Localization of Phospholipase D in Detergent-insoluble, Caveolin-rich Membrane Domains

Abstract

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-1. 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-1. In U937 cells, also, PLD1 is largely excluded from low density Triton-insoluble membrane domains. Expression of recombinant caveolin-1 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-1 82-101 ) modulated the caveolar PLD activity, causing stimulation at concentration of 1-10 M and inhibition at concentrations >10 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-1 expression and of the caveolin scaffolding peptide suggest that in cells that express caveolin-1, 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.

A Role for Caveolae/Lipid Rafts in the Uptake and Recycling of the Endogenous Cannabinoid Anandamide

“…Okamoto et al (41) recently reported the molecular characterization of a phospholipase D isolated from rat heart that is specific for the production of AEA and other N-acylethanolamides. Interestingly, phospholipase D activity has been shown to be enriched in caveolin-rich membranes (42). The biosynthesis of the hydrophobic amide signaling molecule ceramide appears to be compartmentalized to caveolae, establishing a precedence for the localized production of lipid signaling molecules in caveolae (43).…”

Pulmonary phosphatidic acid phosphatase and lipid phosphate phosphohydrolase

“…Sciorra and Morris (224) have recently provided evidence that LPP3 but not LPP1 can generate DAG from PA derived from PLD2 degradation of PC in DIGs in HEK 293 cells. Cell specific localization has also been noted with PLD isoforms, where Czarny et al (45) have observed PLD2 is preferentially targeted over PLD1 to caveolae/rafts in Chinese hamster ovary cells, HaCaT human keratinocytes, and U937 cells. Kim et al (127,128), on the other hand, have localized PLD1 to DIGs in 3Y1 rat fibroblasts and COS7 cells.…”

“…With both cell lines, a proportion of the LPP activity was found associated with the caveolin-enriched domains. Presence of LPP in caveolae/ rafts was anticipated, since studies with other cells have shown that PLD (44,45,127,128) and LPA and S1P receptors (100) can be found associated with DIGs.…”

Protein Kinase Cα Translocates to the Perinuclear Region to Activate Phospholipase D1

“…Some reports indicate that PLD1 also localizes to secretory granules, late endosomes, and lysosomes (18, 20 -22). Cell fractionation studies show that PLD1 activity is restricted to caveolin-enriched membranes in some cell lines (23)(24)(25). There is other evidence that PLD1 might localize at the plasma membrane (26,27).…”