c-Src is required for glial cell line-derived neurotrophic factor (GDNF) family ligand-mediated neuronal survival via a phosphatidylinositol-3 kinase(PI-3K)-dependent pathway

The glial cell line-derived neurotrophic factor (GDNF) family ligands (GFLs ), consisting of GDNF, neurturin, persephin, and artemin, signal via a mult icomponent complex composed of Ret tyrosine kinase and the glycosyl-phospha tidylinositol (GPI) anchored coreceptors GFR alpha1-alpha4. In previous wor k we have demonstrated that the localization of Ret to membrane microdomain s known as lipid rafts is essential for GDNF-induced downstream signaling, differentiation, and neuronal survival. Moreover, we have found that Ret in teracts with members of the Src family kinases (SFK) only when it is locali zed to these microdomains. In the present work we show by pharmacological a nd genetic approaches that Src activity was necessary to elicit optimal GDN F-mediated signaling, neurite outgrowth, and survival. In particular, p60Sr c, but not the other ubiquitous SFKs, Fyn and Yes, was responsible for the observed effects. Moreover, Src appeared to promote neuronal survival via a phosphatidylinositol-3 kinase (PI-3K)-dependent pathway because the PI-3K inhibitor LY294002 prevented GFL-mediated neuronal survival and prevented a ctivated Src-mediated neuronal survival. In contrast, the inhibition of Src activity had no effects on NGF-mediated survival, indicating that the requ irement for Src was selective for GFL-mediated neuronal survival. These dat a confirm the importance of protein-protein interactions between Ret and ra ft-associated proteins in the signaling pathways elicited by GDNF, and the data implicate Src as one of the major signaling molecules involved in GDNF -mediated bioactivity.