INTRODUCTION

Top Abstract Introduction Materials and Methods Results Discussion References

The GDNF family of neurotrophins consists of four members collectively designated GFLs (GDNF family ligands): glial cell-derived neurotrophic factor (GDNF), neurturin, persephin, and artemin. These growth factors play a crucial role in regulating the survival of neurons of the peripheral and central nervous system. The GDNF proteins signal through a multicomponent receptor complex consisting of a ligand-binding GDNF family receptor (GFR), designated the  subunit (GFR), and the proto-oncogenic receptor tyrosine kinase, RET, which forms the  subunit. The four GFR receptors, GFR1, -2, -3, and -4, are linked to the cell membrane via glycosyl-phosphatidylinositol anchors. It was shown that GFR1, -2, -3, and -4 predominantly bind GDNF, neurturin, artemin, and persephin, respectively. RET functions as a common intracellular signal transducing component in conjunction with each of the GFR subunits (1). However, it was recently proposed that the GFR receptors may signal autonomously in the absence of RET (49) and that they target RET to lipid rafts on the cell membrane (47). The GFR and RET receptors are expressed in both distinct and overlapping regions of the developing embryo and the adult mouse. Mice deficient in either GDNF, RET, or GFR1 fail to develop the ureteric bud or undergo metanephric development, demonstrating that this ligand-receptor complex plays a role in kidney morphogenesis. Moreover, the mutant mice lack enteric neurons throughout their digestive tracts (39). The striking similarities in the developmental defects observed in the mutant mice is consistent with the notion that GDNF acts primarily through the GFR1-RET receptor complex (39).

RET has been associated with several human diseases and genetic syndromes. Loss-of-function mutations in RET, designated Hirschsprung disease (HSCR), cause defective intestinal innervation and congenital megacolon (8, 32). Gene rearrangements leading to fusion of the kinase domain of RET with heterologous proteins containing dimerization motifs result in constitutively activated RET proteins. Such fusion proteins are expressed in papillary thyroid carcinomas (PTC) and have been termed RET-PTC (35). Several different RET-PTC genes have been identified in thyroid carcinomas differing in the RET fusion partner. Finally, germ line point mutations in RET result in inherited multiple endocrine neoplasia types 2A and 2B (MEN 2A and MEN 2B) and familial medullary thyroid carcinoma (13). It has been shown that the oncogenic activity of RET in MEN and familial medullary thyroid carcinoma is caused by mutations leading to formation of unpaired cysteines in the extracellular domain which facilitate receptor dimerization and activation (40).

Three RET protein variants (RET9, RET43, and RET51) have been shown to be generated by alternative RNA splicing, leading to the expression of RET isoforms with identical primary structures up to amino acid 1063, followed by unique carboxy-terminal sequences (27). RET51 contains two additional tyrosines (23), one of which, Y1096, functions as a binding site of Grb2 (2). It has been reported that rather than being involved in Ras-mitogen-activated protein (MAP) kinase activation, Y1096 plays an important role in recruiting Gab2-phosphatidylinositol 3-kinase complexes to RET (6). Phosphorylated Y905, Y1015, and Y1062, amino acids common to the three RET protein forms, have been characterized as docking sites for several signaling molecules. pY905 mediates the recruitment of the SH2 domain-containing adapter proteins Grb7 and Grb10 (29, 30). Phospholipase C binds to activated RET via pY1015 (7), and Shc is recruited to activated RET by means of binding of its phosphotyrosine-binding (PTB) domain to pY1062, a tyrosine residue located within a canonical NKLpY (single-letter amino acid code) PTB motif (3, 4, 24). In addition, the PDZ and LIM domain-containing protein Enigma binds to RET via Y1062 in a phosphorylation-independent manner (11, 12). It was demonstrated that Y1062 is essential for the mitogenic and transforming activity of RET-MEN 2A and RET-PTC alleles (4, 46) and for the survival-promoting signal induced by RET-MEN 2A (10).

It is now well established that the Ras-MAP kinase (MAPK) signaling cascade plays a pivotal role in cell mitogenesis and transformation (25) and that the Grb2-Sos complex serves as a link between activated receptor tyrosine kinases (RTKs) and Ras. The Grb2-Sos complex may be recruited to the plasma membrane by direct interaction with activated RTKs or indirectly through membrane-linked docking proteins that are tyrosine phosphorylated in response to RTK stimulation (33, 45). Direct recruitment of the Grb2-Sos complex occurs through the binding of the SH2 domain of Grb2 to the pYXN motif on activated RTKs, as shown for the epidermal growth factor (EGF) receptor (22). However, receptors lacking the pYXN motif, such as the insulin or fibroblast growth factor (FGF) receptors, utilize the docking proteins IRS1 or FRS2, respectively, for recruitment of Grb2-Sos complexes (45). Thus, docking proteins play a dominant role in recruiting the Grb2-Sos complex to the plasma membrane to mediate Ras activation by RTKs which do not bind Grb2 directly (47). In addition, docking proteins play a role in the targeting of signaling molecules to the plasma membrane and in the expansion of the repertoire of signaling pathways activated by RTKs.

In this report, we show that FRS2 proteins are tyrosine phosphorylated in response to activation of the RET receptor. Our data show that the PTB domain of FRS2 binds to pY1062 of RET, an autophosphorylation site which also serves as the binding site for Shc. Accordingly, two natural HSCR-associated mutants of the NKLpY(1062) motif of RET show defective binding to FRS2. FRS2 is constitutively associated with the chimeric RET-PTC oncoproteins, both in a human tumor cell line and in transfected cells. Moreover, overexpression of FRS2 leads to potentiation of MAPK activation by oncogenic RET mutants. These experiments reveal an important role for FRS2 in the normal function of RET and in diseases caused by gain-of-function or loss-of-function RET mutations.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results Discussion References

Expression plasmids. Expression plasmids for FRS2 and its nonmyristylated mutant (G2A) were described previously (21). All RET constructs used in this work, unless otherwise specified, were performed in a background of a RET9 isoform. The RET-PTC3 and RET-PTC3 (Y588F) cDNAs were cloned in the pBABE retroviral vector and in the pCDNA3(Myc-His) vector (Invitrogen, Groningen, The Netherlands) fused in frame at the C terminus with a myc epitope or a His tag (15, 41). The RET plasmids coding for the MEN 2A-associated changes, C634Y and C634R, as well as those coding for the HSCR-associated changes, L1061P and N1059, were previously described (14, 40). The chimeric EGF receptor-RET (E-R) receptor was reported previously (42). The hemagglutinin (HA) epitope-tagged MAPK (HA-ERK2) and myc epitope-tagged p52shc constructs were described previously (14, 16). The RET (C634Y, Y905F), RET (C634Y, Y1062F), RET (C634Y, K758 M), RET (C634Y, Y1015F), and RET (C634Y)-51 (expressing the RET51 form of the MEN 2A-associated C634Y RET mutant) constructs were generated by site-directed mutagenesis using a QuickChange mutagenesis kit (Stratagene, La Jolla, Calif.). The mutations were confirmed by DNA sequencing.

Antibodies and recombinant proteins. Polyclonal anti-RET antibodies were raised against the recombinant kinase domain of the RET protein (42). Anti-FRS2 antibodies have been described previously (21). Anti-phosphotyrosine antibodies (4G10) were from Upstate Biotechnology Inc. (Lake Placid, N.Y.). Rabbit polyclonal anti-MAPK (#9101) and anti-phospho-MAPK (#9102) antibodies were from New England Biolabs (Beverley, Mass.). Anti-Grb2, anti-glutathione S-transferase (GST), anti-tag, and secondary antibodies coupled to horseradish peroxidase were from Santa Cruz Biotechnology (Santa Cruz, Calif.).

Cell culture and transfection. NIH 3T3 fibroblasts were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum and 100 U (each) of penicillin and streptomycin (Gibco BRL, Paisley, Pa.)/ml. NIH 3T3 cells expressing the E-R chimeric receptor have been described previously (42). For EGF stimulation experiments, E-R-expressing cells were serum starved for 36 h prior to stimulation (50 ng/ml, 5 min). Human 293 cells were grown in DMEM supplemented with 10% fetal calf serum and antibiotics. Transient transfections in 293 cells were carried out using LipofectAMINE (Gibco BRL) according to the protocol recommended by the manufacturer. The papillary thyroid carcinoma cells (PTC) and human thyroid cells (HTC) were cultured as described (9). PC Cl 3 is a thyroid epithelial cell line derived from 18-month-old Fischer rats. These cells were cultured in Coon's modified F12 medium supplemented with 5% calf serum (Gibco BRL). For both stable and transient transfections, approximately 5 × 10⁵ PC Cl 3 cells were plated 48 h before transfection. Three hours before transfection the medium was changed to DMEM containing 5% calf serum. Calcium phosphate-DNA precipitates were prepared by standard procedures and were incubated with the cells for 1 h. Then DNA precipitates were removed and cells were incubated in 15% glycerol for 2 min. Finally, the cells were washed with DMEM and incubated for 48 h in Coon's modified F12 medium containing 5% calf serum. PC-PTC3 and PC-PTC3 (Y588F) cells were obtained by expressing the wild type or the Y588F RET-PTC3 mutant in PC Cl 3 cells. Representative mass populations of several hundred clones were obtained by puromycin selection. PC-PTC3 cells transfected with FRS2 were selected with 400 µg of Geneticin (G418) per ml. PC-E-R cells were obtained by transfecting PC Cl 3 cells with the E-R construct (42). PC-PTC3 and PC-PTC3 (Y588F) cells were subjected to transient transfection with FRS2 or p52Shc expression vectors and then processed by cytofluorimetric analysis. Trace amounts of enhanced green fluorescent protein (EGFP) were added to identify transfected cells. For cytofluorimetric analysis cells were fixed in methanol for 1 h at 20°C, rehydrated in phosphate-buffered saline (PBS) for an additional hour at 4°C, and then treated with RNase A (50 µg/ml) for 30 min. Propidium iodide (25 µg/ml) was added to the cells, and samples were analyzed with a FACScan flow cytometer (Becton Dickinson, San Jose, Calif.) interfaced with an Hewlett Packard computer (Palo Alto, Calif.). The percentages of cells in the G₀/G₁, S, and G₂/M phases were determined. The average values from three independent experiments were obtained. [³H]thymidine incorporation assays were performed as described previously (42). Briefly, cells were grown to confluence in 24-well plates (Costar, Acton, Mass.) and then maintained for 24 h in Coon's modified F12 medium containing 1% serum in the presence of 4 µCi of [³H]thymidine (Amersham, Buck, United Kingdom) per ml. [³H]thymidine incorporation was measured in triplicate. The average values from three independent experiments were obtained.

Immunoprecipitation and peptide binding experiments. Cells were solubilized in lysis buffer containing 50 mM HEPES (pH 7.5), 1% (vol/vol) Triton X-100, 150 mM NaCl, 5 mM EGTA, 50 mM NaF, 20 mM sodium pyrophosphate, 1 mM sodium orthovanadate, 2 mM phenylmethylsulfonyl fluoride, and 0.2 µg (each) of aprotinin and leupeptin per ml. Cell lysates were subjected to immunoprecipitation with different antibodies or subjected to pull-down binding assays with purified recombinant proteins immobilized on agarose beads. The protein complexes were washed with the lysis buffer, eluted, and resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Immunoblotting with specific antibodies and enhanced chemiluminescence (ECL; Amersham) were employed for immunodetection of proteins in complexes. Peptides dissolved in the appropriate solvent were added to the cell lysates. Incubation conditions for binding and washing of protein complexes, SDS-PAGE separation, and detection by immunoblotting were performed as detailed above. The peptides used in this study include STWIENKLYGRIS (p1062) and STWIENKLpYGRIS (pp1062), spanning the RET sequence around Y1062, and QISLDNPDpYQQDF (pEGFR), spanning the EGFR sequence that functions as the binding site for the PTB domain of Shc (pY1148) (5). These peptides were synthesized by Neosystem Laboratoire (Strasbourg, France). Subcellular fractionation was performed according to published procedures (8). Cells were homogenized in a hypotonic buffer containing 20 mM HEPES (pH 7.5), 5 mM EGTA, 50 mM NaF, 20 mM sodium pyrophosphate, 1 mM sodium orthovanadate, 2 mM phenylmethylsulfonyl fluoride, and 0.2 µg (each) of aprotinin and leupeptin per ml. The nuclear pellet was removed after centrifugation at 1,000 × g for 5 min at 4°C. The postnuclear fraction was centrifuged at 100,000 × g for 45 min to separate the soluble and particulate fractions.

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

Ligand-stimulated E-R chimeric receptor induces tyrosine phosphorylation of FRS2. Although a large body of evidence has implicated RET in oncogenesis, the underlying mechanisms of signaling through activated RET remain poorly understood. In order to study intracellular signaling through RET, we have generated NIH 3T3 cells which express a chimeric E-R receptor, whereby the extracellular ligand-binding domain of RET was replaced with that of the EGF receptor (42) (Fig. 1A). The parental NIH 3T3 cells express negligible amounts of endogenous EGFRs and no RET receptors. The use of this chimeric receptor allowed the analysis of signal transduction through RET independently of the GFR coreceptors, which have been shown to be capable of autonomous signaling in response to ligand binding (49). Stimulation of NIH 3T3 cells expressing the E-R receptors with EGF has been shown to result in mitogenesis and cell transformation (42). Moreover, stimulation of RET results in tyrosine phosphorylation of Shc and complex formation with Grb2-Sos (3, 4, 24).

View larger version (29K): [in this window] [in a new window]   FIG. 1.   FRS2 coimmunoprecipitates with the activated RET RTK. (A) A schematic representation of RET and the chimeric EGFR-RET receptor. CAD, cadherin homologous domain; CYS, cysteine-rich sequence; TM, transmembrane domain. (B) Quiescent NIH 3T3 cells expressing E-R receptors were left unstimulated or were stimulated with EGF (50 ng/ml, 5 min), and lysates from these cells were immunoprecipitated (IP) with anti-Grb2 antibodies, followed by SDS-PAGE and immunoblotting (IB) with anti-pTyr antibodies. The migration of E-R, Shc, and FRS2 as revealed by immunoblotting with specific antibodies is marked. pY, pY1062. (C) Quiescent parental NIH 3T3 (NIH) and NIH-E-R cells were left unstimulated or were stimulated with EGF (50 ng/ml, 5 min). Lysates from these cells were immunoprecipitated with anti-FRS2 antibodies followed by SDS-PAGE and immunoblotting with anti-pTyr antibodies. (D) Quiescent parental PC Cl 3 (PC) and PC-E-R cells were left unstimulated or were stimulated with EGF (50 ng/ml, 5 min). Lysates were immunoprecipitated with anti-FRS2 antibodies followed by SDS-PAGE and immunoblotting with anti-pTyr antibodies.

The PTB domain of FRS2 binds to pY1062 of RET. We proceeded to investigate the nature of the interaction between FRS2 and RET. To this end, we made use of a constitutively activated RET mutant responsible for the MEN 2A cancer syndrome, RET (C634Y). The use of this mutant rather than the wild-type receptor circumvents the necessity to cotransfect the GFR coreceptors and confines the analysis to RET-mediated signaling. It has been shown that the substitution of C634 with different amino acids (such as Y or R) induces receptor activation through disulfide-linked RET dimerization (40). We also constructed three additional RET mutants in the C634Y background. These include RET (C634Y, Y905F), a mutant with active but reduced kinase activity (18), RET (C634Y, K758 M) (11), a kinase-negative mutant, and RET (C634Y, Y1062F), a mutant unable to recruit Shc (3, 4, 24) (Fig. 2A). RET (C634Y), RET (C634Y, Y905F), RET (C634Y, K758 M), and RET (C634Y, Y1062F) were transiently expressed in human 293 cells, and lysates prepared from these cells were subjected to a pull-down binding assay with the PTB domain of FRS2 expressed as a GST fusion protein and immobilized on agarose beads. GST alone was used as a control. Specifically bound proteins were eluted, resolved by SDS-PAGE, and electroblotted onto nitrocellulose membranes. The binding of RET proteins to the PTB domain of FRS2 was detected by immunoblotting with anti-RET antibodies. This experiment shows that the PTB domain binds to phosphorylated Y1062 on RET (Fig. 2B, left). Alternative splicing at the RET carboxyl terminus generates three protein variants (RET9, RET43, and RET51) with different C termini. Y1062 and residues N terminal to this amino acid are common to RET9, RET51, and RET43 (27). We have examined the possibility of whether the different C termini of the different isoforms influenced complex formation between RET and FRS2. RET (C634Y) and RET (C634Y)-51, expressing the C634Y MEN 2A mutant in the RET9 or RET51 background, respectively, were expressed in 293 cells, and lysates were subjected to a pull-down assay with the PTB domain of FRS2. The two forms of RET (C634Y) showed similar abilities to bind the PTB domain of FRS2 (Fig. 2B, right). These findings are consistent with earlier studies demonstrating that amino acids N terminal to the pTyr residue are essential for specific binding of PTB domains to target sequences (20, 26, 45).

View larger version (30K): [in this window] [in a new window]   FIG. 2.   The PTB domain of FRS2 binds to pY1062 of RET. (A) A schematic diagram of the RET protein denoting residues that were mutated. CAD, cadherin homologous domain; CYS, cysteine-rich sequence; TM, transmembrane domain. (B) 293 cells were transfected with the expression plasmids encoding RET (C634Y), RET (C634Y, Y905F), RET (C634Y, K758 M), RET (C634Y, 1062F) (left-side gel), or RET (C634Y)-51 (right-side gel). The lysates (500 µg) were subjected to a pull-down binding assay with the GST fusion protein of the PTB domain of FRS2 or with GST alone as a control. The protein complexes were resolved by SDS-PAGE followed by immunoblotting (IB) with anti-RET antibodies (upper gel). Equal amounts of total cell lysates from the lysate sample of each mutant were resolved by SDS-PAGE followed by immunoblotting with anti-RET antibodies to demonstrate the comparable expression of the different mutants (lower gel). (C and D) 293 cells were transfected with the expression plasmids encoding RET (C634Y), RET (C634Y, Y1062F), RET (C634Y, K758 M), RET (C634Y, Y1015F), or RET (C634Y, Y905F). One milligram of each lysate sample was immunoprecipitated (IP) with anti-RET antibodies. The immunoprecipitates were recovered with protein A-Sepharose beads and were washed. The beads were then incubated with a purified GST fusion protein of the PTB domain of FRS2 or with GST alone as a control (top section of panel D). The protein complexes were washed and analyzed by SDS-PAGE followed by immunoblotting with anti-GST antibodies (upper gel of panel C and upper gel of panel D). Equal amounts of total cell lysates were stained with anti-RET antibodies to evaluate the expression of the different mutants (lower gels of each panel). (E) 293 cells were transfected with the expression plasmids encoding RET (C634Y) or RET (C634Y, Y1062F). One milligram of each lysate sample was immunoprecipitated with anti-FRS2 antibodies. The immunoprecipitates were resolved by SDS-PAGE followed by immunoblotting with anti-pTyr antibodies (upper gel). Equal amounts of total cell lysates from the lysate sample of each mutant were resolved by SDS-PAGE followed by immunoblotting with anti-RET antibodies to determine the expression of the different mutants (lower gel). pY, pY1062.

HSCR-associated RET mutations impair interaction of RET with FRS2. Many HSCR cases are associated with naturally occurring loss-of-function mutations of the RET RTK. HSCR mutations include deletions, insertions, frameshifts, and nonsense and missense mutations dispersed throughout the RET coding sequence (37). Recently, two distinct mutations of RET were mapped in the vicinity of the binding site for the PTB domains of Shc and FRS2: deletion of Asn 1059 (N1059) and replacement of Leu 1061 by a Pro (L1061P) (14). To evaluate their effects on the interaction between RET and FRS2, we introduced these changes into the constitutively activated MEN 2A-associated RET (C634R) mutant (as schematically shown in Fig. 3A) and analyzed the binding of these mutants with FRS2 in vitro and in living cells. RET (C634R) or the HSCR mutants were expressed in 293 cells, and lysates prepared from the cells were subjected to a pull-down binding assay with the PTB domain of FRS2 expressed as a GST fusion protein and immobilized on agarose beads. The binding of the RET proteins was detected by immunoblotting with anti-RET antibodies. The experiment presented in Fig. 3B demonstrates that the binding of both the HSCR mutants to the PTB domain of FRS2 was significantly impaired, although the binding of the L1061P mutant was reduced to a lesser extent than the binding of the N1059 mutant. Thus, the binding of RET to the PTB domain of FRS2 requires Asn and Leu residues at positions 3 and 1 relative to pY1062, respectively.

View larger version (33K): [in this window] [in a new window]   FIG. 3.   HSCR-associated RET gene mutations impair the binding of FRS2 to activated RET. (A) A schematic diagram of the RET protein denoting residues which have been mutated. CAD, cadherin homologous domain; CYS, cysteine-rich sequence; TM, transmembrane domain. (B) RET (C634R) alone and RET (C634R) bearing the HSCR-associated changes, N1059 or L1061P, were transiently expressed in 293 cells. Cell lysates were subjected to a pull-down binding assay, with the GST fusion protein of the PTB domain of FRS2 immobilized on beads. The protein complexes were eluted, resolved by SDS-PAGE, and immunoblotted (IB) with anti-RET antibodies (upper gel). Equal amounts of total cell lysates were resolved by SDS-PAGE followed by immunoblotting with anti-RET antibodies to evaluate the expression of the different mutants (lower gel). (C) 293 cells were transfected with plasmids encoding RET (C634R) or the mutants bearing the HSCR-associated changes, N1059 or L1061P. One milligram of each lysate sample was immunoprecipitated (IP) with anti-FRS2 antibodies. The immunoprecipitates were eluted and resolved by SDS-PAGE followed by immunoblotting with anti-pTyr antibodies (upper gel). Equal amounts of total cell lysates were resolved by SDS-PAGE, followed by immunoblotting with anti-RET antibodies to evaluate the expression of the different mutants (lower gel). pY, pY1062. (D) 293 cells were transfected with plasmids coding for an epitope-tagged MAPK construct (HA-ERK2) together with RET (C634R) or the HSCR changes, N1059 and L1061P. Each sample (50 µg) was resolved by SDS-PAGE followed by immunoblotting with anti-RET (upper gel) or anti-pMAPK (middle gel) or with the anti-HA epitope antibodies for normalization (MAPK, lower gel). , 293 cells transfected with HA-ERK2 alone. (E) 293 cells were transfected with plasmids encoding RET (C634R) or RET (C634R, Y1062F) in the presence of p52Shc or FRS2-encoding plasmids as indicated (top section). Each lysate sample (50 µg) was resolved by SDS-PAGE followed by immunoblotting with anti-pMAPK (middle section) or with anti-MAPK antibodies for normalization (bottom section). RET, FRS2, and Shc expression was confirmed by immunoblotting (not shown).

View larger version (31K): [in this window] [in a new window]   FIG. 4.   The PTB domain of FRS2 binds to pY1062 of RET. The RET (C634Y) or, as a control, RET (C634Y, Y1062F) proteins were transiently expressed in 293 cells. Equal amounts of the lysate were incubated with the GST fusion proteins of the PTB domains of FRS2 or Shc in the presence of 1, 10, or 50 µM concentrations of the pp1062, p1062 (A), or pEGFR (B) peptides. pp1062 is a phosphorylated RET peptide (STWIENKLpY1062GRIS), and p1062 is the corresponding nonphosphorylated peptide. pEGFR is an EGFR-derived phosphopeptide (QISDNPDpY1148QQDF) which functions as a binding site for the PTB domain of Shc. Bound proteins were eluted and resolved by SDS-PAGE followed by immunoblotting (IB) with anti-RET antibodies. (C) Equal amounts of total cell lysates from the lysate sample of RET (C634Y) or RET (C634Y, Y1062F) were resolved by SDS-PAGE followed by immunoblotting with anti-RET antibodies to evaluate the expression of the different mutants.

Differential effects of FRS2 and Shc overexpression on RET-PTC-induced MAPK response, cell cycle progression, and membrane targeting. PTC is the most common form of thyroid malignancy in humans. The predominant molecular lesion that is associated with these tumors is the fusion of RET with heterologous proteins. The fusion proteins, termed RET-PTC, are expressed in thyroid carcinomas, are cytosolic, and contain coiled-coil domains that are responsible for RET-PTC dimerization and activation (48). Several oncogenic RET-PTC fusion proteins have been characterized (15, 41). The RET-PTC1 and RET-PTC3 oncoproteins result from the fusion of the kinase domain of RET with the coiled-coil motifs of the H4 protein and the RFG gene product, respectively (Fig. 5A) (16, 44). It has been shown that transgenic mice expressing either RET-PTC1 or RET-PTC3 develop thyroid carcinomas (19, 38, 43).

View larger version (30K): [in this window] [in a new window]   FIG. 5.   RET-PTC oncoproteins interact with FRS2. (A) A schematic representation of the wild-type RET protein and the chimeric RET-PTC1 and RET-PTC3 transforming proteins. Tyrosine 1062 of RET and the corresponding tyrosines in RET-PTCs are indicated. CAD, cadherin homologous domain; CYS, cysteine-rich sequence; TM, transmembrane domain. (B) Lysates from normal thyroid epithelial cells (HTC) and papillary thyroid carcinoma cells (PTC) were immunoprecipitated (IP) with anti-FRS2 antibodies followed by SDS-PAGE and immunoblotting (IB) with anti-pTyr antibodies (upper gel). An immunoblot with anti-RET antibodies was performed to show the presence of RET-PTC1 protein in PTC but not HTC cells (lower gel). pY, pY1062. (C) Expression vectors for RET-PTC3 and RET-PTC3 (Y588F) were expressed in 293 cells, and a pull-down assay with GST fusion protein of the PTB domains of FRS2 was performed; untransfected 293 cells () were used as a control. (D) 293 cells were transfected with expression vectors encoding RET-PTC3 and RET-PTC3 (Y588F). One milligram of each lysate sample was immunoprecipitated with anti-FRS2 antibodies. The immunoprecipitates were eluted and resolved by SDS-PAGE followed by immunoblotting with anti-pTyr antibodies (upper gel). Equal amounts of total cell lysates were resolved by SDS-PAGE followed by immunoblotting with anti-RET antibodies to evaluate the expression of the different mutants (lower gel). (E) 293 cells were transfected with expression vectors encoding RET-PTC3 or RET-PTC3 (Y588F). One milligram of each lysate sample was immunoprecipitated with anti-Shc antibodies. The immunoprecipitates were eluted and resolved by SDS-PAGE followed by immunoblotting with anti-pTyr antibodies (upper gel). Equal amounts of total cell lysates were resolved by SDS-PAGE followed by immunoblotting with anti-RET antibodies to evaluate the expression of the different mutants (lower panel).

View larger version (34K): [in this window] [in a new window]   FIG. 6.   FRS2 promotes localization of RET-PTC3 protein to particulate fraction and potentiates RET-PTC-mediated MAPK activation. (A) 293 cells were transfected with RET-PTC3 alone or together with wild-type FRS2 or the G2A nonmyristylated mutant. Cells were homogenized and separated into the postnuclear soluble (C) and particulate membrane (M) fractions. After removal of cell nuclei, about 30% of the total proteins were found in the membrane fraction and 70% were in the cytosolic fraction. Each fraction (50 µg) was subjected to SDS-PAGE and immunoblotted (IB) with anti-RET antibodies or anti-FRS2 antibodies. The efficiency of fractionation was determined by using EGFR and eps15 as specific markers for the membrane and cytosolic fractions, respectively (data not shown). (B) 293 cells were transfected with RET-PTC3 alone or together with a myc-tagged p52Shc expression vector. Cells were homogenized and separated into the postnuclear soluble and particulate membrane fractions. Each fraction (50 µg) was subjected to SDS-PAGE and immunoblotted with anti-RET or anti-myc antibodies. The efficiency of fractionation was determined by using EGFR and eps15 as specific markers for the membrane and cytosolic fractions, respectively (data not shown). (C) 293 cells were transfected with expression vectors encoding epitope-tagged MAPK (HA-ERK2) together with expression vectors for myc- and His-tagged RET-PTC1 or RET-PTC3 alone or in combination with FRS2 as indicated. Each lysate sample (50 µg) was resolved by SDS-PAGE followed by immunoblotting with (i) pMAPK antibodies, (ii) anti-HA antibodies (MAPK), and (iii) anti-His-tagged antibodies (RET-PTC1 and RET-PTC3). FRS2 expression was detected by immunoprecipitation of 200 µg of each sample followed by SDS-PAGE and immunoblotting with anti-FRS2 antibodies. (D) 293 cells were transfected with expression vectors for myc- and His-tagged RET-PTC3 alone or in combination with FRS2 or p52Shc as indicated. Each lysate sample (50 µg) was resolved by SDS-PAGE followed by immunoblotting with pMAPK or MAPK antibodies. RET-PTC3, FRS2, and Shc expression was confirmed by immunoblotting (data not shown).

View larger version (29K): [in this window] [in a new window]   FIG. 7.   Potentiation of RET-PTC3 induced stimulation of S-phase entry of PC Cl 3 thyroid cells by overexpression of FRS2. (A) PC-PTC3 cells (PC Cl 3 cells expressing the RET-PTC3 oncogene) were transfected with the FRS2 expression vector. A representative cell population was designated PC-PTC3-FRS2. Aliquots of 100 µg of each lysate sample from the PC-PTC3 and PC-PTC3-FRS2 cells were resolved by SDS-PAGE, followed by immunoblotting (IB) with anti-RET, anti-FRS2, or anti-pMAPK antibody. (B) PC-PTC3 and PC-PTC3-FRS2 cells were homogenized and separated into the postnuclear soluble (C) and particulate membrane (M) fractions. Each fraction (100 µg) was subjected to SDS-PAGE and immunoblotted with anti-RET antibodies. The efficiency of fractionation was determined by using anti-EGFR and anti-eps15 antibodies as specific markers for the membrane and cytosolic fractions, respectively (data not shown). (C) PC-PTC3 and FRS2-expressing PC-PTC3 cells were harvested after 96 h of serum deprivation and analyzed by flow cytometry. When indicated, PC-PTC3-FRS2 cells were pretreated with 20 µM PD98059 (PD). The percentage of cells in each phase of the cell cycle is depicted in a bar graph. The results are the averages of three independent experiments. (D) PC-PTC3 or PC-PTC3 (Y588F) cells were transiently transfected with expression vectors for FRS2 or p52Shc together with an expression vector for EGFP to identify transfected cells. On average, approximately 70% of transfected cells were EGFP positive (not shown). Cells were harvested after 48 h of serum deprivation (1% serum), and EGFP-positive cells were analyzed by flow cytometry. The results are the averages of three independent experiments.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

The FRS2 docking proteins are major substrates of the FGF and NGF receptors (16, 21). The two members of the FRS2 family (FRS2 and FRS2) are structurally similar, comprising an N-terminal myristylation sequence, a PTB domain, and a C-terminal fragment that contains multiple tyrosine residues that, when phosphorylated, constitute recognition motifs for the SH2 domains of Grb2 and Shp2, leading to the recruitment of Grb2-Sos complexes to the cell membrane (16, 21, 50). The binding of Shp2 to FRS2 results in its own tyrosine phosphorylation followed by complex formation with Grb2. Thus, upon tyrosine phosphorylation, the FRS2 proteins mediate the recruitment of multiple Grb2-Sos complexes both directly and indirectly through Shp2. Compared to other docking proteins, FRS2 and FRS2 show several unique properties. While most docking proteins, such as IRS1, -2, -3, and -4, interact with multiple signaling molecules, such as Grb2, Shp2, Nck, and phosphatidylinositol-3 kinase, the FRS2 proteins mainly appear to be involved in the recruitment of Grb2 and Shp2. In addition, IRS, Dok, and Shc are translocated from the cytosol to the plasma membrane in response to receptor activation, while FRS2 proteins are permanently linked to the plasma membrane via a myrstyl anchor (45).

FRS2 proteins bind directly to the FGF and NGF receptors and, as demonstrated in this report, to RET through their PTB domains. However, while the interaction between the FRS2 proteins and the FGF receptor does not depend on tyrosine phosphorylation, their binding to the NGF receptor or RET requires tyrosine autophosphorylation of these receptors. FRS2 proteins bind specifically to a highly conserved region in the juxtamembrane region of FGFR1, recognizing the sequence KSIPLRRQVTVS (28, 50). In contrast, the binding of FRS2 proteins to Trk (26, 28) or RET is mediated through a canonical PTB domain recognition sequence comprising the NXXpY motif. Accordingly, in this report we show that the PTB domain of FRS2 binds to RET through an NKLpY1062 motif at the C-terminal tail of the receptor. Another docking protein, Shc, also binds to activated Trk and RET receptors at pY490 and pY1062, respectively, by means of its PTB domain. From the characterization of interactions between FRS2 proteins and the FGF, Trk, and RET receptors, it appears that the PTB domains of FRS2 proteins are capable of recognizing relatively diverse sequences in different receptors. Although the -turn structure formed by the NPXpY motif in several binding sites of PTB domains has been shown to be required for high-affinity interaction with PTB domains, both the recognition sites for the PTB domains of FRS2 and FRS2 on FGFR1 and RET lack the -turn-forming motifs. Thus, the PTB domains of FRS2 proteins exhibit ligand-binding specificities that are not restricted to recognition of phosphotyrosine-containing or -turn-forming sequences.

In addition to the recruitment of Shc and FRS2 proteins, it has been shown that Y1062 in RET is required for the binding of Enigma, a protein composed of a PDZ domain followed by three LIM domains. Enigma has been shown to associate directly with RET or the RET-PTC fusion proteins constitutively through its second LIM domain (11, 12). Several studies have demonstrated that Y1062 of RET plays a critical role in mediating the mitogenic effects of wild-type RET as well as the transforming potential of the RET-PTC oncoproteins (4, 11, 12, 46). Recently, two mutations of RET in the vicinity of Y1062, resulting in the deletion of Asn1059 (N1059) or the replacement of Leu1061 by Pro (L1061P), respectively, were identified in HSCR patients (14). Biochemical analysis provided evidence that the recruitment of Shc to the N1059 or L1061P mutants of the receptor was impaired (14). In this report, we demonstrate that the N1059 or L1061P mutations also interfere with the binding of FRS2 to the RET protein. Intriguingly, while most HSCR mutations are heterozygous, the L1061P mutation, which causes only a partial defect in Shc and FRS2 binding and partial attenuation of MAPK response, has been found in the homozygous state in HSCR patients (14).

We have shown that a phosphopeptide corresponding to the binding site of the PTB domain of Shc on the EGF receptor effectively inhibits the binding of the PTB domain of Shc to RET but does not interfere with the binding of FRS2 to the same receptor. This result suggests that the PTB domains of Shc and FRS2 bind to overlapping but not identical sites on RET within the context of pY1062. It would be of interest to identify loss-of-function mutations in RET that show differential binding towards Shc, FRS2, and perhaps also Enigma. The characterization of the binding of Shc, FRS2, and Enigma to such mutants may provide insights into the molecular mechanisms of interaction of these proteins with RET in the vicinity of Y1062.

Gain-of-function mutations in RET genes give rise to constitutively activated receptors, resulting in the MEN 2 class of dominantly inherited cancer syndromes (13). Here, we report that two of the naturally occurring activating point mutations in the MEN 2A syndrome (C634Y and C634R) are constitutively associated with FRS2. As shown previously, Shc is also constitutively associated with and tyrosine phosphorylated by the MEN 2 form of RET. Tyrosine-phosphorylated FRS2 and Shc proteins mediate the recruitment of the Grb2-Sos complexes to the plasma membrane to activate Ras, resulting in activation of the MAPK cascade (45). Thus, FRS2 and Shc may be dominant targets of the activated forms of RET, acting as mediators of RET-induced cell transformation. However, the roles played by FRS2 and Shc in RET signaling and oncogenesis do not appear to be identical. Here we show that overexpression of FRS2, but not Shc, promotes MAPK activation induced by RET-MEN 2A and RET-PTC3. These findings indicate that FRS2 is particularly efficient in coupling RET to the MAPK pathway. This can be due to the ability of FRS2 to recruit multiple Grb2-Sos complexes, the localization of FRS2 at the cell membrane, and its ability to recruit directly Shp2, which also appears to play a role in MAPK stimulation. These findings do not exclude a role for Shc in this process; rather, they indicate that FRS2 plays a more prominent role in mediating cellular signaling of oncogenic RET proteins.

Somatic events that lead to the activation of RET have been extensively documented in human PTC (35). In these cases, chromosomal translocations occur resulting in the fusion of the RET locus with other genes. These rearrangements, which give rise to the RET-PTC oncoproteins, may play a causative role in thyroid cancer. However, the mechanism(s) by which these proteins cause cell transformation is poorly understood. Here we show that FRS2 is recruited by RET-PTC proteins and that FRS2 overexpression potentiates MAP activation and mitogenesis induced by these oncogenic proteins. Biochemical fractionation experiments demonstrate that overexpression of FRS2 leads to accumulation of RET-PTC3 in the particulate fraction. It is likely that the FRS2-RET-PTC complex is localized, at least in part, at the cell membrane due to interactions with FRS2, as FRS2 is linked to the cell membrane by myristylation (21). FRS2-mediated plasma membrane recruitment of RET-PTC proteins is likely to contribute to the activation of signaling cascades that are initiated at the plasma membrane. By contrast, experiments presented in this report show that Shc is unable to recruit RET-PTC3 protein to the particulate cell fraction.

Our findings demonstrate that FRS2 couples RET with the Ras-MAPK signaling cascade. Potentiation of this central signaling cascade can be involved in neoplastic diseases associated with gain-of-function mutations of RET genes. On the other hand, impairment of the link between RET and MAPK takes place in congenital megacolon (HSCR) (14, 39). Accordingly, De Vita et al. demonstrated that the survival signal induced by RET is dependent upon activation of Akt and MAPK (10). It has been shown that enteric neural crest cells undergo apoptosis in the foregut of embryos lacking the RET receptor (39), indicating that impairment in cell survival pathways may be one of the major consequences of RET inactivation in HSCR. Indeed, we have recently demonstrated that FRS2 plays an important role in the control of a cell survival pathway mediated by Gab1 and phosphatidylinositol-3 kinase (29). On the basis of the experiments described in this report revealing a role for FRS2 in signaling via RET, we propose that FRS2 proteins play a pivotal role in coupling RET with the MAPK signaling cascade under normal physiological conditions and by the oncogenic forms of RET.