Signaling complexes and protein-protein interactions involved in the activation of the Ras and phosphatidylinositol 3-kinase pathways by the c-Ret receptor tyrosine kinase

Proximal signaling events and protein-protein interactions initiated after activation of the c-Ret receptor tyrosine kinase by its ligand, glial cell line-derived neurotrophic factor (GDNF), were investigated in cells carryin g native and mutated forms of this receptor. Mutation of Tyr-1062 (Y1062F) in the cytoplasmic tail of c-Ret abolished receptor binding and phosphoryla tion of the adaptor Shc and eliminated activation of Ras by GDNF. Phosphory lation of Erk kinases was also greatly attenuated but not eliminated by thi s mutation. This residual wave of Erk phosphorylation was independent of th e kinase activity of c-Ret. Mutation of Tyr-1096 (Y1096F), a binding site f or the adaptor Grb2, had no effect on Erk activation by GDNF, Activation of phosphatidylinositol-3 kinase (PI3K) and its downstream effector Akt was a lso reduced in the Y1082F mutant but not completely abolished unless Tyr-10 96 was also mutated, Ligand stimulation of neuronal cells induced the assem bly of a large protein complex containing c-Ret, Grb2, and tyrosine-phospho rylated forms of Shc, p85(PI3K), the adaptor Gab2, and the protein-tyrosine phosphatase SHP-2. In agreement with Ras-independent activation of PI3K by GDNF in neuronal cells, survival of sympathetic neurons induced by GDNF wa s dependent on PI3K but was not affected by microinjection of blocking anti -Pas antibodies, which did compromise neuronal survival by nerve growth fac tor, suggesting that Ras is not required for GDNF-induced survival of sympa thetic neurons. These results indicate that upon ligand stimulation, at lea st two distinct protein complexes assemble on phosphorylated Tyr-1062 of c- Ret via Shc, one leading to activation of the Ras/Erk pathway through recru itment of Grb2/Sos and another to the PI3K/Akt pathway through recruitment of Grb2/Gab2 followed by p85(PI3K) and SHP-2. This latter complex can also assemble directly onto phosphorylated Tyr-1096, offering an alternative rou te to PI3K activation by GDNF.