Phospholipase D2: functional interaction with caveolin in low-density membrane microdomains

Low-density detergent-insoluble membrane domains contain caveolin-1 and are enriched in a phospholipase D activity that is not PLD1, Here we show that caveolin-rich fractions, prepared from HaCaT human keratinocytes by either detergent-based or detergent-free methods, contain PLD2, Caveolar membrane PLD activity is stimulated 2-fold by low concentrations (10-30 mu M) of th e caveolin-1 and caveolin-2 scaffolding domain peptides, whereas it is inhi bited at higher concentrations of the peptides, Immunoisolated HA-tagged PL D1 and PLD2 are not stimulated by the peptides, although both enzymes retai n sensitivity to their inhibitory effect. Down-regulation of caveolin-1 exp ression by treatment of the cells with acetyl-leucyl-leucyl-norleucinal dec reased caveolar PLD activity by 50%. Similarly expression of an active form of the sterol regulatory element-binding protein (SREBP1-490) down-regulat ed caveolin-1 expression by 50% and decreased caveolar PLD activity by 60%. These data identify the PLD activity in caveolin-rich membranes as PLD2 an d provide in vivo evidence suggesting that caveolin-1 regulates PLD2 activi ty. (C) 2000 Federation of European Biochemical Societies.