EPHA2 regulates the abundance of amino acid transporter LAT3 through its receptor tyrosine kinase activity

LAT3 (SLC43A1) mediates sodium-independent transport of neutral and branched-chain amino acids and participates in both physiological and pathological processes, including development and cancer. Dysregulation of LAT3 function is associated with disease, yet the regulatory mechanism of LAT3 remains poorly defined. Here, we show that a photo-lysine–based optoproteomics strategy, combined with affinity purification–mass spectrometry, enables systematic mapping of LAT3 membrane-associated interactions in living cells. From this analysis, we identified 167 high-confidence membrane-associated interactors, of which 43 localize to the plasma membrane after excluding LAT3 itself. Among these candidates, EPHA2 was the only receptor kinase identified. Biochemical assays showed that EPHA2 directly interacts with LAT3 and phosphorylates it at tyrosine 251. Inhibition of EPHA2 increased LAT3 protein abundance, indicating that EPHA2 kinase activity negatively regulates LAT3 stability through a post-translational degradation pathway. This study provides a comprehensive LAT3 membrane interactome to unravel the molecular basis of functional regulation.