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NOXO1 phosphorylation on serine 154 is critical for optimal NADPH oxidase 1 assembly and activation

Maya Debbabi1, Yolande Kroviarski, Odile Bournier

  • 1Institut National de Santé et de Recherche Médicale (INSERM) U773, Centre de Recherche Biomédicale Bichat Beaujon CRB3, Paris, France.

FASEB Journal : Official Publication of the Federation of American Societies for Experimental Biology
|January 17, 2013
PubMed

Insights

NADPH oxidase 1 (NOX1) activation in colon cells is triggered by NOXO1 phosphorylation at Ser-154. This phosphorylation enhances NOXO1 interactions, leading to optimal ROS production by NOX1.

Area of Science:

  • Cellular Biology
  • Biochemistry
  • Molecular Biology

Background:

  • NADPH oxidase 1 (NOX1) is crucial for reactive oxygen species (ROS) production in colon epithelial cells.
  • NOX1 activation requires p22(PHOX), NOXO1, NOXA1, and Rac1.
  • The regulation of NOX1, unlike NOX2, is not well understood, with a prevailing view that NOXO1 activation occurs without phosphorylation.

Purpose of the Study:

  • To investigate the role of NOXO1 phosphorylation in NOX1 regulation.
  • To identify the specific site and mechanism of NOXO1 phosphorylation.
  • To determine the functional consequences of NOXO1 phosphorylation on NOX1 assembly and activity.

Main Methods:

  • Utilized human embryonic kidney (HEK) 293 cells and T84 colon epithelial cells.
  • Stimulated NOXO1 phosphorylation using phorbol myristate acetate (PMA) and identified the phosphorylated site (Ser-154) via protein kinase C.
  • Employed site-directed mutagenesis (S154A, S154D) and pulldown assays to assess protein interactions and ROS production.

Main Results:

  • Demonstrated that PMA induces NOXO1 phosphorylation at Ser-154 in HEK 293 cells and that endogenous NOXO1 in T84 cells is also phosphorylated.
  • Showed that Ser-154 phosphorylation significantly enhances NOXO1 binding to NOXA1 and p22(PHOX), and increases NOXO1 colocalization with p22(PHOX).
  • Confirmed that phosphorylation at Ser-154 is essential for optimal ROS production by NOX1, acting as the initial trigger for full NOX1 assembly and activation.

Conclusions:

  • NOXO1 phosphorylation at Ser-154 is a critical and physiologically relevant regulatory step in NOX1 activation.
  • Phosphorylation of NOXO1 by protein kinase C initiates a cascade, enhancing interactions with NOXA1 and p22(PHOX), thereby enabling ROS production.
  • This finding challenges the previous understanding and reveals a tightly regulated mechanism for NOX1 assembly and function.

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