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Regulation of Nox1 activity via protein kinase A-mediated phosphorylation of NoxA1 and 14-3-3 binding

Jun-Sub Kim1, Becky A Diebold, Bernard M Babior

  • 1Department of Immunology, The Scripps Research Institute, La Jolla, California 92037, USA.

The Journal of Biological Chemistry
|October 5, 2007
PubMed

Insights

Protein kinase A (PKA) phosphorylates Nox activator 1 (NoxA1) at specific sites, enhancing its binding to 14-3-3 proteins. This interaction inhibits Nox1-dependent reactive oxygen species (ROS) production by promoting NoxA1 dissociation from the Nox1 complex.

Area of Science:

  • Biochemistry
  • Cell Biology
  • Molecular Signaling

Background:

  • The NADPH oxidase Nox1 system regulates reactive oxygen species (ROS) production, crucial in cellular signaling and immunity.
  • While phosphorylation regulates the related Nox2 system, its role in Nox1 regulation, particularly involving Nox activator 1 (NoxA1), remains largely unexplored.

Purpose of the Study:

  • To investigate the role of protein kinase A (PKA) in regulating Nox1-dependent ROS production through phosphorylation of NoxA1.
  • To elucidate the mechanism by which PKA-mediated phosphorylation affects NoxA1 function and its interaction with other regulatory proteins.

Main Methods:

  • Identification of PKA phosphorylation sites on NoxA1 using transfected human embryonic kidney 293 cells and colon cell lines expressing Nox1.
  • Analysis of NoxA1 complex formation with 14-3-3 proteins following PKA-mediated phosphorylation.
  • Assessment of Nox1-dependent ROS production under conditions of altered cAMP levels and PKA activity.

Main Results:

  • Serine residues 172 and 461 of NoxA1 were identified as key phosphorylation sites for PKA.
  • PKA phosphorylation enhanced the binding of NoxA1 to 14-3-3 proteins, particularly 14-3-3zeta.
  • Elevated cAMP levels (activating PKA) inhibited Nox1-ROS production, while PKA inhibition enhanced it, mediated by NoxA1 phosphorylation and 14-3-3 binding.
  • Phosphorylation and 14-3-3 binding led to NoxA1 dissociation from the Nox1 complex at the plasma membrane, explaining ROS inhibition.

Conclusions:

  • PKA-mediated phosphorylation of NoxA1 at Ser172 and Ser461 is a novel regulatory mechanism for Nox1 activity.
  • This phosphorylation promotes NoxA1 binding to 14-3-3 proteins, leading to NoxA1 dissociation from the Nox1 complex and subsequent inhibition of ROS production.
  • This discovery reveals a new pathway for controlling ROS generation by the Nox1 system and potentially other NoxA1-utilizing Nox family enzymes.

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