Original ContributionNovel redox-dependent regulation of NOX5 by the tyrosine kinase c-Abl
Section snippets
Cell culture and stable expression of NOX5 and Abl proteins in K562 cells
K562 human leukemia cells were grown in RPMI medium supplemented with 10% fetal bovine serum, plus 100 U/ml penicillin and 100 μg/ml streptomycin. Cells in the logarithmic phase of growth were transfected with expression vectors as described previously [17] and stably expressing clones selected in the appropriate antibiotic. Single-cell clones were established by limiting dilution in 96-well plates. The human NOX5β cDNA cloned into the pGEX-2T vector and the HEK293 cell line stably expressing
Hydrogen peroxide induces NOX5 activity
To study the effect of H2O2 on NOX5 activity, we used K562 human myeloid cells ectopically expressing the NOX5 protein (K562/NOX5 cells). Immunoblotting with NOX5 antibody demonstrated a band of ∼75 kDa in the crude membranes of K562/NOX5 cells, but not the K562 parental line (Fig. 1A).
The luminol-based chemiluminescence assay (Diogenes reagent) was used to measure the effect of H2O2 on NOX5 activity, because this reagent selectively detects superoxide anion. We confirmed that this reagent does
Discussion
Here we report that H2O2 positively regulates NOX5 activity by c-Abl through a Ca2+-mediated, redox-dependent signaling pathway and suggest a functional association between NOX5 and c-Abl. H2O2 stimulation of NOX5 was blocked by imatinib mesylate, an inhibitor of c-Abl tyrosine kinase, and by genistein, an inhibitor of Src tyrosine kinases, known activators of c-Abl [31], [32]. However, in K562/NOX5 cells the overexpression of a constitutively active or inactive mutant of Src did not affect the
Acknowledgments
This work was supported by Grants AI020866, AG019519, and AI034879 from the National Institutes of Health. The confocal microscopy studies were performed in the Institutional Optical Imaging Facility of the University of Texas Health Science Center at San Antonio, which is supported by NIH Grants P30-CA054174 (San Antonio Cancer Institute), P30-AG013319 (Nathan Shock Center), and P01-AG019316 (Aging, Oxidative Stress and Cell Death). The authors would like to thank Hanna Abboud and Alain Virion
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