Localization of phospholipase D in detergent-insoluble, caveolin-rich membrane domains - Modulation by caveolin-1 expression and caveolin-1(82-101)

The activation of cellular phospholipase D (PLD) is implicated in vesicular trafficking and signal transduction, Two mammalian PLD forms, designated P LD1 and PLD2, have been cloned, but their cellular localization and functio n are not fully understood. Here, we report that in HaCaT human keratinocyt es, as well as other cell lines, PLD activity is highly enriched in low den sity, Triton X-100-insoluble membrane domains that contain the caveolar mar ker protein caveolin-l. Similar to other PLDs, the PLD activity in these me mbrane domains is stimulated by phosphatidylinositol 4,5-bisphosphate and i s inhibited by neomycin, Immunoblot analysis indicated that caveolin-rich m embrane domains do not contain the PLD1 isoform, Stable transfection of mou se PLD2 in Chinese hamster ovary cells greatly increased PLD activity in th ese domains compared with PLD activity in control Chinese hamster ovary cel ls transfected with vector alone. PLD activity is enriched in low density T riton-insoluble membrane domains also in U937 promonocytes, even though the se cells do not express caveolin-l. In U937 cells, also, PLD1 is largely ex cluded from low density Triton-insoluble membrane domains. Expression of re combinant caveolin-l in v-Src-transformed NIH-3T3 cells resulted in up-regu lation of PLD activity in the caveolin-containing membrane domains. The cav eolin scaffolding peptide (caveolin-l(82-101)) modulated the caveolar PLD a ctivity, causing stimulation at concentration of 1-10 mu M and inhibition a t concentrations >10 mu M. We conclude that a PLD activity, which is likely to represent PLD2, is enriched in low density Triton-insoluble membrane do mains. 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.