Scaffolding Proteins in Macrophage Phagocytosis

巨噬细胞吞噬作用中的支架蛋白

• 批准号: 6987835
• 负责人: Seth Joel Corey
• 金额: $ 29.49万
• 依托单位: UNIVERSITY OF TX MD ANDERSON CAN CTR
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-01-01 至 2008-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6987835
• 关键词: RNA interference; actins; antibody receptor; biological signal transduction; cell adhesion; chemotaxis; cytoskeletal proteins; cytoskeleton; gene induction /repression; genetically modified animals; laboratory mouse; macrophage; molecular site; phagocytosis; protein folding; protein isoforms; protein localization; protein protein interaction; protein structure function

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.

描述（由申请人提供）：Src 激酶 Lyn 通过其 SH3 和 SH2 结构域与少量磷蛋白相互作用，例如 Shc、Cbl 和 Vav，调节细胞周期和细胞骨架。使用 Lyn 的 Unique、SH3 和 SH2 结构域作为酵母双杂交筛选中的诱饵，我们分离出了具有支架蛋白 CIP4 特征的基因。我们已经确定了它的四种亚型。 CIP4 包含一个保守的 N 端结构域、一个推定的卷曲螺旋结构域和一个 C 端 SH3 结构域。 CIP4 之所以特别令人着迷，是因为它能够结合两组不同的细胞骨架蛋白及其激活剂。细胞骨架重组对于正常和肿瘤细胞功能至关重要，需要一组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) 结构域结合微管，并通过其 C 端 SH3 结构域结合富含脯氨酸的蛋白质，例如 WASp。 CIP4 属于在酵母等低等真核生物中发现的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与 Src 激酶和 Cdc42 连接，在白细胞中 Fc 受体或粘附诱导的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 是一种关键的支架蛋白，它将 Lyn 聚集在一起并将 WASp 招募到静止细胞的质膜上，并且在受体接合后，Lyn 被激活，磷酸化 WASp，并且 CIP4 促进激活的 Cdc42 的招募，从而驱动 WASp 展开。 Arp2/3 被激活，肌动蛋白成核开始。这种局部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传导功能。为了解决这些假设，我们提出以下具体目标：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：确定细胞骨架过程中巨噬细胞中 CIP4、Cdc42、Lyn 和 WASp 之间的时间和空间关系集会。