THE PC MOTIF - A NOVEL AND EVOLUTIONARILY CONSERVED SEQUENCE INVOLVEDIN INTERACTION BETWEEN P40(PHOX) AND P67(PHOX), SH3 DOMAIN-CONTAININGCYTOSOLIC FACTORS OF THE PHAGOCYTE NADPH OXIDASE

The superoxide-generating NADPH oxidase, dormant in resting phagocytes , is activated during phagocytosis following assembly of the membrane- integrated protein cytochrome b(558) and cytosolic factors. Among the latter are the three proteins containing Src homology 3(SH3) domains,p 67(phox),p47(phox) and p40(phox). While the first two factors are indi spensable for the activity, p40(phox) is tightly associated with p67(p hox) in resting cells and is suggested to have some modulatory role. H ere we describe a systematic analysis of the interaction between p40(p hox) and p67(phox) using the yeast two-hybrid system and in vitro bind ing assays with recombinant proteins. Both methods unequivocally showe d that the minimum requirements for stable interaction are the C-termi nal region of p40(phox) and the region between the two SH3 domains of p67(phox). This interaction is maintained even in the presence of anio nic amphiphiles used for the activation of the NADPH oxidase, raising a possibility that it mediates constitutive association of the two fac tors in both resting and activated cells. The C-terminal region of p40 (phox) responsible for the interaction contains a characteristic stret ch of amino acids designated as the PC motif, that also exists in othe r signal-transducing proteins from yeast to human. Intensive site-dire cted mutagenesis to the motif in p40(phox) revealed that it plays a cr itical role in the binding to p67(phox). Thus the PC motif appears to represent a novel module for protein-protein interaction used in a var iety of signaling pathways.