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The Tyrosine Phosphatase Shp-2 Mediates Intracellular Signaling Initiated by Ret Mutants

Oncologia Sperimentale E, Istituto Nazionale Tumori (A.D’A., D.C., G.Sa.), Fondazione "G. Pascale," 80131 Naples, Italy; Dipartimento di Biologia e Patologia Cellulare e Molecolare (M.I., M.S.C.), Università di Napoli "Federico II," 80131 Naples, Italy; Farmacologia e Neuroscienze Istituto Nazionale per la Ricerca sul Cancro (T.F., G.Sc.), 16132 Genova, Italy; Sez Farmacologia, Dip. Oncologia (T.F., G.Sc.), Biologia e Genetica, Università di Genova, 16132 Genova, Italy; and Istituto di Endocrinologia ed Oncologia Sperimentale "G. Salvatore" del Consiglio Nazionale delle Ricerche (L.C., V.d.F.), 80131 Naples, Italy

Address all correspondence and requests for reprints to: V. de Franciscis, Istituto di Endocrinologia ed Oncologia Sperimentale "G. Salvatore" del Consiglio Nazionale delle Ricerche via S. Pansini 5, 80131 Naples, Italy.

	Abstract

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The Src homology 2-containing tyrosine phosphatase, Shp-2, is a crucial enzyme that mediates intracellular signaling and is implicated in cell proliferation and differentiation. Here we investigated the involvement of the Shp-2 tyrosine phosphatase in determining the downstream signaling pathways initiated by the Ret oncogene, carrying either the cysteine 634 to tyrosine or the methionine 918 to threonine substitutions. These mutations convert the receptor tyrosine kinase, Ret, into a dominant transforming protein and induce constitutive activation of its intrinsic tyrosine kinase activity leading to congenital and sporadic cancers in neuroendocrine organs. Using the PC12, rat pheochromocytoma cell line, as model system, we show that Shp-2 mediates immediate-early gene expression if induced by either of the mutant alleles. Furthermore, we show that Shp-2 activity is required for Ret^M918T-induced Akt activation. The results indicate that Shp-2 is a downstream mediator of the mutated receptors Ret^C634Y and Ret^M918T, thus suggesting that it may act as a limiting factor in Ret-associated endocrine tumors, in the neoplastic syndromes multiple endocrine neoplasia types 2A and 2B.

	Introduction

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THE RET PROTEIN is a transmembrane receptor of the protein tyrosine kinase family. Four distinct ligands for Ret have recently been identified; they are all polypeptide growth factors belonging to the glial cell line-derived neurotrophic factor (GDNF) family. The biological actions of the GDNF family members are mediated through physical interactions with a multicomponent receptor complex consisting of a common signaling component, the Ret receptor tyrosine kinase, and of one of the four members of the glycosyl-phosphatidylinositol-anchored receptor family, GFR1–4 (1, 2, 3). Germline mutations in the ret gene are responsible for the inheritance of multiple endocrine neoplasia (MEN) type 2A and 2B syndromes, and of familial medullary thyroid carcinoma (FMTC). MEN-2A and MEN-2B are distinct hereditary neoplastic diseases both characterized by the presence of medullary thyroid carcinomas and pheochromocytomas. MEN-2A also features hyperplasia of parathyroid cells, whereas MEN-2B is a more severe disease, being associated with skeletal abnormalities, ganglioneuromas of the intestinal tract, mucosal neuromas, and characterized by an earlier age of tumor onset (4). Missense mutations that cause the creation of an unpaired cysteine residue in the extracellular domain are the most frequent events in FMTC and in MEN-2A syndrome. A single point mutation, which results in a threonine for methionine substitution at codon 918 within the Ret catalytic domain, is the most frequent mutation in MEN-2B syndrome. Both classes of mutations convert Ret into a dominant transforming gene and cause constitutive activation of its intrinsic tyrosine kinase activity (5, 6, 7).

The evidence that inheritance of specific ret mutations predisposes to distinct disease phenotypes, supports the belief that some specific cell types undergo abnormal proliferation depending on the type of Ret activation. Indeed, MEN-2A-associated and MEN-2B-associated Ret mutants differ in their activation mechanisms and substrate specificity (5, 8). However, a description of the molecular mechanism(s) responsible for the specificity of each of these diseases (MEN-2B vs. MEN-2A) is still lacking (6, 7).

Phosphotyrosine phosphatases constitute a family of regulators (either negative or positive) in the intracellular pathways that result in growth factor-specific cell responses, which act to dephosphorylate signaling intermediaries thereby regulating their function. So far, little is known about the involvement of tyrosine phosphatases as effectors involved in Ret-induced intracellular signaling (9, 10, 11).

Shp-2 (previously known as PTP1D, PTP2C, SH-PTP2, SH-PTP3, Syp) is a nontransmembrane phosphotyrosine phosphatase that contains, beside a central catalytic domain, two Src homology 2 domains at the amino terminus and two tyrosine residues located at the carboxy terminus that likely regulate its catalytic activity (12). Shp-2 positively regulates ERKs signaling in response to insulin, fibroblast growth factor, and epidermal growth factor, and serves as signal transducer for several receptor tyrosine kinases, G protein-coupled and cytokine receptors (12, 13, 14, 15, 16, 17, 18, 19). Besides its implication as positive modulator of the Ras/Erk pathway, Shp-2 has been reported to be necessary for Akt activation by several growth factors (20, 21). Furthermore, as recently reported, Shp-2 differently regulates the kinetics and magnitude of signaling cascades initiated by epidermal growth factor receptor or by other tyrosine kinase receptors, this regulation being receptor specific (22).

Recent evidence in a motorneuron cell line, MN1, implicates Shp-2 as a potential key molecule in Ret signaling. Indeed, upon stimulation of Ret^wt, the phosphatidylinositol 3-kinase mediates Ret signaling through a multiprotein complex that includes the docking proteins Gab2 and Shp-2 (9). In this report, we investigated the implication of Shp-2 in determining the intracellular signaling initiated by two different Ret mutants (either associated to MEN-2A or MEN-2B syndromes). The results presented here indicate that, in PC12 cells, Shp-2 activity is required for both Ret mutants signaling but mediates distinct functions according to which Ret mutant is involved.

	Materials and Methods

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Cell culture and transfections
Parental PC12 cells, PC12/wt, PC12/MEN2A, PC12/MEN2B, and PC12-1/wt cell lines, which expressed Ret9^wt, Ret9^C634Y, Ret9^M918T, and Ret9^wt plus GFR-1 proteins, respectively, were cultured as previously described, and when required, with the appropriate selection pressure (23). The PC12/MEN2A and PC12/MEN2B cells were transfected with myc-SH-PTP2 (C/S) plasmid and selected for 15 d in presence of 400 ng/ml of G418 to obtain PC12/MEN2A^{Shp-2 C/S} and PC12/MEN2B^{Shp-2 C/S} cells, respectively. A mass population and individual clones were grown and maintained in selection medium.

The pEBG-mBad comprises the sequence relative to Bad gene fused with glutathione-S-transferase (GST) gene sequence (New England Biolabs, Inc., Beverly, MA). The pCEFL-HA-Akt, contains the Akt gene fused to the hemagglutinin (HA) epitope. Plasmids encoding myc-SH-PTP2 or myc-SH-PTP2 (C/S) were kindly provided by Dr. J. Pessin (University of Iowa, Iowa City, IA). The pfos-CAT (-356 to +109) (24), pEgr-1-CAT (C4) (-1150 to +200) fused to the chloramphenicol acetyl transferase (CAT) gene (23).

Preparation of cell extracts, immunoprecipitation, and immunoblotting
Between 10⁶ and 10⁷ cells were washed twice in ice-cold PBS, then lysed in lysis buffer [50 mM Tris-HCl (pH 8) containing 150 mM NaCl, 1% Nonidet P-40, 2 µg/ml aprotinin, 1 µg/ml pepstatin, 2 µg/ml leupeptin, 1 mM Na₃VO₄]. Protein concentrations were estimated by a modified Bradford assay (Bio-Rad, Hercules, CA). One milligram of total cell lysate was incubated with specific antibodies for 2 h at 4 C and then immunoprecipitated with protein G-plus agarose (Calbiochem, Oncogene Research) overnight at 4 C. Immunoprecipitates were washed five times with the above lysis buffer and boiled in Laemmli buffer for 5 min, then subjected to SDS-PAGE (10% acrylamide) and transferred to polyvinylidene difluoride membrane (Millipore Co., Bedford, MA). The antibodies used were: anti-SH-PTP2 (C-18), anti-RET (C-19), and anti-c-myc (9E10) from Santa Cruz Biotechnology Inc. (Santa Cruz, CA); anti-Gab2 and antiphosphotyrosine monoclonal antibodies (4G10) from Upstate Biotechnology Inc. (Lake Placid, NY); anti-HA from Roche Molecular Biochemicals (Basel, Switzerland); anti-pAkt (Ser473), anti-Bad, anti-pBad-112, and anti-pBad-136 primary antibodies from New England Biolabs Inc., and detected with the ECL system (Amersham Biosciences Corp., Piscataway, NJ). When indicated, filters were stripped in 62.5 mM Tris-HCl (pH 6.7), 100 mM 2-mercaptoethanol, 2% sodium dodecyl sulfate, for 30 min at 55 C. The immunoblots shown are examples of at least three independent experiments.

DNA fragmentation analysis and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) assay
For the extraction of fragmented DNA, 2.5 x 10⁶ cells/sample were incubated in serum-free culture medium for 16 h and 40 h (as indicated), then harvested and lysed in a buffer containing 0.5% Triton X-100, 5 mM Tris-HCl (pH 7.4), 20 mM EDTA. Intact nuclei were removed by centrifugation, and soluble DNA was purified by phenol extraction and ethanol precipitation. Soluble DNA was analyzed by electrophoresis on a 1.2% agarose gel. An equal number of cells was subjected to TUNEL assay following the manufacturer’s instructions (Roche Molecular Biochemicals). Apoptosis was evaluated by Fast Red (Dako Co., Carpinteria, CA) staining.

GST capture
For GST capture, experiments cells were harvested 48 h after transfection, then GST-Bad fusion proteins were purified by incubating 1 mg of cell extracts with glutathione Sepharose (Amersham Biosciences Corp.) for 2 h at 4 C, subjected to SDS-PAGE and immunoblotted.

CAT assay
Cell extracts were harvested 72 h after transfection, and CAT activity was analyzed by thin-layer chromatography, as previously described (23). The individual spots were isolated and counted in a scintillation counter. For each experiment, the percentage of conversion to acetylated cloramphenicol ¹⁴C was then calculated and normalized for the transfection efficiency. Equal transfection efficiency was confirmed for each experimental point by cotransfection with the pSV-Luc reporter plasmid, and measuring of the luciferase activity.

Immunocomplex Akt kinase assay
To appreciate the effects of the Shp-2 (C/S) we transfected PC12 cells at low molar ratio (Ret:Akt = 1:6). These conditions were sufficient to observe the stimulation of pAkt, still enabling to observe the interfering action of Shp-2 (C/S). In these conditions, phosphorylation of Akt was stimulated up to 4-fold over the background by Ret^M918T and less than 1.5-fold by Ret^C634Y. Forty-eight hours after transfection, cells were harvested, and 500 µg of total cell lysate were immunoprecipitated with anti-HA antibody for 2 h at 4 C. Samples were washed three times with kinase buffer [20 mM HEPES (pH 7.4), 10 mM MgCl₂, 10 mM MnCl₂], and incubated in the same buffer containing 60 µg/ml histone 2B from Upstate Biotechnology Inc. (Lake Placid, NY), 1 mM ATP, 1 mM dithiothreitol, and 10 µCi of [-³²P]ATP for 30 min at 25 C and stopped with Laemmli buffer. The expression of Ret mutants upon transient transfections was confirmed indirectly by performing parallel assays of Egr-1-CAT activity. The products of the kinase reactions were separated on SDS-PAGE, blotted, and autoradiographed.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Both Ret^C634Y and Ret^M918T form a complex with Shp-2
Given that GDNF may induce the formation of a multiprotein stable complex in which Shp-2 recruitment to Ret is mediated by Gab2 (9), we first examined whether similar interactions might take place between constitutively active Ret mutants (Ret^C634Y or Ret^M918T) and Shp-2. To this aim, we used PC12-derived cell lines stably transfected with either Ret^wt, Ret^C634Y and Ret^M918T alleles (PC12/wt, PC12/MEN2A, and PC12/MEN2B, respectively). These cell lines express comparable levels of the Ret, Shp-2, and Gab2 proteins (Ref. 25 and Fig. 1). Cell extracts were immunoprecipitated with anti-Gab2 antibodies and analyzed by immunoblotting with anti-pTyr, anti-Ret and anti-Shp-2 antibodies. As shown (Fig. 1A, right upper panel), the active Ret proteins (either Ret^C634Y or Ret^M918T) and Shp-2 were both immunoprecipitated together with Gab2. On the other hand, the unstimulated Ret^wt did not coimmunoprecipitate with Gab2 (first lane). As determined by hybridizing with antiphosphotyrosine antibodies such complex consists of several tyrosine phosphorylated proteins, three of which can be identified as Ret, Shp-2, and Gab2 based on the respective molecular sizes (Fig. 1A, left panel). Furthermore Shp-2 and the active Ret mutants, but not the wild-type inactive Ret, were found in the same immunocomplex (Fig. 1B), though, Gab2-Ret^C634Y complex binds Shp-2 at a much lesser extent compared with Gab2-Ret^M918T (Fig. 1A, right panel). On the other hand, the active mutant Ret proteins, but not Shp-2, coimmunoprecipitate with the p85 regulatory subunit of the PI3Kinase, that has been previously described to be recruited to the active Ret complex. Consistently with previous reports, our results indicate that active Ret is functionally coupled to tyrosine phosphorylated Shp-2, and interaction is likely mediated by Gab2 but not by p85. Whether Ret is itself substrate of Shp-2, and whether molecular partners, other than Gab2, mediate the interactions of Shp-2 with Ret^C634Y is under investigation.

View larger version (40K): [in this window] [in a new window]   FIG. 1. Ret and Shp-2 proteins form a complex with Gab2. A, One milligram of proteins from PC12/wt, PC12/MEN2A, and PC12/MEN2B cells was immunoprecipitated (IP) with anti-Gab2 antibodies and immunoblotted (IB) with antiphosphotyrosine antibodies (left panel), the proteins with a molecular size corresponding to Ret, Gab2, and Shp-2 are marked by the arrows. The filter was then stripped, cut into distinct strips, and each hybridized with anti-Ret, anti-Gab2, and anti-Shp-2 antibodies (right panel). B, Cell extracts (1 mg) from the indicated cell lines were immunoprecipitated with either anti-Shp-2, anti-Ret, or anti-p85 antibodies and immunoblotted with anti-Shp-2 anti-Ret or anti-phosphotyrosine antibodies, as indicated. All cell lines used, express comparable amounts of the Ret protein. Cos7/Shp-2 myc cells are Cos7 cells transfected with the SH-PTP2-myc tagged plasmid. Fifteen micrograms of total protein lysate (wl) were used as positive control of hybridization for Shp-2.

Thus, we used an Egr-1-CAT plasmid in which the CAT expression was driven by the Egr1 promoter. We cotransfected PC12 cells with increasing amounts of an expression construct for Shp-2 together with a construct for Ret^C634Y. Transfecting Shp-2 (wt) did not induce any relevant change of the CAT activity (not shown). In contrast, the Shp-2 (C/S) mutant inhibited the Egr-1 and c-fos-promoter dependent CAT activity (reaching up to 75% of inhibition for the Egr-1 and more than 95% for the c-fos promoter) (Fig. 2A, lanes 2–4; and B, lanes 1 and 2). As shown in Fig. 2, the extent of inhibition of Ret^C634Y effects was similar to that observed following nerve growth factor (NGF) stimulation of Egr-1-CAT (lanes 10 and 12). The persistence of CAT activity in the presence of maximal amounts of the Shp-2 (C/S) mutant suggests that, even though this phosphatase contributes to the positive regulation of downstream signaling, Shp-2-independent pathways are also relevant for Egr1 (but not c-fos) promoter induction by these tyrosine kinase receptors. Upon transfection of the Ret^C634Y together with Shp-2 (C/S), NGF stimulation did not restore the levels of Egr-1-CAT activity, thus indicating that NGF stimulates Shp-2-dependent common substrates (not shown).

View larger version (48K): [in this window] [in a new window]   FIG. 2. Shp-2 is required for Egr-1 promoter induction. A, PC12 cells were transfected with pEgr-1-CAT (2 µg) together with 0.5 µg of LTR-3 vector (lanes 9–12), or pRet9C634Y (lanes 1–4) or pRet9M918T (lanes 5–8). Where indicated, cells were also cotransfected with 2 µg (lanes 2 and 6), 4 µg (lanes 3 and 7) and 8 µg (lanes 4, 8, 11, and 12) of a plasmid encoding Shp-2 (C/S) mutant. As a control, NGF (100 ng/ml) was added 24 h after transfection (lanes 10 and 12). B, PC12 cells were transfected with pfos-CAT (2 µg) together with 0.5 µg of LTR-3 vector (lanes 5–7), or pRet9C634Y (lanes 1 and 2) or pRet9M918T (lanes 3 and 4). Where indicated, cells were also cotransfected with 8 µg (lanes 2, 4, and 7) of a plasmid encoding Shp-2 (C/S) mutant. As a control, NGF (100 ng/ml) was added 24 h after transfection (lanes 6 and 7). In all experiments, total transfected DNA was kept constant by adding increasing amounts of the empty vector. Increasing amounts of the Shp-2 protein were confirmed by immunoblot with anti-myc antibody. The fold values of CAT activity in the presence of Shp-2 (C/S) mutant have been calculated over the control (in the absence of phosphatase) set to 1 (Fig. 2A, lanes 1, 5, and 10; and Fig. 1B, lanes 1 and 6). The results represent an example of three separate transfections performed in duplicate; the results of individual transfection varied by less than 25%.

Shp-2 mediates Ret^M918T-induced cell survival
Given the importance of the PI3K/Akt signal cascade for the transforming activity of Ret (26), we asked whether binding of Shp-2 might be involved in the induction of survival signals triggered by Ret mutants. In agreement with previous reports (26, 27), expressing either Ret^C634Y or Ret^M918T oncogenes protects PC12 cells by trophic withdrawal-induced apoptosis. Using PC12/MEN2A and PC12/MEN2B cell lines we show that the expression of Ret^M918T rescued PC12 and PC12/wt cells from apoptosis more efficiently compared with Ret^C634Y, and further delayed the genomic DNA fragmentation (Fig 3). Because Ret^C634Y protects PC12 cells from serum withdrawal-mediated apoptosis by activating the PI3K/Akt cascade (27), we determined whether Shp-2 activity might be involved in the activation of this pathway. To this aim, we generated stable PC12/MEN2A and PC12/MEN2B derived-cell lines that stably express the Shp-2 (C/S) and analyzed the levels of Akt Ser 473 phosphorylation, as indicative of its activation (28). To this aim, we took advantage of a PC12/wt derivative cell line that expresses GFR1 (PC12-1/wt). In these cells, GDNF causes activation of Ret^wt kinase and neurite outgrowth (data not shown). As shown in Fig. 4A, in PC12-1/wt cells GDNF rapidly induces phosphorylation of endogenous Akt, similarly, in both PC12/MEN2A and PC12/MEN2B cells, Akt phosphorylation was constitutive, although at reduced extent compared with acute stimulation. On the other hand, stable expression of Shp-2 (C/S) in PC12/MEN2A^{Shp-2 C/S} and PC12/MEN2B^{Shp-2 C/S} strongly reduced phosphorylation of Akt compared with parental cells (PC12/MEN2A and PC12/MEN2B cells, respectively), even though the extent of inhibition largely varied among all the clones analyzed (not shown; and Materials and Methods). Therefore, to further confirm the implication of Shp-2 in Akt activation and to avoid possible artifacts caused by the clonal expansion of stable Shp-2 (C/S) transfectants, we performed transient transfections in PC12 cells. However, because only a fraction of cells became transfected (approximately 10% as determined with a GFP expressing vector, not shown), any interference of the exogenous Shp-2 (C/S) on the overall extent of phosphorylation of endogenous Akt could not be appreciated. Therefore, we transfected the PC12 cells with a reporter construct for HA-Akt together with that expressing the mutated Ret protein (either Ret^C634Y or Ret^M918T), and analyzed the levels of Akt Ser 473 phosphorylation. Furthermore, to better appreciate the inhibitory effects of the Shp-2 (C/S) we performed all experiments in nonsaturating conditions of Ret stimulation (Materials and Methods). As shown in Fig. 4B, phosphorylation of Akt was clearly stimulated by Ret^M918T, but remained low upon expression of Ret^C634Y, indicating that this pathway is preferentially activated by the Ret protein carrying the methionine 918 to threonine mutation. Thus, to investigate the involvement of Shp-2 in Ret^M918T signaling we induced Akt activation by expressing Ret^M918T together with the catalytically inactive Shp-2 (C/S) phosphatase. Expression of Shp-2 (C/S) strongly reduced both phosphorylation and kinase activity of Akt (Fig. 4, C and D).

View larger version (19K): [in this window] [in a new window]   FIG. 3. PC12/MEN2A and PC12/MEN2B cells resist to serum starvation at different degrees. A, PC12, PC12/MEN2A, and PC12/MEN2B cells were serum starved for 16 h, and the percent of apoptosis was determined by TUNEL assay. The bar graphs indicate values scored from six different frames analyzed (with at least 20 cells analyzed per frame). B, Soluble DNA was extracted from PC12/wt, PC12/MEN2A, and PC12/MEN2B cells as indicated starved for 16 and 40 h.

View larger version (29K): [in this window] [in a new window]   FIG. 4. Shp-2 is involved in cell survival. A, Equal amounts of total protein extracted from PC12-1/wt cells either untreated or treated with GDNF (50 ng/ml), PC12/MEN2A, PC12/MEN2B, PC12/MEN2AShp-2 C/S, PC12/MEN2BShp-2 C/S cells (as indicated) were analyzed by immunoblot with anti-pAkt, anti-Akt and anti-Myc antibodies. B–F, PC12 cells were transient transfected with Shp-2-C/S plasmid, pRet9C634Y or pRet9M918T (as indicated) together with either HA-Akt (B–D) or with GST-Bad plasmids (E and F). Cells were kept 8 h in serum free medium then harvested. Cell lysates were immunoprecipitated (IP) with anti-HA antibody and immunoblotted (IB) with anti-pAkt (B and C, upper panel), or subjected to immuno kinase assay (D, upper panel). The amount of immunoprecipitated HA-Akt protein was checked by immunoblot of the same membrane with anti-HA antibodies (B–D, lower panel). After purification GST-Bad proteins were immunoblotted with a mixture of anti-pBad112 and -pBad136 antibodies (E and F, upper panel), or single antibodies (not shown). Same filter was hybridized with anti-Bad antibodies (E and F, lower panel). Similar results were obtained by immunoblot of total cell lysates (not shown). The values of pAkt and pBad induction in the presence of Ret or Ret mutants have been calculated using the NIH Image Program, as fold increase over the control (in the absence of Ret stimulation) set to 1, except for panel E, in which the control was not measurable. nm, Not measurable.

Shp-2 mediates GDNF-induced signaling
To further support the implication of Shp-2 in Ret signaling, we addressed the question of the involvement of Shp-2 in GDNF induced intracellular signaling. To this aim, we took advantage of a PC12-1/wt cells. In these cells, GDNF stimulates immediate early gene expression and phosphorylation of endogenous Akt several times (Fig. 4A and data not shown). As shown in Fig. 5A, transfecting the Shp-2 (C/S) mutant inhibited both the Egr-1 and c-fos promoter-dependent CAT activity.

View larger version (25K): [in this window] [in a new window]   FIG. 5. Involvement of Shp-2 in GDNF-induced signaling. A, PC12-1/wt cells were transfected with pEgr-1-CAT (left panel) or pfos-CAT (right panel) together with Shp-2 (C/S), as indicated. 24 h later, GDNF (50 ng/ml) was added for 48 h, as indicated. The fold values of CAT activity in the presence of Shp-2 (C/S) mutant have been calculated over the control (in the absence of phosphatase) set to 1 (see also legend to Fig. 2). B, PC12-1/wt cells were transient transfected with HA-Akt fusion protein together with Shp-2-C/S plasmid, as indicated. Thirty-six hours after the transfections, cells were starved for 12 h and then stimulated 5 min with GDNF (100 ng/ml). Cell lysates were immunoprecipitated (IP) with anti-HA antibodies, and immunoblotted (IB) with anti-pAkt (upper panel) or, to confirm equal loading, anti-HA (lower panel) antibodies.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Here we report that Shp-2 activity mediates multiple signaling pathways initiated by two mutants of the Ret oncogene, each being responsible for a distinct disease: Ret^C634Y causes inheritance of MEN-2A and, rarely, FMTC, whereas Ret^M918T causes inheritance of MEN-2B. Indeed, by using stable Ret transfectants (PC12/MEN2B and PC12/MEN2A) first we show that in PC12 cells Shp-2 may be recruited to a complex with the oncogenic active Ret molecules (either Ret^M918T or Ret^C634Y), but not with unstimulated Ret^wt, and likely involving binding to Gab2. The finding that constitutively active Ret molecules may form a complex with Shp-2, although in good agreement with recent reports of GDNF-dependent Shp-2 interaction with the wild-type Ret (9, 29), looks in contrast with what reported by expressing a different Ret mutant carrying a 9-bp duplication (Ret-9bp) in NIH 3T3 (11). The most plausible interpretation for this discrepancy likely relies on the evidence reported here that binding to Shp-2 would also depend on which Ret mutant allele is involved (i.e. binding to Ret^C634Y is less efficient compared with Ret^M918T). Whether phosphorylation or recruitment of specific Ret substrates may account for these differences is under investigation.

Once recruited to the active Ret, Shp-2 likely interacts with other effector molecules and acts as a positive regulator of downstream signaling. Indeed, in analogy with reports using NGF (30), the induction by Ret of immediate-early gene expression is depressed by the expression of a dominant interfering mutant of Shp-2. Drastic inhibition by the Shp-2 mutant was observed either with GDNF-stimulated Ret^wt or with any of the mutant oncogenes. Given that both Ret mutants require the continuous activation of Ras/Erk pathway to induce the expression of the immediate early genes (25), this indicates that activation of this cascade would be mediated by Shp-2, though the extent depends on the Ret genetic background. This is consistent with several reports that implicate the phosphatase activity of Shp-2 in the activation of the Ras/Erk pathway by several receptor tyrosine kinase (16).

On the other hand, as recently reported, Shp-2 acts to regulate strength and duration of PI3K/Akt cascade in a receptor-specific manner (22). In this context, our results, indicating that the Shp-2 activity is required for the full activation of the Akt pathway specifically if induced by the Ret^M918T mutant, are consistent with the indication that Ret^M918T may recruit specific molecules otherwise poorly recruited by the GDNF-stimulated Ret^wt or by Ret^C634Y (8, 31). Indeed, the threonine for methionine substitution at codon 918 lies within the catalytic domain of Ret and results in a shift in substrate specificity. As a consequence, the mutated Ret^M918T preferentially phosphorylates optimal substrates for Src and Abl (8). On the other hand, a recent report (32) showing a stronger activation of Akt by the Ret^M918T mutant is consistent with our observation of the poor stimulation of this pathway by either GDNF-stimulated Ret^wt or by Ret^C634Y.

The implication of Shp-2 in Ret signaling looks in contrast with the evidence that, in NIH 3T3 cells, the overexpression of Shp-2 did not reduce the autophosphorylation of an active Ret mutant allele (11). Although here we show results obtained with different Ret mutants and cell system, the most plausible interpretation for this apparent discrepancy is that Ret likely neither bind Shp-2 nor is a direct substrate of the phosphatase activity. This interpretation is strongly supported by recent evidences showing that recruitment of Shp-2 to GDNF-stimulated Ret^wt is indirect and mediated by binding to a docking protein, even though the identity of the docking protein involved in Shp-2 binding is not unique and likely depends on the experimental cell system (9, 29). Furthermore, the results shown here were obtained either with GDNF-stimulated Ret^wt or with each of two different Ret mutants, thus indicating that in PC12 cells Shp-2 activity is crucial for Ret downstream signaling, though we cannot exclude that Shp-2 substrate(s) recruited to Ret mutants may be different (or present at different concentrations) in the different cell lines, i.e. in PC12 compared with NIH 3T3 cells. On the other hand, given that the related Shp-1 tyrosine phosphatase has opposite, or no, effects on Ret signaling (Ref. 11 ; and Califano, D., manuscript in preparation), it seems likely that Shp-2 acts in a specific way to mediate Ret signaling.

In summary, the experiments presented demonstrate that in PC12 cells both Ret^M918T and Ret^C634Y mutants may be found in complex with Shp-2, and indicate that this phosphatase mediates distinct functional interactions that depend on which Ret mutant was present in such a complex. These findings makes evident a main molecular difference between these Ret mutants and indicate this phosphatase as a key substrate involved in determining the difference between the MEN-2A and MEN-2B associated diseases.

	Acknowledgments

 
This is dedicated to the memory of Franco Tatò and Giulia Colletta. We thank D. Di Taranto for continuous helping during the work.

	Footnotes

 
This work was supported by the European Union Grant (Contract No. QLG1-CT-2000-00562) and by a Ministero Istruzione Università e Ricerca-Fondo Investimenti Ricerca di Base grant.

Abbreviations: CAT, Chloramphenicol acetyl transferase; FMTC, familial medullary thyroid carcinoma; GDNF, glial cell line-derived neurotrophic factor; GFR1-4, glycosyl-phosphatidylinositol-anchored receptor 1-4 family; GST, glutathione-S-transferase; HA, hematagglutinin; MEN, multiple endocrine neoplasia; NGF, nerve growth factor; Ret, receptor tyrosine kinase; Shp2, Src homology 2-containing tyrosine phosphatase; TUNEL, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling.

Received May 20, 2003.

Accepted for publication June 10, 2003.

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Top Abstract Introduction Materials and Methods Results Discussion References

 

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