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. Using our Ras variant cells and specific pharmacological inhibitors, we are currently extending this analysis to evaluate 1) the kinetics of PI 3-kinase/Akt and Ras/Erk activation and 2) the interactions between these pathways downstream of Ras and PI 3-kinase.

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