Abstract
The signaling mechanisms by which neurotrophic receptors regulate neuronal survival and axonal growth are still incompletely understood. In the receptor tyrosine kinase RET, a receptor for GDNF (glial cell line-derived neurotrophic factor), the functions of the majority of tyrosine residues that become phosphorylated are still unknown. Here we have identified the protein-tyrosine phosphatase SHP2 as a novel direct interactor of RET and the first effector known to bind to phosphorylated Tyr(687) in the juxtamembrane region of the receptor. We show that SHP2 is recruited to RET upon ligand binding in a cooperative fashion, such that both interaction with Tyr(687) and association with components of the Tyr(1062) signaling complex are required for stable recruitment of SHP2 to the receptor. SHP2 recruitment contributes to the ability of RET to activate the PI3K/AKT pathway and promote survival and neurite outgrowth in primary neurons. Furthermore, we find that activation of protein kinase A (PKA) by forskolin reduces the recruitment of SHP2 to RET and negatively affects ligand-mediated neurite outgrowth. In agreement with this, mutation of Ser(696), a known PKA phosphorylation site in RET, enhances SHP2 binding to the receptor and eliminates the effect of forskolin on ligand-induced outgrowth. Together, these findings establish SHP2 as a novel positive regulator of the neurotrophic activities of RET and reveal Tyr(687) as a critical platform for integration of RET and PKA signals. We anticipate that several other phosphotyrosines of unknown function in neuronal receptor tyrosine kinases will also support similar regulatory functions.
Highlights
Brain dopaminergic neurons, much of the initial interest in glial cell line-derived neurotrophic factor (GDNF) was focused on its possible therapeutic effects in Parkinson disease [1, 2]
In agreement with a role of the PI3K/AKT pathway in the neuronal differentiation of PC12 cells by the activities of RET and SHP2, the PI3K inhibitor Ly294002 reduced GDNF-mediated neurite outgrowth (Fig. 5A). These results suggest that recruitment of SHP2 to Tyr687 and activation of the PI3K/AKT pathway are important for the ability of GDNF to induce neuronal differentiation of PC12 cells via RET
Activation of protein kinase A (PKA) Diminishes the Ability of GDNF to Induce PC12 Cell Differentiation via RET—In view of the ability of PKA to affect the recruitment of SHP2 to RET and the importance of SHP2 and Tyr687 for RET-mediated neuronal differentiation, we investigated the effects of PKA activation by forskolin on the ability of GDNF to stimulate neurite outgrowth in PC12 cells expressing wild type and mutant RET molecules
Summary
Brain dopaminergic neurons, much of the initial interest in GDNF was focused on its possible therapeutic effects in Parkinson disease [1, 2]. Recovered from COS cell total lysates by beads containing the mutation (Fig. 2A), thereby validating Tyr687 as a novel site of phosphopeptide, indicating that the interaction was phos- SHP2 binding in RET.
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