Banin, Sharon; (1997) Identification and characterisation of the interaction between Wiskott-Aldrich syndrome protein (WASP) and c-Src kinases. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Receptor-mediated signal transduction requires the assembly of multimeric complexes of signalling proteins. A number of conserved protein domains, such as the SH2, SH3 and PH domains, are involved in mediating specific protein-protein interactions in such complexes. The identification of binding partners for these domains has added considerably to our understanding of signalling pathways and the aim of this work was to identify such binding proteins for the c-Src family of cytoplasmic protein tyrosine kinases, in haematopoietic cells. Affinity chromatography experiments were performed using domains from the c-Src family members as fusion proteins with glutathione S-transferase, to search for SH2 and SH3 binding proteins in the human monoblastic leukaemic cell line, U937. Protein microsequencing identified one of the SH3 binding proteins as WASP, the protein defective in Wiskott-Aldrich syndrome (WAS) and isolated X-linked thrombocytopenia. WAS is an X-linked recessive condition characterised by severe eczema, recurrent infections, thrombocytopenia, impaired humoral and cellular immunity and increased susceptibility to lymphoid malignancies. WASP bound preferentially in vitro to SH3 domains from c-Src family kinases and its expression was restricted to cells of haematopoietic lineages. WASP and various protein kinases were expressed in insect cells using the baculovirus expression system, and co-expression experiments demonstrated a specific interaction between WASP and Fyn in insect cells. A WASP-Fyn complex was also observed in U937 cells. Additional biochemical studies on the WASP-Fyn interaction revealed that WASP is tyrosine phosphorylated when co-expressed with Fyn in insect cells and under these conditions WASP could stimulate the tyrosine kinase activity of Fyn. In vivo phosphorylation of WASP was also observed in a human T-cell line, and data from U937 cells engineered to overexpress WASP, suggest that WASP may stimulate tyrosine kinase activity in the cell. Finally, analysis of the subcellular localisation of WASP in U937 cells using immunocytochemistry with confocal microscopy, showed co-localisation of WASP with Fyn and [alpha]-tubulin. Haematopoietic cells from individuals with WAS exhibit defects in cell morphology and signal transduction. Members of the c-Src family of protein tyrosine kinases, including Fyn are involved in a range of signalling pathways, including those regulating cytoskeletal structure. These data suggest that binding of Fyn to WASP may be a critical event in such signalling pathways in haematopoietic cells.

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