Multiple SH3 domain interactions regulate NADPH oxidase assembly in whole cells.

Abstract

Src homology 3 (SH3) domains mediate specific protein-protein interactions crucial for signal transduction and protein subcellular localization. Upon phagocyte stimulation, two SH3 domain-containing cytosolic components of the NADPH oxidase, p47(phox) and p67(phox), are recruited to the membrane where they interact with flavocytochrome b 558 to form an activated microbicidal oxidase. Deletion analysis of p47(phox) and p67(phox) in transfected K562 cells demonstrated multiple SH3-mediated interactions between p47(phox) and the transmembrane flavocytochrome b 558 and also between the cytosolic components themselves. The core region of p47(phox) (residues 151-284), spanning both SH3 domains, was required for flavocytochrome-dependent translocation and oxidase activity in whole cells. Furthermore, translocation of p67(phox) occurred through interactions of its N-terminal domain (residues 1-246) with p47(phox) SH3 domains. Both of these interactions were promoted by PMA activation of cells and were influenced by the presence of other domains in both cytosolic factors. Deletion analysis also revealed a third SH3 domain-mediated interaction involving the C-termini of both cytosolic factors, which also promoted p67(phox) membrane translocation. These data provide evidence for a central role for p47(phox) in regulation of oxidase assembly through several SH3 domain interactions.