Activation of the phagocyte NADPH oxidase protein p47(phox). Phosphorylation controls SH3 domain-dependent binding to p22(phox).
Activation of phagocyte NADPH oxidase requires interaction between p47(phox) and p22(phox). p47(phox) in resting phagocytes does not bind p22(phox). Phosphorylation of serines in the p47(phox) C terminus enables binding to the p22(phox) C terminus by inducing a conformational change in p47(phox) that unmasks the SH3A domain. We report that an arginine/lysine-rich region in the p47(phox) C terminus binds the p47(phox) SH3 domains expressed in tandem (SH3AB) but does not bind the individual N-terminal SH3A and C-terminal SH3B domains. Peptides matching amino acids 301-320 and 314-335 of the p47(phox) arginine/lysine-rich region block the p47(phox) SH3AB/p22(phox) C-terminal and p47(phox) SH3AB/p47(phox) C-terminal binding and inhibit NADPH oxidase activity in vitro. Peptides with phosphoserines substituted for serines 310 and 328 do not block binding and are poor inhibitors of oxidase activity. Mutated full-length p47(phox) with aspartic acid substitutions to mimic the effects of phosphorylations at serines 310 and 328 bind the p22(phox) proline-rich region in contrast to wild-type p47(phox). We conclude that the p47(phox) SH3A domain-binding site is blocked by an interaction between the p47(phox) SH3AB domains and the C-terminal arginine/lysine-rich region. Phosphorylation of serines in the p47(phox) C terminus disrupts this interaction leading to exposure of the SH3A domain, binding to p22(phox), and activation of the NADPH oxidase.
Research Service, Baltimore Veterans Affairs Medical Center and Marlene and Stewart Greenebaum Cancer Center, University of Maryland School of Medicine, Baltimore, Maryland 21201, USA.
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Kleinberg
D000595:Amino Acid Sequence; D001667:Binding, Competitive; D003573:Cytochrome b Group / Q000378:metabolism; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D026901:Membrane Transport Proteins; D008969:Molecular Sequence Data; D009154:Mutation; D009252:NADPH Dehydrogenase / Q000378:metabolism*; D019255:NADPH Oxidases / Q000378:metabolism*; D009504:Neutrophils; D010446:Peptide Fragments / Q000494:pharmacology; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae; D014162:Transfection; D018909:src Homology Domains / Q000235:genetics
D003573:Cytochrome b Group; D004791:Enzyme Inhibitors; D026901:Membrane Transport Proteins; D010446:Peptide Fragments; D010750:Phosphoproteins; C036888:cytochrome b558; D019255:NADPH Oxidases; C086986:CYBA protein, human; C060505:neutrophil cytosolic factor 1; D009252:NADPH Dehydrogenase