INTERACTIONS BETWEEN THE CYTOSOLIC COMPONENTS P47(PHOX) AND P67(PHOX) OF THE HUMAN NEUTROPHIL NADPH OXIDASE THAT ARE NOT REQUIRED FOR ACTIVATION IN THE CELL-FREE SYSTEM

Activation of the human NADPH oxidase requires the interaction of at least four cytosolic proteins and one membrane bound heterodimeric protein. Src homology 3 (SH3) domains and their proline rich counterstructures have been shown to play an important role in protein-protein interactions. Because it was found that the cytosolic oxidase components p67(phox), p47(phox), and p40(phox) reside in a complex in resting neutrophils, we studied the role of SH3 domains in their interaction by use of an overlay technique. Wild-type and mutated labeled p67(phox) and p47(phox) were used to detect immobilized cytosolic proteins on a protein blot. A specific association of native p67(phox) to blotted p47(phox) and blotted p40(phox) was found. These interactions were not disturbed by deleting the only proline-rich region (amino acids 227-231) in p67(phox). We also found a specific association of native p47(phox) with blotted p67(phox). Deletions in a putative SH3-binding region of p47(phox) completely abrogated the interaction with p67(phox). Other results suggest that the C terminus of p47(phox) exposes this SH3-binding domain for interaction with p67(phox). Similar results were obtained when the binding of cytosolic p67(phox) to wild-type or mutated p47(phox) were studied in solution. Interestingly, mutants of p47(phox) unable to bind to p67(phox) were fully capable of supporting superoxide production under cell-free activation conditions, We conclude that an interaction between the C-terminal proline-rich region of p47(phox) and the second SH3 domain of p67(phox) is not required for oxidase activity in the cell-free assay.