The phagocyte NADPH oxidase is activated during phagocytosis to produce superoxide, a precursor of microbicidal oxidants. The activation involves assembly of membrane-integrated cytochrome b558 comprising gp91(phox) and p22(phox), two specialized cytosolic proteins (p47(phox) and p67(phox)), each containing two Src homology 3 (SH3) domains, and the small G protein Rac. In the present study, we show that the N-terminal SH3 domain of p47(phox) binds to the C-terminal cytoplasmic tail of p22(phox) with high affinity (KD = 0.34 microM). The binding is specific to this domain among several SH3 domains including the C-terminal one of p47(phox) and the two of p67(phox) and requires the Pro156-containing proline-rich sequence but not other putative SH3 domain-binding sites of p22(phox). Replacement of Trp193 by Arg in the N-terminal SH3 domain completely abrogates the association with p22(phox). A mutant p47(phox) with this substitution is incapable of supporting superoxide production under cell-free activation conditions. These findings provide direct evidence that the interaction between the N-terminal SH3 domain of p47(phox) and the proline-rich region of p22(phox) is essential for activation of the NADPH oxidase.

The superoxide-generating NADPH oxidase, dormant in resting phagocytes, is activated during phagocytosis following assembly of the membrane-integrated protein cytochrome b 558 and cytosolic factors. Among the latter are the three proteins containing Src homology 3 (SH3) domains, p67 phox , p47 phox and p40 phox . While the first two factors are indispensable for the activity, p40 phox is tightly associated with p67 phox in resting cells and is suggested to have some modulatory role. Here we describe a systematic analysis of the interaction between p40 phox and p67 phox using the yeast two-hybrid system and in vitro binding assays with recombinant proteins. Both methods unequivocally showed that the minimum requirements for stable interaction are the C-terminal region of p40 phox and the region between the two SH3 domains of p67 phox . This interaction is maintained even in the presence of anionic amphiphiles used for the activation of the NADPH oxidase, raising a possibility that it mediates constitutive association of the two factors in both resting and activated cells. The C-terminal region of p40 phox responsible for the interaction contains a characteristic stretch of amino acids designated as the PC motif, that also exists in other signal-transducing proteins from yeast to human. Intensive site-directed mutagenesis to the motif in p40 phox revealed that it plays a critical role in the binding to p67 phox . Thus the PC motif appears to represent a novel module for proteinϪprotein interaction used in a variety of signaling pathways.

The phagocyte NADPH oxidase is activated during phagocytosis to produce superoxide, a precursor of microbicidal oxidants. The formation of the active oxidase complex at the membrane requires translocation of the Rac GTPase and two specialized cytosolic proteins that harbor SH3 domains, p67 phox and p47

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Steroid receptors are transcription factors that regulate hormone-responsive genes and whose activity is controlled by their interaction with numerous other proteins. Observations reported here reveal that estrogen receptors ␣ and ␤ (ER␣ and ER␤), androgen receptor, and glucocorticoid receptor bind in vitro to vinexin ␣, a multiple SH3 motif-containing protein associated with the cytoskeleton. The SH3 domains are not involved in this interaction. Furthermore, we demonstrate that vinexin ␣ stimulates the ligand-induced transactivation function of these receptors, although it is devoid of intrinsic transcriptional activity when tethered to DNA. In addition, the ectopic coexpression of vinexin ␣ and ER␣ results in a loss of ER␣ phosphorylation on serines and the partial redistribution of vinexin ␣ into the nucleus, where it colocalizes with ER␣. These results establish a new model of transcriptional regulation where components of the cell-cell and cellsubstrate adhesion complexes can regulate the phosphorylation and activity of steroid receptors.Estrogen receptors (ERs) 1 are members of the nuclear receptor (NR) superfamily of transcription factors, which includes the closely related steroid receptors, receptors for thyroid hormone, retinoic acids, fatty acids and their metabolites, vitamin D, oxysterols, and a large number of proteins for which a natural ligand has yet to be identified (for review see Refs.