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 adenocarci noma A549 cells) the role of RhoA and ARF in activation of PLD by phorbol 1 2-myristate 13-acetate, bradykinin, and/or sphingosine 1-phosphate, To expr ess 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 d ouble subgenomic recombinant Sindbis virus Rho19N), cells were infected wit h Sindbis virus, a novel vector that allows rapid, high level expression of heterologous proteins. Expression of C3 toxin or Rho19N increased basal an d decreased phorbol 12-myristate 13-acetate-stimulated PLD activity. Bradyk inin or sphingosine 1-phosphate increased PLD activity with additive effect s that were abolished in cells expressing C3 exoenzyme or Rho19N, In cells expressing C3, modification of Rho appeared to be incomplete, suggesting th e existence of pools that differed in their accessibility to the enzyme. Si milar results were obtained with cells scrape-loaded in the presence of C3; however, results with virus infection were more reproducible. To assess th e role of ARF, cells were incubated with brefeldin A (BFA), a fungal metabo lite that disrupts Golgi structure and inhibits enzymes that catalyze ARF a ctivation 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 e ffect on Rho-stimulated PLD activity, indicating that RhoA action did not i nvolve a BFA-sensitive pathway, A novel PLD activation mechanism, not sensi tive to BFA and involving RhoA, was identified in human airway epithelial c ells by use of a viral infection technique that preserves cell responsivene ss.