Leto T L, Adams A G, de Mendez I
Laboratory of Host Defenses, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892.
Proc Natl Acad Sci U S A. 1994 Oct 25;91(22):10650-4. doi: 10.1073/pnas.91.22.10650.
The NADPH oxidase responsible for generation of superoxide anion and related microbicidal oxidants by phagocytes is assembled from at least five distinct proteins. Two are cytosolic components (p47-phox and p67-phox) that contain Src homology 3 (SH3) domains and associate with a transmembrane cytochrome b558 upon activation. We show here that the SH3 domains of p47-phox bind to proline-rich sequences in p47-phox itself and the p22-phox subunit of cytochrome b558. Binding of the p47-phox SH3 domains to p22-phox was abolished by a mutation in one proline-rich sequence (Pro156-->Gln) noted in a distinct form of chronic granulomatous disease and was inhibited by a short proline-rich synthetic peptide corresponding to residues 149-162 of p22-phox. Expression of mutated p22-phox did not restore oxidase activity to p22-phox-deficient B cells and did not enable p22-phox-dependent translocation of p47-phox to membranes in phorbol ester-stimulated cells. We also show that the cytosolic oxidase components associate with one another through the C-terminal SH3 domain of p67-phox and a proline-rich C-terminal sequence in p47-phox. These SH3 target sites conform to consensus features deduced from SH3 binding sites in other systems. We propose a model in which the oxidase complex assembles through a mechanism involving SH3 domains of both cytosolic proteins and cognate proline-rich targets in other oxidase components.
负责由吞噬细胞生成超氧阴离子及相关杀菌性氧化剂的NADPH氧化酶由至少五种不同的蛋白质组装而成。其中两种是胞质成分（p47-phox和p67-phox），它们含有Src同源3（SH3）结构域，并在激活后与跨膜细胞色素b558结合。我们在此表明，p47-phox的SH3结构域与p47-phox自身以及细胞色素b558的p22-phox亚基中富含脯氨酸的序列结合。在一种特殊形式的慢性肉芽肿病中发现的一个富含脯氨酸的序列（Pro156→Gln）发生突变后，p47-phox的SH3结构域与p22-phox的结合被消除，并且被一种对应于p22-phox第149 - 162位残基的富含脯氨酸的短合成肽所抑制。突变的p22-phox的表达不能将氧化酶活性恢复到p22-phox缺陷的B细胞，也不能使p47-phox在佛波酯刺激的细胞中依赖p22-phox向膜转位。我们还表明，胞质氧化酶成分通过p67-phox的C末端SH3结构域和p47-phox中富含脯氨酸的C末端序列相互结合。这些SH3靶位点符合从其他系统中的SH3结合位点推导出来的共有特征。我们提出了一个模型，其中氧化酶复合物通过一种机制组装，该机制涉及胞质蛋白的SH3结构域和其他氧化酶成分中同源的富含脯氨酸的靶标。
