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 proliferation and differentiation of hematopoietic cells. The authors previously reported cloning and characterization of the receptor tyrosine kinase Tif, also termed Tyro3, Using the yeast 2-hybrid technology, they recently identified that the p85 subunit of phosphatidylinositol 3-kinase (P13 kinase) interacted with the cytoplasmic domain of Tyro3, On treatment with epidermal growth factor (EGF), NIH3T3 cells expressed EGFR/Tyro3 (a fusion receptor with the extracellular domain from epidermal growth factor receptor and the transmembrane and cytoplasmic domains from Tyro3), and EGFR/Tyro3 was rapidly phosphorylated on tyrosine residues. The interaction between Tyro3 and p85 was also confirmed by glutathione S-transferase (GST) pull-down experiments, Co-immunoprecipitation followed by Western blot analysis revealed that P13 kinase was associated with and phosphorylated by the activated Tyro3. Tyro3-associated P13 kinase exhibited an enhanced kinase activity In addition, EGF treatment of EGFR/Tyro3-expressing cells led to enhanced phosphorylation 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 protein S also resulted in phosphorylation of Akt, Soft agar colony assays showed that the addition of EGF to EGFR/Tyro3-transfected cells, but not to the parental NIH3T3 cells, resulted in a concentration-dependent increase in the formation of anchorage-independent colonies. Tyro3-mediated transformation of NIH3T3 cells was significantly blocked by wortmannin, a P13 kinase-specific inhibitor. Results of these combined studies strongly suggested that the oncogenic transforming ability of Tyro3 was mediated at least in part by the P13 kinase pathway.