A region N-terminal to the tandem SH3 domain of p47phox plays a crucial role in the activation of the phagocyte NADPH oxidase

The superoxide-producing NADPH oxidase in phagocytes is crucial for host defence; its catalytic core is the membrane-integrated protein gp91(phox) [also known as Nox2 (NADPH oxidase 2)], which forms a stable heterodimer with p22P(phox). Activation of the oxidase requires membrane translocation of the three cytosolic proteins p47(phox), p67(phox) and the small GTPase Rac. At the membrane, these proteins assemble with the gp91(phox)-p22(phox) heterodimer and induce a conformational change of gp91(phox), leading to Superoxide production. p47(phox) translocates to membranes using its two tandemly arranged SH3 domains, which directly interact with P22(phox), whereas P67(phox) is recruited in a P47(phox)-dependent manner. In the present study, we show that a short region N-terminal to the bis-SH3 domain is required for activation of the phagocyte NADPH oxidase. Alanine substitution for IIe(152) in this region, a residue that is completely conserved during evolution, results in a loss of the ability to activate the oxidase; and the replacement of Thr(153) also prevents oxidase activation, but to a lesser extent. In addition, the corresponding isoleucine residue (IIe(151)) of the p47(phox) homologue Noxol (Nox organizer 1) participates in the activation of non-phagocytic oxidases, such as Nox1 and Nox3. The I152A substitution in p47(phox). however, does not affect its interaction with P22(phox) or with p67(phox). Consistent with this, a mutant P47(phox) (I152A), as well as the wild-type protein, is targeted upon cell stimulation to membranes, and membrane recruitment of P67(phox) and Rac normally occurs in P47(phox) (I152A)-expressing cells. Thus the IIe(152)-containing region of p47(phox) plays a crucial role in oxidase activation, probably by functioning at a process after oxidase assembly.