Autocatalytic phosphorylation of receptor tyrosine kinases (RTKs) enables different context-dependent responses to extracellular signs but comes at the price tag on autonomous ligand-independent activation. the past due 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. Eph receptors constitute the largest subfamily of receptor tyrosine kinases (RTKs) and together with their ephrin ligands function as principal cell guidance cues during CD244 development and disease1 2 3 Phenprocoumon Eph/ephrin interactions normally lead to cell-cell repulsion and sorting yet depending on the cellular context the same pair can elicit opposite effects4 5 Activation of Ephs follows a general RTK framework beginning with ligand binding receptor oligomerization and subsequent trans-autophosphorylation catalysed by the kinase domain6. Tyrosine phosphorylation of two residues in the conserved juxtamembrane segment (JMS) and a third one in the kinase activation loop triggers conformational changes that release inhibitory interactions between the JMS and kinase domain. These regulatory tyrosines can directly enhance Eph catalytic activity by modulating the structure of the kinase domain to allow ATP and substrate to access the active site7 8 9 10 Activation of RTKs by autophosphorylation is thus an autocatalytic system that generates an amplified response to an extracellular signal. However the classical view of RTK activation does not take into account the conformational plasticity of the kinase domain random receptor collisions on membranes and the fraction of enzyme already in an active state11 all of which are factors that can lead to spurious phosphorylation and uncontrolled receptor activity. Indeed EphA2 expression and activity are elevated in clinical specimens of human cancer including that of colon breast prostate and aggressive melanomas12 13 14 This autocatalytic RTK program needs to become counterbalanced from the opposing activity of proteins tyrosine phosphatases (PTPs)15. In keeping with this raised PTP activity in EphA3-positive leukaemia cells maintains a dephosphorylated receptor that provokes adhesion for an ephrin-A5 surface area while PTP inhibition induces receptor phosphorylation producing a repulsive Phenprocoumon response16. Eph-specific PTPs include PTP receptor type O17 the leukocyte common antigen related receptor tyrosine phosphatase (LAR-1)18 and the endoplasmic reticulum-anchored PTP1B19 20 Considering that the catalytic activity of fully active PTPs is up to three orders of magnitude higher than RTKs21 22 a gradient of PTP activity originating in the perinuclear area and declining towards the plasma membrane (PM)-proximal cytoplasm is a prerequisite for allowing RTK signal initiation23 24 However the low PTP activity near the PM would imply that autonomous RTK activation and spurious signals are likely to occur at high surface density25 26 The density of receptors at the cell surface is dynamically controlled through a balance between endocytic uptake and vesicular recycling. Typically ligand binding triggers receptor internalization into Rab5-positive early endosomes that mature into sorting endosomes or multivesicular bodies (MVBs). Sorting endosomes/MVBs gradually develop into late endosomes that are Phenprocoumon enriched in proteins such as Rab7. Fusion of late endosomes with lysosomes leads to receptor degradation and signal attenuation. Ligand-activated RTKs can also recycle back to the PM from peripheral endosomes or from the Rab11-positive pericentriolar recycling endosome (RE) resulting in sustained signalling27 28 This indicates that the endocytic system positioned spatially and temporally between the PM and the lysosomes can control RTK signal duration. Despite ample studies Phenprocoumon on membrane trafficking of ligand-activated RTKs little is known about the role of trafficking in regulating autonomous RTK activity an important facet considering the propensity of RTKs to self-activate. To study how vesicular membrane dynamics differentially control the activity of autonomously-versus ligand-activated EphA2 we designed a genetically encoded fluorescent biosensor for EphA2 that enables the determination of the fraction of Phenprocoumon active receptor in live cells by imaging its fluorescence lifetime. This allows for the quantification of EphA2 autocatalytic response properties whereby we show that a ubiquitin-mediated switch in receptor trafficking.