Threonine phosphorylation regulates the molecular assembly and signaling of EGFR in cooperation with membrane lipids

Abstract

The cytoplasmic domain of the receptor tyrosine kinases (RTKs) plays roles as a phosphorylation enzyme and a protein scaffold but the allocation of these two functions is not fully understood. We here analyzed assembly of the transmembrane (TM)-juxtamembrane (JM) region of EGFR, one of the best studied species of RTKs, by combining single-pair FRET imaging and a nanodisc technique. The JM domain of EGFR contains a threonine residue (Thr654) that is phosphorylated after ligand association. We observed that the TM-JM peptides of EGFR form anionic lipid-induced dimers and cholesterol-induced oligomers. The two forms involve distinct molecular interactions, with a bias towards oligomer formation upon threonine phosphorylation. We further analyzed the functions and oligomerization of whole EGFR molecules, with or without a substitution of Thr654 to alanine, in living cells. The results suggested an autoregulatory mechanism in which Thr654 phosphorylation causes a switch of the major function of EGFR from kinase activation dimers to scaffolding oligomers.