Specificity of p47phox SH3 domain interactions in NADPH oxidase assembly and activation.
The delineation of molecular structures that dictate Src homology 3 (SH3) domain recognition of specific proline-rich ligands is key to understanding unique functions of diverse SH3 domain-containing signalling molecules. We recently established that assembly of the phagocyte NADPH oxidase involves multiple SH3 domain interactions between several oxidase components (p47phox, p67phox, and p22phox). p47phox was shown to play a central role in oxidase activation in whole cells by mediating interactions with both the transmembrane component p22phox and cytosolic p67phox. To understand the specific roles of each SH3 domain of p47phox in oxidase assembly and activation, we mutated critical consensus residues (Tyr167 or Tyr237-->Leu [Y167L or Y237L], W193R or W263R, and P206L or P276L) on each of their binding surfaces. The differential effects of these mutations indicated that the first SH3 domain is responsible for the p47phox-p22phox interaction and plays a predominant role in oxidase activity and p47phox membrane assembly, while the second p47phox SH3 domain interacts with the NH2-terminal domain of p67phox. Binding experiments using the isolated first SH3 domain also demonstrated its involvement in intramolecular interactions within p47phox and showed a requirement for five residues (residues 151 to 155) on its N-terminal boundary for binding to p22phox. The differential effects of nonconserved-site mutations (W204A or Y274A and E174Q or E244Q) on whole-cell oxidase activity suggested that unique contact residues within the third binding pocket of each SH3 domain influence their ligand-binding specificities.
de Mendez
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Laboratory of Host Defenses, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA. imendez@atlas.niaid.hin.gov
Homayounpour
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Leto
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D000595:Amino Acid Sequence; D001665:Binding Sites / Q000235:genetics; D002460:Cell Line; D004789:Enzyme Activation; D006801:Humans; D008024:Ligands; D026901:Membrane Transport Proteins; D008954:Models, Biological; D008969:Molecular Sequence Data; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D009252:NADPH Dehydrogenase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019255:NADPH Oxidases / Q000378:metabolism*; D010750:Phosphoproteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D013481:Superoxides / Q000378:metabolism; D018909:src Homology Domains
D008024:Ligands; D026901:Membrane Transport Proteins; D010750:Phosphoproteins; C079789:neutrophil cytosol factor 67K; D013481:Superoxides; D019255:NADPH Oxidases; C086986:CYBA protein, human; C060505:neutrophil cytosolic factor 1; D009252:NADPH Dehydrogenase