title: Activation of phospholipase D by bradykinin and sphingosine 1-phosphate in A549 human lung adenocarcinoma cells via different GTP-binding proteins and protein kinase C delta signaling pathways.
abstract: Phospholipase D (PLD) is involved in the signaling by many extracellular ligands, and its regulation appears to be quite complex. We investigated the signaling pathways initiated by bradykinin (BK) or sphingosine 1-phosphate (S1P) in A549 cells to define molecular mechanisms responsible for their additive effects on PLD activity. BK and S1P each elicited a sustained increase in phosphatidic acid content through a rapid and transient activation of PLD. The two pathways demonstrated rapid homologous downregulation, but heterologous desensitization was not observed. Action of both agonists required protein kinase C (PKC) activation and Ca(2+) influx but was mediated by different heterotrimeric G proteins. In membranes, inhibition of PKCdelta by rottlerin enhanced BK activation of PLD but inhibited that by S1P. Rottlerin inhibited activation of PLD in nuclei by both BK and S1P. By in situ immunofluorescence or cell fractionation followed by immunoblotting, PLD1 was concentrated primarily in nuclei, whereas the membrane fraction contained PLD2 and PLD1. Moreover, PKCdelta specifically phosphorylated recombinant PLD2, but not PLD1. BK and S1P similarly enhanced RhoA translocation to nuclei, whereas BK was less efficacious than S1P on RhoA relocalization to membranes. Effects of both agonists on the nuclear fraction, which contains only PLD1, are compatible with a RhoA- and PKCdelta-dependent process. In membranes, which contain both PLD1 and PLD2, the stimulatory effect of S1P on PLD activity can best be explained by RhoA- and PKCdelta-dependent activation of PLD1; in contrast, the effects of BK on RhoA translocation and enhancement of BK-stimulated PLD activity by PKC inhibition are both consistent with PLD2 serving as its primary target.
authors.affiliation: Dipartimento di Scienze Biochimiche, Università di Firenze, Viale G.B. Morgagni 50, 50134 Firenze, Italy.
authors.forename: Elisabetta
authors.initials: E
authors.lastname: Meacci
authors.forename: Francesca
authors.initials: F
authors.lastname: Nuti
authors.forename: Serena
authors.initials: S
authors.lastname: Catarzi
authors.forename: Valeria
authors.initials: V
authors.lastname: Vasta
authors.forename: Chiara
authors.initials: C
authors.lastname: Donati
authors.forename: Sylvain
authors.initials: S
authors.lastname: Bourgoin
authors.forename: Paola
authors.initials: P
authors.lastname: Bruni
authors.forename: Joel
authors.initials: J
authors.lastname: Moss
authors.forename: Martha
authors.initials: M
authors.lastname: Vaughan
mesh_terms: D000230:Adenocarcinoma / Q000201:enzymology* / Q000378:metabolism; D001920:Bradykinin / Q000819:agonists / Q000502:physiology*; D002118:Calcium / Q000502:physiology; D004789:Enzyme Activation / Q000502:physiology; D020404:Glycerophospholipids / Q000378:metabolism; D020962:Heterotrimeric GTP-Binding Proteins / Q000502:physiology*; D006801:Humans; D008175:Lung Neoplasms / Q000201:enzymology* / Q000378:metabolism; D008246:Lysophospholipids; D010712:Phosphatidic Acids / Q000378:metabolism; D010739:Phospholipase D / Q000378:metabolism*; D011493:Protein Kinase C / Q000502:physiology*; D051745:Protein Kinase C-delta; D021381:Protein Transport / Q000502:physiology; D015398:Signal Transduction / Q000502:physiology*; D013110:Sphingosine / Q000819:agonists / Q000031:analogs & derivatives* / Q000502:physiology*; D013347:Subcellular Fractions / Q000201:enzymology / Q000378:metabolism; D014316:Tritium; D014407:Tumor Cells, Cultured; D020742:rhoA GTP-Binding Protein / Q000378:metabolism
chemical_list: D020404:Glycerophospholipids; D008246:Lysophospholipids; D010712:Phosphatidic Acids; C051521:phosphatidylethanol; D014316:Tritium; C060506:sphingosine 1-phosphate; C497258:PRKCD protein, human; D011493:Protein Kinase C; D051745:Protein Kinase C-delta; C105606:phospholipase D2; D010739:Phospholipase D; C106801:phospholipase D1; D020962:Heterotrimeric GTP-Binding Proteins; D020742:rhoA GTP-Binding Protein; D013110:Sphingosine; D001920:Bradykinin; D002118:Calcium