Oleate activates PLD2 lipase and GEF activity by modulating membrane microdomain dynamics via S-acylation.

Phospholipase D2 (PLD2) plays critical roles in cellular signaling, membrane dynamics, and cancer progression. Oleate (OA) has been shown to activate PLD2 and promote triple-negative breast cancer (TNBC) cell migration, but the underlying molecular mechanisms remain poorly understood. Using confocal microscopy, lipid raft isolation, and S-acylation assays, we show that OA enhanced PLD2 S-acylation at Cys223 and Cys224, disrupting its lipid raft localization, and consequently increasing its colocalization with PIP(2)-enriched microdomains. Furthermore, we identified PLD2 as a guanine nucleotide exchange factor (GEF) for Cdc42, with its GEF activity regulated by OA-dependent S-acylation and lipid raft dynamics. Mutation of the S-acylation sites or disruption of lipid rafts abolished PLD2-mediated Cdc42 activation and filopodia-like cell protrusion formation. These findings reveal a novel regulatory mechanism by which OA modulates PLD2 activity through S-acylation and membrane microdomain reorganization, providing new insights into the regulation of PLD2 in cell migration and signaling.