SH3 DOMAIN-MEDIATED INTERACTIONS INVOLVING THE PHOX COMPONENTS OF THENADPH OXIDASE

Objective and Design: To derive a model describing the SH3 domain-medi ated assembly of the activated NADPH oxidase. Materials: Recombinant S H3 domain and Pro-rich fusion proteins were used to investigate potent ial co-associations. Methods: Interactions were assessed using biotiny lated overlay assays and the yeast two hybrid system. Association with p47(phox) from cell lysates was examined by immunoblot analysis. Resu lts: The association between p47- and p22(phox) involves the SH3 domai ns of p47(phox) functioning in tandem. The Pro-rich motif in p47(phox) interacts with both p40(phox) and the COOH-terminal SH3 domain of p67 (phox). Conclusions: In the resting cell, the Pro-rich motif of p47(ph ox) interacts with the SH3 domain of p40(phox), which in turn associat es with p67(phox). Upon activation, the p47-p40(phox) regulatory compl ex dissociates, permitting the association of p47(phox) with the COOH- terminal SH3 domain of p67(phox). This complex translocates to the pla sma membrane and associates with cytochrome b(558), via interaction of the tandem SH3 domains of p47(phox) With the p22(phox) Pro-rich motif .