Phagocytosis is an important arm of the lung's host defense against a variety of pathogenic organisms, including Streptococcal pneumonia, Mycobacterium tuberculosis, and Pneumocystis carinii. Defects of phagocytosis have been documented in patients with diabetes mellitus, cirrhosis, uremia, various autoimmune diseases, and AIDS. In addition to its medical significance, phagocytosis serves as a convenient model system to study cytoskeletal dynamics that occur at the leading edge of all eukaryotic cells. During Fc receptor.mediated phagocytosis, pseudopods are elaborated which are enriched in F-actin and numerous tyrosine- phosphorylated proteins. Fc receptor-mediated phagocytosis is blocked by cytochalasins and tyrosine kinase inhibitors; thus, it is dependent on F- actin assembly and one or more tyrosine kinases. The identity of specific tyrosine kinases that are capable of mediating these cytoskeletal changes is unknown. We have developed several model systems to probe specific steps in the formation of pseudopods and engulfment of IgG-coated particles. These employ cell lines transfected with chimeric constructs containing Fc receptor subunits, and chimeric fusion proteins containing the entire coding region of several cytosolic tyrosine kinases. These and other studies have enabled us to identify the protein tyrosine kinase syk as a likely candidate to play a central role in mediating cytoskeletal rearrangements that culminate in phagocytosis. The overall goal of this proposal is to determine how syk becomes activated to signal cytoskeletal alterations and pseudopod formation. We will address the following related hypotheses: (1) Do Src family tyrosine kinases participate directly in phagocytic signaling by activating syk tyrosine kinase? (2) Is syk tyrosine kinase critical for Fc receptor-mediated phagocytosis? (3) Does syk activate downstream pathways that mediate phagocytosis? We will address the role of the Src family tyrosi~e kinases in Specific Aim 1, in which we employ a model system for pseudopod formation: a lymphocyte cell line that expresses a chimerib receptor whose cytosolic domain consists of the y subunit of Fc receptors. We will use lyn-deficient lymphocytes to address the requirement for this tyrosine kinase in pseudopod formation. The spatial relationship between lyn and hck kinases and Fc receptors will be explored. Are lyn and/or hck required for the activation of syk kinase? In Specific Aim 2, we will address the role of syk tyrosine kinase in pseudopod formation using sykdeficient lymphocytes. We will characterize a model system for phagocytosis in COS cells expressing transmembrane fusion proteins bearing syk, and various mutated forms of syk. Finally, in Specific Aim 3 we will explore the mechanisms by which syk signals phagocytosis, and identify possible downstream targets of syk, such as phosphatidylinositol 3-kinase, the small molecular weight GTP-binding protein ras and potentially novel substrates of syk.
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