Topological organization of the cytosolic activating complex of the superoxide-generating NADPH-oxidase. Pinpointing the sites of interaction between p47phoz, p67phox and p40phox using the two-hybrid system

Abstract

Activation of the superoxide-generating NADPH-oxidase in phagocytic cells requires the assembly of a membrane-bound flavocytochrome b and cytosolic factors p47phox and p67phox under the control of the GTP-binding protein, Rac. A novel cytosolic component p40phox was recently identified. Most of the components of the complex contain SH3 domains and/or polyproline motifs which are likely to mediate protein-protein interactions occurring in the formation of the active NADPH-complex. The two-hybrid system was used to explore associations between the cytosolic factors. Various constructs of p47phox, p67phox and p40phox cDNAs coding for functional domains were inserted into two-hybrid system vectors, expressing fusion proteins either with the DNA binding protein Lex A or with the activation domain of Gal 4. The site of interaction of p67phox with p47phox was restricted to the C-terminal SH3 domain of p67phox and to the polyproline motif of p47phox. The polyproline motif of p47phox was also found to mediate interaction with the SH3 domain of p40phox, as well as intramolecular interaction within p47phox. The site of interation of p67phox with p40phox was found to be in the 150 amino acid stretch between the two SH3 domains of p67phox. As the C-terminal tail of p40phox which interacts with p67phox contains neither a SH3 domain nor a polyproline consensus site, it is concluded that a novel type of interaction occurs between p40phox and p67phox. Taken together, the results of the two-hybrid experiments led us to formulate a model for oxidase activation, induced by phosphorylation, in which p40phox tends to prevent spontaneous activation.