressing Ras variants to modulate the level of Ras-GTP, and stimulation with platelet-derived growth factor (PDGF) to activate cell surface receptors, in conjunction with an equilibrium binding model to characterize the interactions among activated receptors, PI 3-kinase, and Ras-GTP [Kaur et al., Biochem. J., 393: 235 (2006)]. We concluded that these molecules form a ternary complex in a cooperative manner, as expected from the plasma membrane localization of receptors and Ras. We have significantly extended this analysis on two fronts. Through quantitative measurements, the activation kinetics of the PI 3-kinase/Akt and Ras/Erk pathways have been surveyed in cells expressing Ras variants or treated with pharmacological inhibitors. Together with kinetic models and Brownian dynamics computations, we have systematically assessed the consistency of the previous Ras-PI 3-kinase interaction model in light of additional time course data, and we have shed light on the interactions between these pathways further downstream.