A new role of Pro-73 of p47phox in the activation of neutrophil NADPH oxidase.
The PX domain of p47phox is thought to be involved in autoinhibition. However, when the domain was deleted, the ability to activate the phagocyte NADPH oxidase was markedly diminished. We have mutated the proline-rich region of the PX domain and examined the mutants for the ability to activate. Substitution of Gln for Pro-73 of p47phox(1-286) (P73Q) resulted in a considerably lower activity than the wild type and P73Q had a much lower affinity for the oxidase complex. Whereas, Gln substitution for Pro-76 (P76Q) showed a slightly enhanced activation and the mutant had a slightly higher affinity for the complex than the wild type. Affinity for p67phox(1-210) was slightly decreased either by P73Q or P76Q. Optimal SDS concentration for the activation was lowered by these mutations. Binding of PX domain with phosphatidylinositol-3,4-bisphosphate was diminished by P73Q mutation. The results in this study suggest that Pro-73 has a role in interaction with the catalytic component cytochrome b558.
Nagasawa
Teruaki
T
Department of Applied Chemistry, Faculty of Engineering, Ehime University, Matsuyama, 790-8577, Ehime, Japan.
Ebisu
Kentaro
K
Inoue
Yasuyuki
Y
Miyano
Kei
K
Tamura
Minoru
M
D020816:Amino Acid Motifs; D002478:Cells, Cultured; D002942:Circular Dichroism; D003573:Cytochrome b Group / Q000378:metabolism; D004789:Enzyme Activation / Q000502:physiology; D006801:Humans; D007700:Kinetics; D009154:Mutation; D019255:NADPH Oxidases / Q000378:metabolism*; D009504:Neutrophils / Q000201:enzymology / Q000378:metabolism*; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism; D010743:Phospholipids / Q000378:metabolism; D010750:Phosphoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011392:Proline / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012967:Sodium Dodecyl Sulfate / Q000737:chemistry; D020744:rac GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism
D003573:Cytochrome b Group; D018129:Phosphatidylinositol Phosphates; D010743:Phospholipids; D010750:Phosphoproteins; D011994:Recombinant Proteins; C060980:phosphatidylinositol 3,4-diphosphate; D012967:Sodium Dodecyl Sulfate; C036888:cytochrome b558; D011392:Proline; D019255:NADPH Oxidases; C060505:neutrophil cytosolic factor 1; D020744:rac GTP-Binding Proteins