Dynamic regulation of autocatalytic EGFR activation
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Date
2017
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Abstract
The dynamics of epidermal growth factor receptor (EGFR) signaling emerge from its recursive interactions with protein tyrosine phosphatases (PTPs) and autocatalytic receptor activation thereby determining cellular behavior including proliferation, migration and differentiation. The autocatalytic activation of EGFR causes an amplification of EGFR phosphorylation in response to extracellular signals, but its trade-off is spontaneous activation at high receptor densities even in the absence of ligand. Structural and molecular dynamics studies identified an allosteric activation mechanism upon EGF-induced receptor activation, but the molecular basis of autocatalytic EGFR activation remains unclear. To better understand autocatalysis we developed a Förster Resonance Energy Transfer (FRET)-based, conformational EGFR indicator (CONEGI) using genetic code expansion to monitor the conformational state of the tyrosine kinase domain (TKD) in living cells and relate it to EGFR activity. We show that EGFR monomers can adopt an active conformation that is stabilized upon Y845 phosphorylation. Since Y845 is an auto-phosphorylation site this creates a positive feedback loop generating an autocatalytic amplification mechanism. To counteract autonomous, autocatalytic EGFR activation in the absence of ligand, intrinsic and extrinsic safeguard mechanisms are required. Intrinsic auto-inhibitory structural features can be overcome by thermal conformational fluctuations allowing a sub-population of EGFR to adopt an active conformation. This autonomous, autocatalytic EGFR activity is counterbalanced by a spatial cycle that suppresses phosphorylation of Y845 on EGFR monomers by vesicular recycling through perinuclear areas with high PTP1B activity. EGF-binding induces receptor dimerization and phosphorylation of the c-Cbl docking Y1045 leading to receptor ubiquitination that targets EGFR for degradation in lysosomes. The re-routing regulates EGFR signaling response by the transit-time to late endosomes where it is switched-off by high PTP1B activity. This ubiquitin-mediated switch from a suppressive cycle to a unidirectional trafficking mode is a uniquely suited solution to suppress spontaneous activation while maintaining responsiveness to EGF.
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EGFR, Autocatalysis, Conformational sensor, Genetic code expansion, Vesicular recycling