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Phospholipase D-mediated activation of IQGAP1 through Rac1 regulates hyperoxia-induced p47phox translocation and reactive oxygen species generation in lung endothelial cells.

Abstract
Phosphatidic acid generated by the activation of phospholipase D (PLD) functions as a second messenger and plays a vital role in cell signaling. Here we demonstrate that PLD-dependent generation of phosphatidic acid is critical for Rac1/IQGAP1 signal transduction, translocation of p47(phox) to cell periphery, and ROS production. Exposure of [(32)P]orthophosphate-labeled human pulmonary artery endothelial cells (HPAECs) to hyperoxia (95% O(2) and 5% CO(2)) in the presence of 0.05% 1-butanol, but not tertiary-butanol, stimulated PLD as evidenced by accumulation of [(32)P]phosphatidylbutanol. Infection of HPAECs with adenoviral constructs of PLD1 and PLD2 wild-type potentiated hyperoxia-induced PLD activation and accumulation of O(2)(.)/reactive oxygen species (ROS). Conversely, overexpression of catalytically inactive mutants of PLD (hPLD1-K898R or mPLD2-K758R) or down-regulation of expression of PLD with PLD1 or PLD2 siRNA did not augment hyperoxia-induced [(32)P]phosphatidylbutanol accumulation and ROS generation. Hyperoxia caused rapid activation and redistribution of Rac1, and IQGAP1 to cell periphery, and down-regulation of Rac1, and IQGAP1 attenuated hyperoxia-induced tyrosine phosphorylation of Src and cortactin and ROS generation. Further, hyperoxia-mediated redistribution of Rac1, and IQGAP1 to membrane ruffles, was attenuated by PLD1 or PLD2 small interference RNA, suggesting that PLD is upstream of the Rac1/IQGAP1 signaling cascade. Finally, small interference RNA for PLD1 or PLD2 attenuated hyperoxia-induced cortactin tyrosine phosphorylation and abolished Src, cortactin, and p47(phox) redistribution to cell periphery. These results demonstrate a role of PLD in hyperoxia-mediated IQGAP1 activation through Rac1 in tyrosine phosphorylation of Src and cortactin, as well as in p47(phox) translocation and ROS formation in human lung endothelial cells.
AuthorsPeter V Usatyuk, Irina A Gorshkova, Donghong He, Yutong Zhao, Satish K Kalari, Joe G N Garcia, Viswanathan Natarajan
JournalThe Journal of biological chemistry (J Biol Chem) Vol. 284 Issue 22 Pg. 15339-52 (May 29 2009) ISSN: 0021-9258 [Print] United States
PMID19366706 (Publication Type: Journal Article, Research Support, N.I.H., Extramural)
Chemical References
  • Cortactin
  • Guanine Nucleotide Exchange Factors
  • IQ motif containing GTPase activating protein 1
  • Mutant Proteins
  • RNA, Small Interfering
  • Reactive Oxygen Species
  • TIAM1 protein, human
  • ras GTPase-Activating Proteins
  • Phosphotyrosine
  • NADPH Oxidase
  • neutrophil cytosolic factor 1
  • src-Family Kinases
  • phospholipase D2
  • Phospholipase D
  • phospholipase D1
  • rac1 GTP-Binding Protein
Topics
  • Biocatalysis
  • Cell Membrane (enzymology)
  • Cortactin (metabolism)
  • Down-Regulation (drug effects)
  • Endothelial Cells (cytology, enzymology)
  • Enzyme Activation
  • Guanine Nucleotide Exchange Factors (metabolism)
  • Humans
  • Hyperoxia (enzymology)
  • Lung (cytology)
  • Mutant Proteins (metabolism)
  • NADPH Oxidase (metabolism)
  • Phospholipase D (metabolism)
  • Phosphotyrosine (metabolism)
  • Protein Binding
  • Protein Transport
  • RNA, Small Interfering (metabolism)
  • Reactive Oxygen Species (metabolism)
  • rac1 GTP-Binding Protein (metabolism)
  • ras GTPase-Activating Proteins (metabolism)
  • src-Family Kinases (metabolism)

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