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
2000; Elsevier BV; Volume: 275; Issue: 50 Linguagem: Inglês
10.1074/jbc.m006908200
ISSN1083-351X
AutoresValérie Besset, Rizaldy P. Scott, Carlos F. Ibáñez,
Tópico(s)Axon Guidance and Neuronal Signaling
ResumoProximal 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 carrying native and mutated forms of this receptor. Mutation of Tyr-1062 (Y1062F) in the cytoplasmic tail of c-Ret abolished receptor binding and phosphorylation of the adaptor Shc and eliminated activation of Ras by GDNF. Phosphorylation of Erk kinases was also greatly attenuated but not eliminated by this mutation. This residual wave of Erk phosphorylation was independent of the kinase activity of c-Ret. Mutation of Tyr-1096 (Y1096F), a binding site for the adaptor Grb2, had no effect on Erk activation by GDNF. Activation of phosphatidylinositol-3 kinase (PI3K) and its downstream effector Akt was also reduced in the Y1062F mutant but not completely abolished unless Tyr-1096 was also mutated. Ligand stimulation of neuronal cells induced the assembly of a large protein complex containing c-Ret, Grb2, and tyrosine-phosphorylated forms of Shc, p85PI3K, 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 was dependent on PI3K but was not affected by microinjection of blocking anti-Ras antibodies, which did compromise neuronal survival by nerve growth factor, suggesting that Ras is not required for GDNF-induced survival of sympathetic neurons. These results indicate that upon ligand stimulation, at least two distinct protein complexes assemble on phosphorylated Tyr-1062 of c-Ret via Shc, one leading to activation of the Ras/Erk pathway through recruitment of Grb2/Sos and another to the PI3K/Akt pathway through recruitment of Grb2/Gab2 followed by p85PI3K and SHP-2. This latter complex can also assemble directly onto phosphorylated Tyr-1096, offering an alternative route to PI3K activation by GDNF. 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 carrying native and mutated forms of this receptor. Mutation of Tyr-1062 (Y1062F) in the cytoplasmic tail of c-Ret abolished receptor binding and phosphorylation of the adaptor Shc and eliminated activation of Ras by GDNF. Phosphorylation of Erk kinases was also greatly attenuated but not eliminated by this mutation. This residual wave of Erk phosphorylation was independent of the kinase activity of c-Ret. Mutation of Tyr-1096 (Y1096F), a binding site for the adaptor Grb2, had no effect on Erk activation by GDNF. Activation of phosphatidylinositol-3 kinase (PI3K) and its downstream effector Akt was also reduced in the Y1062F mutant but not completely abolished unless Tyr-1096 was also mutated. Ligand stimulation of neuronal cells induced the assembly of a large protein complex containing c-Ret, Grb2, and tyrosine-phosphorylated forms of Shc, p85PI3K, 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 was dependent on PI3K but was not affected by microinjection of blocking anti-Ras antibodies, which did compromise neuronal survival by nerve growth factor, suggesting that Ras is not required for GDNF-induced survival of sympathetic neurons. These results indicate that upon ligand stimulation, at least two distinct protein complexes assemble on phosphorylated Tyr-1062 of c-Ret via Shc, one leading to activation of the Ras/Erk pathway through recruitment of Grb2/Sos and another to the PI3K/Akt pathway through recruitment of Grb2/Gab2 followed by p85PI3K and SHP-2. This latter complex can also assemble directly onto phosphorylated Tyr-1096, offering an alternative route to PI3K activation by GDNF. glial cell line-derived neurotrophic factor multiple endocrine neoplasia extracellular signal-regulated kinase phosphatidylinositol-3 kinase superior cervical ganglion nerve growth factor glutathione S-transferase Ras binding domain GDNF family receptor The receptor tyrosine kinase c-Ret is one of the first components in the signaling cascade activated by members of the GDNF1 family, a group of structurally and functionally related polypeptides involved in the control of neuron survival and differentiation, kidney morphogenesis, and spermatogonial cell fate (1Meng X. Lindahl M. Hyvonen M.E. Parvinen M. de Rooij D.G. Hess M.W. Raatikainen-Ahokas A. Sainio K. Rauvala H. Lakso M. Pichel J.G. Westphal H. Saarma M. Sariola H. Science. 2000; 287: 1489-1493Crossref PubMed Scopus (1108) Google Scholar, 2Trupp M. Arenas E. Fainzilber M. Nilsson A.-S. 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F. Neuron, in press.Google Scholar). Several point mutations and chromosomal rearrangements can also activate the c-Ret kinase. In humans, these mutations turn on the oncogenic potential of the c-ret gene, leading to the development of several types of cancers, including multiple endocrine neoplasias type 2A and 2B (MEN2A and MEN2B), familial medullary thyroid carcinomas, and papillary thyroid carcinomas (reviewed in Refs. 12Edery P. Eng C. Munnich A. Lyonnet S. Bioessays. 1997; 19: 389-395Crossref PubMed Scopus (69) Google Scholar and 13Santoro W. Carlomagno F. Melillo R.M. Billaud W. Vecchio G. Fusco A. J. Endocrinol. Invest. 1999; 22: 811-819Crossref PubMed Scopus (32) Google Scholar). Although the c-ret gene has been known for more than a decade, most of our knowledge about its signal transduction capabilities derives from studies of its oncogenic forms, several of which appear to activate unique signaling pathways. Activation of c-Ret initiates many of the same signal transduction pathways activated by other receptor tyrosine kinases including the Ras/Raf pathway, which leads to activation of the mitogen-activated protein kinases Erk1 and Erk2, and the PI3K pathway, which leads to activation of the serine-threonine kinase Akt and cell survival (Ref. 8Trupp M. Scott R. Whittemore S.R. Ibáñez C.F. J. Biol. Chem. 1999; 274: 20885-20894Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholarand references therein). However, it is still unclear how these two pathways are initiated by the c-Ret receptor. A number of adaptor proteins have been implicated in signaling by various oncogenic and ligand-activated forms of c-Ret, including Shc, Grb2, SNT/FRS2, Gab1, Nck, Crk, and p62Dok (8Trupp M. Scott R. Whittemore S.R. Ibáñez C.F. J. Biol. Chem. 1999; 274: 20885-20894Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar, 14Alberti L. Borrello M.G. Ghizzoni S. Torriti F. 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In the case of NGF, both Ras-dependent and Ras-independent mechanisms of PI3K activation are at work, each accounting for roughly 50% of the survival responses of SCG neurons to NGF (32Nobes C.D. Tolkovsky A.M. Eur. J. Neurosci. 1995; 7: 344-350Crossref PubMed Scopus (71) Google Scholar, 33Mazzoni I.E. Said F.A. Aloyz R. Miller F.D. Kaplan D. J. Neurosci. 1999; 19: 9716-9727Crossref PubMed Google Scholar). The role of Ras in the survival responses elicited by GDNF has not been addressed. In the work presented here, we have investigated how the Ras and PI3K pathways couple to the c-Ret receptor when activated by GDNF in cells co-expressing the GFRα1 ligand binding subunit. These studies led us to the characterization of distinct macromolecular complexes that assemble in a ligand-dependent manner onto the activated c-Ret receptor, leading to activation of the Ras/Erk and PI3K/Akt pathways. Fibroblast cell lines were derived from mouse MG87 cells, an NIH3T3 subclone. The M23 cell line was derived by stable transfection of full-length rat GFRα1 into MG87 cells. c-Ret-expressing cell lines were derived by stable transfection of wild type and mutated forms of full-length human c-Ret (long isoform) into M23 cells. MN1 is an immortalized motorneuron cell line generated by cell fusion of mouse embryonic motor neurons with a mouse neuroblastoma (34Salazar-Grueso E. Kim S. Kim H. Neuroreport. 1991; 2: 505-508Crossref PubMed Scopus (82) Google Scholar) and expresses endogenous c-Ret and GFRα1. Recombinant rat GDNF was produced in SF21 insect cells and purified as described previously (35Trupp M. Rydén M. Jörnvall H. Timmusk T. Funakoshi H. Arenas E. Ibáñez C.F. J. Cell Biol. 1995; 130: 137-148Crossref PubMed Scopus (521) Google Scholar). NGF was purchased from Promega. Point mutations in human c-Ret (subcloned in pCDNA3, Invitrogen) were introduced by oligonucleotide-mediated site-directed mutagenesis by the Kunkel method (36Kunkel T. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 488-492Crossref PubMed Scopus (4903) Google Scholar). Cell monolayers in 10-cm plates were changed to serum-free media 16 h before incubation at 37 °C with 50 ng/ml GDNF for the indicated time periods and immediately lysed with 1 ml of ice-cold Nonidet P-40 lysis buffer (10 mm Tris/HCl, pH 7.5, 137 mm NaCl, 2 mm EDTA, 10% glycerol, 1% Nonidet P-40) supplemented with a mixture of protease inhibitors (Roche Molecular Biochemicals) and a mixture of phosphatase inhibitors (1 mm sodium orthovanadate, 20 mm NaF, 10 mmβ-glycerolphosphate). After a 15-min lysis on ice, cell lysates were cleared by centrifugation. Immunoprecipitations were done by 4 °C overnight incubation of cell lysates with antibodies plus 100 μl of protein G-Sepharose bead slurry (Pharmacia Biotech, Uppsala, Sweden). Beads were washed five times with lysis buffer and boiled in SDS/β-mercaptoethanol buffer. Immunoprecipitates were fractionated by SDS-polyacrylamide gel electrophoresis and blotted to polyvinylidene difluoride membranes. Blots were probed with the indicated antibodies followed by alkaline phosphatase-conjugated anti-IgG and developed with the ECF Western detection system (Amersham Pharmacia Biotech). All blots were scanned in a Storm 840 fluorimager (Molecular Dynamics). For reprobing, blots were stripped for 60 min at room temperature in 0.1 m acetic acid, 0.15 m NaCl. Antibodies were obtained from various sources as follows: anti-Shc, anti-Gab1, anti-Gab2, and anti-p85PI3K were from Upstate Biotechnology Inc. (Lake Placid, NY); anti-Grb2, anti-SHP-2, anti-phosphotyrosine, and anti-Ret (long isoform) were from Santa Cruz Biotechnology Inc. (Santa Cruz, CA); anti-Ras was from Transduction Laboratories (Lexington, KY); anti-Erk, anti-P-Erk, anti-Akt, and anti-P-Akt were from New England Biolabs (Hutchin, UK); anti-glutathioneS-transferase (GST) was from Amersham Pharmacia Biotech). The protein kinase C inhibitor Gö6983 (Calbiochem) was used at 120 nm. For PI3K assays, total lysates, prepared from cells treated or not with GDNF (50 ng/ml) for 45 min, were immunoprecipitated with anti-p85PI3K antibodies. After immunoprecipitation and washing, beads were further washed twice in TNE (10 mm Tris HCl, 150 mm NaCl, 5 mmEDTA) containing 0.1 mm sodium orthovanadate and protease inhibitors. Pellets were then incubated for 10 min at 37 °C in 50 μl of TNE supplemented with 20 μg of phosphatidylinositol, 20 mm MgCl2, 0.88 mm ATP, and 30 μCi of [γ-32P]ATP. The reaction was stopped by addition of HCl 6 n, and the radiolabeled lipids were extracted with CHCl3:CH3OH (1:1). Fifty μl of the organic phase were spotted on silicon TLC plates, and lipids were then separated by thin layer chromatography and visualized by autoradiography on phosphorscreens. For Akt kinase assays, after immunoprecipitation and washing, beads were further washed twice in kinase buffer (20 mm HEPES, pH 7.0, 25 mm MgCl2, 1 mmdithiothreitol, 1 mm sodium orthovanadate, 25 mm β-glycerolphosphate) supplemented with protease inhibitors and incubated at room temperature for 20 min in 50 μl of kinase buffer containing 50 μCi of [γ-32P]ATP, 1 mm ATP, and histone B2 (HB2) as substrate. After a 20-min incubation at 30 °C, the reaction was stopped by the addition of an equal volume of SDS/β-mercaptoethanol sample buffer, boiled, fractionated by SDS/polyacrylamide gel electrophoresis and blotted onto polyvinylidene difluoride membranes. The membranes were exposed to phosphorscreens, which were subsequently scanned in a Storm 840 PhosphorImager (Molecular Dynamics) and quantified using ImageQuant software. After exposure, the membranes were probed with different antibodies as above. Plasmids for bacterial expression of GST fusions with amino acids 1 to 149 from the Ras binding domain (RBD) of Raf1 (GST-RBD), full-length p85PI3K (GST-p85PI3KFL), and full-length Grb2 (GST-Grb2FL) were obtained from Stephen Taylor (Cornell University, Ithaca, NY), Jonathan Backer (Albert Einstein College of Medicine, Bronx, NY), and James Bliska (SUNY, Stony Brook, NY), respectively. GST fusions were produced in Escherichia coli and purified by chromatography on glutathione-conjugated-agarose beads (Amersham Pharmacia Biotech). For pull-down assays, the indicated purified GST fusion proteins were incubated with cell lysates overnight at 4 °C together with glutathione-Sepharose beads, followed by washing, SDS/polyacrylamide gel electrophoresis, and Western blotting as above. The activated Ras interaction assay was performed as described previously (37Taylor S.J. Shalloway D. Curr. Biol. 1996; 6: 1621-1627Abstract Full Text Full Text PDF PubMed Scopus (352) Google Scholar) using the pGEX-RBD plasmid encoding a GST-RBD fusion protein. For Far Western blotting, polyvinylidene difluoride blots were blocked overnight at 4 °C, followed by an incubation of 2 h at room temperature with purified GST fusion proteins (1 μg/ml), washed, and then developed with anti-GST antibodies as above. Neuronal survival assays were performed using dissociated cultures of post-natal day 1 (P1) rat SCG. Ganglia were dissociated by 2 incubations of 30 min at 37 °C in phosphate-buffered saline, the first one containing 0.025% trypsin, and the second containing 5 mg/ml collagenase (Sigma) followed by mechanical trituration in neuron basal medium. Neuron basal medium consist of 50% Dulbecco's modified Eagle's medium, 50% Ham's F-12 (Life Technologies, Inc.), and 1 mg/ml bovine serum albumin. Cells were incubated overnight in the presence of 5 ng/ml NGF. This medium was removed on the next day and replaced by fresh medium containing anti-NGF antibodies (Roche Molecular Biochemicals) and GDNF at 100 ng/ml. The PI3K inhibitor LY294002 (Calbiochem) was added together with GDNF (or with NGF at 10 ng/ml when indicated) at 10, 50, or 100 μm in neuron basal medium containing 0.6% methylcellulose to prevent cells from mechanically detaching. Survival was assessed 24 h later by counting phase-bright, neurite-bearing neurons in random fields. Microinjection was carried out using an inverted fluorescence microscope (Axiovert 100, Zeiss) with an Eppendorf transjector and micromanipulator. Cells were injected into the cytoplasm with 0.5 mg/ml anti-Ras-blocking antibodies (Oncogene Research) or 0.5 mg/ml purified guinea pig IgG (Sigma) as a control and with 5 μg/ml neutral red 70-kDa Texas Red Dextran (Molecular Probes) in 0.5× phosphate-buffered saline. Texas Red Dextran-positive cells were counted 24 h later. We generated fibroblast cell lines stably expressing the GFRα1 receptor together with either wild type or mutant versions of the long isoform of human c-Ret. Tyr-1062 and Tyr-1096 have previously been shown to be phosphorylated in oncogenic forms of c-Ret, where they serve as docking sites for the Shc and Grb2 adaptor proteins, respectively. Tyr-981 lies within a consensus binding site for p85PI3K and could therefore be involved in PI3K activation by ligand-activated c-Ret. Fibroblast lines expressing the Y981F, Y1062F, Y1096F, or the double Y1062F/Y1096F c-Ret mutants were generated and tested for ligand-dependent tyrosine phosphorylation. The four c-Ret mutants could be activated by stimulation with GDNF and showed comparable levels of total tyrosine phosphorylation (Fig. 1 A). Mutation of Tyr-1062 abolished binding of Shc to the activated receptor, Shc tyrosine phosphorylation, and Shc binding to Grb2 after GDNF treatment (Fig. 1 B). Grb2, on the other hand, was still able to interact with the Y1062F mutant in a ligand-dependent manner (Fig. 1 C). This interaction was eliminated in the double mutant Y1062F/Y1096F (Fig. 1 C), confirming the capacity of Grb2 to associate with the activated receptor via phosphorylated Tyr-1096 independently of Shc. We then investigated the ability of the c-Ret mutants to activate Ras after GDNF stimulation using a pull-down assay with a fusion construct between GST and the RBD of Raf1 (GST-RBD), which interacts specifically with the GTP-loaded activated form of Ras. In cells expressing the wild type receptor, Ras was activated within 5 min after ligand stimulation, declining back to basal levels by 60 min (Fig. 2 A). No stimulation of Ras activity could be observed in the Y1062F mutant after ligand treatment (Fig. 2 A), indicating that activation of Ras by GDNF depends upon phosphorylation of this residue in c-Ret. The activation of Erk kinases, one of the downstream targets of Ras, was investigated using anti-phospho-Erk antibodies. Rapid Erk phosphorylation was induced by GDNF in cells carrying wild type c-Ret, which lasted up to 45 min after stimulation (Fig. 2 B). In the Y1062F mutant, Erk phosphorylation was greatly attenuated, although a residual, delayed, and transient activation remained 15 min after ligand stimulation (Fig. 2 B). In contrast, neither the time course or intensity of Erk phosphorylation were affected after mutation of Tyr-1096 (Fig. 2 B), confirming that this residue does not participate in activation of the Ras/Erk pathway by c-Ret. Moreover, the residual Erk phosphorylation observed in the Y1062F mutant was not eliminated in the Y1062F/Y1096F double mutant, indicating that it was not mediated via Tyr-1096. Inhibition of protein kinase C could neither block the residual Erk activation in the Y1062F mutant (Fig. 2 C), ruling out the participation of the phospholipase Cγ pathway in this response. A similar transient peak in Erk phosphorylation was also observed in a fibroblast line carrying the kinase-inactive c-Ret mutant Y905F (Fig. 2 C), which replaces a crucial tyrosine in the activation loop of the enzyme. This result suggested that the residual Erk activation remaining in the Y1062F mutant was a c-Ret-independent response. In fact, GDNF-dependent phosphorylation of Erk has been detected in cells expressing GFRα1 receptors in the absence of c-Ret (9Poteryaev D. Titievsky A. Sun Y.F. Thomas-Crusells J. Lindahl M. Billaud M. Arumäe U. Saarma M. FEBS Lett. 1999; 463: 63-66Crossref PubMed Scopus (143) Google Scholar). 2V. Besset, R. P. Scott, and C. F. Ibáñez, unpublished observations.. This novel pathway is mediated by members of the Src kinase family and appears not to involve Ras activation (8Trupp M. Scott R. Whittemore S.R. Ibáñez C.F. J. Biol. Chem. 1999; 274: 20885-20894Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar), in agreement with the present findings. Thus, activation of the Ras/Erk pathway by ligand stimulation of c-Ret is mediated by phosphorylation of Tyr-1062 followed by docking of Shc and Grb2, with little or no participation of the Grb2 binding site at Tyr-1096. These results also indicated that binding of Grb2 to phosphorylated Tyr-1096 may lead to the recruitment of other c-Ret targets distinct from the Ras activator Sos. We then examined the activity of PI3K in wild type and Y1062F mutant c-Ret after GDNF treatment. Ligand stimulation caused a significant increase in PI3K activity in cells carrying wild type c-Ret (Fig. 3 A). Mutation of Tyr-1062 greatly diminished this response but did not abolished it completely; a residual 50% increase over base line could be reproducibly detected after GDNF treatment (Fig. 3 A). Stimulation of the kinase activity of Akt, a target of the PI3K pathway, in response to GDNF was also attenuated, but not totally eliminated, in cells carrying the Y1062F c-Ret mutant, which showed a residual 50% stimulation over base line (Fig. 3 B). In agreement with this, a reduced and delayed stimulation of Akt phosphorylation by GDNF could still be detected in these cells (Fig. 3 C). Together, these data indicated the existence of alternative pathways for PI3K/Akt activation independently of phosphorylation of Tyr-1062 and suggested that Tyr-1096, which bound Grb2 but did not contribute to the Ras pathway, could be involved. Indeed, ligand-dependent phosphorylation of Akt was completely eliminated in the Y1062F/Y1096F double mutant (Fig. 3 C), demonstrating the participation of Tyr-1096 in the activation of the PI3K/Akt pathway by c-Ret. PI3K may be activated through the recruitment of the p85PI3K regulatory subunit to activated receptors. We examined the role of p85PI3K in the activation of PI3K by c-Ret using pull-down assays with a recombinant GST-p85PI3Kfusion protein followed by c-Ret immunoblotting. GST-p85PI3K brought down wild type c-Ret in a ligand-dependent manner (Fig. 4 A), suggesting a capacity of the p85PI3K subunit for interacting with the activated receptor. In agreement with our previous results, we observed a diminished but significant interaction of GST-p85PI3K with Y1062F c-Ret but not with the Y1062F/Y1096F double mutant (Fig. 4 A), underscoring the role of both these tyrosine residues in the recruitment of p85PI3K to the activated receptor. GST-p85PI3K was also able to pull-down the Y981F mutant in a ligand-dependent manner (Fig. 4 A), indicating that this residue does not play a significant role in the activation of PI3K by c-Ret. This was also confirmed by the ability of this mutant to induce normal phosphorylation of Akt upon ligand stimulation (data not shown). In addition to these fibroblast cell lines, GST-p85PI3K could also precipitate c-Ret in a ligand-dependent manner from cells of neuronal origin expressing endogenous c-Ret receptors, including the motorneuron cell line MN1 and the neuroblastoma Neuro2A-α1 (Fig. 4 B). Despite its ability to precipitate c-Ret from lysates of cells stimulated with GDNF, GST-p85PI3K failed to recognize the activated receptor in Far Western overlay assays of c-Ret immunoprecipitates (data not shown), although several other interactors cou
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