Mapping the Domains of Interaction of p40 with Both p47 and p67 of the Neutrophil Oxidase Complex Using the Two-hybrid System

Abstract

The superoxide-generating NADPH oxidase complex in phagocytic cells is constituted of a heterodimeric flavocytochrome b and cytosolic factors, p67phox, p47phox and p40phox as well as a small G protein Rac (for review, see Refs. 1-3). A truncated form of the p40phox cDNA was isolated by a two hybrid screen of a B lymphocyte library using a full length clone of p47phox as target. This truncated form of p40phox consisting of the Src Homology 3 (SH3) domain to the 3' stop codon was also shown to interact with p67phox in the same system. A library of smaller fragments of the truncated p40 cDNA was constructed and screened against either p47phox or p67phox. Results show that the SH3 domain of p40phox is sufficient for interaction with p47phox, whereas the C terminus of p40phox but not its SH3 domain is involved in the interaction with p67phox.

P40phox associates with the neutrophil Triton X-100-insoluble cytoskeletal fraction and PMA-activated membrane skeleton: a comparative study with P67phox and P47phox

“…This suggests that a cytoskeleton docking site for p40 phox could become available after PMA stimulation. A potential docking site is p47 phox , because an interaction between p40 phox and phosphorylated p47 phox has been described [36]; an alternative potential docking site is coronin, as Grogan et al [37] showed that p40 phox binds directly to this protein.…”

Translocation of proteins homologous to human neutrophil p47phox and p67phox to the cell membrane in activated hemocytes of Galleria mellonella

“…The O 2 À generating NADPH oxidase is a multicomponent system consisting of a membrane-bound flavocytochrome b 558 (composed of two subunits, p22 phox and gp91 phox ) [19] and four cytosolic factors, p47 phox , p67 phox , p40 phox and the small G protein, rac 2 [20]. These cytosolic proteins interact with each other [21,22], with rac [23,24] and with the flavocytochrome [25][26][27] through a number of Src homology 3 (SH3), proline-rich, tetratricopeptide repeat, and PC motifs. Using immunological and matrixassisted laser desorption ionisation-time of flight analysis (MALDI-TOF), the presence of homologous proteins to p67 phox and p47 phox were found in insect hemocytes [6] further strengthening the similarities between the oxidative burst pathways in the two cell types.…”

Novel domains in NADPH oxidase subunits, sorting nexins, and PtdIns 3‐kinases: Binding partners of SH3 domains?

“…This is the minimum complex required to generate the microbicidal superoxide anion. An additional cytosolic factor, p40pho", is known also to associate with the NADPH oxidase complex (Wientjes et al, 1993), although its regulatory roles remain uncertain.P40PhoX, p47phox, and p67phoX each contain Src homology 3 (SH3) domains, which mediate multiple associations with proline-rich targets within the NADPH oxidase complex (Fuchs et al, 1995;de Mendez et al, 1996). These domains are also present in a diverse range of kinases, phosphatases, phospholipases, and cytoskeletal proteins, and bind left-handed polyproline type I1 helices (reviewed in Pawson, 1995).…”

“…P40PhoX, p47phox, and p67phoX each contain Src homology 3 (SH3) domains, which mediate multiple associations with proline-rich targets within the NADPH oxidase complex (Fuchs et al, 1995;de Mendez et al, 1996). These domains are also present in a diverse range of kinases, phosphatases, phospholipases, and cytoskeletal proteins, and bind left-handed polyproline type I1 helices (reviewed in Pawson, 1995).…”