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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*

  • Valerie Besset
    Footnotes
    Affiliations
    Division of Molecular Neurobiology, Department of Neuroscience, Karolinska Institute, 17177 Stockholm, Sweden
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  • Rizaldy P. Scott
    Affiliations
    Division of Molecular Neurobiology, Department of Neuroscience, Karolinska Institute, 17177 Stockholm, Sweden
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  • Carlos F. Ibáñez
    Correspondence
    To whom correspondence should be addressed: Div. of Molecular Neurobiology, Dept. of Neuroscience, Karolinska Institute, Berzelius väg 1, 17177 Stockholm, Sweden. Tel.: 46-8-728 7660; Fax: 46-8-33 9548.
    Affiliations
    Division of Molecular Neurobiology, Department of Neuroscience, Karolinska Institute, 17177 Stockholm, Sweden
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  • Author Footnotes
    * This work was supported by Swedish Cancer Society Grant 3474-B97-05XBC, Göran Gustafssons Stiftelsen, European Commission Grant BMH4-97-2157), and the Karolinska Institute.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
    ‡ Supported by a grant from the Wenner Gren Foundation.
Open AccessPublished:December 15, 2000DOI:https://doi.org/10.1074/jbc.M006908200
      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.
      GDNF
      glial cell line-derived neurotrophic factor
      MEN
      multiple endocrine neoplasia
      Erk
      extracellular signal-regulated kinase
      PI3K
      phosphatidylinositol-3 kinase
      SCG
      superior cervical ganglion
      NGF
      nerve growth factor
      GST
      glutathione S-transferase
      RBD
      Ras binding domain
      GFR
      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 (
      • Meng 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.
      ,
      • Trupp M.
      • Arenas E.
      • Fainzilber M.
      • Nilsson A.-S.
      • Sieber B.A.
      • Grigoriou M.
      • Kilkenny C.
      • Salazar-Grueso E.
      • Pachnis V.
      • Arumäe U.
      • Sariola H.
      • Saarma M.
      • Ibáñez C.F.
      ,
      • Durbec P.
      • Marcos-Gutierrez C.V.
      • Kilkenny C.
      • Grigoriou M.
      • Suvanto P.
      • Wartiovaara K.
      • Smith D.
      • Ponder B.
      • Costantini F.
      • Saarma M.
      • Sariola H.
      • Pachnis V.
      ). Binding of GDNF to c-Ret is mediated by a glycosyl phosphatidylinositol-anchored co-receptor subunit termed GFRα1 (
      • Jing S.Q.
      • Wen D.Z., Yu, Y.B.
      • Holst P.L.
      • Luo Y.
      • Fang M.
      • Tamir R.
      • Antonio L.
      • Hu Z.
      • Cupples R.
      • Louis J.C.
      • Hu S.
      • Altrock B.W.
      • Fox G.M.
      ,
      • Treanor J.
      • Goodman L.
      • Desauvage F.
      • Stone D.M.
      • Poulsen K.T.
      • Beck C.D.
      • Gray C.
      • Armanini M.P.
      • Pollock R.A.
      • Hefti F.
      • Phillips H.S.
      • Goddard A.
      • Moore M.W.
      • Buj-Bello A.
      • Davies A.M.
      • Asai N.
      • Takahashi M.
      • Vandlen R.
      • Henderson C.E.
      • Rosenthal A.
      ). Three close mammalian homologues of GDNF have been identified, all of which utilize c-Ret as signaling receptor with the aid of different members (GFRα1 to -4) of the GFRα family of glycosyl phosphatidylinositol-linked co-receptors (reviewed in Refs.
      • Airaksinen M.S.
      • Titievsky A.
      • Saarma M.
      and
      • Baloh R.H.
      • Enomoto H.
      • Johnson Jr., E.M.
      • Milbrandt J.
      ). In the absence of c-Ret, GDNF family ligands may still signal in some cell types expressing GFRα receptors via activation of members of the Src family of cytoplasmic tyrosine kinases in lipid raft microdomains, presumably in collaboration with yet unknown transmembrane proteins (
      • Trupp M.
      • Scott R.
      • Whittemore S.R.
      • Ibáñez C.F.
      ,
      • Poteryaev D.
      • Titievsky A.
      • Sun Y.F.
      • Thomas-Crusells J.
      • Lindahl M.
      • Billaud M.
      • Arumäe U.
      • Saarma M.
      ). GFRα receptors can mediate activation of c-Ret by GDNF when expressed on the surface of the same cell (cis signaling) or when presented in soluble form or immobilized onto agarose beads (transsignaling) (
      • Treanor J.
      • Goodman L.
      • Desauvage F.
      • Stone D.M.
      • Poulsen K.T.
      • Beck C.D.
      • Gray C.
      • Armanini M.P.
      • Pollock R.A.
      • Hefti F.
      • Phillips H.S.
      • Goddard A.
      • Moore M.W.
      • Buj-Bello A.
      • Davies A.M.
      • Asai N.
      • Takahashi M.
      • Vandlen R.
      • Henderson C.E.
      • Rosenthal A.
      ,
      • Yu T.
      • Scully S., Yu, Y.B.
      • Fox G.M.
      • Jing S.Q.
      • Zhou R.P.
      ,

      Paratcha, G., Ledda, F., Baars, L., Coulpier, M., Besset, V., Anders, J., Scott, R., and Ibanez, C. F. Neuron, in press.

      ). 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.
      • Edery P.
      • Eng C.
      • Munnich A.
      • Lyonnet S.
      and
      • Santoro W.
      • Carlomagno F.
      • Melillo R.M.
      • Billaud W.
      • Vecchio G.
      • Fusco A.
      ). 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.
      • Trupp M.
      • Scott R.
      • Whittemore S.R.
      • Ibáñez C.F.
      and 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 (
      • Trupp M.
      • Scott R.
      • Whittemore S.R.
      • Ibáñez C.F.
      ,
      • Alberti L.
      • Borrello M.G.
      • Ghizzoni S.
      • Torriti F.
      • Rizzetti M.G.
      • Pierotti M.
      ,
      • Borrello M.G.
      • Pelicci G.
      • Arighi E.
      • De F.L.
      • Greco A.
      • Bongarzone I.
      • Rizzetti M.
      • Pelicci P.G.
      • Pierotti M.A.
      ,
      • van Weering D.
      • Medema J.P.
      • van Puijenbroek A.
      • Burgering B.M.
      • Baas P.D.
      • Bos J.L.
      ,
      • Rizzo C.
      • Califano D.
      • Colucci-Damato G.L.
      • Devita G.
      • Dalessio A.
      • Dathan N.A.
      • Fusco A.
      • Monaco C.
      • Santelli G.
      • Vecchio G.
      • Santoro M.
      • De Franciscis V.
      ,
      • Arighi E.
      • Alberti L.
      • Torriti F.
      • Ghizzoni S.
      • Rizzetti M.G.
      • Pelicci G.
      • Pasini B.
      • Bongarzone I.
      • Piutti C.
      • Pierotti M.A.
      • Borrello M.G.
      ,
      • Lorenzo M.J.
      • Gish G.D.
      • Houghton C.
      • Stonehouse T.J.
      • Pawson T.
      • Ponder B.
      • Smith D.P.
      ,
      • Ohiwa M.
      • Murakami H.
      • Iwashita T.
      • Asai N.
      • Iwata Y.
      • Imai T.
      • Funahashi H.
      • Takagi H.
      • Takahashi M.
      ,
      • Durick K.
      • Gill G.N.
      • Taylor S.S.
      ,
      • Bocciardi R.
      • Mograbi B.
      • Pasini B.
      • Borrello M.G.
      • Pierotti M.A.
      • Bourget I.
      • Fischer S.
      • Romeo G.
      • Rossi B.
      ,
      • Murakami H.
      • Iwashita T.
      • Asai N.
      • Shimono Y.
      • Iwata Y.
      • Kawai K.
      • Takahashi M.
      ). The Shc adaptor protein has been shown to interact with phosphorylated Tyr-1062 in several oncogenic forms of c-Ret (
      • Arighi E.
      • Alberti L.
      • Torriti F.
      • Ghizzoni S.
      • Rizzetti M.G.
      • Pelicci G.
      • Pasini B.
      • Bongarzone I.
      • Piutti C.
      • Pierotti M.A.
      • Borrello M.G.
      ,
      • Ishiguro Y.
      • Iwashita T.
      • Murakami H.
      • Asai N.
      • Iida K.I.
      • Goto H.
      • Kayakawa T.
      • Takahashi M.
      ). The adaptor Grb2 links Shc to the Ras pathway, and other work has shown that it can also be recruited directly to c-Ret by binding to phosphorylated Tyr-1096 in the tail of the long isoform of this receptor (
      • Alberti L.
      • Borrello M.G.
      • Ghizzoni S.
      • Torriti F.
      • Rizzetti M.G.
      • Pierotti M.
      ). Intriguingly, the short c-Ret isoform, which differs from the long in the 50 C-terminal residues and lacks Tyr-1096, appears to also be able to recruit Grb2 independently of Shc, although the precise mechanism in this case is unknown (
      • Alberti L.
      • Borrello M.G.
      • Ghizzoni S.
      • Torriti F.
      • Rizzetti M.G.
      • Pierotti M.
      ). Whether activation of Ras is coupled to Tyr-1062, Tyr-1096, or to other phosphorylated residues in c-Ret is still unclear. Activation of PI3K, on the other hand, has recently been shown to depend exclusively on phosphorylation of Tyr-1062 in the short isoform of the MEN2A Ret oncoprotein (
      • Segouffin-Cariou C.
      • Billaud M.
      ). How PI3K activation is triggered by ligand-activated c-Ret and whether this is dependent upon Ras activity is unknown.
      The PI3K/Akt pathway is an important regulator of neuronal survival, both in central and peripheral subpopulations (
      • Datta S.R.
      • Dudek H.
      • Tao X.
      • Masters S.
      • Fu H.A.
      • Gotoh Y.
      • Greenberg M.E.
      ,
      • Delpeso L.
      • Gonzalezgarcia M.
      • Page C.
      • Herrera R.
      • Nuñez G.
      ,
      • Kennedy S.G.
      • Wagner A.J.
      • Conzen S.D.
      • Jordan J.
      • Bellacosa A.
      • Tsichlis P.N.
      • Hay N.
      ). Survival of sympathetic neurons of the superior cervical ganglion (SCG) induced by nerve growth factor (NGF) is critically dependent upon an intact PI3K pathway (
      • Crowder R.J.
      • Freeman R.S.
      ,
      • Philpott K.L.
      • Mccarthy M.J.
      • Klippel A.
      • Rubin L.L.
      ). GDNF is also able to activate PI3K and to promote survival of SCG neurons (
      • Creedon D.J.
      • Tansey M.G.
      • Baloh R.H.
      • Osborne P.A.
      • Lampe P.A.
      • Fahrner T.J.
      • Heuckeroth R.O.
      • Milbrandt J.
      • Johnson E.M.
      ), although a causal link between these two events has not been established. 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 (
      • Nobes C.D.
      • Tolkovsky A.M.
      ,
      • Mazzoni I.E.
      • Said F.A.
      • Aloyz R.
      • Miller F.D.
      • Kaplan D.
      ). 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.

      DISCUSSION

      In this study, we have investigated proximal signaling events following activation of the GDNF receptor tyrosine kinase c-Ret. We have focused in elucidating the protein complexes that interact with the receptor, leading to activation of the Ras/Erk and PI3K/Akt pathways. Our results confirm the importance of phosphorylated Tyr-1062 in the cytoplasmic tail of c-Ret for the activation of these two pathways by GDNF, as suggested by previous studies using oncogenic forms of this receptor (
      • Arighi E.
      • Alberti L.
      • Torriti F.
      • Ghizzoni S.
      • Rizzetti M.G.
      • Pelicci G.
      • Pasini B.
      • Bongarzone I.
      • Piutti C.
      • Pierotti M.A.
      • Borrello M.G.
      ,
      • Ohiwa M.
      • Murakami H.
      • Iwashita T.
      • Asai N.
      • Iwata Y.
      • Imai T.
      • Funahashi H.
      • Takagi H.
      • Takahashi M.
      ,
      • Segouffin-Cariou C.
      • Billaud M.
      ).
      In contrast to Tyr-1062, phosphorylation of Tyr-1096 appears to contribute only to activation of the PI3K pathway, with little or no role in activation of Ras and Erk. This was surprising, as Grb2, which binds to phosphorylated Tyr-1096, is a well known link between receptor tyrosine kinases and the Ras pathway. This adaptor can be present in a pre-associated complex with different downstream components, including the Ras activator protein Sos and the Gab1/2 adaptors. The Grb2/Sos and Grb2/Gab complexes are stabilized via interaction between the SH3 domains in Grb2 and proline-rich regions in Sos and Gab. It is possible that the Grb2/Sos complex, although capable of interacting with phosphorylated Shc, may be sterically hindered to bind to phosphorylated Tyr-1096. In agreement with this hypothesis, this residue does not appear to be crucial for the Ras-dependent-transforming activity of either MEN2A or MEN2B Ret oncogenic forms (
      • Asai N.
      • Murakami H.
      • Iwashita T.
      • Takahashi M.
      ). On the other hand, our results indicate that the Grb2·Gab complex binds readily to phosphorylated Tyr-1096 and Shc, linking both Tyr-1062 and -1096 of c-Ret to activation of PI3K through the recruitment of p85PI3K. The lack of residual PI3K activation in the Y1062F mutant of the short isoform of the MEN2A Ret oncoprotein (
      • Segouffin-Cariou C.
      • Billaud M.
      ) could have been due to the absence of Tyr-1096 in this receptor form and suggests differences in the mechanisms of PI3K activation by the two c-Ret isoforms. Alternatively, and because Grb2 appears to also interact directly with the short c-Ret isoform (
      • Alberti L.
      • Borrello M.G.
      • Ghizzoni S.
      • Torriti F.
      • Rizzetti M.G.
      • Pierotti M.
      ), the inability of Y1062F MEN2A to stimulate PI3K activity could indicate differences in the signaling mechanisms of oncogenic versusligand-activated Ret receptors.
      The PI3K/Akt pathway can be activated by at least two distinct mechanisms: (i) binding of the regulatory subunit p85PI3Kto tyrosine phosphorylated residues on the receptor and on docking proteins and ii) binding of the catalytic subunit p110PI3Kto Ras (reviewed in Ref.
      • Wymann M.P.
      • Pirola L.
      ). We have shown that a recombinant GST-p85PI3K associates with c-Ret and that this association as well as Akt phosphorylation is lost in the Y1062F/Y1096F double mutant, linking both Tyr-1062 and -1096 of c-Ret to activation of PI3K through the recruitment of p85PI3K. Although Tyr-981 of c-Ret is present in a consensus sequence (i.e.YXXM), preferentially recognized by the SH2 domains of p85PI3K, we and others (
      • Segouffin-Cariou C.
      • Billaud M.
      ) did not find any evidence of the participation of this residue in the activation of the PI3K/Akt pathway by c-Ret.
      By pull-down and co-immunoprecipitations experiments, we have shown that PI3K is linked to c-Ret through a complex containing Gab1/2, Grb2, Shp-2, and Shc. These results are summarized in the scheme shown in Fig. 8. As evidenced by Far Western blot experiments, Gab interacts directly with p85PI3K and Grb2. Although the interaction of p85PI3K with Gab was found to be stimulated by GDNF-induced phosphorylation, Grb2 binding to Gab was constitutive. Gab proteins possess several proline-rich regions (i.e. PXXP) susceptible to bind the SH3 domain of Grb2. Although such motifs are also present in p85PI3K, we found no evidence for direct binding of Grb2 to p85PI3K by Far Western blot analysis (data not shown). Gab proteins possess several consensus sites for the SH2 domains of SHP-2 (
      • Gu H.
      • Pratt J.C.
      • Burakoff S.J.
      • Neel B.G.
      ). Although p85PI3K could also in principle interact directly with SHP-2 through the YXXM motifs present on the phosphatase, the results of our Far Western experiments indicate that, after stimulation with GDNF, the majority of SHP-2 in the p85PI3Kcomplex is bound to Gab2.
      Figure thumbnail gr8
      Figure 8Macromolecular complexes leading to PI3K activation by c-Ret. The scheme shows protein interactions involved in the complex formed between PI3K and c-Ret upon GDNF stimulation of MN1 cells. For SH2-mediated associations, the consensus binding sites, as reported in the literature, are indicated. Proline-rich motifs are indicated by hatched boxes.
      Our observations implicate for the first time the adaptor protein Gab2 in receptor tyrosine kinase signaling in neuronal cells. This protein had originally been isolated as a main signaling component downstream of cytokine and antigen receptors in hematopoietic cells (
      • Gu H.
      • Pratt J.C.
      • Burakoff S.J.
      • Neel B.G.
      ,
      • Nishida K.
      • Yoshida Y.
      • Itoh M.
      • Fukada T.
      • Ohtani T.
      • Shirogane T.
      • Atsumi T.
      • Takahashi-Tezuka M.
      • Ishihara K.
      • Hibi M.
      • Hirano T.
      ,
      • Zhao C., Yu, D.H.
      • Shen R.
      • Feng G.S.
      ). Similar to its better-characterized homologue Gab1, Gab2 has been reported to bind Grb2 and p85PI3K as well as SHP-2 and to potentiate activation of Erk (
      • Gu H.
      • Pratt J.C.
      • Burakoff S.J.
      • Neel B.G.
      ,
      • Nishida K.
      • Yoshida Y.
      • Itoh M.
      • Fukada T.
      • Ohtani T.
      • Shirogane T.
      • Atsumi T.
      • Takahashi-Tezuka M.
      • Ishihara K.
      • Hibi M.
      • Hirano T.
      ,
      • Zhao C., Yu, D.H.
      • Shen R.
      • Feng G.S.
      ). Unlike Gab1, however, Gab2 is unable to interact directly with the c-Met receptor tyrosine kinase (
      • Schaeper U.
      • Gehring N.H.
      • Fuchs K.P.
      • Sachs M.
      • Kempkes B.
      • Birchmeier W.
      ), one of the main upstream activators of Gab1, suggesting distinct functions for the two proteins. Gab2 is more abundantly expressed in the nervous system than Gab1 (
      • Zhao C., Yu, D.H.
      • Shen R.
      • Feng G.S.
      ), indicating functions in neuronal cells. Gab1 had been found to mediate NGF-induced survival, neurite outgrowth, and DNA synthesis in PC12 cells, where its overexpression induced activation of Akt and Erk kinases (
      • Holgado-Madruga M.
      • Moscatello D.K.
      • Emlet D.R.
      • Dieterich R.
      • Wong A.J.
      ,
      • Korhonen J.M.
      • Said F.A.
      • Wong A.J.
      • Kaplan D.R.
      ). Although we found that Gab1 plays a similar role to that of Gab2 in GDNF signaling in fibroblast cells,
      V. Besset, unpublished observations.
      we were unable to detect this adaptor protein in MN1 cells.
      GDNF promotes survival of newborn rat SCG neurons in vitro(
      • Creedon D.J.
      • Tansey M.G.
      • Baloh R.H.
      • Osborne P.A.
      • Lampe P.A.
      • Fahrner T.J.
      • Heuckeroth R.O.
      • Milbrandt J.
      • Johnson E.M.
      ,
      • Trupp M.
      • Rydén M.
      • Jörnvall H.
      • Timmusk T.
      • Funakoshi H.
      • Arenas E.
      • Ibáñez C.F.
      ,
      • Kotzbauer P.T.
      • Lampe P.A.
      • Heuckeroth R.O.
      • Golden J.P.
      • Creedon D.J.
      • Johnson E.M.
      • Milbrandt J.
      ) and is required for their survival in vivo(
      • Sánchez M.P.
      • Silossantiago I.
      • Frisen J.
      • He B.
      • Lira S.A.
      • Barbacid M.
      ,
      • Moore M.W.
      • Klein R.D.
      • Farinas I.
      • Sauer H.
      • Armanini M.
      • Phillips H.
      • Reichardt L.F.
      • Ryan A.M.
      • Carvermoore K.
      • Rosenthal A.
      ,
      • Pichel J.G.
      • Shen L.Y.
      • Sheng H.Z.
      • Granholm A.C.
      • Drago J.
      • Grinberg A.
      • Lee E.J.
      • Huang S.P.
      • Saarma M.
      • Hoffer B.J.
      • Sariola H.
      • Westphal H.
      ). GDNF induces PI3K activity in SCG neurons (
      • Creedon D.J.
      • Tansey M.G.
      • Baloh R.H.
      • Osborne P.A.
      • Lampe P.A.
      • Fahrner T.J.
      • Heuckeroth R.O.
      • Milbrandt J.
      • Johnson E.M.
      ), and we have shown that neuron survival in response to GDNF is dependent upon activation of this enzyme. Our results indicate that activation of PI3K by GDNF can be mediated independently of Ras by recruitment of p85PI3K to the activated c-Ret receptor. In agreement with this, we show that survival of sympathetic neurons in response to GDNF is dependent upon PI3K activation but is not affected by inhibition of Ras. This is in contrast to NGF, where Ras activity contributes to both PI3K activation and neuron survival in sympathetic as well as sensory neurons (this study and Refs.
      • Mazzoni I.E.
      • Said F.A.
      • Aloyz R.
      • Miller F.D.
      • Kaplan D.
      and
      • Klesse L.J.
      • Parada L.F.
      ). Interestingly, survival of serum-deprived PC12 cells in the presence of NGF does not appear to require Ras activity (
      • Klesse L.J.
      • Meyers K.A.
      • Marshall C.J.
      • Parada L.F.
      ), indicating that the same receptor system may also work differently in different cell types. An important difference between NGF and GDNF signaling is that, whereas in the NGF receptor TrkA both Ras and PI3K pathways depend upon the binding of Shc to a single phosphotyrosine residue (i.e. Tyr-490) (
      • Obermeier A.
      • Bradshaw R.A.
      • Seedorf K.
      • Choidas A.
      • Schlessinger J.
      • Ullrich A.
      ,
      • Stephens R.M.
      • Loeb D.M.
      • Copeland T.D.
      • Pawson T.
      • Greene L.A.
      • Kaplan D.R.
      ), at least two different residues in c-Ret (i.e. Tyr-1062 and -1096) can mediate PI3K activation (Fig. 8). Thus, it may be that Ras plays only a minor contribution in PI3K activation by GDNF or, alternatively, that PI3K activity in GDNF-treated cells is not limiting, so that elimination of the Ras component does not reduce it below the threshold level required to maintain maximal neuronal survival. In agreement with the former possibility, inhibition of Ras activity in the neuroectoderm-derived cell line SKF5 had no significant effect on Akt activation and lamellipodium formation induced by an EGFR/c-Ret chimeric receptor (
      • van Weering D.
      • Derooij J.
      • Marte B.
      • Downward J.
      • Bos J.L.
      • Burgering B.
      ).
      In conclusion, our results indicate that c-Ret can activate the PI3K/Akt pathway via Tyr-1062 and-1096, whereas only Tyr-1062 appears to contribute to activation of the Ras/Erk pathway. Activation of PI3K by GDNF is mediated by the assembly of a large protein complex onto the c-Ret receptor in which the adaptor proteins Shc, SHP-2, Grb2, and Gab2 play a crucial role. Finally, although GDNF-induced survival of SCG neurons depends on PI3K activity, in contrast to NGF, it is independent of Ras. Because sympathetic and other neurons are responsive to both GDNF and NGF (or other neurotrophins), differences in the requirements of signaling components for the biological activities of these ligands may form a molecular basis for the synergistic cooperation of different neurotrophic factors in neuronal survival and differentiation.

      ACKNOWLEDGEMENTS

      We thank Stephen Taylor, James Bliska, and Jonathan Backer for GST fusion plasmids. We thank Ann-Sofie Nilson and Annika Ahlsén for technical assistance, and Xiaoli Li-Ellström for secretarial help.

      REFERENCES

        • Meng 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: 1489-1493
        • Trupp M.
        • Arenas E.
        • Fainzilber M.
        • Nilsson A.-S.
        • Sieber B.A.
        • Grigoriou M.
        • Kilkenny C.
        • Salazar-Grueso E.
        • Pachnis V.
        • Arumäe U.
        • Sariola H.
        • Saarma M.
        • Ibáñez C.F.
        Nature. 1996; 381: 785-789
        • Durbec P.
        • Marcos-Gutierrez C.V.
        • Kilkenny C.
        • Grigoriou M.
        • Suvanto P.
        • Wartiovaara K.
        • Smith D.
        • Ponder B.
        • Costantini F.
        • Saarma M.
        • Sariola H.
        • Pachnis V.
        Nature. 1996; 381: 789-792
        • Jing S.Q.
        • Wen D.Z., Yu, Y.B.
        • Holst P.L.
        • Luo Y.
        • Fang M.
        • Tamir R.
        • Antonio L.
        • Hu Z.
        • Cupples R.
        • Louis J.C.
        • Hu S.
        • Altrock B.W.
        • Fox G.M.
        Cell. 1996; 85: 1113-1124
        • Treanor J.
        • Goodman L.
        • Desauvage F.
        • Stone D.M.
        • Poulsen K.T.
        • Beck C.D.
        • Gray C.
        • Armanini M.P.
        • Pollock R.A.
        • Hefti F.
        • Phillips H.S.
        • Goddard A.
        • Moore M.W.
        • Buj-Bello A.
        • Davies A.M.
        • Asai N.
        • Takahashi M.
        • Vandlen R.
        • Henderson C.E.
        • Rosenthal A.
        Nature. 1996; 382: 80-83
        • Airaksinen M.S.
        • Titievsky A.
        • Saarma M.
        Mol. Cell. Neurosci. 1999; 13: 313-325
        • Baloh R.H.
        • Enomoto H.
        • Johnson Jr., E.M.
        • Milbrandt J.
        Curr. Opin. Neurobiol. 2000; 10: 103-110
        • Trupp M.
        • Scott R.
        • Whittemore S.R.
        • Ibáñez C.F.
        J. Biol. Chem. 1999; 274: 20885-20894
        • Poteryaev D.
        • Titievsky A.
        • Sun Y.F.
        • Thomas-Crusells J.
        • Lindahl M.
        • Billaud M.
        • Arumäe U.
        • Saarma M.
        FEBS Lett. 1999; 463: 63-66
        • Yu T.
        • Scully S., Yu, Y.B.
        • Fox G.M.
        • Jing S.Q.
        • Zhou R.P.
        J. Neurosci. 1998; 18: 4684-4696
        • van Weering D.
        • Derooij J.
        • Marte B.
        • Downward J.
        • Bos J.L.
        • Burgering B.
        Mol. Cell. Biol. 1998; 18: 1802-1811
        • Edery P.
        • Eng C.
        • Munnich A.
        • Lyonnet S.
        Bioessays. 1997; 19: 389-395
        • Santoro W.
        • Carlomagno F.
        • Melillo R.M.
        • Billaud W.
        • Vecchio G.
        • Fusco A.
        J. Endocrinol. Invest. 1999; 22: 811-819
        • Alberti L.
        • Borrello M.G.
        • Ghizzoni S.
        • Torriti F.
        • Rizzetti M.G.
        • Pierotti M.
        Oncogene. 1998; 17: 1079-1087
        • Borrello M.G.
        • Pelicci G.
        • Arighi E.
        • De F.L.
        • Greco A.
        • Bongarzone I.
        • Rizzetti M.
        • Pelicci P.G.
        • Pierotti M.A.
        Oncogene. 1994; 9: 1661-1668
        • van Weering D.
        • Medema J.P.
        • van Puijenbroek A.
        • Burgering B.M.
        • Baas P.D.
        • Bos J.L.
        Oncogene. 1995; 11: 2207-2214
        • Rizzo C.
        • Califano D.
        • Colucci-Damato G.L.
        • Devita G.
        • Dalessio A.
        • Dathan N.A.
        • Fusco A.
        • Monaco C.
        • Santelli G.
        • Vecchio G.
        • Santoro M.
        • De Franciscis V.
        J. Biol. Chem. 1996; 271: 29497-29501
        • Arighi E.
        • Alberti L.
        • Torriti F.
        • Ghizzoni S.
        • Rizzetti M.G.
        • Pelicci G.
        • Pasini B.
        • Bongarzone I.
        • Piutti C.
        • Pierotti M.A.
        • Borrello M.G.
        Oncogene. 1997; 14: 773-782
        • Lorenzo M.J.
        • Gish G.D.
        • Houghton C.
        • Stonehouse T.J.
        • Pawson T.
        • Ponder B.
        • Smith D.P.
        Oncogene. 1997; 14: 763-771
        • Ohiwa M.
        • Murakami H.
        • Iwashita T.
        • Asai N.
        • Iwata Y.
        • Imai T.
        • Funahashi H.
        • Takagi H.
        • Takahashi M.
        Biochem. Biophys. Res. Commun. 1997; 237: 747-751
        • Durick K.
        • Gill G.N.
        • Taylor S.S.
        Mol. Cell. Biol. 1998; 18: 2298-2308
        • Bocciardi R.
        • Mograbi B.
        • Pasini B.
        • Borrello M.G.
        • Pierotti M.A.
        • Bourget I.
        • Fischer S.
        • Romeo G.
        • Rossi B.
        Oncogene. 1997; 15: 2257-2265
        • Murakami H.
        • Iwashita T.
        • Asai N.
        • Shimono Y.
        • Iwata Y.
        • Kawai K.
        • Takahashi M.
        Biochem. Biophys. Res. Commun. 1999; 262: 68-75
        • Ishiguro Y.
        • Iwashita T.
        • Murakami H.
        • Asai N.
        • Iida K.I.
        • Goto H.
        • Kayakawa T.
        • Takahashi M.
        Endocrinol. 1999; 140: 3992-3998
        • Segouffin-Cariou C.
        • Billaud M.
        J. Biol. Chem. 2000; 275: 3568-3576
        • Datta S.R.
        • Dudek H.
        • Tao X.
        • Masters S.
        • Fu H.A.
        • Gotoh Y.
        • Greenberg M.E.
        Cell. 1997; 91: 231-241
        • Delpeso L.
        • Gonzalezgarcia M.
        • Page C.
        • Herrera R.
        • Nuñez G.
        Science. 1997; 278: 687-689
        • Kennedy S.G.
        • Wagner A.J.
        • Conzen S.D.
        • Jordan J.
        • Bellacosa A.
        • Tsichlis P.N.
        • Hay N.
        Genes Dev. 1997; 11: 701-713
        • Crowder R.J.
        • Freeman R.S.
        J. Neurosci. 1998; 18: 2933-2943
        • Philpott K.L.
        • Mccarthy M.J.
        • Klippel A.
        • Rubin L.L.
        J. Cell Biol. 1997; 139: 809-815
        • Creedon D.J.
        • Tansey M.G.
        • Baloh R.H.
        • Osborne P.A.
        • Lampe P.A.
        • Fahrner T.J.
        • Heuckeroth R.O.
        • Milbrandt J.
        • Johnson E.M.
        Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7018-7023
        • Nobes C.D.
        • Tolkovsky A.M.
        Eur. J. Neurosci. 1995; 7: 344-350
        • Mazzoni I.E.
        • Said F.A.
        • Aloyz R.
        • Miller F.D.
        • Kaplan D.
        J. Neurosci. 1999; 19: 9716-9727
        • Salazar-Grueso E.
        • Kim S.
        • Kim H.
        Neuroreport. 1991; 2: 505-508
        • Trupp M.
        • Rydén M.
        • Jörnvall H.
        • Timmusk T.
        • Funakoshi H.
        • Arenas E.
        • Ibáñez C.F.
        J. Cell Biol. 1995; 130: 137-148
        • Kunkel T.
        Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 488-492
        • Taylor S.J.
        • Shalloway D.
        Curr. Biol. 1996; 6: 1621-1627
        • Schaeper U.
        • Gehring N.H.
        • Fuchs K.P.
        • Sachs M.
        • Kempkes B.
        • Birchmeier W.
        J. Cell Biol. 2000; 149: 1419-1432
        • Asai N.
        • Murakami H.
        • Iwashita T.
        • Takahashi M.
        J. Biol. Chem. 1996; 271: 17644-17649
        • Wymann M.P.
        • Pirola L.
        Biochim Biophys Acta. 1998; 1436: 127-150
        • Gu H.
        • Pratt J.C.
        • Burakoff S.J.
        • Neel B.G.
        Mol. Cell. 1998; 2: 729-740
        • Nishida K.
        • Yoshida Y.
        • Itoh M.
        • Fukada T.
        • Ohtani T.
        • Shirogane T.
        • Atsumi T.
        • Takahashi-Tezuka M.
        • Ishihara K.
        • Hibi M.
        • Hirano T.
        Blood. 1999; 93: 1809-1816
        • Zhao C., Yu, D.H.
        • Shen R.
        • Feng G.S.
        J. Biol. Chem. 1999; 274: 19649-19654
        • Holgado-Madruga M.
        • Moscatello D.K.
        • Emlet D.R.
        • Dieterich R.
        • Wong A.J.
        Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12419-12424
        • Korhonen J.M.
        • Said F.A.
        • Wong A.J.
        • Kaplan D.R.
        J. Biol. Chem. 1999; 274: 37307-37314
        • Kotzbauer P.T.
        • Lampe P.A.
        • Heuckeroth R.O.
        • Golden J.P.
        • Creedon D.J.
        • Johnson E.M.
        • Milbrandt J.
        Nature. 1996; 384: 467-470
        • Sánchez M.P.
        • Silossantiago I.
        • Frisen J.
        • He B.
        • Lira S.A.
        • Barbacid M.
        Nature. 1996; 382: 70-73
        • Moore M.W.
        • Klein R.D.
        • Farinas I.
        • Sauer H.
        • Armanini M.
        • Phillips H.
        • Reichardt L.F.
        • Ryan A.M.
        • Carvermoore K.
        • Rosenthal A.
        Nature. 1996; 382: 76-79
        • Pichel J.G.
        • Shen L.Y.
        • Sheng H.Z.
        • Granholm A.C.
        • Drago J.
        • Grinberg A.
        • Lee E.J.
        • Huang S.P.
        • Saarma M.
        • Hoffer B.J.
        • Sariola H.
        • Westphal H.
        Nature. 1996; 382: 73-76
        • Klesse L.J.
        • Parada L.F.
        J. Neurosci. 1998; 18: 10420-10428
        • Klesse L.J.
        • Meyers K.A.
        • Marshall C.J.
        • Parada L.F.
        Oncogene. 1999; 18: 2055-2068
        • Obermeier A.
        • Bradshaw R.A.
        • Seedorf K.
        • Choidas A.
        • Schlessinger J.
        • Ullrich A.
        EMBO J. 1994; 13: 1585-1590
        • Stephens R.M.
        • Loeb D.M.
        • Copeland T.D.
        • Pawson T.
        • Greene L.A.
        • Kaplan D.R.
        Neuron. 1994; 12: 691-705
      1. Paratcha, G., Ledda, F., Baars, L., Coulpier, M., Besset, V., Anders, J., Scott, R., and Ibanez, C. F. Neuron, in press.