Intracellular signaling of the Ufo/Axl receptor tyrosine kinase is mediated mainly by a multi-substrate docking-site

Ufo/Axl belongs to a new family of receptor tyrosine kinases with an extracellular structure similar to that of neural cell adhesion molecules, In order to elucidate intracellular signaling, the cytoplasmic moiety of Ufo/Axl was used to screen an expression library according to the CORT (cloning of receptor targets) method, Three putative Ufo substrates were identified: phospholipase C gamma 1 (PLC gamma), as well as p85 alpha and p85 beta subunits of phosphatidylinositol 3'-kinase (PI3-kinase), Subsequently, chimeric EGFR/Ufo receptors consisting of the extracellular domains of the epidermal growth factor receptor (EGFR) and the transmembrane and intracellular moiety of Ufo were engineered, Using different far-Western blot analyses and coimmunoprecipitation assays, receptor binding of PLC gamma and p85 proteins as well as GRB2, c-src and lck was examined in vitro and in vivo, Competitive inhibition of substrate binding and mutagenesis experiments with EGFR/Ufo constructs revealed C-terminal tyrosine 821 (EILpYVNMDEG) as a docking site for multiple effecters, namely PLC gamma, p85 proteins, GRB2, c-src and Ick, Tyrosine 779 (DGLpYALMSRC) demonstrated an additional, but lower binding affinity for the p85 proteins in vitro, In addition, binding of PLC gamma occurred through tyrosine 866 (AGRpYVLCPST), Moreover, our in vivo data indicate that further direct or indirect binding sites for PLC gamma, GRB2, c-src and lck on the human Ufo receptor may exist.