Abstract
Autocatalytic phosphorylation of receptor tyrosine kinases (RTKs) enables diverse, context-dependent responses to extracellular signals but comes at the price of autonomous, ligand-independent activation. Using a conformational biosensor that reports on the kinase activity of the cell guidance ephrin receptor type-A (EphA2) in living cells, we observe that autonomous EphA2 activation is suppressed by vesicular recycling and dephosphorylation by protein tyrosine phosphatases 1B (PTP1B) near the pericentriolar recycling endosome. This spatial segregation of catalytically superior PTPs from RTKs at the plasma membrane is essential to preserve ligand responsiveness. Ligand-induced clustering, on the other hand, promotes phosphorylation of a c-Cbl docking site and ubiquitination of the receptor, thereby redirecting it to the late endosome/lysosome. We show that this switch from cyclic to unidirectional receptor trafficking converts a continuous suppressive safeguard mechanism into a transient ligand-responsive signalling mode.
Highlights
Autocatalytic phosphorylation of receptor tyrosine kinases (RTKs) enables diverse, context-dependent responses to extracellular signals but comes at the price of autonomous, ligand-independent activation
The linkers were designed to form an antiparallel coiled-coil helix that: (1) constrains the orientation of mCitrine with respect to the insertion point, (2) minimally perturbs the juxtamembrane segment (JMS) and (3) positions the mCitrine on ‘a stalk’ outside the body of EphA2 to avoid hindrance of EphA2 interactions (Fig. 1a). Conformational changes in this Linker optimized Intramolecular-Forster Resonance Energy Transfer (FRET) sensor for EphA2 (LIFEA2) alter the FRET efficiency between the two fluorescent proteins (FP), which can be quantified via the fluorescence lifetime (t) of mCitrine using fluorescence lifetime imaging microscopy (FLIM)[29]
This was confirmed by photobleaching monomeric cherry (mCherry) in live Cos-7 cells, where the resultant t of LIFEA2 approached that of mCitrine (Fig. 1b)
Summary
A genetically encoded conformational biosensor for EphA2. To investigate the dependence of EphA2 activation on its local density on cellular membranes, we engineered a genetically encoded Forster Resonance Energy Transfer (FRET) biosensor that reports its active conformation. From the ligand-induced interaction of a LIFEA2 variant that only has the mCitrine JMS insertion with mCherrylabelled dimeric SH2 domain of pp60src (dSH2-mCherry)[31] we conclude that the drop in LIFEA2 fluorescence lifetime reflects a conformational transition to a state that has a phosphorylated JMS (Fig. 1e). This was reflected in the phosphorylation profile of LIFEA2 that followed its conformational activation kinetics (Fig. 1f). LIFEA2 maintained its native autophosphorylation activity as well as its capacity to inhibit the PI3K/Akt and Ras/ERK signalling pathways[32,33], but
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