Scaffolding Proteins in Macrophage Phagocytosis

描述（由申请人提供）：Src激酶林恩通过其SH 3和SH 2结构域与少量磷蛋白（如Shc、Cbl和Vav）相互作用，这些磷蛋白调节细胞周期和细胞骨架。使用林恩的独特，SH 3和SH 2结构域作为酵母双杂交筛选的诱饵，我们分离出一个具有支架蛋白CIP 4特征的基因。我们已经确定了它的四个异构体。CIP 4含有保守的N-末端结构域、推定的卷曲螺旋结构域和C-末端SH 3结构域。CIP 4特别吸引人的研究是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，正常以及肿瘤细胞功能所必需的，需要一套复杂的蛋白质相互作用。CIP 4结合Src激酶和RhoGT 3 Cdc 42的活化状态。此外，CIP 4通过其N-末端Fes/CIP 4同源（FCH）结构域结合微管，并通过其C-末端SH 3结构域结合富含脯氨酸的蛋白质如WASp。CIP 4属于在低等真核生物如酵母中发现的新描述的蛋白质家族。在哺乳动物中，我们假设CIP 4通过提供支架并将细胞骨架系统连接到Src激酶和Cdc 42，在白细胞中Fc受体或粘附诱导的整合素信号转导中起重要作用。所产生的细胞骨架重排是细胞扩散、迁移和功能活化所必需的。我们假设CIP 4是一个关键的支架蛋白，它将林恩聚集在一起，并将WASp募集到静止细胞的质膜上，并且在受体接合时，林恩被激活，磷酸化WASp，CIP 4促进激活的Cdc 42的募集，从而驱动WASp展开。Arp 2/3被激活，肌动蛋白成核开始。这种局部化的细胞骨架组装有助于吞噬体/足状体的形成。我们进一步假设SH 3结构域和h-插入片段介导CIP 4亚型之间不同的信号传导功能。为了解决这些假设，我们提出了以下具体目标：具体目标1：确定CIP 4结构域对细胞骨架功能的贡献，具体目标2：确定巨噬细胞中CIP 4，Cdc 42，林恩和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.