(390i) Regulation of Epidermal Growth Factor Receptor Phosphorylation and Endocytosis Dynamics by Protein Tyrosine Phosphatases

Cell signaling pathways initiated by the epidermal growth factor receptor (EGFR) are broadly involved in normal physiology and pathology.  Phosphorylated EGFR levels, which dictate receptor signaling strength and persistence, are the net result of two opposing processes, auto-phosphorylation of cytoplasmic tyrosines by the receptor kinase and dephosphorylation of those tyrosines by protein tyrosine phosphatases (PTPs).  Whereas the processes leading to tyrosine phosphorylation have been studied in great detail, relatively little is known about the kinetics of EGFR tyrosine dephosphorylation or the coupling of this process to other receptor-level phenomena which depend upon receptor phosphorylation, such as endocytosis.  Here, we developed a novel mechanistic model of EGFR phosphorylation dynamics to determine the rates of EGFR dephosphorylation at the plasma membrane and in the cell interior and applied the model to generate predictions for the effects of these dephosphorylation kinetics on EGFR endocytosis dynamics.  Our model was constructed to allow for the possibility of different rates of receptor dephosphorylation at the plasma membrane and cell interior and accounted for interactions of EGFR with itself (dimerization and phosphorylation), ATP, kinase inhibitors, PTPs, and adapter proteins involved in receptor trafficking processes.  To determine rate constants for EGFR tyrosine dephosphorylation at the plasma membrane and cell interior, the model was regressed against a diverse set of experimental data for receptor phosphorylation response to EGFR ligands, PTP inhibitors, and EGFR kinase inhibitors as well as data for steady EGFR phosphorylation levels in the absence of ligands.  Our regression results led to the novel conclusion that EGFR dephosphorylation is significantly more rapid at the plasma membrane than in the cell interior, a finding which contradicts the classical view of EGFR regulation by PTPs and several previous computational studies of EGFR phosphorylation dynamics which were developed by considering data for receptor response to ligand alone.  In fact, our model results suggest that the process of EGFR dephosphorylation is so rapid that receptor tyrosines may go through a complete phosphate cycle (phosphorylation and dephosphorylation) as many as thirteen times per minute.  Thus, our model results suggest that, as with other kinase-phosphatase systems, EGFR and its associated PTPs participate in a “futile cycling” process wherein phosphate cycling occurs on a time scale much smaller than that for other important receptor processes such as endocytosis and degradation.  Since the most dominant pathways for EGFR endocytosis require phosphorylation of key EGFR tyrosines, a feature encoded within our model, our results further suggest that regulation of EGFR by PTPs at the plasma membrane may play a significant role in determining rates of ligand-mediated EGFR endocytosis.  In fact, for an EGFR expression level characteristic of epithelial cells, our model predicted that PTP activity at the plasma membrane may reduce the rate constant for ligand-mediated EGFR endocytosis by up to six-fold from the value it would otherwise be in the absence of PTP activity at the plasma membrane.  This predicted effect of PTPs on EGFR endocytosis could be reduced by the inclusion of the trafficking adapter protein referred to previously in the model calculations.  Experimental measurements of the effects of PTPs on EGFR endocytosis were made for cultured fibroblasts and epithelial cells using radiolabeled EGFR ligands or antibodies and inhibitors of PTP activity.  Overall, comparison between our measurements and model calculations suggested that PTP activity at the plasma membrane influences EGFR endocytosis kinetics but that the enhanced binding of adapter proteins in the context of PTP inhibition reduces the observed effect from the maximum predicted effect.