Wiskott-Aldrich Syndrome (WAS) is an X-linked immunodeficiency disorder with dermatologic, hematologic and immunologic manifestations. The WAS gene ha been identified and, except for its association with Nck and Cdc42, very little is known about the biological function of its protein product WASP. Elucidation of the function of WASP is critical for understanding the molecular basis of the pathology of WAS. Using the yeast two hybrid system we have identified a novel gene, WIP (WASP- Interacting Protein), whose protein products interact with WASP. WIP displays homology to the yeast protein verprolin which is involved in maintaining the integrity of the cytoskeleton. Preliminary studies show that WIP associates with Nck. More importantly, overexpression of a truncated form of WIP containing the WASP-interacting domain causes disruption of actin stress fibers in CHO cells and a decrease in the microvilli in the human B cell line BJAB lymphocytes. We propose to: (1) Characterize WIP and perform a structural analysis of WIP-WASP interaction. We will a) determine the cellular localization of WIP; b) identify the domains and amino acid residues of WIP and WASP that are important for their interaction; c) investigate the association of WIP with WASP, Nck and Cdc42 in a multimeric complex and e) examine the interaction of WIP and WASP-related proteins. (2) Examine the structure and function of WIP with the cytoskeleton. We will a) examine the effect of overexpression of WIP and WIP mutants on the cytoskeleton; b) investigate the interaction of WIP with cytoskeletal proteins and c) determine if WIP complements verprolin deficient yeast. (3) Examine the role of WIP in signal transduction. We will a) analyze WIP phosphorylation and b) examine the interaction with rho GTPases, and the Nck associated enzymes PAK65 and PRK2. (4) Generate and analyze mice deficient in WIP. We will a) determine the organization of the murine WIP gene, b) generate and analyze WIP deficient mice. The results of the proposed studies should provide insights into the function of WIP and into the molecular pathology of the Wiskott-Aldrich syndrome.
Showing the most recent 10 out of 110 publications
