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Research ArticleArticle

Calcium/Calmodulin-Dependent Kinase II Mediates the Phosphorylation and Activation of NADPH Oxidase 5

Deepesh Pandey, Jean-Philippe Gratton, Ruslan Rafikov, Stephen M. Black and David J. R. Fulton
Molecular Pharmacology September 2011, 80 (3) 407-415; DOI: https://doi.org/10.1124/mol.110.070193
Deepesh Pandey
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Jean-Philippe Gratton
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Ruslan Rafikov
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Stephen M. Black
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David J. R. Fulton
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Abstract

Excessive synthesis of reactive oxygen species contributes to the pathology of many human diseases and originates from changes in the expression and posttranslational regulation of the transmembrane NADPH oxidases (Noxes). Nox5 is a novel Nox isoform whose activity is regulated by intracellular calcium levels. We have reported that the activity and calcium-sensitivity of Nox5 can also be modulated by direct phosphorylation. However, the kinases that phosphorylate Nox5 have not been identified, and thus, the goal of this study was to determine whether calcium-activated kinases such as calcium/calmodulin-dependent kinase II (CAMKII) are involved. We found that Nox5 activity in bovine aortic endothelial cells was suppressed by two doses of the CAMKII inhibitor 2-(N-[2-hydroxyethyl])-N-(4-methoxybenzenesulfonyl)amino-N-(4-chlorocinnamyl)-N-methylamine (KN-93). In cotransfected COS-7 cells, wild-type and constitutively active CAMKII, but not a dominant-negative, robustly increased basal Nox5 activity. The ability of CAMKII to increase Nox5 activity was also observed with fixed calcium concentrations in an isolated enzyme activity assay. CAMKII did not elevate intracellular calcium or activate other Nox enzymes. In vitro phosphorylation assays revealed that CAMKII can directly phosphorylate Nox5 on Thr494 and Ser498 as detected by phosphorylation state-specific antibodies. Mass spectrometry (MS) analysis revealed the phosphorylation of additional, novel sites at Ser475, Ser502, and Ser675. Of these phosphorylation sites, mutation of only Ser475 to alanine prevented CAMKII-induced increases in Nox5 activity. The ability of CAMKIIα to phosphorylate Ser475 in intact cells was supported by the binding of Nox5 to phosphoprotein-affinity columns and via MS/MS analysis. Together, these results suggest that CAMKII can positively regulate Nox5 activity via the phosphorylation of Ser475.

Footnotes

  • This work was supported in part by the Cardiovascular Discovery Institute at the Medical College of Georgia; the National Institutes of Health National Heart, Lung, and Blood Institute [Grants HL085827, HL092446, HL60190, HL67841]; and an American Heart Association Established Investigator Award.

  • Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.

    doi:10.1124/mol.110.070193.

  • ABBREVIATIONS:

    Nox
    NADPH oxidase
    CAMKII
    calcium/calmodulin-activated kinase II
    ROS
    reactive oxygen species
    BAEC
    bovine aortic endothelial cell
    MS
    mass spectrometry
    MS/MS
    tandem mass spectrometry
    PMA
    phorbol 12-myristate 13-acetate
    siRNA
    small interfering RNA
    PAGE
    polyacrylamide gel electrophoresis
    TFA
    trifluoroacetic acid
    WT
    wild type
    HA
    hemagglutinin
    RFP
    red fluorescent protein
    MOPS
    3-(N-morpholino)propanesulfonic acid
    MALDI-TOF
    matrix-assisted laser desorption ionization/time of flight
    KN-93
    2-(N-[2-hydroxyethyl])-N-(4-methoxybenzenesulfonyl)amino-N-(4-chlorocinnamyl)-N-methylamine
    KN-92
    2-[N-(4-methoxybenzenesulfonyl)]amino-N-(4-chlorocinnamyl)-N-methylbenzylamine, phosphate
    CHCA
    α-cyano-4-hydroxycinnamic acid
    L-012
    8-amino-5-chloro-7-phenylpyrido[3,4-d]pyridazine-1,4-(2H,3H) dione.

  • Received November 27, 2010.
  • Accepted June 2, 2011.
  • Copyright © 2011 The American Society for Pharmacology and Experimental Therapeutics
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Molecular Pharmacology: 80 (3)
Molecular Pharmacology
Vol. 80, Issue 3
1 Sep 2011
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Research ArticleArticle

Calcium/Calmodulin-Dependent Kinase II Mediates the Phosphorylation and Activation of NADPH Oxidase 5

Deepesh Pandey, Jean-Philippe Gratton, Ruslan Rafikov, Stephen M. Black and David J. R. Fulton
Molecular Pharmacology September 1, 2011, 80 (3) 407-415; DOI: https://doi.org/10.1124/mol.110.070193

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Research ArticleArticle

Calcium/Calmodulin-Dependent Kinase II Mediates the Phosphorylation and Activation of NADPH Oxidase 5

Deepesh Pandey, Jean-Philippe Gratton, Ruslan Rafikov, Stephen M. Black and David J. R. Fulton
Molecular Pharmacology September 1, 2011, 80 (3) 407-415; DOI: https://doi.org/10.1124/mol.110.070193
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