Intracellular signal transduction by SH3 domain-containing proteins

在本项目中，我们研究了含有两个SH3结构域的p47^<phox>和p67^<phox>激活吞噬细胞NADPH氧化酶的分子机制，得到如下结果。(1) p47^<phox>的SH3结构域通过与其c端区分子内相互作用而被掩盖。这种相互作用被花生四烯酸或p47^<phox> c端三个丝氨酸残基的磷酸化所破坏，从而使该结构域与氧化酶的酶核细胞色素b_<558>结合。分子间相互作用作为氧化酶激活的开关。(2)两个SH3结构域由10个氨基酸残基连接而成。连接体的长度在分子内和分子间相互作用中都起着重要的作用。(3)激活的另一个开关是将小GTPase Rac转化为gtp结合的激活状态，从而导致与p67^<phox>相互作用。这种结合通过后一种蛋白的TPR结构域介导。(4)我们开发了一种在细胞水平上检测gtp结合Rac的新方法。通过这个过程，我们发现用g1偶联受体激动剂刺激人中性粒细胞的Rac激活需要磷酸肌苷3-激酶。(5)在p47P^<phox>的n端发现了一个新的模块，称为PB2结构域，积极调节氧化酶的激活。(6)氧化酶调节因子p40o ^<phox>与p67^<phox>组成相关，这是通过本项目中发现的两个模块之间的新型相互作用介导的：p67PhOx的pb1结构域和p40^<phox>的PC motif。

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>.