Scaffolding Proteins in Macrophage Phagocytosis

描述(申请人提供)：通过其SH3和SH2结构域，Src激酶Lyn与少量调节细胞周期和细胞骨架的磷酸蛋白相互作用，如Shc、Cb1和Vav。利用Lyn的独特结构域SH3和SH2作为诱饵，在酵母双杂交筛选中分离到一个具有支架蛋白CIP4特征的基因。我们已经确定了它的四种亚型。CIP4含有一个保守的N-末端结构域、一个可能的螺旋线圈结构域和一个C-末端SH3结构域。CIP4之所以特别吸引人研究，是因为它能够结合两组不同的细胞骨架蛋白及其激活剂。细胞骨架重组对正常和肿瘤细胞功能都是必不可少的，需要一系列复杂的蛋白质相互作用。CIP4结合了Src激酶和RhoGTPase CDC42的激活状态。此外，CIP4通过其N端的Fes/CIP4同源(FCH)结构域与微管结合，并通过其C端的SH3结构域与富含Pro的蛋白质(如黄蜂)结合。CIP4属于一个新描述的蛋白质家族，发现于酵母等低等真核生物中。在哺乳动物中，我们假设CIP4在Fc受体或黏附诱导的白细胞整合素信号转导中发挥重要作用，它提供了一个支架，并将细胞骨架系统与Src激酶和CDc42联系起来。由此产生的细胞骨架重排是细胞扩散、迁移和功能激活所必需的。我们假设CIP4是一种关键的支架蛋白，它将Lyn聚集在静止细胞的质膜上，并将黄蜂招募到质膜上，当受体结合时，Lyn被激活，使黄蜂磷酸化，CIP4促进激活的CDc42的招募，从而推动黄蜂的展开。Arp2/3被激活，肌动蛋白开始成核。这种局部的细胞骨架组装有助于吞噬小体/足小体的形成。我们进一步假设SH3结构域和h-插入在CIP4亚型之间调节不同的信号功能。为了解决这些假设，我们提出了以下特定目标：特定目标1：确定CIP4结构域对细胞骨架功能的贡献；特定目标2：确定巨噬细胞中CIP4、CDC42、Lyn和Wasp在细胞骨架组装过程中的时空关系。

DESCRIPTION (provided by applicant): Through its SH3 and SH2 domains, the Src kinase Lyn interacts with a small number of phosphoproteins, such as Shc, Cbl, and Vav, which regulate cell cycle and the cytoskeleton. Using Lyn's Unique, SH3, and SH2 domains as bait in a yeast two-hybrid screen, we isolated a gene with features of a scaffolding protein, CIP4. We have identified its four isoforms. CIP4 contains a conserved N-terminal domain, a putative coiled-coil domain, and a C-terminal SH3 domain. What makes CIP4 particularly fascinating to study is its ability to bind two sets of distinct cytoskeletal proteins and their activators. Cytoskeletal reorganization, essential for normal as well as neoplastic cellular function, requires a complex set of protein interactions. CIP4 binds Src kinases and activated states of the RhoGTPase Cdc42. Also, CIP4 binds microtubules through its N-terminal Fes/CIP4 Homologous (FCH) domain and proline-rich proteins such as WASp through its C-terminal SH3 domain. CIP4 belongs to a newly described family of proteins found in lower eukaryotes such yeast. In mammals, we hypothesize that CIP4 plays an important role in Fc Receptor or adhesion-induced integrin signal transduction in leukocytes by providing a scaffold and linking the cytoskeletal system to Src kinases and Cdc42. The resulting cytoskeletal rearrangement is required for cell spreading, migration and functional activation. We hypothesize that CIP4 is a critical scaffolding protein that brings together Lyn and recruits WASp to the plasma membrane in the quiescent cell, and that upon receptor engagement, Lyn becomes activated, phosphorylates WASp, and CIP4 facilitates the recruitment of activated Cdc42 which drives WASp unfolding. Arp2/3 becomes activated, and actin nucleation commences. This localized cytoskeletal assembly contributes to phagosome/podosome formation. We further hypothesize that the SH3 domain and the h-insert mediate different signaling functions among the CIP4 isoforms. To address these hypotheses, we propose the following specific aims: Specific Aim 1: Determine the contribution of the CIP4 domains on cytoskeletal function, and Specific Aim 2: Determine the temporal and spatial relationships among CIP4, Cdc42, Lyn and WASp in macrophages during cytoskeletal assembly.