A region N-terminal to the tandem SH3 domain of p47phox plays a crucial role in the activation of the phagocyte NADPH oxidase.
The superoxide-producing NADPH oxidase in phagocytes is crucial for host defence; its catalytic core is the membrane-integrated protein gp91phox [also known as Nox2 (NADPH oxidase 2)], which forms a stable heterodimer with p22phox. Activation of the oxidase requires membrane translocation of the three cytosolic proteins p47phox, p67phox and the small GTPase Rac. At the membrane, these proteins assemble with the gp91phox-p22phox heterodimer and induce a conformational change of gp91phox, leading to superoxide production. p47phox translocates to membranes using its two tandemly arranged SH3 domains, which directly interact with p22phox, whereas p67phox is recruited in a p47phox-dependent manner. In the present study, we show that a short region N-terminal to the bis-SH3 domain is required for activation of the phagocyte NADPH oxidase. Alanine substitution for Ile152 in this region, a residue that is completely conserved during evolution, results in a loss of the ability to activate the oxidase; and the replacement of Thr153 also prevents oxidase activation, but to a lesser extent. In addition, the corresponding isoleucine residue (Ile155) of the p47phox homologue Noxo1 (Nox organizer 1) participates in the activation of non-phagocytic oxidases, such as Nox1 and Nox3. The I152A substitution in p47phox, however, does not affect its interaction with p22phox or with p67phox. Consistent with this, a mutant p47phox (I152A), as well as the wild-type protein, is targeted upon cell stimulation to membranes, and membrane recruitment of p67phox and Rac normally occurs in p47phox (I152A)-expressing cells. Thus the Ile152-containing region of p47phox plays a crucial role in oxidase activation, probably by functioning at a process after oxidase assembly.
Taura
Masahiko
M
Department of Biochemistry, Kyushu University Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan.
Miyano
Kei
K
Minakami
Reiko
R
Kamakura
Sachiko
S
Takeya
Ryu
R
Sumimoto
Hideki
H
D000818:Animals; D001692:Biological Transport / Q000235:genetics / Q000502:physiology; D016466:CHO Cells; D019556:COS Cells; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002522:Chlorocebus aethiops; D006224:Cricetinae; D003412:Cricetulus; D006801:Humans; D007532:Isoleucine / Q000235:genetics / Q000502:physiology; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D000074624:NADPH Oxidase 1; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D009504:Neutrophils / Q000378:metabolism; D010586:Phagocytes / Q000201:enzymology*; D010750:Phosphoproteins / Q000737:chemistry / Q000378:metabolism / Q000502:physiology; D011485:Protein Binding / Q000235:genetics / Q000502:physiology; D013329:Structure-Activity Relationship; D013912:Threonine / Q000235:genetics / Q000502:physiology; D020744:rac GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D018909:src Homology Domains / Q000235:genetics / Q000502:physiology*
D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D010750:Phosphoproteins; C079789:neutrophil cytosol factor 67K; D007532:Isoleucine; D013912:Threonine; C056558:CYBB protein, human; D000074624:NADPH Oxidase 1; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases; C402799:NOX1 protein, human; C431570:Nox3 protein, human; C060505:neutrophil cytosolic factor 1; D020744:rac GTP-Binding Proteins