SH3 domain-mediated interactions involving the phox components of the NADPH oxidase.
OBJECTIVE
To derive a model describing the SH3 domain-mediated assembly of the activated NADPH oxidase.


METHODS
Recombinant SH3 domain and Pro-rich fusion proteins were used to investigate potential co-associations.


METHODS
Interactions were assessed using biotinylated overlay assays and the yeast two hybrid system. Association with p47phox from cell lysates was examined by immunoblot analysis.


RESULTS
The association between p47- and p22phox involves the SH3 domains of p47phox functioning in tandem. The Prorich motif in p47phox interacts with both p40phox and the COOH-terminal SH3 domain of p67phox.


CONCLUSIONS
In the resting cell, the Pro-rich motif of p47phox interacts with the SH3 domain of p40phox, which in turn associates with p67phox. Upon activation, the p47-p40phox regulatory complex dissociates, permitting the association of p47phox with the COOH-terminal SH3 domain of p67phox. This complex translocates to the plasma membrane and associates with cytochrome b558, via interaction of the tandem SH3 domains of p47phox with the p22phox Pro-rich motif.
Wilson
L
L
Yamanouchi Research Institute, Oxford, UK.
Butcher
C
C
Finan
P
P
Kellie
S
S
D001667:Binding, Competitive; D001710:Biotin; D003573:Cytochrome b Group / Q000235:genetics / Q000378:metabolism; D004789:Enzyme Activation / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic / Q000235:genetics; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D018922:HL-60 Cells / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D026901:Membrane Transport Proteins; D016297:Mutagenesis, Site-Directed; D009252:NADPH Dehydrogenase / Q000378:metabolism; D019255:NADPH Oxidases / Q000235:genetics / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D015003:Yeasts / Q000235:genetics; D018909:src Homology Domains
D003573:Cytochrome b Group; D026901:Membrane Transport Proteins; D010750:Phosphoproteins; D011993:Recombinant Fusion Proteins; C085072:neutrophil cytosol factor 40K; C079789:neutrophil cytosol factor 67K; D001710:Biotin; C036888:cytochrome b558; D019255:NADPH Oxidases; C086986:CYBA protein, human; C060505:neutrophil cytosolic factor 1; D009252:NADPH Dehydrogenase; D005982:Glutathione Transferase