The most widely studied signaling system in cell physiology is arguably the regulatory switch governing proliferation and programmed cell death. Not surprisingly, mitogens such as platelet-derived growth factor (PDGF) trigger proliferation, through the Ras/Erk pathway, as well as protection from cell death, through the phosphoinositide 3-kinase (PI3K)/Akt pathway. Confounding the analysis of these pathways are the numerous crosstalk interactions between them, which suggest that cell life and death are co-regulated. A quantitative understanding of crosstalk in signaling networks is thought to be a major hurdle in the design of molecular therapeutics targeting intracellular signaling proteins, with implications for cancer, wound healing, and immune cell regulation. Employing PDGF-stimulated signaling in NIH 3T3 fibroblasts as a model system, the magnitudes and kinetics of Ras/Erk and PI3K/Akt signaling will be manipulated through specific genetic and pharmacological interventions, and crosstalk will be assessed by measuring the sensitivities of other intermediates to those changes for various levels of receptor stimulation. This general strategy is termed crosstalk titration. Central to these efforts are quantitative, high-throughput cell biochemical assays for the levels of PDGF beta-receptor phosphorylation, Ras-GTP, Akt kinase activity, and Erk kinase activity. With extensive signaling data for various PDGF concentrations and intracellular manipulations, a mathematical model of the signaling network will be formulated to predict the outcomes of more complex intervention strategies. Finally, for conditions found to perturb the signaling network, the corresponding effects on cell proliferation and survival will be assessed.
