Mapping of Grb2 binding site in N-WASP.

Abstract

Cancer metastasis is the leading cause of death worldwide. Cancer cells form invadopodia, actin-rich membrane protrusions capable of degrading extracellular matrix to invade and migrate into blood vessels and connective tissues away from the primary site of tumor. Epidermal growth factor which stimulates cancer metastasis binds to its receptor and the tyrosine phosphorylated epidermal growth factor receptor binds Src Homology (SH) 2 domain of Growth factor receptor-bound protein 2 (GRB2). This triggers the binding of SH3 domains of GRB2 to proline-rich regions on Neural Wiskott-Aldrich syndrome protein (N-WASP). GRB2 is an effector of N-WASP, stimulating actin assembly via N-WASP-Arp2/3 complex and results in formation of invadopodia. These invadopodia-related elements could be potential target in cancer therapeutic treatments. The aim of this project is to map the binding site of GRB2 on N-WASP using Yeast Two-Hybrid Assay. GAL4 BD plasmid fused with N-WASP peptides and GAL4 AD plasmid constructed with GRB2 were cotransformed into PJ69-4A yeast cells and their growth tested on synthetically defined medium (SD). It was found that larger N-WASP constructs bind more efficiently than smaller constructs. Moreover, the binding site of GRB2 on N-WASP is postulated to be around amino acid 271-286 and 317-356 of the proline-rich regions.