Phospholipase D1 in Caveolae:  Regulation by Protein Kinase Cα and Caveolin-1

Abstract

Caveolae are small plasma membrane invaginations that have been implicated in cell signaling, and caveolin is a principal structural component of the caveolar membrane. Previously we have demonstrated that protein kinase Calpha (PKCalpha) directly interacts with phospholipase D1 (PLD1), activating the enzymatic activity of PLD1 in the presence of phorbol 12-myristate 13-acetate (PMA) [Lee, T. G., et al. (1997) Biochim. Biophys. Acta 1347, 199-204]. In this study, using a detergent-free procedure for the purification of a caveolin-enriched membrane fraction (CEM) and immunoblot analysis, we show that PLD1 is enriched in the CEMs of 3Y1 rat fibroblasts. Purified PLD1 directly bound to a glutathione S-transferase-caveolin-1 fusion protein in in vitro binding assays. The association of PLD1 with caveolin-1 could be completely eliminated by preincubation of PLD1 with an oligopeptide corresponding to the scaffolding domain (amino acids 82-101) of caveolin-1, indicating that caveolin-1 interacts with PLD1 through the scaffolding domain. The peptide also inhibited PKCalpha-stimulated PLD1 activity and the interaction between PLD1 and PKCalpha with an IC50 of 0.5 microM. PMA elicits translocation of PKCalpha to the CEMs, inducing PLD activation through the interaction of PKCalpha with PLD1 in the CEMs. Caveolin-1 also coimmunoprecipitated with PLD1 in the absence of PMA, and the amounts of coimmunoprecipitated caveolin-1 decreased in response to treatment with PMA. Taken together, our results suggest a new mechanism for the regulation of the PKCalpha-dependent PLD activity through the molecular interaction between PLD1, PKCalpha, and caveolin-1 in caveolae.

Phospholipases D1 and D2 Coordinately Regulate Macrophage Phagocytosis

“…The lack of detectable PLD1 in COS cells was not due to a failure of the Ab to detect monkey PLD1 protein, because endogenous PLD1 was readily detected in primary monkey brain tissue and comigrated with baculovirus-expressed recombinant human PLD1. The lack of PLD1 in COS cells has been previously reported with a different anti-PLD1 Ab (48).…”

Pulmonary phosphatidic acid phosphatase and lipid phosphate phosphohydrolase

“…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.…”

“…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.…”

“…However, the possibility that another PA-degrading activity is present must still be investigated. S. H. Ryu's group (127,128) has recently proposed a mechanism for the control of PLD1 activity through molecular interactions among PLD1, PKC-␣, and caveolin-1. With this mechanism, PLD1 activation arises through PKC-␣-dependent phosphorylation in caveolae.…”

Evidence for Heme Oxygenase‐1 Association with Caveolin‐1 and ‐2 in Mouse Mesangial Cells

“…Recent studies have shown that many receptors, signal proteins and enzymes are localized in the caveolae by being anchored through caveolins. These include the epidermal growth factor receptor (7), estrogen receptor (8), insulin receptor (9), Grb 7 (10), c-Src (11), Gprotein (12), protein kinase C (13), phospholipase D (14), nitric oxide synthases (NOS) including the inducible NOS (iNOS) (15,16) and cyclooxygenase-2 (COX-2) (17), among others. Among these proteins known to be associated with caveolins, the expression of NO producing iNOS and prostaglandin producing COX-2 increases by the same oxidative stimuli like the ischemia-reperfusion (5) or exposures to heavy metals (6), heat shock (18) and NO (19) that also cause the induction of HO-1.…”