A regulated adaptor function of p40phox: distinct p67phox membrane targeting by p40phox and by p47phox.
In the phagocytic cell, NADPH oxidase (Nox2) system, cytoplasmic regulators (p47(phox), p67(phox), p40(phox), and Rac) translocate and associate with the membrane-spanning flavocytochrome b(558), leading to activation of superoxide production. We examined membrane targeting of phox proteins and explored conformational changes in p40(phox) that regulate its translocation to membranes upon stimulation. GFP-p40(phox) translocates to early endosomes, whereas GFP-p47(phox) translocates to the plasma membrane in response to arachidonic acid. In contrast, GFP-p67(phox) does not translocate to membranes when expressed alone, but it is dependent on p40(phox) and p47(phox) for its translocation to early endosomes or the plasma membrane, respectively. Translocation of GFP-p40(phox) or GFP-p47(phox) to their respective membrane-targeting sites is abolished by mutations in their phox (PX) domains that disrupt their interactions with their cognate phospholipid ligands. Furthermore, GFP-p67(phox) translocation to either membrane is abolished by mutations that disrupt its interaction with p40(phox) or p47(phox). Finally, we detected a head-to-tail (PX-Phox and Bem1 [PB1] domain) intramolecular interaction within p40(phox) in its resting state by deletion mutagenesis, cell localization, and binding experiments, suggesting that its PX domain is inaccessible to interact with phosphatidylinositol 3-phosphate without cell stimulation. Thus, both p40(phox) and p47(phox) function as diverse p67(phox) "carrier proteins" regulated by the unmasking of membrane-targeting domains in distinct mechanisms.
Ueyama
Takehiko
T
Laboratory of Molecular Pharmacology, Biosignal Research Center, Kobe University, Kobe 657-8501, Japan.
Tatsuno
Toshihiko
T
Kawasaki
Takumi
T
Tsujibe
Satoshi
S
Shirai
Yasuhito
Y
Sumimoto
Hideki
H
Leto
Thomas L
TL
Saito
Naoaki
N
D000818:Animals; D019556:COS Cells; D002462:Cell Membrane / Q000276:immunology*; D002522:Chlorocebus aethiops; D006801:Humans; D051379:Mice; D009154:Mutation; D019255:NADPH Oxidases / Q000378:metabolism*; D010587:Phagocytosis; D010588:Phagosomes / Q000737:chemistry / Q000276:immunology*; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism; D010750:Phosphoproteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary / Q000235:genetics; D021381:Protein Transport; D017452:Receptors, IgG / Q000276:immunology; D017422:Sequence Analysis, DNA
D018129:Phosphatidylinositol Phosphates; D010750:Phosphoproteins; D017452:Receptors, IgG; C085072:neutrophil cytosol factor 40K; C079789:neutrophil cytosol factor 67K; C055525:phosphatidylinositol 3-phosphate; D019255:NADPH Oxidases; C060505:neutrophil cytosolic factor 1