Characterization of intracellular signals via tyrosine 1062 in RET activated by glial cell line-derived neurotrophic factor

Glial cell line derived neurotrophic factor (GDNF) signals through a multic omponent receptor complex consisting of RET receptor tyrosine kinase and a member of GDNF family receptor alpha (GPR alpha). Recently, it was shown th at tyrosine 1062 in RET represents a binding site for SHC adaptor proteins and is crucial for both RAS/mitogen activated protein kinase (MAPK) and pho sphatidylinositol 3-kinase (PI3K)/AKT signaling pathways. In the present st udy, we characterized how these two pathways diverge from tyrosine 1062, us ing human neuroblastoma and primitive neuroectodermal tumor cell lines expr essing RET at high levels. In response to GDNF stimulation, SHC bound to GA B1 and GRB2 adaptor proteins as well as RET, and SHC and GAB1 were highly p hosphorylated on tyrosine. The complex formation consisting of SHC, GAB1 an d GRB2 was almost abolished by replacement of tyrosine 1062 in RET with phe nylalanine. Tyrosine-phosphorylated GAB1 was also associated with p85 subun it of PI3-K, resulting in PI3-K and AKT activation, whereas SHC-GRB2-SOS co mplex was responsible for the RAS/ERK signaling pathway. These results sugg ested that the RAS and PI3K pathways activated by GDNF bifurcate mainly thr ough SHC bound to tyrosine 1062 in RET. Furthermore, using luciferase repor ter-gene assays, we found that the RAS/ERK and PI3-K signaling pathways are important for activation of CREB and NF-kappa B in GDNF-treated cells, res pectively.