Phosphorylation of Nox1 Regulates Association with NoxA1 Activation Domain
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 the 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 (PKC-βI) mediates phosphorylation of Nox1 in response to tumor necrosis factor-α (TNF-α). siRNA-mediated silencing of PKC-βI abolished TNF-α-mediated reactive oxygen species (ROS) production and vascular smooth muscle cell (VSMC) migration. Site-directed mutagenesis and isothermal titration calorimetry indicated that PKC-βI phosphorylates Nox1 at T429. Moreover, Nox1 T429 phosphorylation facilitated the association of Nox1 with the NoxA1 activation domain and was necessary for NADPH oxidase complex assembly, ROS production, and VSMC migration.
Conclusions: We conclude that PKC-βI phosphorylation of T429 regulates activation of Nox1 NADPH oxidase.
- Received April 25, 2014.
- Revision received September 12, 2014.
- Accepted September 16, 2014.