Effect of Rho and ADP-ribosylation factor GTPases on phospholipase D activity in intact human adenocarcinoma A549 cells.
Phospholipase D (PLD) has been implicated as a crucial signaling enzyme in secretory pathways. Two 20-kDa guanine nucleotide-binding proteins, Rho and ADP-ribosylation factor (ARF), are involved in the regulation of secretion and can activate PLD in vitro. We investigated in intact (human adenocarcinoma A549 cells) the role of RhoA and ARF in activation of PLD by phorbol 12-myristate 13-acetate, bradykinin, and/or sphingosine 1-phosphate. To express recombinant Clostridium botulinum C3 exoenzyme (using double subgenomic recombinant Sindbis virus C3), an ADP-ribosyltransferase that inactivates Rho, or dominant-negative Rho containing asparagine at position 19 (using double subgenomic recombinant Sindbis virus Rho19N), cells were infected with Sindbis virus, a novel vector that allows rapid, high level expression of heterologous proteins. Expression of C3 toxin or Rho19N increased basal and decreased phorbol 12-myristate 13-acetate-stimulated PLD activity. Bradykinin or sphingosine 1-phosphate increased PLD activity with additive effects that were abolished in cells expressing C3 exoenzyme or Rho19N. In cells expressing C3, modification of Rho appeared to be incomplete, suggesting the existence of pools that differed in their accessibility to the enzyme. Similar results were obtained with cells scrape-loaded in the presence of C3; however, results with virus infection were more reproducible. To assess the role of ARF, cells were incubated with brefeldin A (BFA), a fungal metabolite that disrupts Golgi structure and inhibits enzymes that catalyze ARF activation by accelerating guanine nucleotide exchange. BFA disrupted Golgi structure, but did not affect basal or agonist-stimulated PLD activity, i.e. it did not alter a rate-limiting step in PLD activation. It also had no effect on Rho-stimulated PLD activity, indicating that RhoA action did not involve a BFA-sensitive pathway. A novel PLD activation mechanism, not sensitive to BFA and involving RhoA, was identified in human airway epithelial cells by use of a viral infection technique that preserves cell responsiveness.
Meacci
E
E
Pulmonary-Critical Care Medicine Branch, NHLBI, National Institutes of Health, Bethesda, Maryland 20892, USA.
Vasta
V
V
Moorman
J P
JP
Bobak
D A
DA
Bruni
P
P
Moss
J
J
Vaughan
M
M
D036002:ADP Ribose Transferases / Q000378:metabolism; D020727:ADP-Ribosylation Factors; D000230:Adenocarcinoma / Q000201:enzymology*; D000262:Adenylyl Cyclases / Q000378:metabolism*; D001905:Botulinum Toxins; D001920:Bradykinin / Q000494:pharmacology; D020126:Brefeldin A / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000378:metabolism*; D020690:GTPase-Activating Proteins; D006056:Golgi Apparatus / Q000378:metabolism; D006801:Humans; D008246:Lysophospholipids; D010739:Phospholipase D / Q000378:metabolism*; D013110:Sphingosine / Q000031:analogs & derivatives / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014407:Tumor Cells, Cultured; D020742:rhoA GTP-Binding Protein
D020690:GTPase-Activating Proteins; D008246:Lysophospholipids; C085379:rho GTPase-activating protein; D020126:Brefeldin A; C060506:sphingosine 1-phosphate; D036002:ADP Ribose Transferases; C063007:exoenzyme C3, Clostridium botulinum; D010739:Phospholipase D; D001905:Botulinum Toxins; D019204:GTP-Binding Proteins; D020727:ADP-Ribosylation Factors; D020742:rhoA GTP-Binding Protein; D000262:Adenylyl Cyclases; D013110:Sphingosine; D013755:Tetradecanoylphorbol Acetate; D001920:Bradykinin