Direct interaction of actin with p47(phox) of neutrophil NADPH oxidase.
The cell-free activation of human neutrophil NADPH oxidase is enhanced by actin, and actin filaments formed during activation are suggested to stabilize the oxidase. In an attempt to elucidate the mechanism, we examined the protein-protein interactions between actin and cytosolic components of the oxidase. Far-Western blotting using recombinant phox proteins showed that both alpha- and beta-actin interacted with p47(phox) and rac1, and weakly with rac2. A deletion mutant of p47(phox) proved that its C-terminal region was essential for the interaction. The dissociation constant (K(d)) for interaction between actin and p47(phox) was estimated to be 0.45 microM by surface plasmon resonance, and that between actin and rac1 or rac2 was 1.7 or 4.6 microM, respectively. Far-Western blotting using cytosol as a target showed an interaction between actin and endogenous p47(phox) and rac proteins. These results suggest that actin can directly interact with p47(phox) and possibly with rac in the cells.
Tamura
M
M
Department of Applied Chemistry, Ehime University, Matsuyama, Ehime, 790-8577, Japan. miketamu@en3.ehime-u.ac.jp
Kai
T
T
Tsunawaki
S
S
Lambeth
J D
JD
Kameda
K
K
D000199:Actins / Q000378:metabolism*; D001665:Binding Sites; D006801:Humans; D019255:NADPH Oxidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D009504:Neutrophils / Q000201:enzymology* / Q000378:metabolism; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism; D017384:Sequence Deletion / Q000235:genetics; D020349:Surface Plasmon Resonance; D013816:Thermodynamics; D020744:rac GTP-Binding Proteins / Q000378:metabolism; D020830:rac1 GTP-Binding Protein / Q000378:metabolism; D000097766:RAC2 GTP-Binding Protein
D000199:Actins; D019255:NADPH Oxidases; D010750:Phosphoproteins; D011994:Recombinant Proteins; D020744:rac GTP-Binding Proteins; D020830:rac1 GTP-Binding Protein; D000097766:RAC2 GTP-Binding Protein; C060505:neutrophil cytosolic factor 1