Scaffolding Proteins in Macrophage Phagocytosis

描述（由申请人提供）：Src激酶Lyn通过其SH3和SH2结构域与少量磷酸化蛋白相互作用，如Shc、Cbl和Vav，这些磷酸化蛋白调节细胞周期和细胞骨架。利用Lyn's Unique， SH3和SH2结构域作为诱饵，在酵母双杂交筛选中分离出一个具有支架蛋白特征的基因CIP4。我们已经确定了它的四种同工异构体。CIP4包含一个保守的n端结构域、一个假定的线圈结构域和一个c端SH3结构域。CIP4特别吸引人的是它结合两组不同的细胞骨架蛋白及其激活剂的能力。细胞骨架重组是正常细胞和肿瘤细胞功能所必需的，需要一系列复杂的蛋白质相互作用。CIP4结合Src激酶和RhoGTPase Cdc42的激活状态。此外，CIP4通过其n端Fes/CIP4同源（FCH）结构域与微管结合，并通过其c端SH3结构域与富含脯氨酸的蛋白质（如WASp）结合。CIP4属于在酵母等低等真核生物中发现的一个新描述的蛋白质家族。在哺乳动物中，我们假设CIP4通过提供支架并将细胞骨架系统连接到Src激酶和Cdc42，在Fc受体或粘附诱导的白细胞整合素信号转导中发挥重要作用。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是一个关键的支架蛋白，它将Lyn聚集在一起并将WASp招募到静止细胞的质膜上，当受体接合时，Lyn被激活，使WASp磷酸化，CIP4促进活化Cdc42的招募，从而推动WASp展开。Arp2/3被激活，肌动蛋白成核开始。这种局部的细胞骨架组装有助于吞噬体/足体的形成。我们进一步假设SH3结构域和h-插入介导CIP4亚型之间不同的信号功能。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CIP4结构域对细胞骨架功能的贡献；具体目标2：确定巨噬细胞在细胞骨架组装过程中CIP4、Cdc42、Lyn和WASp之间的时空关系。

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.