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 p47phox, thereby activating the oxidase

Activation of the superoxide-producing phagocyte NADPH oxidase requires int eraction between p47(phox) and p22(phox), which is mediated via the SH3 dom ains of the former protein. This interaction is considered to be induced by exposure of the domains that are normally masked by an intramolecular inte raction with the C-terminal region of p47(phox). Here we locate the intramo lecular SH3-binding site at the region of amino acid residues 286-340, wher e Ser-303, Ser-304, and Ser-328 that are among several serines known to bec ome phosphorylated upon cell stimulation exist. Simultaneous replacement of the three serines in p47(phox) with aspartates or glutamates, each mimicki ng phosphorylated residues, is sufficient for disruption of the intramolecu lar interaction and resultant access to p22(phox). Th, triply mutated prote ins are also capable of activating the NADPH oxidase without in vitro activ ators such as arachidonate under cell-free conditions. In a whole-cell syst em where expression of the wild-type p47(phox) reconstitutes the stimulus-d ependent oxidase activity, substitution of the kinase-insensitive residue a lanine for Ser-328 as well as for Ser-303/Ser-304 leads to a defective prod uction of superoxide. These findings suggest that phosphorylation of the th ree serines in p47(phox) induces a conformational change to a state accessi ble to p22(phox), thereby activating the NADPH oxidase.