Original Contribution
Novel redox-dependent regulation of NOX5 by the tyrosine kinase c-Abl

https://doi.org/10.1016/j.freeradbiomed.2007.11.020Get rights and content

Abstract

We investigated the mechanism of H2O2 activation of the Ca2+-regulated NADPH oxidase NOX5. H2O2 induced a transient, dose-dependent increase in superoxide production in K562 cells expressing NOX5. Confocal studies demonstrated that the initial calcium influx generated by H2O2 is amplified by a feedback mechanism involving NOX5-dependent superoxide production and H2O2. H2O2 NOX5 activation was inhibited by extracellular Ca2+ 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 Ca2+-dependent manner. In contrast to H2O2, the Ca2+ 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 H2O2 treatment. Furthermore, H2O2-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 Ca2+-mediated, redox-dependent signaling pathway and suggest a functional association between NOX5 NADPH oxidase and 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

References (47)

  • A.J. Sbarra et al.

    The biochemical basis of phagocytosis: metabolic changes during the ingestion of particles by polymorphonuclear leukocytes

    J. Biol. Chem.

    (1959)
  • L.A. Blatter et al.

    Intracellular diffusion, binding, and compartmentalization of the fluorescent calcium indicators indo-1 and fura-2

    Biophys. J.

    (1990)
  • B.B. Brasher et al.

    c-Abl has high intrinsic tyrosine kinase activity that is stimulated by mutation of the Src homology 3 domain and by autophosphorylation at two distinct regulatory tyrosines

    J. Biol. Chem.

    (2000)
  • D. Mizuchi et al.

    BCR/ABL activates Rap1 and B-Raf to stimulate the MEK/Erk signaling pathway in hematopoietic cells

    Biochem. Biophys. Res. Commun.

    (2005)
  • R.S. BelAiba et al.

    NOX5 variants are functionally active in endothelial cells

    Free Radic. Biol. Med.

    (2007)
  • B. Burlando et al.

    Ca2+ is mobilized by hydroxyl radical but not by superoxide in RTH-149 cells: the oxidative switching-on of Ca2+ signaling

    Cell Calcium

    (2005)
  • K.M. Lounsbury et al.

    Calcium signaling and oxidant stress in the vasculature

    Free Radic. Biol. Med.

    (2000)
  • O. Hantschel et al.

    Myristoyl/phosphotyrosine switch regulates c-Abl

    Cell

    (2003)
  • R.A. Van Etten

    Cycling, stressed-out and nervous: cellular functions of c-Abl

    Trends Cell. Biol.

    (1999)
  • M.E. Ginn-Pease et al.

    Redox signals and NF-κB activation in T cells

    Free Radic. Biol. Med.

    (1998)
  • M. Sundaresan et al.

    Requirement for generation of H2O2 for platelet-derived growth factor signal transduction

    Science

    (1995)
  • A.M. Zafari et al.

    Role of NADH/NADPH oxidase-derived H2O2 in angiotensin II-induced vascular hypertrophy

    Hypertension

    (1998)
  • H.S. Park

    Cutting edge: direct interaction of TLR4 with NAD(P)H oxidase 4 isozyme is essential for lipopolysaccharide-induced production of reactive oxygen species and activation of NF-kappa B

    J. Immunol.

    (2004)
  • Cited by (0)

    View full text