Abstract
The phagocyte NADPH oxidase is activated during phagocytosis to produce superoxide, a precursor of microbicidal oxidants. The activation involves assembly of membrane-integrated cytochrome b558 comprising gp91(phox) and p22(phox), two specialized cytosolic proteins (p47(phox) and p67(phox)), each containing two Src homology 3 (SH3) domains, and the small G protein Rac. In the present study, we show that the N-terminal SH3 domain of p47(phox) binds to the C-terminal cytoplasmic tail of p22(phox) with high affinity (K(D) = 0.34 μM). The binding is specific to this domain among several SH3 domains including the C-terminal one of p47(phox) and the two of p67(phox) and requires the Pro156-containing proline-rich sequence but not other putative SH3 domain-binding sites of p22(phox). Replacement of Trp193 by Arg in the N-terminal SH3 domain completely abrogates the association with p22(phox). A mutant p47(phox) with this substitution is incapable of supporting superoxide production under cell-free activation conditions. These findings provide direct evidence that the interaction between the N-terminal SH3 domain of p47(phox) and the proline-rich region of p22(phox) is essential for activation of the NADPH oxidase.
| Original language | English |
|---|---|
| Pages (from-to) | 22152-22158 |
| Number of pages | 7 |
| Journal | Journal of Biological Chemistry |
| Volume | 271 |
| Issue number | 36 |
| DOIs | |
| Publication status | Published - Sep 25 1996 |
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All Science Journal Classification (ASJC) codes
- Biochemistry
- Molecular Biology
- Cell Biology
Cite this
Assembly and activation of the phagocyte NADPH oxidase : Specific interaction of the N-terminal Src homology 3 domain of p47(phox) with p22(phox) is required for activation of the NADPH oxidase. / Sumimoto, Hideki; Hata, Kenichiro; Mizuki, Kazuhito; Ito, Takashi; Kage, Yohko; Sakaki, Yoshiyuki; Fukumaki, Yasuyuki; Nakamura, Michio; Takeshige, Koichiro.
In: Journal of Biological Chemistry, Vol. 271, No. 36, 25.09.1996, p. 22152-22158.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Assembly and activation of the phagocyte NADPH oxidase
T2 - Specific interaction of the N-terminal Src homology 3 domain of p47(phox) with p22(phox) is required for activation of the NADPH oxidase
AU - Sumimoto, Hideki
AU - Hata, Kenichiro
AU - Mizuki, Kazuhito
AU - Ito, Takashi
AU - Kage, Yohko
AU - Sakaki, Yoshiyuki
AU - Fukumaki, Yasuyuki
AU - Nakamura, Michio
AU - Takeshige, Koichiro
PY - 1996/9/25
Y1 - 1996/9/25
N2 - The phagocyte NADPH oxidase is activated during phagocytosis to produce superoxide, a precursor of microbicidal oxidants. The activation involves assembly of membrane-integrated cytochrome b558 comprising gp91(phox) and p22(phox), two specialized cytosolic proteins (p47(phox) and p67(phox)), each containing two Src homology 3 (SH3) domains, and the small G protein Rac. In the present study, we show that the N-terminal SH3 domain of p47(phox) binds to the C-terminal cytoplasmic tail of p22(phox) with high affinity (K(D) = 0.34 μM). The binding is specific to this domain among several SH3 domains including the C-terminal one of p47(phox) and the two of p67(phox) and requires the Pro156-containing proline-rich sequence but not other putative SH3 domain-binding sites of p22(phox). Replacement of Trp193 by Arg in the N-terminal SH3 domain completely abrogates the association with p22(phox). A mutant p47(phox) with this substitution is incapable of supporting superoxide production under cell-free activation conditions. These findings provide direct evidence that the interaction between the N-terminal SH3 domain of p47(phox) and the proline-rich region of p22(phox) is essential for activation of the NADPH oxidase.
AB - The phagocyte NADPH oxidase is activated during phagocytosis to produce superoxide, a precursor of microbicidal oxidants. The activation involves assembly of membrane-integrated cytochrome b558 comprising gp91(phox) and p22(phox), two specialized cytosolic proteins (p47(phox) and p67(phox)), each containing two Src homology 3 (SH3) domains, and the small G protein Rac. In the present study, we show that the N-terminal SH3 domain of p47(phox) binds to the C-terminal cytoplasmic tail of p22(phox) with high affinity (K(D) = 0.34 μM). The binding is specific to this domain among several SH3 domains including the C-terminal one of p47(phox) and the two of p67(phox) and requires the Pro156-containing proline-rich sequence but not other putative SH3 domain-binding sites of p22(phox). Replacement of Trp193 by Arg in the N-terminal SH3 domain completely abrogates the association with p22(phox). A mutant p47(phox) with this substitution is incapable of supporting superoxide production under cell-free activation conditions. These findings provide direct evidence that the interaction between the N-terminal SH3 domain of p47(phox) and the proline-rich region of p22(phox) is essential for activation of the NADPH oxidase.
UR - http://www.scopus.com/inward/record.url?scp=0029806925&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0029806925&partnerID=8YFLogxK
U2 - 10.1074/jbc.271.36.22152
DO - 10.1074/jbc.271.36.22152
M3 - Article
C2 - 8703027
AN - SCOPUS:0029806925
VL - 271
SP - 22152
EP - 22158
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
SN - 0021-9258
IS - 36
ER -