The galectin-3 carbohydrate recognition domain maintains plasma membrane phosphatidylserine required for KRAS function

KRAS is a small GTPase essential for cellular proliferation, differentiation, and survival. It is predominantly localized to the plasma membrane (PM), where it forms nanoclusters essential for signal transduction. This PM binding and nanoclustering strictly depend on phosphatidylserine (PtdSer), an anionic phospholipid enriched in the inner PM leaflet. Galectin-3 (Gal-3), a β-galactoside-binding lectin implicated in cell growth, migration and tumor progression, has been shown to regulate KRAS PM binding and nanoclustering. Here, we demonstrate that Gal-3 knockout (KO) or inhibition of its carbohydrate recognition domain (CRD) depletes KRAS and PtdSer from the PM. Exogenous PtdSer supplementation fully restores PM PtdSer and partially restores KRAS PM binding, indicating that Gal-3 promotes KRAS PM binding in part by maintaining PM PtdSer content. Total cellular PtdSer, PM PI4P, cholesterol and GM3 are unchanged, excluding these established regulators of PM PtdSer. Instead, Gal-3 CRD inhibition disrupts the cellular balance of sphingomyelin (SM) and ceramide, alters intracellular SM distribution, and impairs the endocytic and recycling transport of PtdSer, SM, and epidermal growth factor receptor (EGFR). Together, our findings demonstrate that Gal-3 CRD activity sustains PM PtdSer content through endocytic trafficking, providing the PM lipid composition required for KRAS PM spatial organization.