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 (NADPHoxidase 2)], which forms a stable heterodimer with p22^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^pho6x-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 Ile¹⁵² 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 Thr¹⁵³ also prevents oxidase activation, but to a lesser extent. In addition, the corresponding isoleucine residue (Ile¹⁵⁵) of the p47^phox homologue Noxo1 (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 thewild-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 Ile¹⁵²-containing region of p47^phox plays a crucial role in oxidase activation, probably by functioning at a process after oxidase assembly.