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• W2008767841 abstract "Dominant-activating mutations in the RET (rearranged during transfection) proto-oncogene, a receptor tyrosine kinase, are causally associated with the development of multiple endocrine neoplasia type 2A (MEN2A) syndrome. Such oncogenic RET mutations induce its ligand-independent constitutive activation, but whether it spreads identical signaling to ligand-induced signaling is uncertain. To address this question, we designed a cellular model in which RET can be activated either by its natural ligand, or alternatively, by controlled dimerization of the protein that mimics MEN2A dimerization. We have shown that controlled dimerization leaves proximal RET signaling intact but impacts substantially on the tuning of the distal AKT kinase activation (delayed and sustained). In marked contrast, distal activation of ERK remained unaffected. We further demonstrated that specific temporal adjustment of ligand-induced AKT activation is dependent upon a lipid-based cholesterol-sensitive environment, and this control step is bypassed by MEN2A RET mutants. Therefore, these studies revealed that MEN2A mutations propagate previously unappreciated subtle differences in signaling pathways and unravel a role for lipid rafts in the temporal regulation of AKT activation. Dominant-activating mutations in the RET (rearranged during transfection) proto-oncogene, a receptor tyrosine kinase, are causally associated with the development of multiple endocrine neoplasia type 2A (MEN2A) syndrome. Such oncogenic RET mutations induce its ligand-independent constitutive activation, but whether it spreads identical signaling to ligand-induced signaling is uncertain. To address this question, we designed a cellular model in which RET can be activated either by its natural ligand, or alternatively, by controlled dimerization of the protein that mimics MEN2A dimerization. We have shown that controlled dimerization leaves proximal RET signaling intact but impacts substantially on the tuning of the distal AKT kinase activation (delayed and sustained). In marked contrast, distal activation of ERK remained unaffected. We further demonstrated that specific temporal adjustment of ligand-induced AKT activation is dependent upon a lipid-based cholesterol-sensitive environment, and this control step is bypassed by MEN2A RET mutants. Therefore, these studies revealed that MEN2A mutations propagate previously unappreciated subtle differences in signaling pathways and unravel a role for lipid rafts in the temporal regulation of AKT activation. The RET 4The abbreviations used are:RETrearranged during transfectionDRMsdetergent-resistant membranesERKextracellular signal regulated kinaseGDNFglial cell line-derived neurotrophic factorGFRαglial cell line-derived neurotrophic factor receptor αMBCmethyl-β-cyclodextrineMEN2multiple endocrine neoplasia type 2PI3Kphosphatidylinositol 3 kinasePLCγphospholipase C γShcSrc homology and collagen proteinTUNELterminal deoxynucleotidyltransferase-mediated dUTP nick end-labelingGFLGDNF family ligandsHAhemagglutininMTT3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromideAPAP20187. 4The abbreviations used are:RETrearranged during transfectionDRMsdetergent-resistant membranesERKextracellular signal regulated kinaseGDNFglial cell line-derived neurotrophic factorGFRαglial cell line-derived neurotrophic factor receptor αMBCmethyl-β-cyclodextrineMEN2multiple endocrine neoplasia type 2PI3Kphosphatidylinositol 3 kinasePLCγphospholipase C γShcSrc homology and collagen proteinTUNELterminal deoxynucleotidyltransferase-mediated dUTP nick end-labelingGFLGDNF family ligandsHAhemagglutininMTT3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromideAPAP20187. proto-oncogene is located on chromosome 10q11.2 and encodes a receptor tyrosine kinase with four cadherin-related motifs and a cysteine-rich domain in the extracellular domain (1Airaksinen M.S. Saarma M. Nat. Rev. Neurosci. 2002; 3: 383-394Crossref PubMed Scopus (1445) Google Scholar). It associates with ligand-specific co-receptors known as GFRαs (GDNF, glial-cell-line-derived neurotrophic factor, family receptors α), to form functional receptors for the GFL (GDNF family ligands). In the current view, homodimeric GFL binding induces a GFL2-GFRα2-RET2 complex (2Leppanen V.M. Bespalov M.M. Runeberg-Roos P. Puurand U. Merits A. Saarma M. Goldman A. EMBO J. 2004; 23: 1452-1462Crossref PubMed Scopus (52) Google Scholar). RET dimerization leads to increased receptor kinase activity and autophosphorylation of cytoplasmic tyrosine residues, which serve as docking sites for Src homology 2 (SH2)- and phospho-tyrosine-binding domain-containing proteins, such as Shc or phospholipase Cγ (3Ichihara M. Murakumo Y. Takahashi M. Cancer Lett. 2004; 204: 197-211Crossref PubMed Scopus (162) Google Scholar). These proteins then recruit additional effector molecules, resulting in the assembly of signaling complexes and the activation of intracellular signaling pathways, including the Ras/extracellular-regulated kinase (ERK) and phosphoinositide 3-kinase (PI3K)/AKT pathways. GFL-mediated signaling pathways are involved in the development and maintenance of a broad spectrum of neuronal subpopulations (1Airaksinen M.S. Saarma M. Nat. Rev. Neurosci. 2002; 3: 383-394Crossref PubMed Scopus (1445) Google Scholar). rearranged during transfection detergent-resistant membranes extracellular signal regulated kinase glial cell line-derived neurotrophic factor glial cell line-derived neurotrophic factor receptor α methyl-β-cyclodextrine multiple endocrine neoplasia type 2 phosphatidylinositol 3 kinase phospholipase C γ Src homology and collagen protein terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling GDNF family ligands hemagglutinin 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide AP20187. rearranged during transfection detergent-resistant membranes extracellular signal regulated kinase glial cell line-derived neurotrophic factor glial cell line-derived neurotrophic factor receptor α methyl-β-cyclodextrine multiple endocrine neoplasia type 2 phosphatidylinositol 3 kinase phospholipase C γ Src homology and collagen protein terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling GDNF family ligands hemagglutinin 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide AP20187. Recently, membrane microdomains, or lipid rafts, have been shown to profoundly influence the functional impact of GDNF-stimulated RET downstream signaling (4Tansey M.G. Baloh R.H. Milbrandt J. Johnson Jr., E.M. Neuron. 2000; 25: 611-623Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar, 5Paratcha G. Ledda F. Baars L. Coulpier M. Besset V. Anders J. Scott R. Ibanez C.F. Neuron. 2001; 29: 171-184Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar). Lipid rafts are suggested to be lateral microdomains in membranes of living cells, enriched in sphingolipids, cholesterol, and specific membrane proteins. They are characterized by higher order and by having a lower buoyant density than bulk plasma membranes (6Simons K. Toomre D. Nat. Rev. Mol. Cell. Biol. 2000; 1: 31-39Crossref PubMed Scopus (5149) Google Scholar, 7Sprong H. van der Sluijs P. van Meer G. Nat. Rev. Mol. Cell. Biol. 2001; 2: 504-513Crossref PubMed Scopus (476) Google Scholar). Although uncertainties about the precise molecular nature of rafts remain (8Edidin M. Annu. Rev. Biophys. Biomol. Struct. 2003; 32: 257-283Crossref PubMed Scopus (1134) Google Scholar, 9Munro S. Cell. 2003; 115: 377-388Abstract Full Text Full Text PDF PubMed Scopus (1325) Google Scholar, 10Sharma P. Varma R. Sarasij R.C. Ira Gousset K. Krishnamoorthy G. Rao M. Mayor S. Cell. 2004; 116: 577-589Abstract Full Text Full Text PDF PubMed Scopus (710) Google Scholar, 11Kusumi A. Koyama-Honda I. Suzuki K. Traffic. 2004; 5: 213-230Crossref PubMed Scopus (333) Google Scholar), compelling evidence indicates that lipid rafts can coalesce into larger and more stable structures where proteins can segregate to perform functions (12Golub T. Wacha S. Caroni P. Curr. Opin. Neurobiol. 2004; 14: 542-550Crossref PubMed Scopus (135) Google Scholar, 13Rajendran L. Simons K. J. Cell Sci. 2005; 118: 1099-1102Crossref PubMed Scopus (468) Google Scholar). With regard to GDNF-stimulated RET signaling, it has been shown that at steady state, GFRα1, but not RET, is a resident of lipid raft-related detergent-resistant membranes (DRMs). GDNF stimulation was demonstrated to trigger RET recruitment and activation into DRMs. This was strongly correlated with downstream signaling, cell survival, and differentiation (4Tansey M.G. Baloh R.H. Milbrandt J. Johnson Jr., E.M. Neuron. 2000; 25: 611-623Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar). Furthermore, Paratcha et al. (5Paratcha G. Ledda F. Baars L. Coulpier M. Besset V. Anders J. Scott R. Ibanez C.F. Neuron. 2001; 29: 171-184Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar) showed that RET association with DRMs may influence the nature of the intracellular signaling. Together, these studies demonstrate a connection between RET association with DRMs and RET signaling and support a role for lipid rafts in controlling GDNF-stimulated RET signaling. Mutations in the RET proto-oncogene have been identified as causative for human papillary thyroid carcinoma, multiple endocrine neoplasia (MEN) type 2A and 2B, and familial medullary thyroid carcinoma (14Hansford J.R. Mulligan L.M. J. Med. Genet. 2000; 37: 817-827Crossref PubMed Scopus (208) Google Scholar). MEN2A is an autosomal dominant cancer syndrome, characterized by medullary thyroid carcinoma, pheochromocytoma, and hyperplasia of the parathyroid gland. MEN2A mutations were identified in the cysteine-rich region, and ∼90% of those mutations affect codon 634 (most frequently a cysteine to arginine change) (15Eng C. J. Clin. Oncol. 1999; 17: 380-393Crossref PubMed Google Scholar). They result in a constitutive activation of RET through formation of covalently linked dimers of the receptor, independent of GFL (16Asai N. Iwashita T. Matsuyama M. Takahashi M. Mol. Cell. Biol. 1995; 15: 1613-1619Crossref PubMed Google Scholar, 17Santoro M. Carlomagno F. Romano A. Bottaro D.P. Dathan N.A. Grieco M. Fusco A. Vecchio G. Matoskova B. Kraus M.H. Di Fiore P.P. Science. 1995; 267: 381-383Crossref PubMed Scopus (796) Google Scholar). Dimerization occurs early during the maturation process and results in additional activation of incompletely glycosylated intracellular RET precursors (18Cosma M.P. Cardone M. Carlomagno F. Colantuoni V. Mol. Cell. Biol. 1998; 18: 3321-3329Crossref PubMed Scopus (51) Google Scholar). Whether MEN2A signaling is identical to GFL-triggered signaling is difficult to answer because activation of the constitutive MEN2A protein cannot be triggered in resting cells, thus precluding a qualitative and quantitative comparison with the GFL-inducible signaling pathways. Consequently, the current model to explain how MEN2A RET mutants promote tumorigenesis only considers the permanent nature of MEN2A signaling. To address this issue, we have generated a chimeric RET-Fv protein that can be alternatively activated in the same cell, either with the natural ligand GDNF or with a synthetic bivalent dimerizing ligand (19Clackson T. Yang W. Rozamus L.W. Hatada M. Amara J.F. Rollins C.T. Stevenson L.F. Magari S.R. Wood S.A. Courage N.L. Lu X. Cerasoli Jr., F. Gilman M. Holt D.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 10437-10442Crossref PubMed Scopus (418) Google Scholar). The latter mimicked ligand-independent RET dimerization by MEN2A mutants. This induced activation of RET specifically resulted in altered AKT activation but not ERK activation. Furthermore, we demonstrated that a lipid-based cholesterol-sensitive environment regulates the tuning of GDNF-induced AKT activation, suggesting a role of lipid rafts. Finally, we showed that MEN2A mutants escape from this control step. Therefore, these studies revealed complex alterations in oncogenic signaling by MEN2A RET mutants. Antibodies, Reagents, and DNA Constructs—Phospho-tyrosine (4G10), hemagglutinin (HA), flotillin, and human transferrin receptor antibodies were from Upstate Biotechnology (Lake Placid, NY), Covance Research (Berkeley, CA), BD Transduction Laboratories, and Zymed Laboratories (South San Francisco, CA), respectively. Protein-specific anti-phospho antibodies were from Cell Signaling Technology (Beverly, MA). Anti-RET was described elsewhere (20Pasini A. Geneste O. Legrand P. Schlumberger M. Rossel M. Fournier L. Rudkin B.B. Schuffenecker I. Lenoir G.M. Billaud M. Oncogene. 1997; 15: 393-402Crossref PubMed Scopus (87) Google Scholar). Anti-phospho-RETs (Tyr-1015 or Tyr-1062) were generously provided by Dr. Massimo Santoro (21Salvatore D. Barone M.V. Salvatore G. Melillo R.M. Chiappetta G. Mineo A. Fenzi G. Vecchio G. Fusco A. Santoro M. J. Clin. Endocrinol. Metab. 2000; 85: 3898-3907Crossref PubMed Scopus (56) Google Scholar). Lipofectamine 2000, human GDNF, protease inhibitors, monensin, and brefeldin A were from Invitrogen, Promega Corp. (Madison, WI), Roche Diagnostics, BD Biosciences, and Calbiochem, respectively. MTT, Triton X-100, methyl-β-cyclodextrin (MBC), and anisomycin were from Sigma. AP20187 (AP) was provided by Ariad Pharmaceuticals (Cambridge, MA). The HA-tagged AP-binding domain (Fv) was subcloned from the Ariad pC4-Fv1E vector, into the ApaI/XbaI sites of the pcDNA3.1(+) vector (Invitrogen). The Ret9 cDNA (20Pasini A. Geneste O. Legrand P. Schlumberger M. Rossel M. Fournier L. Rudkin B.B. Schuffenecker I. Lenoir G.M. Billaud M. Oncogene. 1997; 15: 393-402Crossref PubMed Scopus (87) Google Scholar) was mutated by PCR to suppress the stop codon and subcloned into the HindIII/XbaI sites of the pcDNA-Fv vector. The HA-GFRα1 cDNA kindly provided by Dr. Carlos Ibanez (22Trupp M. Raynoschek C. Belluardo N. Ibanez C.F. Mol. Cell. Neurosci. 1998; 11: 47-63Crossref PubMed Scopus (162) Google Scholar) was subcloned into the BamHI/EcoRI sites of the pBabe retroviral vector (23Morgenstern J.P. Land H. Nucleic. Acids Res. 1990; 18: 3587-3596Crossref PubMed Scopus (1898) Google Scholar). Cell Culture and Transfections—Rat-1 cells, immortalized rat fibroblasts expressing neither Ret nor GFRα1, were maintained in complete medium: Dulbecco's modified Eagle's medium with 10% fetal bovine serum and antibiotics (all from Sigma). Cells were transfected with Ret-Fv- and GFRα1-containing vectors with the use of Lipofectamine according to manufacturer's recommendations. Stably transfected cells were selected in complete medium with G418 (1 mg/ml) and/or puromycin (2 μg/ml) (both from Sigma). Rat-1 cell clones transformed by RET MEN2A mutants (24Segouffin-Cariou C. Billaud M. J. Biol. Chem. 2000; 275: 3568-3576Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar) were maintained in complete medium with puromycin (2 μg/ml). MEN2A cells were further transfected with GFRα1, and stable clones (termed Mα) were selected with G418 (1 mg/ml) and puromycin (2 μg/ml). Stimulations and Immunoblotting—Equal numbers of cells were seeded in plates to achieve ∼60% confluence in 24 h and serum-starved for 4 h prior to stimulation. Cells were stimulated for the indicated period of time with either 10 ng/ml GDNF or 100 nm AP20187 in serum-free medium and lysed at 4 °C for 20 min in radioimmune precipitation buffer (150 mm NaCl, 50 mm Tris-HCl (pH 7.2), 1% Triton X-100, 1% Sodium deoxycholate, 0.05% SDS, 4 mm NaVO4, 5 mm EGTA, and protease inhibitors). Precleared lysates (10 min at 13,000 rpm) were subjected to SDS-PAGE and Western blotting using the relevant antibody as described previously (25Vidalain P.O. Azocar O. Servet-Delprat C. Rabourdin-Combe C. Gerlier D. Manie S. EMBO J. 2000; 19: 3304-3313Crossref PubMed Scopus (165) Google Scholar). Densitometry of the blots was assessed using a Fluor-S multi-imager system (Bio-Rad). DRM Assay—Cells were scrapped, rinsed once with phosphate-buffered saline, and lysed at 3 mg of protein/ml in a final volume of 0.5 ml of ice-cold TNE buffer (150 mm NaCl, 25 mm Tris-HCl (pH 7.2), 4 mm NaVO4, 5 mm EGTA, and protease inhibitors) containing 0.5% Triton X-100. Lysates were homogenized five times through a 23-gauge needle and incubated with constant agitation for 30 min at 4 °C. They were then mixed with 1 ml of 60% saccharose in TNE buffer containing 0.1% Triton X-100 and transferred to ultracentrifuge tubes. The samples were overlaid by a saccharose gradient (2 ml of 30% saccharose followed by 1 ml of 25, 20, 17.5, 15, 12.5, and 5% saccharose in TNE buffer containing 0.1% Triton X-100) and centrifuged for 16 h at 39,000 rpm in a Beckman SW41 rotor at 4 °C. The gradient was fractionated into 1-ml fractions (see Fig. 6B, 1.15-ml fractions) from the top of the tube, except for the last one that contained 1.5 ml. Aliquots of each fraction were analyzed by SDS-PAGE and immunoblotting. Cellular Cholesterol Depletion and Brefeldin A/Monensin Treatment—Cells prepared as described above were incubated in serum-free medium containing MBC (10 mm) and 50 mm Hepes for 20 min at 37 °C with constant agitation. Cells were then rinsed twice in phosphate-buffered saline, stimulated, and lysed to isolate DRMs. To abrogate RET protein maturation, cells were treated or not with 2.5 μg/ml brefeldin A and 0.66 μl/ml monensin (Golgi stop) in serum-free Dulbecco's modified Eagle's medium for 6 h and further stimulated with AP for the indicated period of time, before being processed for immunoblotting analysis. Cell Growth and TUNEL Assays—Cells were plated at 500 cells/well in 96-well plates in complete medium with GDNF (10 ng/ml) or AP (100 nm), the growth medium being changed every 3 days. Every 2 days, cells were harvested, and an MTT test was realized for each condition of stimulation; 30 μl of MTT (7.5 mg/ml in phosphate-buffered saline) was added to each well, and cells were incubated for 4 h at 37°C. The medium was then replaced with 100 μl of Me2SO with 0.04 n HCl. OD was read at 492 nm. The measured absorbance is proportional to the number of live cells present. For TUNEL assay, Mα cells were plated on coverslips at 7 × 104 cells/well in Dulbecco's modified Eagle's medium containing 1% fetal bovine serum, overnight, and stimulated with 50 ng/ml GDNF for 1 h in the same medium before being incubated with anisomycin (1 μg/ml) for 5 h. A TUNEL assay was performed according to the supplier's instructions (in situ cell death detection kit, Roche Diagnostics). Controlled Homodimerization and Activation of RET—The inducible GDNF/GFRα1-independent activation system of RET is controlled by the synthetic bivalent dimerizing ligand AP. It is based on the ability of a fusion protein (RET-Fv), containing RET linked to an AP-binding domain (Fv), to be induced to homodimerize in the presence of AP, leading to RET activation (Fig. 1A). Both RET-Fv and GFRα1 were stably expressed in Rat-1 fibroblastic cells so that RET-Fv can be activated alternatively with GDNF or AP treatment. Fig. 1A shows the expression of RET-Fv and GFRα1 in representative Rat-1 fibroblast clones. The two RET species correspond to the incompletely glycosylated protein present in the endoplasmic reticulum (162 kDa, major form in Rat-1 cells) and to the fully glycosylated protein expressed at the plasma membrane (182 kDa) (26van Weering D.H. Moen T.C. Braakman I. Baas P.D. Bos J.L. J. Biol. Chem. 1998; 273: 12077-12081Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). Similarly, the 65-kDa form of GFRα1 was expressed on the cellular surface, whereas the 55-kDa form was not (not shown) and was assumed to correspond to an immature GFRα1 product. Stimulation of RET-Fv/GFRα1-expressing cells with an optimal concentration of AP or GDNF for 15 min induced a comparable activation of mature 182-kDa RET proteins, as detected with an antibody specific to phosphorylated autocatalytic residue Y905RET (Fig. 1B, upper panel). However, in contrast to GDNF, AP diffused into the cytoplasm and then bound to and activated the immature 162-kDa Ret protein. This is similar to what is typically observed with MEN2A RET mutants in which both the mature and the immature forms of RET are activated. Despite the additional activation of the immature form of RET, a comparable increase in tyrosine phosphorylation of cellular proteins was detected after either GDNF or AP stimulation (Fig. 1B, lower panel). It would appear that the addition of the Fv module to the cytoplasmic tail of RET does not obviously alter RET activation and signaling since a similar pattern of tyrosine phosphorylation was observed when cells were transfected with wild type RET instead of RET-Fv and stimulated with GDNF (Fig. 1B). Taken together, these results indicated that homodimerization of RET can lead to its efficient activation without the requirement for GDNF/GFRα1 complex. They also suggested that activation of the intracellular immature 162-kDa Ret protein does not contribute much to the RET-dependent pattern of tyrosine phosphorylation. GDNF/GFRα1-independent RET Activation Leads to an Altered Cellular Outcome—Next, we investigated whether activation of RET by AP or GDNF can differently affect biological outcomes. RET-Fv/GFRα1-expressing cells were chronically stimulated with either GDNF or AP, and the growth rate was monitored. Neither AP nor GDNF stimulated growth rate during the first 6 days of culture (Fig. 2). After 8 days of culture, both non-stimulated and GDNF-stimulated cells grew in an even monolayer and displayed normal contact inhibition, as shown on representative May-Grunwald-Giemsa staining of cells after 10 days of culture. As a consequence, these cells stopped growing. In contrast, AP-stimulated cells began to overgrow one another, indicating that they had lost the ability to be contact inhibited. These results indicated the existence of specific AP-mediated cellular outcome(s), suggesting that GDNF/GFRα1-independent RET activation leads to alterations of intracellular signaling pathways. GDNF/GFRα1-independent RET Activation Impacts on AKT Regulation—We next tested whether activation of RET by AP or GDNF can translate into different, or differently regulated, RET-mediated signaling pathways. We first monitored the extent of RET autophosphorylation using two other antibodies specific to phosphorylated Y1015RET and Y1062RET. Fig. 3A shows that phosphorylation over a time course of these autocatalytic residues was comparably achieved by GDNF or AP stimulation. Together with the comparable phosphorylation of Y905RET depicted in Fig. 1, these results indicate that AP or GDNF stimulation leads to similar activation of RET tyrosine kinase. It is of note that RET phosphorylation in Rat-1 cells remained high after 2 h of stimulation and that it could last for 6 h before declining (see Fig. 6A). Similar results were obtained with wild type RET instead of RET-Fv (not shown), indicating that the Fv-binding module is not involved in the long lasting phosphorylation of RET in Rat-1 cells. Y1015RET and Y1062RET are binding sites for PLCγ and Shc/Dok4–5/IRS-1/FRS-2, respectively. The bound molecular partners are in turn phosphorylated by RET (3Ichihara M. Murakumo Y. Takahashi M. Cancer Lett. 2004; 204: 197-211Crossref PubMed Scopus (162) Google Scholar). Fig. 3A shows that both Shc and PLCγ were phosphorylated in a comparable manner following GDNF or AP stimulation and that their kinetics of phosphorylation paralleled those of RET. These results implied that RET activation by AP or GDNF does not influence proximal RET signaling, i.e. autophosphorylation of RET and the resulting phosphorylation of the molecular partners Shc and PLCγ. We next examined the activation of downstream pathways by looking at ERK and AKT phosphorylation. GDNF induced a transient phosphorylation of ERK and AKT (Fig. 3). Therefore, both Ras/ERK and PI3K/AKT signaling pathways can be attenuated even with the prolonged phosphorylation of RET observed in Rat-1 cells. As compared with GDNF, AP stimulated ERK phosphorylation to a similar amplitude and duration. However, in marked contrast, the kinetics of AP-stimulated AKT phosphorylation was delayed and sustained for several hours (Fig. 3, A and B). Kinetic alteration of AKT phosphorylation was detected on both serine 473 and threonine 308, which additively activate AKT (28Brazil D.P. Yang Z.Z. Hemmings B.A. Trends Biochem. Sci. 2004; 29: 233-242Abstract Full Text Full Text PDF PubMed Scopus (719) Google Scholar). It should be emphasized that AP does not directly affect AKT activation since no alteration of AKT phosphorylation could be detected following GDNF treatment of cells expressing RET (instead of RET-Fv) in the presence of AP (not shown). These results indicated that GDNF/GFRα1-independent RET activation translates into differently regulated AKT activation, without really affecting ERK activation. Neither Signaling from GFRα1 Alone nor from Immature Forms of RET Contributes to AKT Phosphorylation—Since GDNF has been reported to signal independently of RET via GFRα1 (29Trupp M. Scott R. Whittemore S.R. Ibanez C.F. J. Biol. Chem. 1999; 274: 20885-20894Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar, 30Poteryaev D. Titievsky A. Sun Y.F. Thomas-Crusells J. Lindahl M. Billaud M. Arumae U. Saarma M. FEBS Lett. 1999; 463: 63-66Crossref PubMed Scopus (143) Google Scholar), it was important to evaluate whether signaling originating from GFRα1 alone could contribute to AKT and ERK phosphorylation in Rat-1 cells. We thus made use of Rat-1 clones expressing GFRα1 only and have been unable to detect any phosphorylation of AKT and ERK following GDNF stimulation of these clones (Fig. 4A). Next, we evaluated whether AP-activated immature 162-kDa forms of RET-Fv were involved in the differential phosphorylation of AKT. To address this issue, cells were treated with brefeldin A and monensin to abrogate RET maturation and delivery to the plasma membrane (31Jung T. Schauer U. Heusser C. Neumann C. Rieger C. J. Immunol. Methods. 1993; 159: 197-207Crossref PubMed Scopus (903) Google Scholar). The combined use of the two protein maturation blocking drugs was favored because under our experimental conditions, each drug alone was not efficient enough to fully prevent apparition of mature RET proteins (not shown). Cellular treatment for 6 h with the two drugs efficiently blocked the expression of the plasma membrane-localized mature forms of RET while increasing the expression of the endoplasmic reticulum-associated immature 162-kDa form of RET (Fig. 4B). Under these conditions, AP stimulation of cells could still induce phosphorylation of the immature form. Interestingly, phosphorylation of Shc was also detectable, indicating that activated endoplasmic reticulum-associated immature 162-kDa forms of RET can recruit molecular partners. However, these activated forms of RET made no detectable contribution to AKT and ERK activation. These data, together with the observation that additional activation of immature forms of RET did not modify the tyrosine phosphorylation pattern (Fig. 1B), strongly suggest that immature forms of RET do not contribute to the signaling pathways monitored here. GDNF/GFRα1-independent RET Activation Does Not Associate with DRMs—GDNF/GFRα1-stimulated RET associates with the raft-related DRMs, and this association has been correlated with the control of downstream signaling (4Tansey M.G. Baloh R.H. Milbrandt J. Johnson Jr., E.M. Neuron. 2000; 25: 611-623Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar, 5Paratcha G. Ledda F. Baars L. Coulpier M. Besset V. Anders J. Scott R. Ibanez C.F. Neuron. 2001; 29: 171-184Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar). Thus, to gain an insight into the mechanism(s) responsible for the differential regulation of AKT activation, we asked whether AP stimulation could also induce the association of RET with DRMs. As expected, GDNF stimulation induced an association of mature forms of RET-Fv with DRMs (Fig. 5A). Flotillin and transferrin receptor were used as positive and negative markers, respectively, of DRM-associated proteins. Although RET must associate with GFRα1to be activated by GDNF, more than 60% of phosphorylated RET-Fv was recovered in soluble fractions (Fig. 5B). This does not seem to reflect a large movement of RET out of DRMs after recruitment and activation since RET remained associated with DRMs for several hours after the beginning of the stimulation in Rat-1 cells (Fig. 5B, and see also Fig. 6A). These results suggested that RET association with DRMs is weaker than GFRα1 association and that it can be partly disrupted by non-ionic detergents. In marked contrast to these results, RET association with DRMs was not evident upon AP stimulation. Similarly, RET proteins carrying a representative MEN2A mutation at Cys-634 (24Segouffin-Cariou C. Billaud M. J. Biol. Chem. 2000; 275: 3568-3576Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar) were not recovered in the DRM fractions (Fig. 5A). These results clearly demonstrated a difference in the association (or the strength of association) of RET with DRMs between GDNF-dependent and GDNF/GFRα1-independent RET activation. RET Association with DRMs Correlates with Specific Regulation of AKT—The above results raised the possibility that tight regulation of AKT, but not ERK, phosphorylation is linked to DRM association of RET. This hypothesis was tested in two different ways. First, association of proteins with DRMs has been shown to be sensitive to the cholesterol-depleting drug MBC (32Xavier R. Brennan T. Li Q. McCormack C. Seed B. Immunity. 1998; 8: 723-732Abstract Full Text Full Text PDF PubMed Scopus (837) Google Scholar). Preincubation of Rat-1 cells for 20 min with 10 mm MBC before stimulation did not affect RET phosphorylation and its" @default.
• W2008767841 created "2016-06-24" @default.
• W2008767841 creator A5007907872 @default.
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• W2008767841 creator A5044022453 @default.
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• W2008767841 date "2005-11-01" @default.
• W2008767841 modified "2023-10-13" @default.
• W2008767841 title "Inducible Dimerization of RET Reveals a Specific AKT Deregulation in Oncogenic Signaling" @default.
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