Intracellular signal transduction by SH3 domain-containing proteins

本课题研究了<phox><phox>含有两个SH 3结构域的p47^和p67^激活吞噬细胞NADPH氧化酶的分子机制，得到以下结果。(1)p47 α的SH 3结构域<phox>通过与其C-末端区域的分子内相互作用被掩蔽。这种相互作用被花生四烯酸或p47^C-末端三个丝氨酸残基的磷酸化破坏<phox>，这使得结构域与氧化酶的酶核心细胞色素B_结合<558>。分子间的相互作用起着氧化酶激活的开关的作用。(2)两个SH 3结构域串联排列，具有10个氨基酸残基的接头。接头的长度在分子内和分子间相互作用中起重要作用。(3)激活的另一个开关是小GTP酶Rac转化为GTP结合的活性状态，这导致与p67 α的相互作用<phox>。这种结合通过后者蛋白的TPR结构域介导。(4)我们已经开发了一种新的方法来检测GTP结合的Rac在细胞水平上。使用这个程序，我们发现，Rac激活人中性粒细胞刺激G1偶联受体激动剂需要磷酸肌醇3-激酶。(5)在p47 β 2的N-末端发现了一个新的模块，<phox>命名为PB 2结构域，其正调控氧化酶活化。(6)氧化酶调节剂p40 α<phox>与p67 α组成性结合<phox>，这是通过本项目中所描述的两个模块之间的新型相互作用介导的：p67 PhOx的PB 1结构域和p40 α的PC基序<phox>。

In this project we have studied molecular mechanism by which p47^<phox> and p67^<phox>, each containing two SH3 domains, activate the phagocyte NADPH oxidase, to obtain the following results. (1) The SH3 domains of p47^<phox> are masked via intramolecular interaction with its C-terminal region. The interaction is disrupted by arachidonic acid or by phosphorylation of three serine residues in the p47^<phox> C-terminus, which allows the domains to bind to cytochrome b_<558>, the enzymatic core of the oxidase. The intermolecular interaction functions as a switch for the oxidase activation. (2) The two SH3 domains are tandemly arranged with a linker of 10 amino acid residues. The length of the linker plays an important role in both intramolecular and intermolecular interactions. (3) Another switch for the activation is conversion of the small GTPase Rac to the GTP-bound active state, which leads to interaction with p67^<phox>. This binding is mediated via the TPR domain of the latter protein. (4) We have developed a novel method to detect the GTP-bound Rac at a cell level. Using this procedure, we find that Rac activation of human neutrophils stimulated with G1-coupled receptor agonists requires phosphoinositide 3-kinase. (5) Discovered is a novel module in the N-terminus of p47P^<phox>, designated PB2 domain, that positively regulates the oxidase activation. (6) The oxidase regulator p4O^<phox> constitutively associates with p67^<phox>, which is mediated via a novel type of interaction between two modules that are discoverd in this project : the PB 1 domain of p67PhOx and the PC motif of p40^<phox>.