Cloning and characterization of Drosophila Nedd4 as a putative binding partner of the proline-rich region of Inscuteable

grantor: University of TorontoIn 'Drosophila', the protein Inscuteable has been identified as controlling several aspects of asymmetric mitosis in neuroblasts and muscle progenitor cells. In an effort to elucidate how Inscuteable performs these roles, a PY motif-containing region of Inscuteable was used as bait in yeast two hybrid and expression screens for protein:protein interaction partners, in hopes of finding WW domain-containing proteins involved in mediating Inscuteable's function. Clone 211, the 'Drosophila' homologue of ' epsin', was cloned in a yeast two hybrid screen. Thorough investigation of 211 was not pursued because the interaction detected could not be duplicated in 'in vitro' mixing experiments. The 'Drosophila' homologue of the E3 ubiquitin ligase protein 'Nedd4', ' dNedd4', was cloned in an expression screen. My results show that dNedd4 and Inscuteable can interact in a WW domain:PY motif dependent fashion, and that Inscuteable is a potential ubiquitination substrate, suggesting a possible role for dNedd4 in regulating Inscuteable.M.Sc