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 aid p67(phox).