Novel redox-dependent regulation of NOX5 by the tyrosine kinase c-Abl.
We investigated the mechanism of H(2)O(2) activation of the Ca(2+)-regulated NADPH oxidase NOX5. H(2)O(2) induced a transient, dose-dependent increase in superoxide production in K562 cells expressing NOX5. Confocal studies demonstrated that the initial calcium influx generated by H(2)O(2) is amplified by a feedback mechanism involving NOX5-dependent superoxide production and H(2)O(2). H(2)O(2) NOX5 activation was inhibited by extracellular Ca(2+) chelators, a pharmacological inhibitor of c-Abl, and overexpression of kinase-dead c-Abl. Transfected kinase-active GFP-c-Abl colocalized with vesicular sites of superoxide production in a Ca(2+)-dependent manner. In contrast to H(2)O(2), the Ca(2+) ionophore ionomycin induced NOX5 activity independent of c-Abl. Immunoprecipitation of cell lysates revealed that active GFP-c-Abl formed oligomers with endogenous c-Abl and that phosphorylation of both proteins was increased by H(2)O(2) treatment. Furthermore, H(2)O(2)-induced NOX5 activity correlated with increased localization of c-Abl to the membrane fraction, and NOX5 proteins could be coimmunoprecipitated with GFP-Abl proteins. Our data demonstrate for the first time that NOX5 is activated by c-Abl through a Ca(2+)-mediated, redox-dependent signaling pathway and suggest a functional association between NOX5 NADPH oxidase and c-Abl.
El Jamali
Amina
A
Department of Medicine, University of Texas Health Science Center, San Antonio, TX 78229-3900, USA. AkoulouzeBik@uthscsa.edu
Valente
Anthony J
AJ
Lechleiter
James D
JD
Gamez
Maria J
MJ
Pearson
Doran W
DW
Nauseef
William M
WM
Clark
Robert A
RA
D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D025461:Feedback, Physiological; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D047468:Immunoprecipitation; D015759:Ionomycin / Q000494:pharmacology; D020014:K562 Cells; D008565:Membrane Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D000074664:NADPH Oxidase 5; D019255:NADPH Oxidases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D010084:Oxidation-Reduction; D010766:Phosphorylation; D016315:Proto-Oncogene Proteins c-abl / Q000378:metabolism*; D017382:Reactive Oxygen Species / Q000378:metabolism; D013481:Superoxides / Q000378:metabolism; D014443:Tyrosine / Q000378:metabolism
D008565:Membrane Proteins; D017382:Reactive Oxygen Species; C485184:enhanced green fluorescent protein; D013481:Superoxides; D049452:Green Fluorescent Proteins; D014443:Tyrosine; D015759:Ionomycin; D000074664:NADPH Oxidase 5; D019255:NADPH Oxidases; C431571:NOX5 protein, human; D016315:Proto-Oncogene Proteins c-abl; D002118:Calcium