ASSEMBLY OF THE PHAGOCYTE NADPH OXIDASE - BINDING OF SRC HOMOLOGY-3 DOMAINS TO PROLINE-RICH TARGETS

NADPH oxidase responsible for generation of superoxide anion and relat ed microbicidal oxidants by phagocytes is assembled from at least five distinct proteins. Two are cytosolic components (p47-phox and p67-pho x) that contain Src homology 3 (SH3) domains and associate with a tran smembrane cytochrome b(558) upon activation. We show here that the SH3 domains of p47-phox bind to proline-rich sequences in p47-phox itself and the p22-phox subunit of cytochrome b(558). Binding of the p47-pho x SH3 domains to p22-phox was abolished by a mutation in one proline-r ich sequence (pro(156) --> Gln) noted in a distinct form of chronic gr anulomatous disease and was inhibited by a short proline-rich syntheti c peptide corresponding to residues 149-162 of p22-phox. Expression of mutated p22-phox did not restore oxidase activity to p22-phox-deficie nt B cells and did not enable p22-phox-dependent translocation of p47- phox to membranes in phorbol ester-stimulated cells. We also show that the cytosolic oxidase components associate with one another through t he C-terminal SH3 domain of p67-phox and a proline-rich C-terminal seq uence in p47-phox. These SH3 target sites conform to consensus feature s deduced from SH3 binding sites in other systems. We propose a model in which the oxidase complex assembles through a mechanism involving S H3 domains of both cytosolic proteins and cognate proline-rich targets in other oxidase components.