Protein-tyrosine phosphatase SHP2 contributes to GDNF neurotrophic activity through direct binding to phospho-Tyr687 in the RET receptor tyrosine kinase

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.

FIGURE 1.

Identification of SHP2 as a direct interactor of phospho-Tyr⁶⁸⁷ in RET. A, micropanning of phage particles expressing the N-terminal SH2 domain of SHP2 against different phosphopeptides derived from RET. Results are expressed as particle-forming units (pfu)/ml. B, peptide pulldown of COS cell lysates analyzed by SHP2 immunoblotting (IB). Endogenous (left) as well as overexpressed SHP2 (right) could be recovered by pulldown with the phospho-Tyr⁶⁸⁷ peptide but not with its unphosphorylated form. C, stimulation of in vitro SHP2 phosphatase activity by phospho-Tyr⁶⁸⁷ peptide. Phosphatase activity is expressed in arbitrary units after normalization. The non-phosphorylated peptide had no effect. The phosphatase inhibitor Na₃VO₄ reduced SHP2 phosphatase activity to background levels. D, pulldown of RET constructs from COS cell lysates with a GST fusion protein containing the N-terminal SH2 domain of SHP2. The blot was probed with anti-RET antibodies. Wild type, but not Y687F mutant, interacted with the N-SH2 domain of SHP2. GST was used as negative control.

FIGURE 2.

SHP2 binding determinants in RET and regulation by PKA activity. A, analysis of co-immunoprecipitation (IP) of SHP2 with constitutively activated MEN2A constructs of RET9 and RET51 isoforms in COS cells. The blots (IB) show representative examples of a series of experiments. The histogram summarizes SHP2/RET interaction results for the RET51 isoform from three independent experiments. Results are expressed as the average ± S.D. of normalized RET levels in SHP2 immunoprecipitates. *, p < 0.05 (versus WT). B, analysis of RET and SHP2 co-immunoprecipitation with components of the Tyr¹⁰⁶² signaling complex, Shc and Gab2. Immunoblotting of total cell lysates (bottom) shows comparable levels of RET51 MEN2A proteins. C, interaction of endogenous SHP2 and RET in the motoneuron cell line MN1. Prior to SHP2 immunoprecipitation, cells had been serum-starved and stimulated with GDNF and soluble GFRα1 for 20 min as indicated. Pretreatment with forskolin for 2 h resulted in a reduction of SHP2/RET co-immunoprecipitation. The histogram summarizes the results of the average ± S.D. of four independent experiments.

FIGURE 3.

Mutation of Tyr⁶⁸⁷ in RET impairs GDNF-dependent AKT signaling. A, analysis of RET tyrosine autophosphorylation in response to GDNF stimulation in stably transfected fibroblast cell lines expressing wild type or Y687F mutant RET51. RET immunoprecipitates were probed with anti-phosphotyrosine antibodies. The histogram shows the average results in arbitrary units normalized to total RET levels from three experiments using two different sets of wild type and mutant RET-expressing cell clones. No significant differences were observed between Y687F and wild type. B, activation of downstream signaling pathways by wild type and Y687F mutant RET51 following GDNF stimulation of stably transfected fibroblast cell lines. Total cell lysates were analyzed with the indicated phospho-specific antibodies. Reprobing against total Akt shows equal loading in all lanes. The blots (IB) show representative examples of several experiments. The histogram shows average results of Akt phosphorylation in arbitrary units normalized to total Akt levels from four experiments. *, p < 0.05, **, p < 0.005.

FIGURE 4.

Mutation of Tyr⁶⁸⁷ affects the ability of RET to induce neuronal differentiation of PC12 cells. A, neuronal differentiation of PC12 cells expressing constitutively activated MEN2A RET51 constructs. Cells displaying neuritic extensions longer than 2 cell diameters were counted at 3 and 6 days post-transfection. The results were normalized to the total number of transfected (i.e. GFP-expressing) PC12 cells in each field and are expressed as the average ± S.D. of triplicate determinations. *, p < 0.05 versus WT. Similar results were obtained in three additional experiments. B, ligand-mediated neuronal differentiation of PC12 cells expressing wild type and mutant RET51 constructs. Differentiation of cells transiently transfected with the indicated constructs was induced by stimulation with GDNF and soluble GFRα1 in serum-free medium. The micrographs show fields of GFP-expressing cells transfected with different RET constructs under control conditions and following ligand stimulation. C, immunoblot (IB) of lysates from transfected PC12 cells indicating similar amounts of RET for each construct and condition. D, percentage of differentiated PC12 cells for each condition and mutant construct after 3 days of treatment. Results are expressed as average ± S.D. of triplicate determinations. *, p < 0.05 versus WT + GDNF. Similar results were obtained in three additional experiments.

FIGURE 5.

SHP2 inhibition and PKA activation reduce neuronal differentiation of PC12 cells in response to GDNF-mediated RET activation. A, neuronal differentiation of PC12 cells expressing WT or mutant RET51 constructs co-transfected with dominant negative SHP2 constructs N-SH2 or C459S or treated with the PI3K inhibitor Ly294002. Under the conditions of this experiment, Ly294002 did not have any effect on cell survival. Results are expressed as the average ± S.D. of triplicate determinations. *, p < 0.05 versus WT + GDNF. Similar results were obtained in two additional experiments. B, neuronal differentiation of PC12 cells expressing the indicated wild type or mutant RET51 constructs following treatment with GDNF/GFRα1, forskolin, or their combination. Results are expressed as the average ± S.D. of triplicate determinations. *, p < 0.05 versus WT + GDNF. Similar results were obtained in two additional experiments.

FIGURE 6.

Signaling through Tyr⁶⁸⁷ is required for RET-mediated neurite outgrowth in sympathetic neurons. A, representative micrographs of dsRed-expressing SCG neurons 30 h after microporation with the indicated constructs maintained in the presence of 1 ng/ml NGF. Examples from wells maintained in only 100 and 1 ng/ml NGF are also shown. B, camera lucida drawings depicting the longest neurites of transfected SCG neurons in 10 different fields superimposed onto each other. C, neurite outgrowth in SCG neurons transfected with wild type or Y687F mutant MEN2A RET constructs. Neurite outgrowth was measured as the length of the longest neurite in each transfected neuron and normalized to the average values obtained with 100 ng/ml NGF. Results are expressed as the average ± S.D. of three independent experiments each performed in triplicate. *, p < 0.05 versus WT (corresponding solid bars).

FIGURE 7.

Signaling through Tyr⁶⁸⁷ contributes to RET-mediated survival of sympathetic neurons. A, photomicrographs of SCG neurons co-transfected with dsRed and the indicated RET constructs. SCGs were grown in NGF overnight and then switched to medium containing NGF-blocking antibodies. Arrows denote dead cells. B, survival of SCG neurons transfected with wild type or Y687F mutant RET constructs or GFP as negative control. Survival is indicated as a percentage of cell number relative to DIV1 (which was arbitrarily set to 100%). Results are expressed as the average ± S.D. of three independent experiments each performed in quadruplicate. *, p < 0.05 versus WT at DIV3.