Localization of phospholipase D in detergent-insoluble, caveolin-rich membrane domains -: Modulation by caveolin-1 expression and caveolin-182-101

The activation of cellular phospholipase D (PLD) is implicated in vesicular trafficking and signal transduction, Two mammalian PLD forms, designated PLD1 and PLD2, have been cloned, but their cellular localization and function are not fully understood. Here, we report that in HaCaT human keratinocytes, as well as other cell lines, PLD activity is highly enriched in low density, Triton X-100-insoluble membrane domains that contain the caveolar marker protein caveolin-l. Similar to other PLDs, the PLD activity in these membrane domains is stimulated by phosphatidylinositol 4,5-bisphosphate and is inhibited by neomycin, Immunoblot analysis indicated that caveolin-rich membrane domains do not contain the PLD1 isoform, Stable transfection of mouse PLD2 in Chinese hamster ovary cells greatly increased PLD activity in these domains compared with PLD activity in control Chinese hamster ovary cells transfected with vector alone. PLD activity is enriched in low density Triton-insoluble membrane domains also in U937 promonocytes, even though these cells do not express caveolin-l. In U937 cells, also, PLD1 is largely excluded from low density Triton-insoluble membrane domains. Expression of recombinant caveolin-l in v-Src-transformed NIH-3T3 cells resulted in up-regulation of PLD activity in the caveolin-containing membrane domains. The caveolin scaffolding peptide (caveolin-l(82-101)) modulated the caveolar PLD activity, causing stimulation at concentration of 1-10 mu M and inhibition at concentrations >10 mu M. We conclude that a PLD activity, which is likely to represent PLD2, is enriched in low density Triton-insoluble membrane domains. The effects of caveolin-l expression and of the caveolin scaffolding peptide suggest that in cells that express caveolin-l, PLD may be targeted to caveolae, The possible functions of PLD in the dynamics of caveolae and related domains and in signal transduction processes are discussed.