Transforming activity of receptor tyrosine kinase Tyro3 is mediated, at least in part, by the PI3 kinase-signaling pathway

Protein tyrosine phosphorylation is an integral part of cytokine-induced pr oliferation and differentiation of hematopoietic cells. The authors previou sly reported cloning and characterization of the receptor tyrosine kinase T if, also termed Tyro3, Using the yeast 2-hybrid technology, they recently i dentified that the p85 subunit of phosphatidylinositol 3-kinase (P13 kinase ) interacted with the cytoplasmic domain of Tyro3, On treatment with epider mal growth factor (EGF), NIH3T3 cells expressed EGFR/Tyro3 (a fusion recept or with the extracellular domain from epidermal growth factor receptor and the transmembrane and cytoplasmic domains from Tyro3), and EGFR/Tyro3 was r apidly phosphorylated on tyrosine residues. The interaction between Tyro3 a nd p85 was also confirmed by glutathione S-transferase (GST) pull-down expe riments, Co-immunoprecipitation followed by Western blot analysis revealed that P13 kinase was associated with and phosphorylated by the activated Tyr o3. Tyro3-associated P13 kinase exhibited an enhanced kinase activity In ad dition, EGF treatment of EGFR/Tyro3-expressing cells led to enhanced phosph orylation of Akt, a downstream component of P13 kinase, Treatment of NIH3T3 cells expressing a full length of rat Tyro-3, but not NIH3T3 cells, with p rotein S also resulted in phosphorylation of Akt, Soft agar colony assays s howed that the addition of EGF to EGFR/Tyro3-transfected cells, but not to the parental NIH3T3 cells, resulted in a concentration-dependent increase i n the formation of anchorage-independent colonies. Tyro3-mediated transform ation of NIH3T3 cells was significantly blocked by wortmannin, a P13 kinase -specific inhibitor. Results of these combined studies strongly suggested t hat the oncogenic transforming ability of Tyro3 was mediated at least in pa rt by the P13 kinase pathway.