Interactions between the cytosolic components p47phox and p67phox 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 p67phox, p47phox, and p40phox 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 35S-labeled p67phox and p47phox were used to detect immobilized cytosolic proteins on a protein blot. A specific association of native p67phox to blotted p47phox and blotted p40phox was found. These interactions were not disturbed by deleting the only proline-rich region (amino acids 227-231) in p67phox. We also found a specific association of native p47phox with blotted p67phox. Deletions in a putative SH3-binding region of p47phox completely abrogated the interaction with p67phox. Other results suggest that the C terminus of p47phox exposes this SH3-binding domain for interaction with p67phox. Similar results were obtained when the binding of cytosolic p67phox to wild-type or mutated p47phox were studied in solution. Interestingly, mutants of p47phox unable to bind to p67phox 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 p47phox and the second SH3 domain of p67phox is not required for oxidase activity in the cell-free assay.
Leusen
J H
JH
Central Laboratory of the Netherlands Red Cross Blood Transfusion Service, Amsterdam.
Fluiter
K
K
Hilarius
P M
PM
Roos
D
D
Verhoeven
A J
AJ
Bolscher
B G
BG
D001483:Base Sequence; D002474:Cell-Free System; D003600:Cytosol / Q000201:enzymology; D004789:Enzyme Activation; D006801:Humans; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009247:NADH, NADPH Oxidoreductases / Q000097:blood*; D009252:NADPH Dehydrogenase / Q000096:biosynthesis / Q000097:blood* / Q000302:isolation & purification; D019255:NADPH Oxidases; D009504:Neutrophils / Q000201:enzymology*; D009838:Oligodeoxyribonucleotides; D010750:Phosphoproteins / Q000096:biosynthesis / Q000097:blood* / Q000302:isolation & purification; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D017384:Sequence Deletion; D013481:Superoxides / Q000378:metabolism
D009838:Oligodeoxyribonucleotides; D010750:Phosphoproteins; D011993:Recombinant Fusion Proteins; C079789:neutrophil cytosol factor 67K; D013481:Superoxides; D009247:NADH, NADPH Oxidoreductases; D019255:NADPH Oxidases; C060505:neutrophil cytosolic factor 1; D009252:NADPH Dehydrogenase