Mechanism for phosphorylation-induced activation of the phagocyte NADPH oxidase protein p47(phox). Triple replacement of serines 303, 304, and 328 with aspartates disrupts the SH3 domain-mediated intramolecular interaction in p47(phox), thereby activating the oxidase.
Activation of the superoxide-producing phagocyte NADPH oxidase requires interaction between p47(phox) and p22(phox), which is mediated via the SH3 domains of the former protein. This interaction is considered to be induced by exposure of the domains that are normally masked by an intramolecular interaction with the C-terminal region of p47(phox). Here we locate the intramolecular SH3-binding site at the region of amino acid residues 286-340, where Ser-303, Ser-304, and Ser-328 that are among several serines known to become phosphorylated upon cell stimulation exist. Simultaneous replacement of the three serines in p47(phox) with aspartates or glutamates, each mimicking phosphorylated residues, is sufficient for disruption of the intramolecular interaction and resultant access to p22(phox). The triply mutated proteins are also capable of activating the NADPH oxidase without in vitro activators such as arachidonate under cell-free conditions. In a whole-cell system where expression of the wild-type p47(phox) reconstitutes the stimulus-dependent oxidase activity, substitution of the kinase-insensitive residue alanine for Ser-328 as well as for Ser-303/Ser-304 leads to a defective production of superoxide. These findings suggest that phosphorylation of the three serines in p47(phox) induces a conformational change to a state accessible to p22(phox), thereby activating the NADPH oxidase.
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Department of Molecular Biology, Kyushu University Graduate School of Medical Science, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Tokyo 108-8639, Japan.
Nunoi
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Ito
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Sumimoto
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D019943:Amino Acid Substitution; D004789:Enzyme Activation; D006801:Humans; D020014:K562 Cells; D016297:Mutagenesis, Site-Directed; D019255:NADPH Oxidases; D010586:Phagocytes / Q000201:enzymology*; D010750:Phosphoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D011487:Protein Conformation; D018909:src Homology Domains
D010750:Phosphoproteins; D019255:NADPH Oxidases; C060505:neutrophil cytosolic factor 1