A reaction-diffusion model predicts the intracellular length scale over which EGFR-initiated GAB1-SHP2 complexes persist

Abstract

AbstractActivation of receptor tyrosine kinases (RTKs) leads to the assembly of multi-membered protein complexes connected by phosphotyrosine-SH2 domain linkages. However, these linkages are relatively weak and reversible, which allows complex disassembly to occur on a time scale that permits phosphatases to dephosphorylate complex members and thereby regulate complex persistence. Here, we generated a computational reaction-diffusion model to predict the length scale over which membrane-bound RTKs can regulate the maintenance of such protein complexes through the intermediary action of diffusible cytoplasmic kinases. Specifically, we show that the RTK EGFR can activate SRC family kinases (SFKs) to maintain the association of SHP2 with phosphorylated GAB1, which activates SHP2, throughout the entire cell volume. This finding is dependent on the ability of SFKs to be activated by EGFR at the plasma membrane and subsequently diffuse through the cytosol, as altering the model topology to permit only SFK activation at the plasma membrane reduces the length scale of GAB1-SHP2 association. Modifying the model topology to neglect GAB1 binding to cytosolic and EGFR-bound GRB2 had little effect on this length scale. Indeed, a model sensitivity analysis identified protein diffusion, SFK inactivation, and GAB1 dephosphorylation as the processes that most strongly control the distance over which GAB1-SHP2 persists distal from EGFR. A model scaling analysis likewise predicted that the length scale of GAB1-SHP2 association is greatly extended compared to that of SFK activation and that GAB1-SHP2 complexes persist throughout the cell volume. Furthermore, the same processes identified in the model sensitivity analysis appeared in the length scale estimate for GAB1-SHP2 association. In vitro experiments using proximity ligation assay and immunofluorescence against GAB1-SHP2 and EGFR, respectively, suggested that GAB1-SHP2 complexes are distributed throughout cells and exist distally from EGFR during EGF stimulation. Overall, our results suggest that GAB1-SHP2 complexes—and thus active SHP2—can persist distally from EGFR due to re-phosphorylation of GAB1 throughout the cytosol by EGFR-activated SFKs.