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Abstract: Activation of the phagocyte NADPH oxidase requires the regulatory proteins p47 phox and p67 phox , each harboring two SH3 domains. p67 phox interacts with p47 phox via simultaneous binding of the p67 phox C-terminal SH3 domain to both the proline-rich region (PRR) of amino acid residues 360–369 and its C-terminally flanking region of p47 phox ; the role of the interaction in oxidase regulation has not been fully understood. Here we show that the p47 phox –p67 phox interaction is disrupted not only by deletion of the PRR but also by substitution for basic residues in the extra-PRR (K383E/K385E). The substitution impaired oxidase activation partially in vitro and much more profoundly in vivo, indicating the significance of the p47 phox extra-PRR. Replacement of Ser-379 in the extra-PRR, a residue known to undergo phosphorylation in stimulated cells, by aspartate attenuates the interaction and thus results in a defective superoxide production, suggesting that phosphorylation of Ser-379 is involved in oxidase regulation. [Copyright &y& Elsevier]

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, p67phox and p47phox. Another SH3-domain-containing protein p40 phox, which is constitutively associated with p67phox in phagocytes, also enters the complex upon cell stimulation. Here we describe how we cloned mouse cDNAs encoding p40 phox and its partner in phagocytes, p67phox. Both p40 phox and p67phox comprise several protein-binding modules that are structurally and functionally well conserved between mouse and human, indicating their nature as adaptor proteins. We have also systematically investigated expression of the gene for p40 phox in comparison with those for p67phox and p47phox. Distributions of the mRNAs for the three proteins among tissues are similar, with the most abundant expression in the spleen. The messages are abundant not only in phagocytic cells, but also in B cell lineage. The p40 phox gene, but not the other two, is expressed in some types of cells such as plasma cells and T lymphocytes. Furthermore, in situ hybridization analysis shows that the p40 phox mRNA is distributed in neuronal cells of mouse brain, providing evidence that one of the genes for the specialized oxidase factors is expressed in neurons. These observations raise the possibility that the adaptor protein p40 phox plays a heretofore unsuspected role via interacting with other proteins in the cells that do not express p67phox or p47phox. [ABSTRACT FROM AUTHOR]

The superoxide-generating NADPH oxidase, dormant in resting phagocytes, is activated during phagocytosis following assembly of the membrane-integrated protein cytochrome b558 and cytosolic factors. Among the latter are the three proteins containing Src homology 3 (SH3) domains, p67phox, p47phox and p40 phox. While the first two factors are indispensable for the activity, p40 phox is tightly associated with p67phox in resting cells and is suggested to have some modulatory role. Here we describe a systematic analysis of the interaction between p40 phox and p67phox 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 p67phox. 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 protein-protein interaction used in a variety of signaling pathways. [ABSTRACT FROM AUTHOR]

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 alpha and beta (ERalpha and ERbeta), androgen receptor, and glucocorticoid receptor bind in vitro to vinexin alpha, a multiple SH3 motif-containing protein associated with the cytoskeleton. The SH3 domains are not involved in this interaction. Furthermore, we demonstrate that vinexin alpha 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 alpha and ERalpha results in a loss of ERalpha phosphorylation on serines and the partial redistribution of vinexin alpha into the nucleus, where it colocalizes with ERalpha. These results establish a new model of transcriptional regulation where components of the cell-cell and cell-substrate adhesion complexes can regulate the phosphorylation and activity of steroid receptors.