In an early step in the assembly of the phagocyte NADPH oxidase, p47-phox translocates from the cytosol to the membrane, mediated by engagement of the N-termini of two p47-phox Src homology 3 (SH3) domains with a proline-rich region (PRR) in the p22-phox subunit of cytochrome b558. In response to phagocyte activation, several serine residues in a C-terminal arginine/lysine-rich domain of p47-phox are phosphorylated, leading to changes in the conformation of p47-phox and exposure of its N-terminal SH3 domain that is normally masked by internal association with the arginine/lysine-rich domain. We report that triple alanine substitutions at Asp-217, Glu-218 and Glu-223 in a short sequence that links the tandem p47-phox SH3 domains unmasked the N-terminal SH3 domain, similar to the effects of aspartic acid substitutions at Ser-310 and Ser-328 in the arginine/lysine-rich region. Recombinant p47-phox proteins with mutations in either the linker region or the arginine/lysine-rich domain were active in the absence of arachidonic acid stimulation in a cell-free NADPH oxidase system consisting of recombinant p67-phox, Rac1–guanosine 5′-[γ-thio]triphosphate and neutrophil membranes. Supplementing neutrophil membranes with phosphoinositides or other negatively charged phospholipids markedly enhanced cell-free superoxide generation by these p47-phox mutants in the absence of arachidonic acid, to levels equivalent to those generated by wild-type p47-phox following arachidonic acid activation. This enhancement may be related to recruitment to the membrane of p47-phox mediated by a novel secondary phox homology (PX) domain binding site that broadly recognizes phospholipids. No specific enhancement by specific phosphorylated phosphatidylinositols was found to suggest a dominant role for the p47-phox primary PX domain binding site. Truncated p47-phox S310D S328D lacking the C-terminal PRR was inactive in the cell-free system without arachidonic acid, but was fully active with arachidonic acid. This suggests that activation of NADPH oxidase in an arachidonate-free cell-free system requires association of the p47-phox C-terminal PRR with the p67-phox C-terminal SH3 domain.
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Research Article|
July 01 2003
Properties of phagocyte NADPH oxidase p47-phox mutants with unmasked SH3 (Src homology 3) domains: full reconstitution of oxidase activity in a semi-recombinant cell-free system lacking arachidonic acid
Guihong PENG;
Guihong PENG
Research Service, Veterans Affairs Maryland Health Care System-Baltimore and Department of Medicine, University of Maryland School of Medicine, Baltimore, MD 21201, U.S.A.
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Jin HUANG;
Jin HUANG
Research Service, Veterans Affairs Maryland Health Care System-Baltimore and Department of Medicine, University of Maryland School of Medicine, Baltimore, MD 21201, U.S.A.
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Mellonie BOYD;
Mellonie BOYD
Research Service, Veterans Affairs Maryland Health Care System-Baltimore and Department of Medicine, University of Maryland School of Medicine, Baltimore, MD 21201, U.S.A.
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Michael E. KLEINBERG
Michael E. KLEINBERG
1
Research Service, Veterans Affairs Maryland Health Care System-Baltimore and Department of Medicine, University of Maryland School of Medicine, Baltimore, MD 21201, U.S.A.
1To whom correspondence should be addressed: University of Maryland Greenebaum Cancer Center, Rm N9E05, UMH, 22 South Greene St, Baltimore, MD 21201 U.S.A. (e-mail mkleinbe@umaryland.edu).
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Publisher: Portland Press Ltd
Received:
October 18 2002
Revision Received:
March 11 2003
Accepted:
March 21 2003
Accepted Manuscript online:
March 21 2003
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London ©2003
2003
Biochem J (2003) 373 (1): 221–229.
Article history
Received:
October 18 2002
Revision Received:
March 11 2003
Accepted:
March 21 2003
Accepted Manuscript online:
March 21 2003
Citation
Guihong PENG, Jin HUANG, Mellonie BOYD, Michael E. KLEINBERG; Properties of phagocyte NADPH oxidase p47-phox mutants with unmasked SH3 (Src homology 3) domains: full reconstitution of oxidase activity in a semi-recombinant cell-free system lacking arachidonic acid. Biochem J 1 July 2003; 373 (1): 221–229. doi: https://doi.org/10.1042/bj20021629
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