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Updated: Apr 23, 2026
Oligopeptide Competition Assay for Phosphorylation Site Determination
Published on: May 18, 2017
Phosphorylation of Nox1 regulates association with NoxA1 activation domain
Jennifer Streeter1, Brandon M Schickling1, Shuxia Jiang1
1From the Departments of Internal Medicine (B.M.S., S.J., B.S., W.H.T., F.J.M.), Microbiology (J.C.D.H.), Anatomy and Cell Biology (J.S.), Biochemistry (L.G.), and Protein Crystallography Facility (L.G.), University of Iowa, Iowa City; and Veterans Affair Medical Center, Iowa City, IA (F.J.M.).
Rationale:
Activation of Nox1 initiates redox-dependent signaling events crucial in the pathogenesis of vascular disease. Selective targeting of Nox1 is an attractive potential therapy, but requires a better understanding of the molecular modifications controlling its activation.
Objective:
To determine whether posttranslational modifications of Nox1 regulate its activity in vascular cells.
Methods And Results:
We first found evidence that Nox1 is phosphorylated in multiple models of vascular disease. Next, studies using mass spectroscopy and a pharmacological inhibitor demonstrated that protein kinase C-beta1 mediates phosphorylation of Nox1 in response to tumor necrosis factor-α. siRNA-mediated silencing of protein kinase C-beta1 abolished tumor necrosis factor-α-mediated reactive oxygen species production and vascular smooth muscle cell migration. Site-directed mutagenesis and isothermal titration calorimetry indicated that protein kinase C-beta1 phosphorylates Nox1 at threonine 429. Moreover, Nox1 threonine 429 phosphorylation facilitated the association of Nox1 with the NoxA1 activation domain and was necessary for NADPH oxidase complex assembly, reactive oxygen species production, and vascular smooth muscle cell migration.
Conclusions:
We conclude that protein kinase C-beta1 phosphorylation of threonine 429 regulates activation of Nox1 NADPH oxidase.
Insights
Protein kinase C-beta1 phosphorylates Nox1 at threonine 429, a key step in activating Nox1 NADPH oxidase. This finding is crucial for understanding vascular disease pathogenesis and developing targeted therapies.
Area of Science:
- Biochemistry
- Molecular Biology
- Cardiovascular Research
Background:
- Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 1 (Nox1) activation drives redox signaling in vascular disease pathogenesis.
- Targeting Nox1 offers a potential therapeutic strategy, necessitating a deeper understanding of its activation mechanisms.
Purpose of the Study:
- To investigate the role of posttranslational modifications in regulating Nox1 activity within vascular cells.
- To identify specific modifications and the enzymes responsible for Nox1 activation.
Main Methods:
- Mass spectrometry and pharmacological inhibition to identify kinases.
- Site-directed mutagenesis and isothermal titration calorimetry to pinpoint phosphorylation sites.
- Small interfering RNA (siRNA) to assess the functional impact of kinase inhibition.
Main Results:
- Nox1 phosphorylation was observed in vascular disease models.
- Protein kinase C-beta1 (PKCβ1) was identified as the kinase mediating Nox1 phosphorylation in response to tumor necrosis factor-α (TNF-α).
- PKCβ1 inhibition or silencing abolished TNF-α-induced reactive oxygen species (ROS) production and vascular smooth muscle cell (VSMC) migration.
- PKCβ1 phosphorylates Nox1 at threonine 429 (T429).
- Nox1 T429 phosphorylation is essential for NoxA1 binding, NADPH oxidase complex assembly, ROS production, and VSMC migration.
Conclusions:
- PKCβ1-mediated phosphorylation of Nox1 at T429 is a critical regulatory step for Nox1 NADPH oxidase activation.
- This mechanism provides a novel target for therapeutic intervention in vascular diseases driven by Nox1 activity.
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