Mechanism of PLD interaction with kinases and Rac: Role on phagocyte chemotaxis

描述(由申请人提供)：炎症、伤口修复和血管生成都有一个共同的初始生理事件，即细胞迁移或趋化。相关的病理过程，如慢性炎症、动脉粥样硬化和缺血/再灌注损伤，也严重依赖于细胞的趋化作用。趋化是血液中性粒细胞的主要功能之一，本实验室首次证实了磷脂酶D(PLD)在趋化和细胞极化中的作用。新的初步数据为PLD在这一过程中可能遵循的机制奠定了基础：PLD中存在影响酪氨酸磷酸化和活性的SH2结构域；小GTP酶rac2(调节肌动蛋白聚合)与PLD的关联；以及PLD来源的PA上调参与趋化作用的蛋白质核糖体S6激酶(S6K)。这一建议的中心假设是，新描述的SH2结构域上酪氨酸磷酸化对PLD的调节，以及它与信号分子rac2和S6K的相互作用，是中性粒细胞趋化的关键决定因素。为此，我们提出了以下三个具体目标：(1)。探讨PLD2介导的中性粒细胞趋化的SH2依赖的上下游机制。我们的目标是测试序贯模型：(A)在FMLP或IL-8刺激中性粒细胞时，酪氨酸激酶Syk/Src磷酸化PLD2；(B)Grb2通过其SH2结构域与PLD2结合；(C)PLD2-Grb2复合体招募运动蛋白WASP，使F-肌动蛋白聚合在可移动的中性粒细胞的前沿。我们将使用磷酸蛋白分析/蛋白质组学和实时显微镜来研究这一目标。(2)。研究RAC和PLD之间是否存在通过肌动蛋白聚合调节中性粒细胞趋化的关联。我们的目标是证明：(A)PLD1(或PLD2)和rac2形成一种蛋白质-蛋白质复合体，用于不同地激活这两种PLD亚型；以及(B)在体内的趋化过程中，rac2通过肌动蛋白对PLD进行调控。我们将实时使用FRET化学计量显微镜和骨髓来源的敲除(rac2-/-)中性粒细胞。(3)。目的：探讨PLD来源的PA在趋化过程中对S6激酶的调节作用。我们将：(A)阐明PA与S6K结合的位置；(B)确定S6K是否是趋化所必需的；以及(C)证明在没有mTOR激活的情况下，PLD增强S6K导致趋化。我们将使用多肽阵列设置和S6K的顺序截断来研究这一目标。这一提议的意义在于，中性粒细胞在试图摧毁慢性炎症中的入侵病原体时，会释放裂解酶，从而对健康组织造成损害。从这些目标的成功实现中获得的信息将有助于理解受调控的细胞迁移的细节。这将使我们能够开发策略和试剂，以加速在动脉粥样硬化或缺血/再灌注损伤等心血管相关病理中看到的有害炎症的消退。项目简介：中性粒细胞释放裂解酶，旨在摧毁入侵的病原体。然而，这些强大的物质也会对健康组织造成巨大的、不可逆转的损害，就像心脏病发作后的缺血一样。这个想法是，如果一个人能阻止中性粒细胞到达损伤部位，就可以防止对健康组织的进一步损害。防止中性粒细胞到来的一个有效方法是损害使它们移动的能力：趋化性。这一建议试图阐明趋化作用的分子机制及其与细胞骨架细胞器的基本分子的相互作用，这是免疫防御的关键生命功能所必需的。

DESCRIPTION (provided by applicant): Inflammation, wound repair and angiogenesis have in common an initial physiological event of cell migration or chemotaxis. Related pathological processes, such as chronic inflammation, atherosclerosis and ischemia/reperfusion injury, are also heavily dependent on cell chemotaxis. Chemotaxis is one of the main functions of blood neutrophils and our laboratory has demonstrated for the first time a role for phospholipase D (PLD) in chemotaxis and cell polarization. New preliminary data serve to form the basis for the mechanism that PLD might follow in this process: the existence of SH2 domains in PLD that affect tyrosyl phosphorylation and activity; an association of the small GTPase, Rac2 (that regulates actin polymerization) with PLD; and the upregulation of a protein involved in chemotaxis, ribosomal S6 kinase (S6K) by PLD-derived PA. The central hypothesis of this proposal is that PLD regulation by tyrosyl phosphorylation in newly-described SH2 domains, and its interaction with signaling molecules Rac2 and S6K, are crucial determinants for neutrophil chemotaxis. To investigate this, we are proposing the following 3 Specific Aims: (1). To investigate the SH2-dependent upstream and downstream mechanisms of PLD2- mediated neutrophil chemotaxis. The goal is to test the sequential model: (a) upon FMLP or IL-8 stimulation of neutrophils, tyrosine kinases Syk/Src phosphorylate PLD2; (b) Grb2 binds to PLD2 through its SH2 domain; and (c) the PLD2-Grb2 complex recruits the motile protein WASP, enabling F-actin polymerization in the leading edges of the mobile neutrophil. We will use phosphoprotein analysis/proteomics and real time microscopy to study this Aim. (2). To investigate whether an association between Rac and PLD exists that regulates neutrophil chemotaxis via actin polymerization. The goal is to demonstrate that: (a) PLD1 (or PLD2) and Rac2, form a protein-protein complex that serves to differentially activate the two PLD isoforms; and (b) Rac2 regulation of PLD occurs via actin during chemotaxis in vivo. We will use FRET stoichiometry microscopy in real time and bone marrow-derived knockout (Rac2-/-) neutrophils. (3). To investigate whether PLD-derived PA regulates S6 kinase during chemotaxis. We will: (a) elucidate the site of PA binding to S6K; (b) determine whether S6K is needed for chemotaxis; and (c) demonstrate that PLD enhances S6K leading to chemotaxis, in the absence of mTOR activation. We will use peptide array settings and sequential truncation of S6K to study this Aim. The significance of this proposal is that neutrophils can cause damage to healthy tissues as they release lytic enzymes in attempting to destroy invading pathogens in chronic inflammation. The information obtained from successful achievement of these Aims will help understand the details of regulated cell migration. This will allow us to develop strategies and reagents to hasten the resolution of harmful inflammation seen in such cardiovascular-related pathologies as atherosclerosis or ischemia/reperfusion injury. PROJECT NARRATIVE: Neutrophils release lytic enzymes intended to destroy invading pathogens. However, these powerful substances also cause great and irreversible damage to healthy tissues as in the case of ischemia after a heart attack. The idea is that if one can prevent the arrival of neutrophils to the site of injury, further damage of healthy tissue can be prevented. An effective way to prevent the arrival of neutrophils is to compromise the very capability that makes them move: chemotaxis. This proposal seeks to elucidate the molecular mechanism chemotaxis and its interplay with fundamental molecules of the cytoskeletal cellular machinery, as necessary for the key life function of immune defense.