The Signaling Adapters Fibroblast Growth Factor Receptor Substrate 2 ...

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Endocrinology 144(3):922–928 Copyright © 2003 by The Endocrine Society doi: 10.1210/en.2002-221002

The Signaling Adapters Fibroblast Growth Factor Receptor Substrate 2 and 3 Are Activated by the Thyroid TRK Oncoproteins VALERIA RANZI, SUSAN O. MEAKIN, CLAUDIA MIRANDA, PIERA MONDELLINI, MARCO A. PIEROTTI*, AND ANGELA GRECO* Department of Experimental Oncology (V.R., C.M., P.M., M.A.P., A.G.), Operative Unit “Molecular Mechanisms of Cancer Growth and Progression,” Istituto Nazionale Tumori, 20133 Milan, Italy; and Laboratory of Neural Signaling (S.O.M.), Cell Biology Group, Robarts Research Institute, London, Ontario N6A 5K8, Canada The thyroid TRK oncogenes are generated by chromosomal rearrangements juxtaposing the neurotrophic tyrosine receptor kinase type 1 (NTRK1) tyrosine kinase domain to foreign activating sequences. TRK oncoproteins display a constitutive tyrosine kinase activity resulting in the capability to transform NIH3T3 cells. The TRK oncoproteins’ signal transduction has been in part elucidated, and it involves several signal transducers activated by the NGF-stimulated NTRK1 receptor. In this paper, we investigate the role of FRS2 and FRS3, two related adapter proteins activated by fibroblast growth factor and NTRK1 receptors, in the signaling of the thyroid TRK-T1 and TRK-T3 oncogenes. By a combination of in vitro and in vivo assays, we demonstrate that both fibro-

blast growth factor receptor substrate (FRS)2 and FRS3 are recruited and activated by TRK-T1 and TRK-T3. Interaction studies using different TRK-T3 mutants indicate that FRS3 is recruited by the same tyrosine residue interacting with Shc and FRS2. Expression studies show different expression patterns of the FRS adapters in normal and tumor thyroid samples: FRS3 is expressed in both normal and thyroid tumor samples, whereas FRS2 is not expressed in normal thyroid but is differentially expressed in some tumors. Altogether, our data indicate that the FRS2 and FRS3 adapters may have a role in thyroid carcinogenesis triggered by TRK oncogenes. (Endocrinology 144: 922–928, 2003)

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(FRS2 and FRS3) are two related adapter proteins, sharing 49% sequence identity, that are activated by the FGF and NTRK1 receptors (6 –9). FRS proteins contain myristylation anchors and PTB (phospho-tyrosine binding) domains in the N termini and large regions with multiple tyrosine phosphorylation sites at their C termini, which, upon phosphorylation, form the binding sites for growth factor receptor binding protein 2 and Src homology tyrosine phosphatase 2 (7, 10). Developmental studies have shown that FRS3 is expressed in a much more tissue-restricted manner than FRS2, suggesting that the two adapters may serve different functions (11). FRS2 binds both FGF and NTRK1 receptors through the PTB domain; however, it recognizes two different primary structures in the two different receptors, in a phosphorylation-independent and -dependent manner, respectively. The phosphorylation-dependent binding of FRS2 to NTRK1 involves the tyrosine residue 490 of the receptor that also functions as binding site for Shc (9). In vitro studies have shown a competition between FRS2 and Shc for the binding to Y490, suggesting that this might regulate the neurotrophin-dependent proliferation and/or differentiation (8). Although the role of FRS2 in NGF-induced neuronal differentiation is defined, its involvement in the proliferation induced by activated NTRK1 tyrosine kinase has not been elucidated yet. This seems of peculiar interest on account of a growing list of cells in which activated NTRK1 tyrosine kinase activity triggers a mitogenic pathway. This includes not only thyroid tumors carrying rearranged TRK oncogenes, but also breast, prostate, and pancreatic tumors in which the NGF/NTRK1 receptor pathway has been pro-

OMATIC REARRANGEMENTS OF the neurotrophic tyrosine receptor kinase type 1 (NTRK1) gene, encoding the receptor tyrosine kinase for nerve growth factor (NGF), have been detected in a consistent fraction of human papillary thyroid carcinomas and produce chimeric proteins (1). In our laboratory, several thyroid-specific TRK oncogenes, named TRK, TRK-T1, TRK-T2, and TRK-T3, have been isolated (1–3). All of them contain a variable portion of NTRK1, including the tyrosine kinase domain, and differ in the 5⬘activating sequences, contributed by tropomyosin, translocated promoter region, and TRK fused gene (TFG) genes, respectively. The activity of rearranged TRK oncogenes recapitulates that of NGF-stimulated wild-type NTRK1 receptor. In fact, similarly to the activated receptor counterpart, TRK oncogenes induce transformation of NIH3T3 mouse fibroblasts and differentiation of rat pheochromocytoma PC12 cells (4). Analysis of TRK oncogenes’ signal transduction, performed in NIH3T3 foci, has detected the involvement of several signal transducers recruited by the NGFstimulated NTRK1 receptor, such as Src homologous and collagen (Shc), growth factor receptor binding protein 2, phospholipase C (PLC)␥, extracellular signal regulated kinases (ERKs), and c-Jun N-terminal kinase (Ref. 5; and our unpublished data). Fibroblast growth factor (FGF) receptor substrate 2 and 3 Abbreviations: FGF, Fibroblast growth factor; FRS, FGF receptor substrate; GST, glutathione-S-transferase; NGF, nerve growth factor; NTRK1, neurotrophic tyrosine receptor kinase type 1; PLC, phospholipase C; PTB, phospho-tyrosine binding; Shc, Src homologous and collagen; TFG, TRK fused gene.

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posed to have a role (12–14). With respect to FRS3, little is known about its contribution to the different biological effects of the NTRK1 receptor. In this study, we have investigated the involvement of FRS2 and FRS3 in the transforming pathway triggered by TRK oncogenes in mouse fibroblasts, with the aim to elucidate the role of the two adapters in thyroid carcinogenesis. Materials and Methods Plasmid constructs Expression vector containing cDNA of FRS3 inserted in pCDNA3.1 myc tagged has been reported (Dixon, S. J., and S. O. Meakin, in preparation). The expression plasmids containing TRK-T1 and TRK-T3 oncogenes have been previously described (2, 3). The TRK-T3 pointmutants T3/Y291F, T3/Y586F, T3/Y291/586F and T3/R774P were constructed by site-directed mutagenesis and have been described (15, 16). Wild-type and mutated TRK-T3 cDNAs were inserted into the pRC/CMV expression vector. The glutathione-S-transferase (GST)FRS2(PTB) and GST-FRS3(PTB) plasmids have been described (8) (Dixon, S. J., and S. O. Meakin, manuscript in preparation). The GSTPLC␥ and GST-Shc(PTB) have been previously reported (5). GST fusion proteins were purified according to standard procedures.

Cell culture and transient transfection NIH3T3 cell lines expressing TRK oncogenes and 293T cell line were cultured in DMEM supplemented, respectively, with 5% calf serum and 10% fetal calf serum. 293T cells were seeded in 10-cm dishes and transfected with the CaPO4 method using 2 ␮g of TRK-T3 cDNA, together with 10 ␮g of FRS3 cDNA. The day after transfection, the cells were washed, incubated in medium supplemented with 10% serum for 5– 6 h, and then starved in 0.5% serum medium overnight before harvesting.

K252a treatment K252a (Calbiochem, La Jolla, CA) was dissolved in dimethylsulfoxide at the concentration of 1 mm and kept in the dark at ⫺20 C. The solution was diluted with medium just before use and added to the cells to a final concentration of 200 nm.

Immunoprecipitation, pull-down, and Western blot analysis Cells were lysed with PLCLB buffer (50 mm HEPES, 150 mm NaCl, 10% glycerol, 1% Triton X-100, 1.5 mm MgCl2, 1 mm EGTA, 10 mm Na4P2O7, 100 mm NaF) supplemented with aprotinin, leupeptin, phenylmethylsulfonyl fluoride, and Na3VO4. One milligram of cell extracts was precipitated with anti-TRK (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), anti-myc (Roche, Basel, Switzerland) antibodies, or p13suc1 agarose (Upstate Biotechnology, Inc., Rochester, NY). In pull-down experiments, 800 ␮g of cell extracts were incubated with 5 ␮g GSTFRS2(PTB) or GST-FRS3(PTB) fusion protein conjugated to gluthationeSepharose. For competition assays 200 ␮g of GST-FRS3(PTB) was digested with 3.5 cleavage units of thrombin (Sigma, St. Louis, MO). Samples were centrifuged for 5 min at 4 C at 1500 rpm. Soluble FRS3(PTB) domain, contained in the supernatant, was quantified by SDS-PAGE and Comassie staining. Twenty-five micrograms of soluble PTB domain were incubated with NF797 lysates (800 ␮g) at 4 C for 90 min. Afterward, incubation with Sepharose-conjugated GSTFRS2(PTB) or GST-PLC␥ fusion proteins was performed. The precipitates were washed three times with HNTG buffer (20 mm HEPES, 150 mm NaCl, 0.1% Triton X-100, 10% glycerol) and suspended in Laemmli sample buffer. Protein samples were electrophoresed on SDS-PAGE (8.5%), transferred onto nitrocellulose filters, and immunoblotted with the antiphosphotyrosine (Upstate Biotechnology, Inc.), anti-TRK, antimyc, or anti-FRS2 antibodies (Upstate Biotechnology, Inc.). The immunoreactive bands were visualized using horseradish peroxidaseconjugated secondary antibody and enhanced chemiluminescence (Amersham Pharmacia Biotech, Buckinghamshire, UK).

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RT-PCR RNA from tumor or normal thyroid samples, as well as normal kidney, was retro-transcribed by reverse transcription reaction performed with the SuperScript kit (Life Technologies, Inc.). The product of reverse transcription was subjected to nested PCR amplification. The FRS2-specific fragment was amplified by using a forward primer FRS2A localized in the IV exon of the FRS2 gene (5⬘-CCCCGATATCCCTCAATTTGGAGATGCTTCATCCCATCCGTCAAG-3⬘) and a reverse primer FRS2R localized in the V exon (5⬘-TTGTCATCTTCATCCCTACTTAGCTTG-3⬘). The PCR product was then subjected to another PCR amplification using primers FRS2C (5⬘-GAGATGCTTCATCCCATCCGTCAAG-3⬘) and FRS2D (5⬘-TGAGGTACTGGTGGATGTCAGACG-3⬘), both localized in the same exon of FRS2A and FRS2R, respectively, and capable to amplify a 486-nucleotide fragment. The FRS3-specific fragment was amplified using a forward primer FRS3A (5⬘-AGCCACCCAATGCTCTAGGCTACA-3⬘) localized in part in the IV exon and in part in the V exon, and a reverse primer FRS3R (5⬘-GTGGGGGCAGGTTCTCATAGTGCA-3⬘) localized in exon VI. The PCR product was then subjected to another PCR amplification using the FRS3C (5⬘-GTCTCCAGCTTTTCCAATGGCTG-3⬘) and the FRS3D (5⬘CGGTGGGCAAAGGTTCCAGC-3⬘), localized in the same exons of FRS3A and FRS3R, respectively. The amplified fragment consists of 515 nucleotides. For the first amplification, the same PCR conditions for both FRS2 and FRS3 were used: 95 C for 5 min, then 35 cycles (95 C for 30 sec, 67 C for 30 sec, 72 C for 45 sec) and a final extension at 72 C for 7 min. The same conditions were used for the second amplification, except for the annealing temperatures: 69 C for FRS2 and 60 C for FRS3. For the amplification of the housekeeping P0 mRNA, the following oligonucleotides were used: P0F (5⬘-CCGGAATTCAGGGAAGACAGGGCGACCTGG-3⬘) and P0R (5⬘-CGCGGATCC(CT)C(GT)GAT(AG)GCCTTGCGCATCAT-3⬘). After a denaturation at 95 C 5 min, 40 PCR cycles were performed (95 C for 30 sec, 56 C for 30 sec, 72 C for 60 sec) followed by a final extension at 72 C for 7 min. The amplified fragment is 250 bp long. PCRs were performed in a final volume of 25 ␮l containing 1⫻ PCR buffer (Perkin-Elmer, Foster City, CA), 0.2 mm each deoxyribonucleotide triphosphate, 1.5 mm MgCl2, 0.5 ␮m each primer, 0.65 U of Taq polymerase (Perkin-Elmer). The PCR products were electrophoresed on 2% agarose gel and visualized by ethidium bromide staining.

Results TRK oncoproteins interact in vitro with FRS adapters

In this study, we used two previously described thyroid TRK oncogenes (Fig. 1). TRK-T1 is activated by sequences of the translocated promoter region gene encoding a protein of the nuclear envelope (17). The TRK-T1 protein is made of 503 amino acids, 310 of which are contributed by NTRK1 (2). TRK-T3 oncogene is activated by TFG, a novel gene on chromosome 3, and encodes a 592-amino-acid protein containing 399 residues of NTRK1 (3). In addition to the activating sequences, TRK-T1 and TRK-T3 differ also in the portion contributed by NTRK1, which is bigger in TRK-T3 and includes the transmembrane domain. In particular, TRK-T1 lacks the KFG motif, which has been shown to be involved in the interaction of NTRK1 with the FRS2 adapter (8, 18). We investigated the TRK oncoproteins interaction with FRS2 and FRS3 adapters by GST pull-down experiments using the GST-FRS2(PTB) and GST-FRS3(PTB) fusion proteins containing the PTB domain of FRS2 and FRS3, respectively. Cell extracts from foci NF861 and NF797, derived from the transfection of NIH3T3 with TRK-T1 and TRK-T3 oncogenes, respectively, were incubated for 4 h with the GST fusion proteins conjugated to Sepharose beads. Western blot analysis of the eluted complexes with anti-TRK antibodies showed that both TRK proteins were able to form complexes with both GST-fusion proteins (Fig. 2A). The association was

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abolished by treatment with K252a, a specific inhibitor of TRK kinase activity (19). No association with GST only was detected. As a control, expression of the TRK oncoproteins in the cell extracts used for the pull-down experiments was determined by Western blot analysis with anti-TRK antibodies (Fig. 2A, right panel). To investigate whether TRK-T1 and TRK-T3 oncogenes differ in the capacity to bind FRS adapters, GST pull-down experiments were performed using two different incubation times: long (4 h, as in the previous experiment) and short (90


min). After a long incubation, both oncoproteins were pulled down by both FRS PTB domains, as previously observed (Fig. 2A). Different results were obtained using a short incubation time (Fig. 2B). Both oncoproteins interacted with GST-FRS3(PTB). On the contrary, only TRK-T3, but not TRKT1, was detected in the GST-FRS2(PTB) complexes. However, the TRK-T1 oncoprotein was efficiently isolated from cell lysates, previously treated with GST-FRS2(PTB), by a subsequent 90-min incubation with either GST-FRS3(PTB) (Fig. 2B, lower panel) or GST-SHC(PTB) (data not shown). These data demonstrate that the both TRK-T1 and TRK-T3 bind to the FRS3 PTB domain with similar capability. On the contrary, the FRS2 binding capability of TRK-T1 is reduced with respect to TRK-T3. This difference might be related to the KFG motif, which in TRK-T1 has been removed by the rearrangement. FRS proteins are activated by TRK oncogenes

FIG. 1. Schematic representation of NTRK1, TRK-T1, and TRK-T3 proteins. In the NTRK1 scheme, the transmembrane (TM) domain, the KFG motif, the tyrosine kinase (TK) domain, the tyrosine residues of the activation loop (Y670, 674, 675), and those responsible for binding Shc (Y490) and PLC␥ (Y785) are indicated. In the TRK-T1 and TRK-T3 schemes, the corresponding tyrosine residues and the portions contributed by NTRK1 and by activating genes TPR and TFG are indicated.

We next investigated the activation of FRS2 and FRS3 in cells expressing TRK oncogenes. To study FRS2 activation, we used NIH3T3 cells transformed by TRK-T1 or TRK-T3 (NF861 and NF797), or expressing the wild-type NTRK1 receptor (E25) and stimulated by NGF as control. All the cell lines were serum deprived and treated or not with the K252a inhibitor. Cell extracts were precipitated with the FRS2interacting p13suc1 protein conjugated to agarose beads, and the eluted complexes analyzed by Western blot with antiphosphotyrosine and anti-FRS2 antibodies, to detect the FRS2 phosphorylation and expression level, respectively (Fig. 3A). As previously reported (8), FRS2 was detected as multiple forms, and only the highest one was tyrosine phosphorylated. In cells expressing NTRK1 and TRK oncogenes, FRS2 phosphorylation was significantly increased with respect to the basal level detected in wild-type NIH3T3 cells. Treatment with the K252a inhibitor, which abrogated NTRK1 and TRK oncoprotein phosphorylation, reduced

FIG. 2. In vitro binding of TRK oncoproteins to FRS2 and FRS3 PTB domains. A, Extracts of NF861, NF797, and NIH3T3 cell lines treated or not with K252a were incubated for 4 h with Sepharose-conjugated GST-FRS2(PTB) and GST-FRS3(PTB) fusion proteins or with GST only. Complexes were separated on SDS-PAGE and hybridized with anti-phosphotyrosine (anti-pTyr) antibodies. To show expression levels of the oncoproteins, the same lysates were immunoprecipitated and hybridized with anti-TRK antibodies. B, In vitro binding of TRK oncoproteins to FRS2 and FRS3 PTB domains after a short incubation time. The same cell lysates described in A were incubated with GST-FRS3(PTB) (left) or with GST-FRS2(PTB) (middle) for 90 min and immunoblotted with antiphosphotyrosine antibodies. In the right panel, cell extracts previously incubated with GST-FRS2(PTB) were pulled-down with GST-FRS3(PTB) and blotted as above.


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FIG. 3. In vivo interaction of TRK oncoproteins with FRS2 and FRS3 adapters. A, Activation of FRS2 in NIH3T3 cell lines expressing NTRK1 (E25), TRK-T1 (NF861), and TRK-T3(NF797). Cells were serum starved in 0.5% calf serum for 48 h, then treated overnight with K252a where indicated. Cell lysates were incubated with p13suc1 conjugated to agarose beads; the eluted complexes were immunoblotted with antiphosphotyrosine (anti-pTyr). The blots were then stripped and hybridized with anti-FRS2 antibodies to show expression level of the FRS2 protein. As control, the same lysates were immunoprecipitated with anti-TRK antibodies and immunoblotted with anti-pTyr antibodies. B, Interaction of FRS2 with TRK oncogenes in living cells. TRK-T1 and TRK-T3 cDNAs were transiently transfected into 293T cells. The lysates were pulled-down with p13suc1 agarose beads, and the eluted complexes were immunoblotted with anti-TRK antibodies. As control, the expression of TRK oncoproteins was detected by immunoprecipitation and Western blot with anti-TRK antibodies. C, In vivo interaction of FRS3 with TRK-T1 and TRK-T3 oncoproteins. 293T cells were transiently transfected with TRK oncogene cDNAs together with myc-FRS3. The lysates were immunoprecipitated with an anti-myc antibody and immunoblotted with anti-myc (first panel), with antiphosphotyrosine (second panel), with anti-TRK (third panel) antibodies. In the bottom two panels, the TRK phosphorylation and expression are shown.

FRS2 phosphorylation to background level in cells expressing the NTRK1 receptor and the TRK oncogenes. We investigated the capability of TRK oncoproteins to interact in vivo with FRS2 by performing coprecipitation experiments. Human 293T cells were transiently transfected with TRK-T1 and TRK-T3 oncogenes; cell extracts were precipitated with the FRS2-interacting protein p13suc1 conjugated to agarose beads, as above described, and the eluted complexes analyzed by Western blot. Hybridization with anti-TRK antibodies detected TRK-T1 and TRK-T3 oncoproteins in p13suc1agarose complexes (Fig. 3B), demonstrating that endogenous FRS2 interacts in vivo with TRK oncoproteins. Because FRS3 does not interact with p13suc1 (20) and antibodies to FRS3 are not yet available, we used transfection studies in 293T cells to examine the interaction between the TRK oncoproteins and a myc-tagged FRS3 construct. As shown in Fig. 3C, expression of myc-FRS3 generates multiple bands with an apparent molecular weight of 60 – 65K. These forms are consistently observed and likely reflect differences

in posttranslational modifications (Dixon, S. J., and S. O. Meakin, unpublished observations). Hybridization of the same blot with antiphosphotyrosine antibodies showed that the FRS3 protein is phosphorylated when coexpressed with TRK oncoproteins. FRS3 phosphorylation is abrogated by treatment with K252a, indicating its strict dependence on TRK activation. Western blot analysis with anti-TRK antibodies detected TRK proteins in anti-myc immunocomplexes, indicating a direct interaction between the FRS3 adapter and the oncoproteins. The difference in FRS3 phosphorylation in cells expressing TRK-T1 and TRK-T3 might reflect variable oncoproteins activity. On the whole, these results indicate that the FRS3 adapter, similarly to FRS2, is a substrate of TRK oncoproteins. Search for the TRK-T3 residue interacting with FRS3

In an attempt to further characterize the FRS3 interaction with the TRK kinase, we analyzed the in vitro binding to the

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GST-FRS3(PTB) fusion protein of the following TRK-T3 mutants: T3/Y291F, in which the tyrosine docking site for Shc and FRS2 (corresponding to the tyrosine 490 of the human wild-type NTRK1) was mutated to phenylalanine; T3/774, carrying the congenital insensitivity to pain with anhidrosisassociated R774P mutation that abrogates the tyrosine kinase activity (15); T3/Y586F, in which the tyrosine docking site for PLC␥ (corresponding to tyrosine 785 of human wild-type NTRK1) was mutated to phenylalanine; T3/Y291/586F, carrying the mutation of both Shc and PLC␥ interaction sites. Wild-type and mutated TRK-T3 constructs were transfected into 293T cells. Cell extracts were incubated with the GSTFRS3(PTB) fusion protein, and the eluted complexes analyzed by Western blot hybridization with anti-TRK antibodies (Fig. 4A). Similarly to T3/WT, the T3/Y586F mutant formed complexes with the FRS3 PTB domain, suggesting that the tyrosine 586 is not involved in the interaction. The unphosphorylated T3/R774P protein failed to interact with FRS3 PTB, confirming that the interaction between TRK-T3 and FRS3 requires the oncoprotein phosphorylation. The lack of interaction of FRS3-PTB domain with T3/Y291F and T3/Y291/586F proteins indicates that TRK-T3 recruits FRS3


through the same tyrosine residue involved in the interaction with Shc and FRS2. This latter finding is further demonstrated by pull-down experiments in the presence of an excess of soluble FRS3(PTB). As shown in Fig. 4B, when cell extracts were preincubated with soluble FRS3(PTB), the TRK-T3 binding to GST-FRS2(PTB) was abrogated, whereas that to GST-PLC␥ was unaffected. Expression of FRS transcripts in normal and tumor thyroid tissues

To explore the role of the interaction between TRK oncoproteins and FRS proteins in TRK-induced thyroid tumorigenesis, we investigated the expression of the two adapters in five normal thyroids and in eight papillary thyroid tumors (Fig. 5). Tumor samples have been previously characterized: three carried the activation of TRK, and three the activation of Ret; none of the two oncogenes was detected in the remaining tumors. We performed RT-PCR using oligonucleotide primers able to amplify a 486-bp fragment of FRS2 mRNA and a 515-bp fragment of FRS3 mRNA. As control, the amplification of the 250-bp fragment of the housekeeping

FIG. 4. A, In vitro interaction of GST-FRS3(PTB) with wild-type and mutated TRK-T3 proteins. The TRK-T3 cDNAs were transiently transfected in 293T cells as described in Materials and Methods. Cell lysates were incubated with the GST-FRS3(PTB) fusion protein conjugated to Sepharose beads. The eluted complexes were analyzed by Western blot analysis with anti-TRK antibodies (top panel). The same cell lysates were immunoprecipitated with anti-TRK and blotted with anti-TRK (middle panel) and antiphosphotyrosine (bottom panel) antibodies, to show the expression and phosphorylation of the TRK-T3 proteins. B, Competition assay. NF797 cell lysates were incubated or not for 90 min with soluble FRS3(PTB) domain. Afterward, an in vitro binding assay with GST-FRS2(PTB) or GST-PLC␥ Sepharose-conjugated fusion proteins was performed. Eluted complexes were analyzed by Western blot with antiphosphotyrosine antibodies. NIH3T3 cell lysate was used as control.

FIG. 5. Expression analysis of FRS2 and FRS3 in normal and tumor thyroid tissues. Amplification by nested RT-PCR of FRS2 and FRS3 mRNA from normal and tumor thyroid samples was performed as described in Materials and Methods. Kidney tissue was used as positive control. P0 is a housekeeping gene used as control of retrotranscription. Tumor samples carrying TRK or RET oncogenes are indicated.


P0 mRNA (21) was performed. In all the normal thyroids, no expression of FRS2 was detected. On the contrary, the 515-bp FRS3-specific fragment was detected in all the samples. In some cases, a slightly bigger fragment, identified as an unspliced precursor by nucleotide sequence (data not shown), was also detected. With respect to the tumor samples, FRS3 expression was detected in all of them. Expression of FRS2 was detected in three out of seven samples, independently from the presence of TRK activation. All of the FRS2-negative samples were analyzed by Southern blot of PCR products with an FRS2 oligonucleotide as probe; also, this assay failed to detect FRS2 expression (data not shown). These data indicate that, in papillary thyroid tumors, FRS3 expression is maintained, whereas FRS2 expression is more variable. Discussion

We have investigated the potential of both FRS2 and FRS3 to contribute to the mitogenic signal triggered by TRK-T1 and TRK-T3 oncoproteins, with the final aim to assess a possible role of FRS2 and FRS3 in thyroid carcinogenesis. We demonstrated the involvement of these transducers, investigating the capability of their PTB domains to bind to the oncoproteins by in vitro binding assays. Moreover, we investigated the interaction of the FRS adapters with TRK oncoproteins in vivo, by coimmunoprecipitation assays. Then, we determined the TRK-T3 tyrosine residue involved in the interaction with FRS3. Finally, we investigated the physiological relevance of these studies analyzing FRS mRNA expression in normal and tumor thyroid samples. In vitro binding assays using isolated PTB domains fused to GST have shown that both TRK-T1 and TRK-T3 oncoproteins interact with FRS2 and FRS3 transducers, after a long incubation time. This interaction was completely abolished by K252a treatment, suggesting its dependence on the TRK kinase activity. Using a short incubation time, no interaction between FRS2 and TRK-T1 was observed, at variance with TRK-T3. Most likely this is due to the lack of the KFG motif in the TRK-T1 oncoprotein. The KFG motif has been previously shown to interfere with NTRK1 binding to FRS2: its deletion reduced FRS2 binding but did not completely abolish it (8, 18). However, when an in vivo p13suc1 pull-down assay was performed, a similar ability of the two TRK oncoproteins to interact with and to activate FRS2 was observed. Therefore, the reduced TRK-T1 capability to bind isolated FRS2(PTB) domain is compensated, in vivo, by the length of interaction and/or oncoprotein expression level, so that no reduction of FRS2 activation is observed. The characterization of FRS3/TRK interaction in living cells supported the notion that FRS3 is a substrate for TRK oncoproteins. In fact, cotransfection experiments showed that FRS3 is phosphorylated in the presence of both TRK-T1 and TRK-T3, and it is dependent on their activity. By using different TRK-T3 mutants and two different experimental approaches, we provide evidence that FRS3 interacts with TRK-T3 tyrosine 291, corresponding to tyrosine 490 of NTRK1, which also recruits Shc and FRS2. We have recently shown that the mutation of the tyrosine 291 abrogates the TRK-T3 oncogenic activity (16). Based on our results, such abrogation might be due to the lack of binding not


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only to Shc and FRS2, but also to FRS3. Our data would indicate that FRS3 binds tyrosine 490 of NTRK1, corresponding to tyrosine 291 of TRK-T3. However, because the interaction of FRS3 with TRK-T3 may be influenced by the oncogene-activating sequences, more investigations will be required to define the modalities of NTRK1 interaction with FRS3 and the relationship among Shc, FRS2, and FRS3. In a previous study, McDougall et al. (11) have shown that, despite their extensive sequence identity, FRS2 and FRS3 have different expression patterns in developing tissues during mouse embryogenesis. These observations indicate that FRS2/FRS3 may serve some overlapping and/or independent roles in different tissues. To investigate the relevance of FRS2 and FRS3 interaction with TRK oncoproteins in the context of thyroid tumorigenesis, we analyzed the expression of FRS mRNAs in normal and tumor thyroid samples. The results indicate that FRS3 is expressed in normal thyroid, in agreement with expression studies in mouse (McDougall, K., and S. O. Meakin, manuscript in preparation), and such expression is maintained in tumor specimens. On the contrary, FRS2 was not expressed in the five normal thyroid samples, as previously observed in mouse (McDougall, K., and S. O. Meakin, manuscript in preparation), whereas it was aberrantly expressed in 50% of the thyroid tumors analyzed. No correlation between FRS2 expression and presence of TRK and RET oncogenes was observed, indicating that it might represent an event of the malignant transformation process, rather than be associated with a particular oncogenic type. In this context, it would be interesting to investigate the existence of any correlation between FRS2 expression and thyroid tumor clinical/pathological features. All together, our data suggest that FRS2 and FRS3 transducers are involved in the thyroid tumorigenesis induced by TRK oncogenes, and they might represent targets for therapeutic approaches aimed to block the oncoprotein signaling. However, more investigations are necessary to unveil the role of FRS2 and FRS3 roles in thyroid carcinogenesis driven by TRK oncoproteins. Acknowledgments The authors thank Miss Cristina Mazzadi for secretarial assistance. Received September 25, 2002. Accepted December 2, 2002. Address all correspondence and requests for reprints to: Angela Greco, Department of Experimental Oncology, Istituto Nazionale Tumori, 1 Via Venezian, 20133 Milan, Italy. E-mail: angela.greco@ istitutotumori.mi.it. This work was supported by the Italian Association for Cancer Research. * M.A.P. and A.G. are senior co-authors.

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