Phosphorylated p40^PHOX as a Negative Regulator of NADPH Oxidase

Abstract

The leukocyte NADPH oxidase catalyzes the production of O(2)(-) from oxygen at the expense of NADPH. Activation of the enzyme requires interaction of the cytosolic factors p47(PHOX), p67(PHOX), and Rac2 with the membrane-associated cytochrome b(558). Activation of the oxidase in a semirecombinant cell-free system in the absence of an amphiphilic activator can be achieved by phosphorylation of the cytosolic factor p47(PHOX) by protein kinase C. Another cytosolic factor, p40(PHOX), was recently shown to be phosphorylated on serine and threonine residues upon activation of NADPH oxidase, but both stimulatory and inhibitory roles were reported. In the present study, we demonstrate that the addition of phosphorylated p40(PHOX) to the cell-free system inhibits NADPH oxidase activated by protein kinase C-phosphorylated p47(PHOX), an effect not observed with the unphosphorylated p40(PHOX). Moreover phosphorylated p40(PHOX) inhibits the oxidase if added before or after full activation of the enzyme. Direct mutagenesis of protein kinase C consensus sites enables us to conclude that phosphorylation of threonine 154 is required for the inhibitory effect of p40(PHOX) to occur. Although the phosphorylated mutants and nonphosphorylated mutants are still able to interact with both p47(PHOX) and p67(PHOX) in pull-down assays, their proteolysis pattern upon thrombin treatment suggests a difference in conformation between the phosphorylated and nonphosphorylated mutants. We postulate that phosphorylation of p40(PHOX) on threonine 154 leads to an inhibitory conformation that shifts the balance toward an inhibitory role and blocks oxidase activation.

A cascade of Ca2+/calmodulin-dependent protein kinases regulates the differentiation and functional activation of murine neutrophils

“…The lack of effects on gp91 phox gene expression suggests that cascade overactivation can antagonize functions of NADPH oxidase components via post-translational modifications. Indeed, serine phosphorylation regulates the activities of several components, including p47 phox , p67 phox and Rap1, and threonine phosphorylation of p40 phox inhibits NADPH oxidase function [45][46][47][48]. Despite this antagonistic role, the cascade is required in mature neutrophils but CKLiK expression levels may be insufficient to positively regulate NADPH oxidase components in the absence of CaMKKα activities.…”

Reconstitution of Chemotactic Peptide-Induced Nicotinamide Adenine Dinucleotide Phosphate (Reduced) Oxidase Activation in Transgenic COS-phox Cells

“…production. However, there are also published reports indicating that p40 phox is an inhibitory molecule for NADPH oxidase activation, in both K562 cells (48) and cell-free assays (49,50). In light of the controversy surrounding the exact role of p40 phox in NADPH oxidase activation, we sought to examine this cytosolic protein, which is absent from the epithelial COS-phox cells.…”

A Regulated Adaptor Function of p40phox: Distinct p67phoxMembrane Targeting by p40phoxand by p47phox

“…There are reports that phosphorylation occurs within p40 phox at Thr154 and Ser315 (Bouin et al, 1998) and that phosphorylation of Thr154, but not Ser315, inhibits Nox2 activation (Lopes et al, 2004). Therefore, we made mutants of GFP-p40 phox that could not be phosphorylated [GFPp40 phox (T154A) and GFP-p40 phox (S315A)] or mimic the phosphorylated states [GFP-p40 phox (T154D) and GFP-p40 phox (S315D)].…”

“…There are, however, no reports of p40 phox defects or deficiencies resulting in CGD. There is some controversy on the precise function of p40 phox , because both Nox2-inhibitory (Sathyamoorthy et al, 1997;Vergnaud et al, 2000;Lopes et al, 2004) and Nox2-supporting effects of p40 phox (Tsunawaki et al, 1996;Cross, 2000;Ellson et al, 2001b;Kuribayashi et al, 2002;He et al, 2004) have been reported. However, recent studies in p40 phox -deficient mice or in Fc␥IIA receptor-reconstituted cells indicate p40 phox is an essential component of the Nox2 system (Ellson et al, 2006;Suh et al, 2006).…”

“…These data suggest that AA induces conformational changes in p40 phox that render the PX domain accessible to bind to early endosomes, as occurs in AA-treated p47 phox to expose its SH3 domains to bind to p22 phox (Shiose and Sumimoto, 2000;Zhao et al, 2002). It is intriguing that GFP-p40 phox and GFP-p47 phox , but not GFP-p67 phox , have the ability to bind to the membrane only after AA stimulation, which likely induces conformational change in both proteins.There are reports that phosphorylation occurs within p40 phox at Thr154 and Ser315 (Bouin et al, 1998) and that phosphorylation of Thr154, but not Ser315, inhibits Nox2 activation (Lopes et al, 2004). Therefore, we made mutants of GFP-p40 phox that could not be phosphorylated [GFPp40 phox (T154A) and GFP-p40 phox (S315A)] or mimic the phosphorylated states [GFP-p40 phox (T154D) and GFP-p40 phox (S315D)].…”