Hydrogen peroxide stimulates tyrosine phosphorylation of focal adhesion kinase in vascular endothelial cells.
Reactive oxygen species (ROS) are implicated in the pathophysiology of several vascular disorders including atherosclerosis. Although the mechanism(s) of ROS-induced vascular damage remains unclear, there is increasing evidence for ROS-mediated modulation of signal transduction pathways. Exposure of bovine pulmonary artery endothelial cells to hydrogen peroxide (H(2)O(2)) enhanced tyrosine phosphorylation of 60- to 80- and 110- to 130-kDa cellular proteins, which were determined by immunoprecipitation with specific antibodies focal adhesion kinase (p125(FAK)) and paxillin (p68). Brief exposure of cells to a relatively high concentration of H(2)O(2) (1 mM) resulted in a time- and dose-dependent tyrosine phosphorylation of FAK, which reached maximum levels within 10 min (290% of basal levels). Cytoskeletal reorganization as evidenced by the appearance of actin stress fibers preceded H(2)O(2)-induced tyrosine phosphorylation of FAK, and the microfilament disruptor cytochalasin D also attenuated the tyrosine phosphorylation of FAK. Treatment of BPAECs with 1,2-bis(2-aminophenoxy)ethane-N,N,N', N'-tetraacetic acid-AM attenuated H(2)O(2)-induced increases in intracellular Ca(2+) but did not show any consistent effect on H(2)O(2)-induced tyrosine phosphorylation of FAK. Several tyrosine kinase inhibitors, including genistein, herbimycin, and tyrphostin, had no detectable effect on tyrosine phosphorylation of FAK but attenuated the H(2)O(2)-induction of mitogen-activated protein kinase activity. We conclude that H(2)O(2)-induced increases in FAK tyrosine phosphorylation may be important in H(2)O(2)-mediated endothelial cell activation.
Division of Pulmonary and Critical Care Medicine, Department of Medicine, Johns Hopkins School of Medicine, Baltimore, Maryland 21224, USA. svepa@welchlink.welch.jhu.edu
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S
Vepa
W M
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Scribner
N L
NL
Parinandi
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D
English
J G
JG
Garcia
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V
Natarajan
D000199:Actins / Q000502:physiology; D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002417:Cattle; D015815:Cell Adhesion Molecules / Q000378:metabolism*; D002478:Cells, Cultured; D003598:Cytoskeletal Proteins / Q000378:metabolism; D004351:Drug Resistance; D004730:Endothelium, Vascular / Q000166:cytology / Q000201:enzymology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D051416:Focal Adhesion Protein-Tyrosine Kinases; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D051419:Paxillin; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D011493:Protein Kinase C / Q000502:physiology; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011651:Pulmonary Artery / Q000166:cytology / Q000201:enzymology; D014443:Tyrosine / Q000378:metabolism*
D000199:Actins; D015815:Cell Adhesion Molecules; D003598:Cytoskeletal Proteins; D004791:Enzyme Inhibitors; D051419:Paxillin; D010750:Phosphoproteins; D014443:Tyrosine; D006861:Hydrogen Peroxide; D011505:Protein-Tyrosine Kinases; D051416:Focal Adhesion Protein-Tyrosine Kinases; D011493:Protein Kinase C; D002118:Calcium