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Only the Substitution of Methionine 918 with a Threonine and Not with Other Residues Activates RET Transforming Potential¹

Centro di Endocrinologia ed Oncologia Sperimentale del Consiglio Nazionale delle Ricerche (A.M.C., G.S., G.D., F.C., N.A.D., R.V., R.M., M.S.), c/o Dipartimento di Biologia e Patologia Cellulare e Molecolare, Facoltà di Medicina e Chirurgia, Università degli Studi di Napoli "Federico II", via S. Pansini 5, 80131 Napoli-Italy and the Dipartimento di Medicina Sperimentale e Clinica (A.F.), Facoltà di Medicina e Chirurgia di Catanzaro, Università degli Studi di Reggio Calabria, via T. Campanella 5, 88100 Catanzaro-Italy

Address all correspondence and requests for reprints to: Massimo Santoro, Centro di Endocrinologia ed Oncologia Sperimentale del C.N.R., Facoltà di Medicina e Chirurgia, via S. Pansini 5, 80131 Napoli-Italy.

	Abstract

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Specific point-mutations of the RET receptor tyrosine kinase protooncogene are responsible for the inheritance of multiple endocrine neoplasia type 2A (MEN2A) and 2B (MEN2B), and familial medullary thyroid carcinoma (FMTC). MEN2B is caused by the substitution of methionine 918 by a threonine in the tyrosine kinase (TK) domain of RET. This mutation converts RET into a dominant transforming oncogene. We have substituted Met918 with four different residues and found that RET acquired transforming activity only when Met918 was substituted with a threonine. However, also when serine and valine, but not leucine or phenylalanine, were inserted in position 918, the RET TK function was activated and induced, especially in the case of the RET(918Ser), immmediate-early response genes. We conclude that the preservation of Met918 is critical for the control of RET kinase. However, only when a threonine residue is present in position 918, does RET efficiently couple with a transforming pathway.

	Introduction

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RET ENCODES a tyrosine-kinase (RTK) transmembrane receptor (Ret) (1), which is involved in the differentiation of neural crest-derived cell lineages (2). This is consistent with the recent demonstration that Ret is part of a multiprotein complex that serves as a receptor for a neurotrophic molecule, named GDNF (3). Accordingly, RET is involved in diseases affecting neural crest cells. Loss-of-function mutations of RET are involved in the pathogenesis of Hirschsprung’s disease (HSCR) (4, 5, 6, 7). Point mutations of RET are responsible for the autosomal dominant cancer syndromes, multiple endocrine neoplasia type 2A and 2B (MEN2A and MEN2B), and familial medullary thyroid carcinoma (FMTC). The mutations more frequently associated with MEN2A and FMTC consist in substitutions of specific extracellular cysteines (8, 9). Conversely, MEN2B is caused by the substitution of methionine 918, located in the TK domain of RET, with a threonine residue (10, 11, 12).

A gain-of-function of RET is believed to trigger these tumoral syndromes. Indeed, RET-MEN2A and RET-MEN2B alleles both act as dominant oncogenes in NIH 3T3 cells (13). The mechanisms by which these mutations activate the transforming potential of RET are different. In the case of MEN2A, a disulfide-bond-mediated homodimerization of Ret molecules results in constitutive activation of their TK function (13). Very little, on the contrary, is known about the activating mechanism of RET-MEN2B (Met918Thr), which causes the more complex and dramatic phenotype of MEN2B with respect to MEN2A, and which is frequently associated with sporadic cases of medullary thyroid carcinomas (MTC) (11).

Methionine 918 is highly conserved in RTKs, whereas a threonine is observed in the corresponding position of cytoplasmic protein tyrosine kinases (14). We have previously reported that the MEN2B mutation activates Ret catalytic function and alters its substrate specificity. Indeed, Ret-MEN2B protein is constitutively phosphorylated on tyrosine residues, and two-dimensional gel electrophoretic analyses (13) and optimal substrate studies (15) demonstrated that the substrate specificity of Ret-MEN2B differs from that of wild-type Ret molecules. In agreement with the hypothesis that altered kinase activity is responsible for MEN2B activation, Iwashita et al. demonstrated that autophosphorylation of different tyrosine residues is required for the catalytic activity of wild-type RET and RET-MEN2B, that Tyr905 is crucial for the function of wild-type RET and that two tyrosine residues (Tyr864 and Tyr952), on the contrary, are important for RET-MEN2B (16).

To elucidate further the role of the Met918-to-Thr mutation, we investigated the effects of different amino acid substitutions of RET methionine 918.

	Materials and Methods

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Engineering of expression vectors
The LTR-RET eukaryotic expression vector encoding the short isoform (1,072 amino acids) of the Ret protein and the LTR-RET-MEN2A- and LTR-RET-MEN2B-encoding RET mutants carrying a Cys634Tyr and a Met918Thr mutation, respectively, are described elsewhere (13). Fragments containing the required mutation were amplified by recombinant PCR using LTR-RET as a template (17). Briefly, two primary PCR reactions (a left and a right reaction) were performed, using standard PCR conditions (AmpliTaq, Perkin Elmer-Cetus Co., NJ). This yielded two products overlapping in the sequence corresponding to the reverse primer of the left PCR and the forward primer of the right PCR, and the mutations were introduced as part of these overlapping PCR primers. Ten nanograms of the purified PCR products of the two primary PCR reactions were annealed and amplified with 20 secondary PCR cycles, using the 5'- and 3'-most oligonucleotides, as primers. The forward and reverse internal overlapping primers, containing the mutations, were as follows (codon 918 is in parentheses): forward (nucleotides 2938–2958): 5'-GTTAAATGG(ATG)GCAATTGAA-3'; [in this primer (ATGMet) was replaced by (AGT), (GTG), (TTT), or (CTG) to generate RET(918Ser), RET(918Val), RET(918Phe) and RET(918Leu), respectively]; reverse (nucleotides 2958–2938): 5'-TTCAATTGC(CAT)CCATTTAAC-3' [the (CATMet) triplet was replaced by the required anticodon]. The external primers were: 5'-CTCGTTCATCGGGACTTGGC-3' (forward; nucleotides 2803–2822) and 5'-CCATCCGGTGGCCGGTCTTCAG-3' (reverse; nucleotides 3106–3085). The recombinant PCR products were cloned in the pT7Blue T vector (Novagen, WI) and completely sequenced using the Sequenase Kit (United States Biochemical). Finally, the fragments containing the mutations were excised by digestion with BglII and BclI and cloned in the LTR-RET or LTR-RET-MEN2A plasmids. The resulting plasmids were sequenced in both strands of the regions that underwent genetic manipulations to verify that the predicted structures had been generated and that no additional mutations had been introduced during the cloning steps. The pNGFI-A-CAT vector contains sequences from position -1150 to +200 of the NGFI-A promoter, fused to the chloramphenicol acetyl transferase (CAT) gene (18).

Cell culture and transfection experiments
NIH 3T3 cells were grown in DMEM supplemented with 10% FCS and were transfected using the calcium phosphate precipitation method as described elsewhere (13). Transformed foci were scored at 3 weeks. Transforming efficiency was calculated in focus-forming units per picomole of added DNA after normalization for the efficiency of colony formation in parallel dishes subjected to marker selection in mycophenolic acid. Mass populations and, in some cases, individual cell clones of transfected NIH 3T3 cells were obtained exploiting the capacity of the E. coli gpt gene of the vector to confer resistance to mycophenolic acid. PC12 cells were grown in RPMI 1640 (Life Technologies, Gaithersburg, MD) supplemented with 10% horse serum and 5% FCS. For transient transfection assays, cells were plated at 3 x 10⁵ cells in 60-mm-diameter tissue culture dishes 24–36 h before transfection. Transfection was performed using the lipofectin reagent according to the manufacturer’s instructions (Life Technologies). The pNGFI-A-CAT plasmid was chosen as a target because of its very low basal level of activity in PC12 cells (lower than 0.5% of chloramphenicol conversion) (19). All transfections were carried out with 2 µg of reporter plasmid together with 1 µg of the RET constructs or the empty LTR control vector. Cell extracts were prepared 60 h after transfection and CAT activity was analyzed by TLC with 95% chloroform and 5% methanol, as previously described (19). Each experimental point was cut from the TLC plate and counted. For each experiment, the percentage of conversion to the acetylated form of chloramphenicol [¹⁴C] was calculated and the results of four experiments, made in duplicate, were expressed as relative promoter induction. Differences of the activating capability between RET-MEN2B, RET(918Ser), wild-type RET and the other RET mutants were assessed by Student’s t test.

Protein studies
Immunoprecipitation and immunoblotting experiments were performed as in Santoro et al. (13). Briefly, cells were lysed in a buffer containing 50 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES; pH 7.5), 1% (vol/vol) Triton X-100, 50 mM NaCl, 5 mM ethylene glycol-bis(ß-amino-ethyl ether) N, N, N', N' tetraacetic acid (EGTA), 50 mM NaF, 20 mM sodium pyrophosphate, 1 mM sodium vanadate, 2 mM phenylmethylsulphonyl fluoride, 0.2 µg each of aprotinin and leupeptin per ml. Lysates were clarified by centrifugation at 10,000 x g for 15 min and the supernatant processed for immunoblotting or immunoprecipitation. Protein concentration was estimated with the Bio-Rad kit. Antibodies included a polyclonal antibody to the Ret tyrosine-kinase domain and the 4G10 anti-pTyr monoclonal antibody (Upstate Biotechnology, Lake Placid, NY). Immunoblots were subsequently stained with appropriate secondary antibodies and revealed with the Amersham ECL system. For the immunocomplex kinase assay, 3 mg of cell lysates were immunoprecipitated with anti-Ret antibodies; the assay was performed as previously reported (13) with the only difference that 20 µg of myelin basic protein (MBP, Sigma Chemical Co., St. Louis, MO) were used as a substrate (6).

	Results

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Biological activities of Ret molecules carrying substitutions of Met918
To analyze the role of the MEN2B mutation (Met918Thr), substitutions of Met918 (to Ser, Val, Phe, and Leu) were introduced into the LTR-RET. The structure of RET and the introduced mutations are schematically shown in Fig. 1. The transforming ability of these constructs was evaluated by a focus forming assay in NIH 3T3 cells. An example of this assay is shown in Fig. 2, and the results of three independent transfections are summarized in Table 1. Only when Met918 was replaced by Thr [RET(918Thr), i.e. RET-MEN2B] did RET acquired a readily detectable oncogenic potential. None of the newly generated RET(918Ser), RET(918Val), RET(918Phe) and RET(918Leu) constructs was endowed with transforming activity. To investigate whether the substitution of Met918 could inactivate RET function, we introduced the four generated mutations in the RET-MEN2A construct. RET-MEN2A is a constitutively active RET mutant in which the extracellular Cys634 is replaced by a tyrosine residue (13). Introduction of Ser, Val, Phe, or Leu in place of Met918 did not alter the transforming ability of RET-MEN2A (Table 1).

View larger version (9K): [in this window] [in a new window]   Figure 1. Schematic representation of RET mutants analyzed in this study. A schematic representation of the Ret protein (short isoform) and of the substitutions of the Ret Met918, analyzed in this study.

View larger version (57K): [in this window] [in a new window]   Figure 2. Transforming activity of RET constructs. An example of a focus assay in NIH 3T3 cells. NIH 3T3 cells were transfected with 1 µg of each construct and transformed foci were scored after 2 weeks. These results were confirmed in three independent assays.

We next investigated the ability of the different RET constructs to activate the expression of the pNGFI-A-CAT plasmid in PC12 cells. NGFI-A is an immediate-early response gene, whose expression is rapidly induced upon growth factor treatment of PC12 cells (18). We previously reported that activation of this promoter is part of the differentiative effect exerted by activated RET versions in PC12 cells (19, 20). Moreover, this assay is more sensitive than the NIH 3T3 transfection assay because pNGFI-A-CAT responds, with a detectable activation, even to the expression of wild-type RET (7, 20). An example of one CAT assay and the bar graphs showing the average results of four independent transfections, each performed in duplicate, are reported in Fig. 3. In agreement with earlier findings (7, 20), transfection of RET-MEN2A and RET(918Thr), i.e. RET-MEN2B, resulted in a marked induction of CAT activity [for RET-MEN2B, 31 (±4.0)-fold (mean ± SEM, n = 4) and thus, about 4- to 5-fold above that of wild-type RET, which was 8.2 (±1.3)-fold)]. RET(918Phe) and RET(918Leu) did not significantly differ from wild-type RET, with respect to CAT induction. In contrast, RET(918Ser) induced CAT activity 21 (±3.1)-folds and thus, resulted, on average, about 3-fold more active than wild-type RET (P < 10^-6).

View larger version (32K): [in this window] [in a new window]   Figure 3. Induction of the NGFI-A promoter by wild-type and mutant RET versions. PC12 cells were transfected with 2 µg of pNGFI-A-CAT and 1 µg of the different RET constructs. Sixty hours after transfection, total proteins were isolated and promoter induction was determined by CAT assay. A representative CAT assay and the bar graphs of the average induction of four separate experiments, each performed in duplicate, are reported. CAT activities are shown as fold increases above the basal activity. The differences between RET-MEN2B and RET(918Ser), and between RET(918Ser) and wild-type RET were assessed by the t test and the obtained P values are reported.

View larger version (28K): [in this window] [in a new window]   Figure 4. Expression and tyrosine kinase activity of RET mutants. A and B, in vivo phosphorylation. Five hundred micrograms of total lysate from cells expressing the different RET constructs were immunoprecipitated with a polyclonal antibody to Ret. One-half of the immunoprecipitate was immunoblotted with anti-Ret (anti-Ret) and one half with a monoclonal antibody to phosphotyrosine (anti-pTyr). As reported, Ret products consist in immature approximately 145 kDa and mature cell surface approximately 160-kDa isoforms; the mol mass of the two protein forms are indicated. C, in vitro kinase activity. Three milligrams of total lysate were immunoprecipitated with anti-Ret antibodies and incubated with labeled ATP and 20 µg of MBP. The phosphorylation of MBP is shown. The results are typical and representative of three independent experiments.

	Discussion

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To investigate the role of RET methionine 918, which is affected by the MEN2B mutation, we have substituted it with four residues (Ser, Val, Phe, or Leu). We demonstrate that preservation of Met918 is crucial for the correct regulation of the Ret kinase activity. In fact, the introduction of a serine residue at position 918 was able to activate the intrinsic Ret catalytic ability. In addition, low levels of tyrosine kinase activity were also observed when a valine residue was inserted at position 918. In agreement with this finding, the 918Ser RET mutant clearly activated immediate-early gene transcription. Two possibilities could be envisaged to account for the activation of the TK function of RET by these mutations. In the case of 918Ser, as in the case of 918Thr, serine or threonine could be target sites for phosphorylation by other kinases and the eventual phosphorylation could result in activation of Ret kinase activity. An analysis performed using the PROSITE compilation of sequence patterns (21) revealed that either serine or threonine, in position 918 of Ret, formed potential casein kinase II (CK-2) phosphorylation sites (Ser/Thr-X-X-Glu/Asp) (22). It is intriguing that also the natural threonine, mapping in cytoplasmic kinases (like c-src and c-yes) in a position corresponding to Ret residue 918, is a potential CK-2 phosphorylation site. Indeed, still incompletely characterized c-yes (23) and c-src (24) peptides were found to be substrates for CK-2. Alternatively, other structural features of residue 918 could activate the Ret TK function. For instance, by the Garnier (25) and Novotny (26) methods (Santoro, M., unpublished results), we observed that the introduction, in place of Met918, of a serine or a threonine, which are characterized by lateral chains smaller than that of methionine, reduced the probability that the involved protein stretch assume an helical conformation. These structural alterations could influence the TK activity of Ret.

Whatever the case, the protein domain where Met918 maps is extremely important for the regulation of catalytic function of several kinases. In fact, this residue maps in a loop whose topology is essential for the regulation of the function of other kinases, e.g. cAPK (cAMP-dependent protein kinase), CDK2 (cyclin-dependent kinase) and ERK2 (MAP kinase) (27). The crystal structure of the tyrosine kinase domain of the insulin receptor confirmed the importance of this TK subdomain. Indeed, the protein loop that includes Met918 is required to maintain the kinase in the inactive conformation in the absence of ligand (28). Thus, it is conceivable that substitutions of this crucial residue may alter this regulatory mechanism.

The results reported here allowed us to dissociate the two effects so far attributed to the MEN2B mutation, i.e. the activation of the TK function, and the acquisition of a transforming ability by RET. Indeed, the induction of the TK function observed in the Ret(918Ser) mutant and, in part, in the Ret(918Val) mutant, was not sufficient to confer transforming ability. Only the substitution of methionine 918 with a threonine rendered Ret able to trigger efficiently a transforming pathway; this underscores the importance of the ability of Thr918 to specifically affect Ret coupling with additional substrates, as predicted by molecular models suggesting that the corresponding protein domain serves as a binding surface for the phosphorylatable tyrosine in the substrate molecule (12). In addition, here we show that the transforming ability of a RET activated by an extracellular mutation (MEN2A), which simulates the action of a ligand, was not abrogated by substitution of Met918 with different residues. Thus, a methionine at position 918 is not an absolute requirement for RET functions; the presence of a threonine at that position probably gives the capability to phosphorylate other still uncharacterized substrates important for its transforming ability. We suggest that the cellular systems and constructs described here may help to identify such molecules.

	Acknowledgments

 
We are grateful to Prof. G. Vecchio for his support during the course of this work and to Dr. V. de Franciscis for his help with the PC12 experiments. We are indebted to Prof. P. Erto for his help in the statistical analysis and we thank Dr. M.V. Chao for the pNGFI-A plasmid.

	Footnotes

 
¹ This study was supported by the Associazione Italiana per la Ricerca sul Cancro (AIRC), and by the Progetto Finalizzato Applicazioni Cliniche della Ricerca Oncologica, Sottoprogetto 2, Biologia Molecolare of the C.N.R..

Received September 11, 1996.

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This Article Abstract Full Text (PDF) Purchase Article View Shopping Cart Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Copyright Permission Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Cirafici, A. M. Articles by Santoro, M. Search for Related Content PubMed PubMed Citation Articles by Cirafici, A. M. Articles by Santoro, M.