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Endocrinology Vol. 142, No. 10 4441-4447
Copyright © 2001 by The Endocrine Society

ARTICLES

Inhibition of Ret Oncogene Activity by the Protein Tyrosine Phosphatase SHP1

Anita M. Hennige, Reiner Lammers, Wolfgang Höppner, Dorit Arlt, Volker Strack, Reinhard Teichmann, Fausto Machicao, Axel Ullrich, Hans-Ulrich Häring and Monika Kellerer

Universität Tübingen (A.M.H., R.L., V.S., F.M., H.-U.H., M.K.), Medizinische Klinik und Poliklinik IV, D-72076 Tübingen, Germany; Institut für Hormon- und Fortpflanzungsforschung (W.H., D.A.), D-22529 Hamburg, Germany; Chirurgische Universitätsklinik (R.T.), D-72076 Tübingen, Germany; and Max-Planck-Institut für Biochemie (A.U.), D-82152 Martinsried, Germany

Address all correspondence and requests for reprints to: Dr. Monika Kellerer, University of Tübingen, Medizinische Klinik und Poliklinik IV, Otfried-Müller-Strasse 10, D-72076 Tübingen, Germany. E-mail: makellerer{at}med.uni-tuebingen.de


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Germline mutations in the Ret protooncogene give rise to the inherited endocrine cancer syndromes MEN types 2A and 2B and familiar medullary thyroid carcinoma. Although it is well accepted that the constitutive active tyrosine kinase of Ret oncogenes ultimately leads to malignant transformation, it is not clear whether a decrease in the autophosphorylation of oncogenic Ret forms can affect the mitogenic and transforming activities of Ret. Potential modulators of the tyrosine kinase activity of Ret could be tyrosine phosphatases that are expressed in human thyroid tissue. Therefore, we investigated the impact of the tyrosine phosphatases SHP1 and SHP2 on the intrinsic tyrosine kinase activity and oncogenic potency of Ret with a 9-bp duplication in the cysteine-rich domain (codons 634–636), which was described in a patient with MEN type 2A recently. SHP1 and SHP2 were stably overexpressed in NIH3T3 fibroblasts together with Ret-9bp. Coexpression of SHP1 with Ret-9bp reduced the autophosphorylation of Ret-9bp by 19 ± 7% (P = 0.01, n = 4), whereas no effect was seen with SHP2. Furthermore, Ret-9bp could be coimmunoprecipitated with SHP1 but not with SHP2 antibodies. Suppression of the Ret-9bp tyrosine kinase activity by SHP1 caused a decrease in activation of Erk2 (extracellular signal-regulated kinase) and abolished PKB/Akt (protein kinase B) phosphorylation. In addition, diminished Ret-9bp autophosphorylation led to reduced phosphorylation of the transcription factor jun-D. Finally, the inhibitory effect on Ret-9bp signaling resulted in a 40–60% reduction of [3H]thymidine incorporation and in reduced ability of NIH3T3 cells to form colonies in soft agar. In conclusion, the data suggest that SHP1 caused a moderate reduction of Ret autophosphorylation, which led to a strong suppression of the Ret oncogene activity.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
MULTIPLE ENDOCRINE NEOPLASIA type 2 and familial medullary thyroid carcinoma are inherited diseases that arise from different mutations in the Ret protooncogene (1, 2, 3). The Ret gene encodes a transmembrane receptor with intrinsic tyrosine kinase activity (4). Several Ret ligands have been described that induce rapid and transient activation of the receptor tyrosine kinase (5, 6, 7, 8, 9, 10).

Most of the MEN type 2A mutations were identified in the cysteine-rich extracellular domain of Ret. Conversion of the Ret protooncogene into the oncogenic form is provoked by these mutations, which cause dimerization and ligand-independent activation of the intrinsic tyrosine kinase (11). It has been suggested that constitutive activation of the intrinsic tyrosine kinase is responsible for oncogenic transformation (11). Therefore, it would be interesting to know whether a reduction of Ret autophosphorylation could change intracellular signaling and oncogenic transformation. This might be important information for a potential therapeutic use of specific inhibitors for the Ret tyrosine kinase. Among the candidates for the modulation of Ret tyrosine kinase activity are protein tyrosine phosphatases. A role of phosphatases in the regulation of endocrine tumors has already been indirectly suggested by studies using somatostatin or its analogs. These compounds have been used therapeutically for endocrine cancers such as carcinoids, and it has been demonstrated that somatostatin could activate the protein tyrosine phosphatase SHP1 in several cell lines (12, 13, 14, 15). In addition, there is evidence that the tyrosine phosphatase SHP2 is part of a signaling complex induced after activation of the Ret protooncogene (16). Therefore, we tried to elucidate the effect of the protein tyrosine phosphatase SHP1 and the homologous phosphatase SHP2 on signaling of the Ret-9bp oncogene. We used Ret-9bp (repeat of codons 634–636), which was described in a patient with MEN type 2A (17). This 9-bp duplication caused ligand-independent activation of the intrinsic tyrosine kinase of Ret and functions like many other Ret mutations in the cysteine-rich domain (18, 19). Our data show that the tyrosine phosphatase SHP1 caused a moderate reduction in the autophosphorylation of the Ret-9bp mutant in NIH3T3 cells. This inhibition of Ret-9bp phosphorylation reduced specifically Erk2- (extracellular signal-regulated kinase) and PKB/Akt- (protein kinase B) activity and resulted in partial conversion of the oncogenic phenotype.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Cell culture reagents and FCS were purchased from Life Technologies, Inc.(Karlsruhe, Germany). Aprotinin, phenylmethylsulfonyl fluoride, Na3VO4, and Triton X-100 were from Sigma (Munich, Germany). Nitrocellulose was from Sartorius (Göttingen, Germany). All other reagents were of the best grade commercially available. Visualization of immunocomplexes after immunoblotting was performed with the nonradioactive enhanced chemiluminescence system and Hyperfilm-ECL from Amersham Pharmacia Biotech (Braunschweig, Germany). Antibodies used in this study are commercially available: the polyclonal anti-RET (C-19)-G, the anti-SHP1 and anti-SHP2 antibodies, and the monoclonal antiphosphotyrosine antibody (PY99) were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Anti-phospho MAPK (Erk), anti-phospho-Akt1/PKB {alpha} (Ser473), and anti-phospho-c-jun (Ser73) were purchased from Upstate Biotechnology, Inc. (Hamburg, Germany).

RT-PCR
Total RNA was isolated from human thyroid and a tumor metastasis of a medullary thyroid carcinoma using the optimized phenol guanidinium isothiocyanate extraction method (peqGold triFast, PeqLab, Erlangen, Germany). Subsequently, 1 µg of total RNA was reverse transcribed using avian myeloblastoma virus reverse transcriptase. PCR primer sequences were 5'-caacatcgtgtaggacaacc-3' upstream and 5'-gcagagttcgagaactaagc-3' downstream for SHP1 and 5'-ggtgaatgcggctgacattg-3' upstream and 5'-tcattgaacgtggcctccag-3' downstream for SHP2, yielding a 267-bp product for SHP2 and a 368-bp product for SHP1. Cycling parameters for SHP2 were 5 min at 95 C followed by 35 cycles of 1 min at 57 C, 1 min at 72 C, and 5 min at 72 C. Cycling parameters for SHP1 were 5 min at 95 C followed by 35 cycles of 5 min at 95 C, 1 min at 61 C, 1 min at 72 C, and 5 min at 72 C.

Cell culture and transfection
Mouse NIH3T3 cells were grown in DMEM containing 10% FCS. Stable transfection of NIH3T3 fibroblasts was performed by the calcium phosphate precipitation method as described (20). Cells were grown in 60-mm dishes to 70% confluence. Plasmid DNA [Ret-wt or Ret-9bp in pRc/CMV2 vector, SHP1, or catalytically inactive SHP1 mutant (SHP1*) created by a point mutation at position 455cys to ser in pRK5 vector] plus pSV2neo were mixed with CaCl2 and 2x transfection buffer. G418-resistant colonies were selected in DMEM plus G418 (500 µg/ml). The expression levels of the proteins in different clones were compared by immunoblotting.

KELLY cells (human neuroblastoma cell line; European Collection of Cell Cultures reference number 92110411) were purchased from the German Collection of Microorganisms and Cell Cultures (DSMZ) (Braunschweig, Germany). Cells were cultured in RPMI 1640 with 2% glutamine and 10% FCS in 150-mm culture dishes to 80% confluence. After overnight starvation with 0.5% FCS, cells were stimulated with 50 ng/ml glial cell line-derived neurotrophic factor (GDNF){alpha} for 10 min.

Immunoblot analysis and immunoprecipitation
NIH3T3 cells overexpressing Ret-wt or Ret-9bp were lysed on ice with 50 mM HEPES buffer containing 150 mM NaCl, 1.5 mM MgCl2, 1 mM EGTA, 10% glycerol, 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, 10 mg/liter aprotinin, 0.4 mM orthovanadate, 10 mM Na4P2O7, and 100 mM NaF. Insoluble material was centrifuged at 10,000 x g for 10 min at 4 C. The supernatant was boiled for 5 min in Laemmli sample buffer (reducing), and proteins were separated by SDS-PAGE (7.5%). For immunoprecipitation, samples were tumbled for 12 h at 4 C with the polyclonal antibody anti-Ret, anti-SHP1, or anti-SHP2 (in dilutions of 1:500) and protein A-Sepharose. The immunoprecipitate was washed four times with ice-cold lysis buffer containing 0.1% Triton X-100. Immuncomplexes were boiled and dissociated after addition of 10 µl of Laemmli buffer, and proteins were separated by 7.5% SDS-PAGE. Proteins were transferred to nitrocellulose by electroblotting (transfer buffer, 48 mM Tris-HCl, pH 7.5, 0.04% SDS, 39 mM glycine, and 20% methanol). After transfer, the membranes were blocked with NET buffer (150 mM NaCl, 5 mM EDTA, 50 mM Tris, 0.05% Triton X-100, and 0.25% gelatin, pH 7.4) for 1 h. Subsequently, filters were incubated with the first antibody overnight at 4 C. The membranes were washed four times with NET buffer before incubating with horseradish peroxidase-conjugated antimouse IgG or antirabbit IgG for 1 h at room temperature. Visualization of immunocomplexes was performed by enhanced chemiluminescence.

Determination of [3H]thymidine incorporation
To measure [3H]thymidine incorporation, cells were grown to confluence on six-well culture plates and subsequently starved for 24 h in DMEM containing 5 mM glucose and 0.5% FCS. [3H]Thymidine (0.5 µCi/ml) was added for 4 h. The dishes were rinsed twice with ice-cold PBS and once with 10% trichloroacetic acid. After 20 min, dishes were washed once with ice-cold 10% trichloroacetic acid, cells were lysed with 500 µl of 0.2 N NaOH/1% SDS, and the lysates were neutralized with 0.5 ml of 0.2 N HCl. Incorporated radioactivity was determined by liquid scintillation counting.

Soft agar transformation assay
NIH3T3 cells (104/ml) stably expressing RET-wt or RET-9bp with or without SHP1 were seeded in culture medium (DMEM) containing 10% FCS and 0.4% agar. Colonies were photographed 3 wk after plating.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Oncogenic transformation of Ret results in constitutive autophosphorylation. In this study, we investigated whether modulation of the intrinsic tyrosine kinase activity by the protein tyrosine phosphatases SHP1 and SHP2 could change the signaling properties and the oncogenic potency of Ret-9bp. These tyrosine phosphatases were chosen because they are expressed in human thyroid as well as in a tumor metastasis of a medullary thyroid carcinoma (Fig. 1Go), and recently, evidence was presented that SHP2 is part of a signaling complex induced by ligand stimulation of c-Ret, although no direct association with c-Ret has been demonstrated (16). Moreover, it has been reported that somatostatin and its analogs, which are used as therapeutic agents for different endocrine tumors, can activate the tyrosine phosphatase SHP1 (12, 13, 14, 15). To investigate potential interaction between the Ret-9bp oncogene and the tyrosine phosphatases SHP1 and SHP2 as well as the functional consequences of such a protein interaction on the Ret oncogene, we established stably transfected NIH3T3 cell lines expressing the Ret-9bp oncogene together with SHP1 or SHP2. Different clones expressing equal amounts of Ret-9bp together with SHP1 or SHP2 were screened. To evaluate the autophosphorylation of Ret-9bp in the presence or absence of SHP1 or SHP2, aliquots of total cell lysates were immunoprecipitated with anti-Ret antibody and run on SDS-PAGE. Separated proteins were transferred to nitrocellulose filters and blotted with the antibodies indicated in Fig. 2Go. As shown at the bottom of that figure, Ret protein could only be detected in transfected NIH3T3 cells and not in parental cells, suggesting very low expression of endogenous Ret protein. Ret was detected as a double band (Fig. 2Go) corresponding to two different glycosylated forms, as described previously (21, 22). Overexpression of Ret-9bp led to ligand-independent activation of the tyrosine kinase, which was not observed with Ret-wt. This is in agreement with other studies demonstrating constitutive activation of Ret carrying mutations in the cysteine-rich domain (11). Coexpression of SHP1 with Ret-9bp caused a 19 ± 7% reduction (P = 0.01, n = 4, t test) of Ret-9bp autophosphorylation (Fig. 2AGo). In contrast, no reduction of Ret-9bp phosphorylation was observed by coexpression with the tyrosine phosphatase SHP2 (Fig. 2BGo).



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  		Figure 1. RT-PCR product of SHP1 and SHP2 mRNA from normal human thyroid tissue and medullary thyroid carcinoma metastasis. RT-PCR products were obtained as described in Materials and Methods. Lane 1 (normal thyroid) and lane 2 (medullary thyroid carcinoma) show the 267-bp product corresponding to the transcript of SHP2. Lane 5 (normal thyroid) and lane 6 (medullary thyroid carcinoma) show a 368-bp product corresponding to the transcript of SHP1. As a control, H2O was used in the probes of lanes 3 and 7. Lane 4, Mol wt markers.

 


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  		Figure 2. Expression and tyrosine phosphorylation of Ret protein-expressing NIH3T3 cells. NIH3T3 fibroblasts were stably transfected with expression plasmids encoding Ret-wt/Ret-9bp and the tyrosine phosphatases SHP1 or SHP2. Whole cell lysates from cells expressing SHP1 (A) or SHP2 (B) with or without Ret-9bp were immunoprecipitated (IP) with anti-Ret-antibody ({alpha}-Ret). Immunocomplexes were separated by 7.5% SDS-PAGE, and phosphorylation of Ret proteins was analyzed by immunoblotting with an antiphosphotyrosine antibody ({alpha}-Ptyr, top panels). Phosphorylation was visualized using enhanced chemiluminescence. Tyrosine phosphorylation of four different experiments was quantified by scanning densitometry and expressed as percent of phosphorylation measured after immunoblotting of cell lysates from cells not overexpressing SHP1 or SHP2. To determine the amount of Ret protein, nitrocellulose filters were reprobed with anti-Ret antibodies ({alpha}-Ret, bottom panels). Two differentially glycosylated Ret species of 150 and 170 kDa were identified. A representative immunoblot of four different experiments is shown. par, NIH3T3 parental cells; wt, NIH3T3 overexpressing wild-type Ret; 9bp, NIH3T3 overexpressing the Ret-9bp mutant; 9bp/SHP, NIH3T3 cells coexpressing Ret-9bp and SHP.

 
To study whether the reduction in Ret-9bp tyrosine phosphorylation was induced by direct association of SHP1 with Ret-9bp, we performed coimmunoprecipitation studies. NIH3T3 cells stably expressing SHP1 or SHP2 alone or together with Ret-9bp were lysed, and the phosphatases were immunoprecipitated with the appropriate antibodies. The presence of SHP1 and SHP2 protein was controlled by reblotting with the appropriate antibody (Fig. 3BGo). Coimmunoprecipitated proteins were measured by immunoblotting. Fig. 3AGo shows that a 150- and 170-kDa tyrosine-phosphorylated protein was coimmunoprecipitated with SHP1 (lanes 2 and 3 represent two different cell clones expressing SHP1 together with Ret-9bp). Tyrosine phosphorylation of SHP1 is also indicated in Fig. 3AGo. Because phosphorylation of SHP1 was seen only in Ret-9bp-expressing cells, it can be suggested that SHP1 represents a direct substrate for the tyrosine kinase of the Ret oncogene. The immunoblot with Ret antibody shown on Fig. 3CGo confirms that the phosphoproteins at 150 and 170 kDa indeed correspond to the Ret protein. As a control, these 150- and 170-kDa bands were not found in immunoprecipitates from NIH3T3 cells not expressing Ret (Fig. 3AGo, lane 1). Results from SHP2-expressing cells are shown on the right. In contrast to SHP1, Ret-9bp could not be coimmunoprecipitated with SHP2 (Fig. 3Go, A and C, lane 5).



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  		Figure 3. Coimmunoprecipitation of Ret proteins together with SHP1 or SHP2. Serum-starved NIH3T3 cells overexpressing SHP1 (lane 1) or SHP2 (lane 4) alone or together with Ret-9bp (lanes 2, 3, and 5) were lysed, and equal amounts of proteins were used for immunoprecipitation with polyclonal anti-SHP1 or anti-SHP2 antibodies. Immunocomplexes were washed and boiled with Laemmli buffer. Proteins were separated on a 7.5% SDS-polyacrylamide gel and transferred to nitrocellulose filters. Phosphorylation of immunoprecipitated Ret protein was detected by immunoblotting with a monoclonal antiphosphotyrosine antibody ({alpha}-Ptyr; A). The same filters were reprobed with the polyclonal anti-SHP1 or anti-SHP2 antibodies ({alpha}-SHP1 and {alpha}-SHP2; B). Coimmunoprecipitated Ret protein was detected by reblotting of the nitrocellulose with an polyclonal anti-Ret antibody ({alpha}-Ret; C).

 
Because we could demonstrate that SHP1 was specifically able to reduce Ret-9bp autophosphorylation and to form a tight complex with Ret-9bp protein, we next tried to determine whether the decrease of Ret-9bp autophosphorylation by SHP1 affected intracellular signaling elements. Two major signaling routes of the oncogene are activation of the MAPK pathway and stimulation of PI3K and PKB/Akt (23, 24, 25, 26, 27). Therefore, we determined the impact of SHP1 coexpression on these signaling routes. Elements from the MAPK pathway (i.e. Erk1 and Erk2) as well as PKB/Akt activity and phosphorylation of transcription factors c-jun and jun-D were tested in NIH3T3 cells overexpressing Ret-9bp alone or together with SHP1. Phosphorylation of Erk was measured by immunoblotting with a site-specific phosphotyrosine antibody. The immunoblot in Fig. 4Go shows phosphorylated Erk1 at 44 kDa and Erk2 at 42 kDa. In contrast to Erk1, which is phosphorylated under all conditions, Erk2 phosphorylation was detected only in cell lysates of Ret-9bp-expressing cells (Fig. 4Go, lane 2). Coexpression of SHP1 with Ret-9bp specifically caused a reduction of Erk2 phosphorylation (Fig. 4Go, lane 3) of 79% (determined by scanning densitometry), whereas Erk1 phosphorylation was not affected. To further investigate whether this effect was induced by SHP1, we performed the same experiments with NIH3T3 cells expressing SHP1*. It can be seen from Fig. 4Go (lane 4) that expression of the mutant form of SHP1 could not induce the reduction of Erk2 tyrosine phosphorylation.



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  		Figure 4. Phosphorylation of Erk in NIH3T3 cells overexpressing Ret and SHP1. Parental NIH3T3 cells (par) or cells overexpressing Ret wild type (wt), Ret-9bp (9bp), SHP1, or catalytically inactive SHP1* were lysed, and proteins were subjected to 7.5% SDS-PAGE. Subsequently, immunoblotting with an anti-phospho-Erk antibody was performed. Tyrosine-phosphorylated Erk1 (p44) and Erk2 (p42) are shown on a representative immunoblot from four different experiments.

 
Because SHP1 coexpression caused suppression of Erk2, we tested whether signaling elements downstream in the MAPK pathways were affected as well. It has been discussed that downstream of the MAPK, c-jun is activated by Ret oncogenes (26, 28). In addition to these studies, we could show that jun-D is activated by Ret-9bp as well (19). Therefore, we tested whether SHP1 had an effect on Ret-9bp-stimulated c-jun and jun-D phosphorylation, which reflects the activation status of these transcription factors. Fig. 5Go indicates that phosphorylation of c-jun and jun-D was increased in Ret-9bp-expressing NIH3T3 cells (lane 2). Cooverexpression of SHP1 in these cells led to only a slight inhibition of c-jun phosphorylation and to a 37% reduction (determined by scanning densitometry) of jun-D phosphorylation (lane 3). Together, these data suggest that an approximately 19% inhibition of Ret-9bp autophosphorylation by SHP1 has specific effects on the MAPK pathway by specific inhibition of Erk2 phosphorylation. Moreover, jun-D phosphorylation was reduced but c-jun was almost not affected.



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  		Figure 5. Ret-dependent phosphorylation of c-jun and jun-D in NIH3T3 cells. Total cell lysates from NIH3T3 fibroblasts overexpressing equal amounts of the indicated proteins were subjected to 10% SDS-PAGE, and proteins were transferred to nitrocellulose membranes. Phosphorylation of c-jun and jun-D was tested by immunoblotting with anti-phospho-c-jun antibodies that cross-react with jun-D. To visualize differences in c-jun phosphorylation, two different times of exposure are shown (top, 30 sec; bottom, 5 min) using the enhanced chemiluminescence system.

 
Recently, it has been demonstrated that cell transformation induced by Ret oncogene Cys634 was strongly dependent on PKB/Akt activation (25). This is in agreement with our observation with the Ret-9bp mutation (19). To study whether SHP1 affects this signaling pathway as well, we measured the phosphorylation and thereby the activation status of PKB/Akt with a site-specific antibody, detecting the phosphoinositide-dependent kinase-dependent serine phosphorylation site at position 473. The immunoblot with the anti-PKB/Akt antibody showed increased phosphorylation of PKB/Akt in lysates that were prepared from Ret-9bp-expressing cells (Fig. 6Go, lane 3) compared with lysates from NIH3T3 cells expressing Ret-wt (lane 2) or parental cells (lane 1). This Ret-9bp-dependent PKB/Akt activation was completely prevented by cooverexpression of SHP1 (lane 4). The inhibitory role of SHP1 on Ret-9bp signaling was further confirmed in cells stably coexpressing catalytically inactive SHP1 with Ret-9bp. Because this SHP1* mutant was not able to reduce Ret-9bp-induced PKB/Akt phosphorylation (lane 5), we suggest that the intact catalytic domain of the phosphatase is required for PKB/Akt inhibition.



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  		Figure 6. Phosphorylation of PKB/Akt in Ret-9bp-expressing cells cotransfected with SHP1 or catalytically inactive SHP1*. NIH3T3 cells overexpressing equal amounts of Ret proteins (lanes 2–5) together with SHP1 (lane 4) or mutant SHP1* (lane 5) and untransfected cells (lane 1) were lysed and separated by 7.5% SDS-PAGE. PKB/Akt phosphorylation was detected by immunoblotting with an anti-phospho-Akt1 antibody specifically recognizing phosphorylated Ser473. Serine phosphorylation of Akt in NIH3T3 cells is shown on a representative immunoblot from four different experiments. The effect of coexpression of Ret-9bp with SHP1 or mutant SHP1* on Akt1 phosphorylation is shown in lanes 4 and 5.

 
Having demonstrated an inhibition of Ret-9bp intracellular signaling pathways in NIH3T3 cells coexpressing SHP1, we tried to elucidate whether this could affect the mitogenic and transforming activity of Ret-9bp. Therefore, we determined [3H]thymidine incorporation as well as the ability of NIH3T3 fibroblasts to form colonies in soft agar. Fig. 7AGo shows quantitative data from four different [3H]thymidine incorporation studies. NIH3T3 fibroblasts overexpressing Ret-9bp revealed a 6-fold higher thymidine incorporation rate compared with Ret-wt. Cooverexpression of SHP1 (clones 6 and 19) reduced the thymidine incorporation rate to 40% and 60%, respectively (P < 0.001, n = 4). The Ret and SHP1 protein amounts were detected by immunoblotting (Fig. 7Go, B and C). More pronounced inhibition (60%) was found in NIH3T3 cells with higher expression levels of SHP1 (clone 6). As a control, no inhibitory effect on thymidine incorporation was observed with the catalytically inactive SHP1 (Fig. 7AGo, cell clones 18 and 20).



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  		Figure 7. [3H]Thymidine incorporation rate in NIH3T3 cells overexpressing Ret-9bp and SHP1 or catalytically inactive SHP1*. NIH3T3 cells overexpressing Ret and SHP1 or mutant SHP1* were incubated for 4 h with 0.5 µCi/ml [3H]thymidine. Cells were washed, and trichloroacetic acid-precipitable radioactivity was measured in a liquid scintillation counter. Results in A are mean values ± SD of four experiments performed in duplicate. A t test was performed for statistical analysis. Aliquots of whole cell lysates were subjected to SDS-PAGE to determine the protein expression level of Ret and SHP1 (B and C).

 
Finally, we studied the transforming activity of Ret-9bp- and SHP1-expressing cells. NIH3T3 cells overexpressing Ret-wt or Ret-9bp with or without SHP1 were seeded in soft agar and cultured for 3 wk. It can be seen in Fig. 8Go (representative for three different experiments) that the ability to form colonies in soft agar was clearly reduced in NIH3T3 cells coexpressing Ret-9bp with SHP1, suggesting that SHP1 caused suppression of Ret-9bp-dependent cell transformation.



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  		Figure 8. Transforming activity of NIH3T3 cells overexpressing Ret-wt, Ret-9bp, or Ret-9bp together with SHP1 or SHP1*. Colony formation was tested in soft agar containing NIH3T3 cells (104/ml) stably expressing Ret-wt or Ret-9bp together with SHP1 or the catalytically inactive SHP1*. Colonies were photographed 3 wk after plating. Results were confirmed in four independent assays.

 
Because we studied RET oncogene interaction with SHP1 in transfected NIH3T3 cells, we tried to confirm the interaction of Ret and SHP1 in a more physiological cell background. Therefore, we performed additional studies in the human neuroblastoma cell line KELLY. In this cell line, we demonstrated endogenous Ret as well as SHP1 protein expression by immunoprecipitation with the appropriate antibody (Fig. 9Go, A and B). In addition, SHP1 could be coimmunoprecipitated with Ret in the basal as well as the GDNF{alpha}-stimulated condition. In agreement with our results from constitutively activated Ret-9bp in NIH3T3 cells (Fig. 3AGo), we demonstrated GDNF{alpha}-dependent tyrosine phosphorylation of SHP1. In summary, these results confirm that SHP1 protein is endogenously expressed in human neuroblastoma cells. Furthermore, SHP1 is associated with Ret and phosphorylated in a GDNF{alpha}-dependent manner in KELLY neuroblastoma cells. Therefore, SHP1 may be an important target in the regulation of Ret-induced tumors.



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  		Figure 9. Ret and SHP1 interaction in the human neuroblastoma cell line KELLY. KELLY cells were grown in 150-mm culture dishes and stimulated with or without GDNF{alpha} (50 ng, 10 min) as indicated. The cell lysates from one culture dish were immunoprecipitated either with polyclonal anti-Ret antibody (A), anti-SHP1 antibody (B), or Ig as a control. Immunocomplexes were washed and boiled with Laemmli buffer. Proteins were separated on a 7.5% SDS-polyacrylamide gel and transferred to nitrocellulose filters. Protein expression of Ret and SHP1 in KELLY cells was detected by immunoblotting with the appropriate antibody as indicated. The filter containing Ret immunoprecipitates was reprobed with the polyclonal anti-SHP1 antibody ({alpha}-SHP1) and a monoclonal antiphosphotyrosine antibody ({alpha}-pTyr), which are seen in the bottom panels of A.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Like many other oncogenes, Ret-9bp is characterized by constitutive active intrinsic tyrosine kinase, which results in cellular transformation (19). In this study, we investigated the impact of the protein tyrosine phosphatases SHP1 and SHP2 on Ret-9bp oncogene activity. We found evidence for a negative regulatory role of SHP1 on autophosphorylation and on the transforming ability of Ret-9bp, whereas no effect was seen with SHP2. Thus, the dephosphorylation of Ret-9bp seems to be rather specific for SHP1. Because SHP1 antibodies were able to coimmunoprecipitate Ret-9bp with SHP1, it is suggested that dephosphorylation of Ret-9bp occurs by a direct association of SHP1 with Ret-9bp. Evidence is presented that several tyrosine kinase receptors are able to activate SHP1 through phosphorylation on Tyr538 (29, 30). In addition, we were able to demonstrate a direct association of Ret-9bp with SHP1 as well as Ret oncogene-dependent tyrosine phosphorylation of SHP1. Therefore, SHP1 might be activated directly by Ret-9bp, and subsequently, the Ret-9bp oncogene seems to be dephosphorylated by SHP1. Because SHP1 causes only a moderate reduction of Ret-9bp tyrosine phosphorylation (approximately 20%) and a more pronounced inhibition of the downstream intracellular signaling proteins Erk2 and PKB/Akt, it can be speculated that SHP1 dephosphorylates at a crucial tyrosine site of Ret. Several tyrosine phosphorylation sites of Ret have been identified, with an important role of Tyr1062 for binding of SHC and the regulatory subunit of PI3K and for transforming ability (25, 31). Therefore, it can be assumed that SHP1 affects only Tyr1062 phosphorylation, which is important for intracellular adaptor protein binding. This could also explain why a small decrease in autophosphorylation of Ret-9bp has a rather large impact on signal transduction.

To evaluate the influence on intracellular signaling, we investigated two major signaling pathways, i.e. the MAPK and the PKB/Akt pathways, which are known to be activated by oncogenic forms of Ret (19, 23, 24, 25, 26, 27, 32). From these studies, it appears that the reduction of Ret-9bp phosphorylation induced by SHP1 has marked inhibitory effects on Erk2 as well as PKB/Akt phosphorylation. In contrast to Erk2, phosphorylation of Erk1 was not induced by Ret-9bp. In summary, the almost complete reduction in Erk2 and PKB/Akt phosphorylation suggests that the common adaptor site at Tyr1062 of Ret-9bp is affected by SHP1.

Furthermore, we evaluated whether the inhibition of Ret-9bp signaling affects [3H]thymidine incorporation and the transforming ability of NIH3T3 cells. Our data show that a moderate reduction in Ret-9bp autophosphorylation induced by SHP1 led to a significant inhibition of [3H]thymidine incorporation and reduced the ability of cells to form colonies. Because both PKB/Akt- and MAPK-dependent signaling pathways are affected by SHP1, it can be assumed that both signaling routes are important for mitogenic activity as well as for cell transformation. Further studies using specific inhibitors will help to evaluate the contribution of each of these signaling pathways for proliferation and malignant transformation.

A role of SHP1 in endocrine tumor cells has already been described (33). Like several other endocrine tumors, medullary thyroid carcinomas are characterized by high expression of somatostatin receptors (34). Evidence is given that activation of certain somatostatin receptor isoforms results in activation and membrane translocation of SHP1 (12, 13, 14). Therefore, a role of SHP1 in the regulation of endocrine tumors has been suggested. In this study, we demonstrated that SHP1 has far reaching consequences on Ret-9bp oncogene signaling. In addition, other investigators have shown activation of SHP1 by somatostatin receptor subtype 2 in NIH3T3 cells (35). Therefore, it can be speculated that somatostatin analogs might be useful therapeutic tools in endocrine tumors expressing high levels of SHP1 together with the somatostatin receptor subtype 2. To assess such a physiological role of SHP1 for the regulation of endocrine tumors in humans, more studies investigating the SHP1 and somatostatin receptor expression levels in tumor tissue from these patients are necessary.


   Footnotes
 
This work was supported by the German Research Foundation (DFG Ke-553-5 and Ke-553-6).

Abbreviation: GDNF, Glial cell line-derived factor.

Received January 16, 2001.

Accepted for publication June 28, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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