Scaffolding Proteins in Macrophage Phagocytosis

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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.