TOPOLOGICAL ORGANIZATION OF THE CYTOSOLIC ACTIVATING COMPLEX OF THE SUPEROXIDE-GENERATING NADPH-OXIDASE - PINPOINTING THE SITES OF INTERACTION BETWEEN P47PHOX, P67PHOX AND P40PHOX USING THE 2-HYBRID SYSTEM

Activation of the superoxide-generating NADPH-oxidase in phagocytic ce lls requires the assembly of a membrane-bound flavocytochrome b and cy tosolic factors p47phox and p67phox under the control of the GTP-bindi ng protein, Rac. A novel cytosolic component p4Ophox was recently iden tified. Most of the components of the complex contain SH3 domains and/ or polyproline motifs which are likely to mediate protein-protein inte ractions occurring in the formation of the active NADPH-complex. The t wo-hybrid system was used to explore associations between the cytosoli c factors. Various constructs of p47phox, p67phox and p40phox cDNAs co ding for functional domains were inserted into two-hybrid system vecto rs, 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 p6 7phox and to the polyproline motif of p47phox. The polyproline motif o f p47phox was also found to mediate interaction with the SH3 domain of p40phox, as well as intramolecular interaction within p47phox. The si te of interaction of p67phox with p40phox was found to be in the 150 a mino acid stretch between the two SH3 domains of p67phox. As the C-ter minal tail of p40phox which interacts with p67phox contains neither a SH3 domain nor a polyproline consensus site, it is concluded that a no vel type of interaction occurs between p40phox and p67phox. Taken toge ther, the results of the two-hybrid experiments led us to formulate a model for oxidase activation, induced by phosphorylation, in which p40 phox tends to prevent spontaneous activation.