Nox activator 1 (NoxA1) is a homologue of p67(phox) that acts in conjunction 
with Nox organizer 1 (NoxO1) to regulate reactive oxygen species (ROS) 
production by the NADPH oxidase Nox1. The phosphorylation of cytosolic 
regulatory components by multiple kinases plays important roles in assembly and 
activity of the phagocyte NADPH oxidase (Nox2) system, but little is known about 
regulation by phosphorylation in the Nox1 system. Here we identify Ser(172) and 
Ser(461) of NoxA1 as phosphorylation sites for protein kinase A (PKA). A 
consequence of this phosphorylation was the enhancement of NoxA1 complex 
formation with 14-3-3 proteins. Using both a transfected human embryonic kidney 
293 cell Nox1 model system and endogenous Nox1 in colon cell lines, we showed 
that the elevation of cAMP inhibits, whereas the inhibition of PKA enhances, 
Nox1-dependent ROS production through effects on NoxA1. Inhibition of Nox1 
activity was intensified by the availability of 14-3-3zeta protein, and this 
regulatory interaction was dependent on PKA-phosphorylatable sites at Ser(172) 
and Ser(461) in NoxA1. We showed that phosphorylation and 14-3-3 binding induce 
the dissociation of NoxA1 from the Nox1 complex at the plasma membrane, 
suggesting a mechanism for the inhibitory effect on Nox1 activity. Our data 
establish that PKA-phosphorylated NoxA1 is a new binding partner of 14-3-3 
protein(s) and that this forms the basis of a novel mechanism regulating the 
formation of ROS by Nox1 and, potentially, other NoxA1-regulated Nox family 
members.