Specificity of p47(phox) SH3 domain interactions in NADPH oxidase assembly and activation

The delineation of molecular structures that dictate Src homology 3 (SH3) domain recognition of specific proline-rich ligands is key to understanding unique functions of diverse SH3 domain-containing signalling molecules. We recently established that assembly of the phagocyte NADPH oxidase involves multiple SH3 domain interactions between several oxidase components (p47(phox), p67(phox), and p22(phox)). p47(phox) was shown to play a central role in oxidase activation in whole cells by mediating interactions with both the transmembrane component p22(phox) and cytosolic p67(phox). To understand the specific roles of each SH3 domain of p47(phox) in oxidase assembly and activation, we mutated critical consensus residues (Tyr(167) or Tyr(237)-->Leu [Y167L or oxidase assembly and activation, we mutated critical consensus residues (Tyr Y237L], W193R or W263R, and P206L or P276L) on each of their binding surfaces. The differential effects of these mutations indicated that the first SH3 domain is responsible for the p47(phox)-p22(phox) interaction and plays a predominant role in oxidase activity and p47(phox) membrane assembly, while the second p47(phox) SH3 domain interacts with the NH2-terminal domain of p67(phox). Binding experiments using the isolated first SH3 domain also demonstrated its involvement in intramolecular interactions within p47(phox) and showed a requirement for five residues (residues 151 to 155) on its N-terminal boundary for binding to p22(phox), The differential effects of nonconserved-site mutations (W204A or Y274A and E174Q or E244Q) on whole-cell oxidase activity suggested that unique contact residues within the third binding pocket of each SH3 domain influence their ligand-binding specificities.