A new mechanism of EGFR activation identified by Phos-tag band shift analysis

Abstract

It has yet to be established whether ligand-unoccupied receptors remain on the plasma membrane or have any functions following a stimulation with ligand. We demonstrated that the minimal activation of EGFR tyrosine kinase by low concentration of EGF is sufficient to fully activate downstream MAPK pathways. Phos-tag band shift assay revealed that most of the remaining EGFR molecules were efficiently phosphorylated at serine/threonine residues by activated p38. The non-canonical p38 phosphorylation of the C-tail around Ser-1015 induced the clathrin-mediated endocytosis of unliganded EGFR monomers, which occurred slightly later than the canonical endocytosis of ligand-bound EGFR dimers via tyrosine autophosphorylation. Endocytosed EGFR via the non-canonical pathway was largely recycled back to the plasma membrane as functional receptors, whereas p38-independent populations were mainly sorted for lysosomal degradation. These results demonstrated that ligand-activated EGFR signaling controls unliganded receptors by feedback phosphorylation, identifying the dual-mode regulation of the endocytic trafficking dynamics of EGFR.