Interactions between cytosolic components of the NADPH oxidase: p40^phox interacts with both p67^phox and p47^phox

The NADPH oxidase of neutrophils and other bone-marrow-derived phagocytic cells is a multi-component system consisting of a flavocytochrome b in the plasma membrane and at least four cytosolic proteins. Three of the cytosolic proteins contain src homology 3 (SH3) domains, two each in p47^phox and p67^phox, and one in p40^phox. All three translocate from the cytosol to the flavocytochrome in the membrane upon stimulation of the cells. A small G-protein, p21^rac, is also involved in activation of the oxidase. The three cytosolic phox proteins occur as a complex in the cytosol and the strongest interaction appeared to be between p67^phox and p40^phox. We have investigated the interaction between p40^phox and the other two cytosolic phox proteins by in vitro binding assays. An affinity-bead approach was used as well as a biosensor technique (surface plasmon resonance). We observed the strongest attachment between p40^phox and p67^phox where the binding was between the N-terminal half of p67^phox and the C-terminal half of p40^phox, and did not appear to involve SH3 domains and proline-rich sequences. p40^phox also bound p47^phox but more weakly than it did p67^phox.