Kinetic Analysis of PDGF Receptor Signaling Pathways in Fibroblasts

Abstract

Living cells perceive information about their surroundings and decode it in order to decide how to respond. All of this information flow is processed through intracellular signaling pathways. However, research on these intracellular processes is difficult because signaling pathways are connected to each other in numerous ways. Due to the complicated nature of the interactions among signaling molecules, the overall signal transduction network has hardly been investigated quantitatively or systematically. In order to reveal the underlying control of the signaling network, we designed mechanistic experiments to quantitatively measure the activity of signaling molecules and formulated a mathematical model consistent with these data. In this manner, we approached the study of the platelet-derived growth factor (PDGF) receptor mediated phosphoinositide (PI) 3-kinase/Akt signaling pathway and crosstalk between Ras and PI 3-kinase.

The linear signaling pathway, PDGF receptor/PI 3-kinase/Akt, was systematically investigated by using biochemical assays and mathematical modeling. We find that PDGF receptor phosphorylation shows positive cooperativity with respect to PDGF concentration, and the receptor dimerization process is consistent with association of two 1:1 ligand-receptor intermediate complexes. The kinetics of receptor phosphorylation are transient at high PDGF concentrations. Activation of PI 3-kinase, and thus Akt, are saturated with respect to the number of activated receptors. At higher PDGF concentrations, the kinetics of 3¡¯ PI production are governed by the first-order turnover rate constant and those of Akt are further controlled by its deactivation rate constant.

To investigate the crosstalk between Ras and PI 3-kinase, we systematically designed experiments to assess the effect of Ras on PI 3-kinase activation and developed a simple mathematical model to describe the molecular assembly of complexes involving PDGF receptors, PI 3-kinase, and Ras. From the experimental results, we find that the Ras effectively enhances the affinity of PI 3-kinase towards the receptor. In our model, this can only be explained if a ternary receptor/PI 3-kinase/Ras complex forms in two steps, where the second step is greatly enhanced through induced proximity and possibly allosteric effects.