A new role of Pro-73 of p47 in the activation of neutrophil NADPH oxidase

Assembly of the phagocyte NADPH oxidase

“…In stimulated intact PMNs, agonist-dependent phosphorylation of several serine residues within the polybasic region of p47phox mediates a conformational change in p47phox, unfolding the protein and exposing the now interactive SH3 domains of p47phox to eventually interact stably with PRRs on p22phox, thereby resulting in translocation to the membrane and activity of a functional oxidase (Shiose and Sumimoto 2000;Ago et al 2003). NMR data confirm a structural shift in the PX domain after phosphorylation, releasing the PX domain from its intramolecular interaction with the carboxy SH3 domain and thereby freeing it to interact with target membrane lipids (Nagasawa et al 2003). However, data from the X-ray structure of the autoinhibited form of p47phox (amino acids suggest that both SH3 domains are required to maintain the inactive, folded state, with the pair creating a groove that accommodates the amino terminus of the polybasic region ( 296 RGAPPRRSS 304 ) (Groemping et al 2003).…”

“…Earlier studies in the cellfree system ) and in transfected cells (de Mendez et al 1996) demonstrated that deletion of the PX domain from p47phox decreases the specific activity of the resulting oxidase, thus suggesting the domain contains residues that positively influence the activation, activity, or both of the assembled oxidase. Mutagenesis within the PXXP domain modifies the activity of truncated p47phox constructs, their capacity to bind to phospholipid vesicles, and their affinity for a truncated p67phox construct (Nagasawa et al 2003). Whereas the P76Q mutant has slightly higher activity and more avidly binds phospholipids and the p67phox construct than does wild-type p47phox, the P73Q mutant is significantly more active by all three criteria, suggesting that this residue within the PX domain interacts directly or indirectly with the catalytically active flavocytochrome b 558 .…”

“…Investigators have employed a broad array of techniques to examine potentially important interactions for oxidase assembly, including interpolating structural relationships from the molecular defects underlying the biochemical disorder in newly identified patients with CGD (Leusen et al 1994b(Leusen et al , 2000Dusi et al 1998;Yu et al 1999;Stasia et al 2002;Heyworth et al 2003), employing phage display libraries to identify specific sequences in each component that might mediate protein-protein interactions (Burritt et al 1995(Burritt et al , 1998(Burritt et al , 2001(Burritt et al , 2003DeLeo et al 1995a), characterizing the functional consequences of supplementing broken cell systems with mutant or chimeric proteins (Nisimoto et al 1997(Nisimoto et al , 1999Hata et al 1998;Toporik et al 1998;Shiose and Sumimoto 2000;Alloul et al 2001;Ebisu et al 2001;Miyano et al 2001Miyano et al , 2003Dang et al 2003;Nagasawa et al 2003;Peng et al 2003) or synthetic peptides (Nauseef et al 1993;Morozov et al 1998;Nathan 2003), using analytical techniques to monitor conformational changes during assembly (Swain et al 1997;Paclet et al 2000;Foubert et al 2002), examining protein-protein and protein-lipid interactions using in vitro overlays (de Mendez et al 1997;Han et al 1998;Kami et al 2002) and yeast two-hybrid systems (Wilson et al 1997), and analyzing the subcellular translocation and functional reconstitution of wild-type and defined mutant components expressed in heterologous systems …”

Atypical Membrane-embedded Phosphatidylinositol 3,4-Bisphosphate (PI(3,4)P2)-binding Site on p47 Phox Homology (PX) Domain Revealed by NMR

“…It has been reported that the mutation Pro-73 abrogates phosphoinositide recognition and interferes with the in vitro activity of NADPH oxidase (46), while the mutation Arg-70 prevents membrane translocation in HEK293 cells (47).…”

Characterization and expression of NADPH oxidase in LPS-, poly(I:C)- and zymosan-stimulated trout (Oncorhynchus mykiss W.) macrophages

“…In order to interact with Nox, p47phox utilizes the Phox homology (PX) domain, a N-terminal region of p47phox that binds to phosphatidylinositol (3,4)P 2 and phosphatidic acid [29][30][31]. In trout p47phox the PxxP motif ( 70 RIIPHLPAP) in the PX domain that regulates the phosphoinositide binding in mammals [32], is also highly conserved, with the minor substitution of His 74 by glutamate. Several of the residues (Fig.…”