title: 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.
abstract: 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 b (558). 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'-[gamma-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.
authors.affiliation: Research Service, Veterans Affairs Maryland Health Care System-Baltimore and Department of Medicine, University of Maryland School of Medicine, Baltimore, MD 21201, USA.
authors.forename: Guihong
authors.initials: G
authors.lastname: Peng
authors.forename: Jin
authors.initials: J
authors.lastname: Huang
authors.forename: Mellonie
authors.initials: M
authors.lastname: Boyd
authors.forename: Michael E
authors.initials: ME
authors.lastname: Kleinberg
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D016718:Arachidonic Acid / Q000378:metabolism*; D002474:Cell-Free System; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D019255:NADPH Oxidases; D010750:Phosphoproteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D017384:Sequence Deletion; D018909:src Homology Domains
chemical_list: D010750:Phosphoproteins; D021122:Protein Subunits; D011994:Recombinant Proteins; D016718:Arachidonic Acid; D019255:NADPH Oxidases; C060505:neutrophil cytosolic factor 1