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.
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