Role of Dok1 in Cell Signaling Mediated by RET Tyrosine Kinase*

Using a yeast two-hybrid screen, we identified Dok1 as a docking protein for RET tyrosine kinase. Dok1 bound more strongly to RET with a multiple endocrine neoplasia (MEN) 2B mutation than RET with a MEN2A mutation and was highly phosphorylated in the cells expressing the former mutant protein. Analysis by site-directed mutagenesis revealed that tyrosine 361 in mouse Dok1 represents a binding site for the Nck adaptor protein and tyrosines 295, 314, 361, 376, 397, and 408 for the Ras-GTPase-activating protein. We replaced tyrosine 361 or these six tyrosines with phenylalanine (designated Y361F or 6F) inDok1 and introduced the mutant Dok1 genes into the cells expressing the wild-type RET or RET-MEN2B protein. Overexpression of Dok1 or Dok1-Y361F, but not Dok1–6F, suppressed the Ras/Erk activation induced by glial cell line-derived neurotrophic factor or RET-MEN2B, implying that this inhibitory effect requires the Ras-GTPase-activating protein binding to Dok1. In contrast, overexpression of Dok1, but not Dok1-Y361F or Dok1–6F, enhanced the c-Jun amino-terminal kinase (JNK) and c-Jun activation. This suggested that the association of Nck to tyrosine 361 in Dok1 is necessary for the JNK and c-Jun activation by glial cell line-derived neurotrophic factor or RET-MEN2B. Because a high level of the JNK phosphorylation was observed in the cells expressing RET-MEN2B, its strong activation via Nck binding to Dok1 may be responsible for aggressive properties of medullary thyroid carcinoma developed in MEN 2B.

RET is a tyrosine kinase receptor that plays an important role in the development of the enteric nervous system and the kidney (1,2). It has been demonstrated that members of the glial cell line-derived neurotrophic factor (GDNF) 1 family including GDNF, neurturin, artemin, and persephin represent the RET ligands. The RET activation by these neurotrophic factors are mediated by their binding to glycosylphosphatidylinositol-anchored co-receptors termed GDNF family receptor ␣ 1-4 (GFR␣ 1-4) (1,2). GDNF, neurturin, artemin, and persephin use GFR␣ 1, GFR␣ 2, GFR␣ 3, and GFR␣ 4 as the preferred receptors, respectively, and play specific roles in vivo through these preferred ligand-receptor complex formation. For example, Gdnf-or Gfr␣1-deficient mice had the phenotype quite similar to that of Ret-deficient mice, exhibiting the defects of enteric neurons as well as renal agenesis or dysgenesis (3)(4)(5)(6)(7)(8). In Nrtn-or Gfra2-deficient mice, the parasympathetic innervation was markedly reduced in the lachrymal and submandibular glands and the intestine (9,10). Gfr␣3-deficient mice showed severe defect of the superior cervical ganglion (11).
RET mutations are responsible for development of several human diseases including Hirschsprung's disease, multiple endocrine neoplasia (MEN) type 2A and 2B, familial medullary thyroid carcinoma (FMTC), and papillary thyroid carcinoma (PTC) (1,2,12). Loss-of-function mutations of RET lead to the development of Hirschsprung's disease (13,14), a malformation characterized by the absence of autonomous enteric neurons. On the other hand, gain-of-function mutations of RET contribute to the development of human neoplastic diseases including MEN 2A, MEN 2B, FMTC, and PTC (12,15,16). MEN 2A, MEN 2B, and FMTC are caused by germ-line point mutations of RET, and PTC is caused by its somatic rearrangement. MEN 2A and MEN 2B share the clinical feature of medullary thyroid carcinoma (MTC) and pheochromocytoma, and FMTC is characterized by the development of MTC alone. In addition, ϳ10 -30% of MEN 2A patients develop parathyroid hyperplasia, whereas MEN 2B patients show a more complex phenotype including ganglioneuromatosis of the gastrointestinal tract, mucosal neuroma, and marfanoid habitus. MTCs developed in MEN 2B appear more aggressive than those in MEN 2A and FMTC.
In this study, to further elucidate the activation mechanism of intracellular signaling through RET receptor, we tried to identify the molecules that interact with phosphorylated RET by a yeast two-hybrid screen. We identified Dok1, the phosphotyrosine-binding (PTB) domain of which was responsible for the binding to tyrosine 1062 in RET in response to GDNF. Tyrosine 361 in mouse Dok1 bound the Nck adaptor protein, and six tyrosines with the YXXP motif bound Ras-GAP, leading to the JNK and c-Jun activation and attenuation of the Erk1/2 activation, respectively. In addition, we found enhanced JNK activation in the cells expressing RET-MEN2B that may result from a high level of Dok1 phosphorylation.

EXPERIMENTAL PROCEDURES
Yeast Two-hybrid Screening-An ϳ1.2-kbp EcoRI-XhoI cDNA fragment encoding the cytoplasmic domain of human RET with a MEN2B mutation (M918T) was amplified by PCR and fused to the sequence encoding the GAL4 DNA binding domain (DB) in the pAS2-1 vector (CLONTECH). The recombinant plasmid was co-transfected into the Y190 strain of Saccharomyces cerevisiae with two-hybrid library derived from fetal human brain cDNA (CLONTECH). The transformants were plated on selective medium lacking histidine, tryptophan, and leucine with 30 mM 3-amino-1,2,4-triazole. From ϳ5.7 ϫ 10 6 independent clones, 77 colonies were found to grow on the selective medium, and to be positive for ␤-galactosidase expression. Plasmids isolated from His(Ϫ) and ␤-galactosidase-positive clones were used to transform Escherichia coli, HB101. To eliminate false positives, the plasmids isolated from bacteria were used to co-transform yeast with pAS2-1 (no insert) or pAS2-1-RETc containing the cytoplasmic domain of human RET. Transformants were grown in selective medium without leucine, tryptophan, and histidine. Positive clones were identified by the ␤-galactosidase activity filter assay. These clones were further characterized by sequencing and analyzed for gene homology by the on-line Blast search engine.
Plasmid Constructs-Wild-type human RET or RET-MEN2B cDNA was introduced into the Rc/CMV expression plasmid (Invitrogen). Fulllength mouse wild-type Dok1 or Dok1 mutant cDNAs were amplified by PCR with introduction of appropriate restriction enzyme sites in the primer. The cDNA fragments were introduced into the pFLAG-CMV2 expression vector (Sigma), thereby fusing the Dok1 cDNAs with the FLAG sequence. All Dok1 mutations were introduced into doublestranded DNA by using the QuikChange site-directed mutagenesis kit (Stratagene) according to the instructions of the manufacturer. The sequences of all constructs were confirmed by DNA sequencing.
Cell Lines-NIH 3T3 mouse fibroblast and SK-N-MC human primitive neuroectodermal tumor cell lines were grown in Dulbecco's modified Eagle's medium supplemented with 8% calf serum. NIH 3T3 and SK-N-MC cell lines transfected with the expression plasmids containing wildtype RET or mutant RET cDNA were described previously (19,31).
Antibodies-Anti-Nck polyclonal antibody and anti-phosphotyrosine (PY) monoclonal antibody were purchased from Upstate Biotechnology Inc. Anti-Dok1 and anti-Erk1/2 polyclonal antibodies, and anti-Ras-GAP, anti-JNK, and anti-GAL4 DNA-binding domain monoclonal antibodies were purchased from Santa Cruz Biotechnology Inc. Anti- with human DOK1 in yeast two-hybrid system. Transformants were assayed for ␤-galactosidase activity filter assay. pAS, pAS2-1 vector with the GAL4 DNA binding domain; pACT, pACT2 vector with the GAL4 activating domain. D, binding of GST-DOK1 fusion protein to RET in vitro. The lysates from NIH 3T3 cells expressing RET with a MEN2A (cysteine 634 3 arginine, C634R) mutation or from SK-N-MC cells expressing wild-type RET untreated or treated with GDNF (100 ng/ml) for 15 min were incubated with the GST-DOK1 fusion protein immobilized on glutathione-agarose beads. Bound proteins were separated on SDS-8% polyacrylamide gels and subjected to immunoblotting with anti-RET antibody. 175-and 155-kDa RET proteins are indicated. phosphoErk1/2, anti-stress-activated protein kinase/JNK, anti-Akt, anti-phosphoAkt, anti-c-Jun, and anti-phosphoJun polyclonal antibodies were purchased from New England Biolabs, Inc. Anti-FLAG monoclonal antibody (M2) was purchased from Sigma. Anti-RET antibody was developed as described previously (23).
Immunoprecipitation and Immunoblotting-Cells were grown subconfluently in 100-mm dishes and serum-starved for 12 h. Then they were lysed in lysis buffer (20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Nonidet P-40, 1 mM sodium orthovanadate) supplemented with one tablet of complete protease inhibitor mixture (Roche Diagnostics) per 50 ml and 1 mM phenylmethylsulfonyl fluoride (PMSF). The lysates were clarified by centrifugation (15,000 ϫ g) for 30 min, and the supernatants were incubated with 5 g of antibodies for 3 h at 4°C. The resulting immunocomplexes were collected with Protein A-or Protein G-Sepharose (Sigma) and washed four times with lysis buffer. The complexes were eluted in sodium dodecyl sulfate (SDS) sample buffer (50 mM Tris-HCl, pH 6.8, 5 mM EDTA, 2% SDS 10% glycerol, 20 g/ml bromphenol blue) by boiling for 5 min and subjected to SDS-polyacrylamide gel electrophoresis. Separated proteins were transferred to polyvinylidene difluoride membranes (Nihon Millipore Kogyo) and reacted with the antibodies. The reaction was examined by enhanced chemiluminescence detection kit (ECL, Amersham Biosciences) according to the instructions of the manufacturer.
Pull-down Assay Using Glutathione S-Transferase (GST) Fusion Proteins-To generate the GST fusion proteins with each domain of Dok1, cDNA fragments of mouse Dok1 were amplified by PCR and cloned into the pGEX-3X vector (Amersham Biosciences) as described previously (34). These constructs were used to transform E. coli to produce the GST fusion proteins that were purified using glutathione-agarose beads.
The cells expressing wild-type RET and mutant RET were lysed in ice-cold lysis buffer (20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Nonidet P-40, 1 mM sodium orthovanadate) supplemented with one tablet of complete protease inhibitor mixture/50 ml and 1 mM PMSF, and the resulting cell lysates were clarified by centrifugation. The supernatants were incubated with 5 g of immobilized GST or GST fusion proteins for 3 h at 4°C and washed with lysis buffer four times. The proteins bound to GST fusion proteins were eluted by boiling in SDS-sample buffer, resolved by SDS-polyacrylamide gel electrophoresis, and immunoblotted with anti-RET or anti-FLAG antibody.
Luciferase Assay-For serum response element (SRE) reporter-gene assay, SK-N-MC cells were cultured in 24-well tissue culture plates 20 -24 h prior to transfection. The cells were co-transfected with 150 ng of SRE-reporter plasmid, 50 ng of phRL-TK plasmid carrying the Renilla luciferase gene (Promega), 600 ng of pCMV-RET(M918T), and 1 g of various mutant Dok1 expression constructs (CMV-Dok1s), CMV-Shc or CMV-RasN17 construct by LipofectoAMINE2000 methods (Invitrogen) according to the specifications of the manufacturer. The total amounts of transfected DNAs were kept constant by including the appropriate empty vector where needed. The cells were cultured for 24 h after transfection and serum-starved for the following 24 h. Then, they were lysed and the resulting extracts were assayed for the luciferase activity using a Promega kit. To investigate the c-Jun-mediated gene expression by RET-MEN2B in SK-N-MC cells, the cells were plated on 24-well culture plates 24 h before transfection. The cells were transfected with 150 ng of GAL4-responsive pFR-Luc reporter plasmid (Stratagene), 50 ng of phRL-TK plasmid, 400 ng of pcDNA-GAL4 DNA binding domain-c-Jun fusion construct (pcDNA-GAL4DB-c-Jun), 600 ng of pCMV-RET(M918T), and 1 g of various Dok1 expression constructs (CMV-Doks) or CMV-Shc construct by LipofectAMINE2000 methods. The cells were harvested 48 h after transfection and assayed for the luciferase activity.
To investigate the effect of GDNF stimulation on the luciferase activity, SK-N-MC(RET) cells were co-transfected with 150 ng of SRE reporter gene, 50 ng of phRL-TK, and 1.2 g of pCMV-Dok1 constructs. For assays of c-Jun-mediated gene expression, the cells were transfected with 150 ng of pFR-Luc reporter plasmid, 50 ng of phRL-TK plasmid, 600 ng of pcDNA-GAL4DB-c-Jun fusion construct, and 1 g of various Dok1 expression constructs (CMV-Doks), or CMV-Shc construct. Twenty-four hours after transfection, GDNF (100 ng/ml) was added for 24 h and luciferase assays were performed. Co-transfection with the phRL-TK plasmid carrying the Renilla luciferase gene was used to normalize all luciferase values. All experiments were repeated three times.
Ras Activation Assay-The GST fusion proteins including the Rasbinding domain of c-Raf (c-RafRBD) were generated in E. coli as described above and used to measure the amount of GTP-bound Ras in the cells. Briefly, GDNF-treated or untreated SK-N-MC cells expressing RET were lysed on ice for 10 min in lysis buffer (50 mM Tris-HCl, pH 7.5, 200 mM NaCl, 2 mM MgCl 2 , 10 mM NaF, 1% Nonidet P-40, 1 mM sodium orthovanadate, 250 M PMSF, and 2 g/ml aprotinin), and the resulting lysates were centrifuged at 15,000 ϫ g at 4°C for 30 min. The supernatant was added to 5 g of GST-c-RafRBD fusion proteins bound to glutathione-agarose beads and incubated at 4°C for 60 -120 min with gentle rotation. The beads were washed four times in lysis buffer and boiled in SDS sample buffer. The amount of GTP-bound Ras was analyzed by immunoblotting with anti-Ras monoclonal antibody (Signal Transduction).

RESULTS
Identification of Dok1 as a RET-binding Protein-We performed a yeast two-hybrid screening to identify the proteins that can interact with the intracellular domain of RET in a phosphotyrosine-dependent manner. A human RET cDNA corresponding to the intracellular domain (amino acids 718 -1114) with or without a MEN2B mutation (methionine 918 3 threonine, M918T) was fused in frame to the DNA binding domain of GAL4 transcription factor in the pAS2-1 plasmid as a bait (Fig. 1A). Western blot analyses with anti-GAL4-DB and anti-RET antibodies showed that 62-kDa fusion proteins were detected in the lysates from yeast transformed by the bait plasmid (Fig. 1B). As expected, the level of tyrosine phosphorylation of the GAL4DB-RET(MEN2B) protein was much higher than that of the GAL4DB-RET(wild-type) protein (Fig.  1B). Approximately 5.7 ϫ 10 6 clones of human fetal brain cDNA inserted in the pACT2 vector were screened, and 77 independent positive clones interacting with GAL4DB-RET(MEN2B) were identified (Fig. 1C). Among them, nucleotide sequences of 14 clones matched cDNA sequence of the human DOK1 gene. They included 5Ј non-coding and coding regions of the human DOK1 cDNA (ϳ1.9 kbp).
To confirm the interaction between human DOK1 and activated RET by a pull-down assay, a full-length DOK1 protein was produced in E. coli as a GST fusion protein. The GST- DOK1 fusion protein was incubated with the lysates of NIH3T3 cells expressing human RET with a MEN2A (cysteine 634 3 arginine, C634R) or MEN2B mutation. The results showed that the fusion protein interacted with RET-MEN2A (Fig. 1D) as well as RET-MEN2B (data not shown). In addition, when the lysate from SK-N-MC human primitive neuroectodermal tumor cells transfected the wild-type RET gene was used for the pull-down assay, the GST-DOK1 fusion protein interacted with 175-kDa RET phosphorylated by GDNF (Fig. 1D).
Strong Association of Dok1 with RET with MEN2B Mutation-Dok1 consists of an amino-terminal pleckstrin homology (PH) domain, a PTB domain, and a COOH-terminal tail (CT) harboring at least seven potential tyrosine phosphorylation sites ( Fig. 2A) (35,36). To determine which domain of Dok1 mediates the interaction with RET, we produced the GST fusion proteins containing PH domain (Dok1-PH), PTB domain (Dok1-PTB), COOH-terminal tail (Dok1-CT), PH and PTB domains (Dok1-PH/PTB), or PTB and COOH-terminal tail (Dok1-PTB/CT). As shown in Fig. 2B, the GST fusion proteins containing the PTB domain were able to strongly bind the RET-MEN2B protein. In addition, because it was reported that two arginine residues at codons 207 and 208 in the Dok1 PTB domain are crucial for its interaction with phosphotyrosine (37), we replaced these two residues with alanine (Dok1-AA in Fig. 2A) and performed the pull-down assay. As expected, this mutation markedly impaired the interaction between RET-MEN2B and Dok1 (Fig. 2B), indicating that the PTB domain of Dok1 is responsible for the interaction with activated RET.
To confirm the interaction between Dok1 and RET in the cells, the lysates from NIH3T3 cells expressing RET with MEN2A(C634R) or MEN2B(M918T) mutation were immunoprecipitated with anti-Dok1 antibody, followed by immunoblotting with anti-RET, anti-phosphotyrosine or anti-Dok1 antibody. As shown in Fig. 3A, RET and Dok1 were coprecipitated from both lysates, although the RET-MEN2B protein was more efficiently coprecipitated with Dok1 than the RET-MEN2A protein was. In addition, the level of tyrosine phosphorylation of Dok1 in NIH-RET(MEN2B) cells was higher than that in NIH-RET(MEN2A) cells (Fig. 3A). Because tyrosine 1062 in RET was reported to be responsible for interaction of the Dok-5 PTB domain (22), we replaced tyrosine 1062 with phenylalanine (Y1062F). When the lysates from NIH3T3 cells expressing RET-MEN2A or RET-MEN2B with the Y1062F mutation were used for immunoprecipitation, coprecipitation of RET with Dok1 was hardly detected (Fig. 3A), confirming the importance of tyrosine 1062 for Dok1 binding.
To further investigate whether Dok1 interacts more strongly with RET-MEN2B than RET-MEN2A, we transfected the FLAG expression plasmid containing mouse full-length Dok1 cDNA into NIH3T3, NIH-RET(MEN2A), and NIH-RET(MEN2B) cells. Immunoprecipitation experiments with anti-FLAG antibody showed that the amount of coprecipitated RET-MEN2B protein was larger than that of coprecipitated RET-MEN2A protein (Fig. 3B). Moreover, when the FLAGtagged Dok1-AA or PH domain-deleted Dok1 (Dok1-delPH) plasmid was transfected into NIH-RET(MEN2B) cells, coprecipitation of RET with Dok1-AA was hardly detected (Fig. 3C). On the other hand, wild-type Dok1 and Dok1-delPH were coprecipitated with RET at similar levels, indicating that the PH domain is unnecessary for the Dok1 binding to RET in the cells (Fig. 3C).
FLAG-tagged mutant genes were transfected into NIH-RET(MEN2B) cells, and the association of RET with mutant Dok1 protein was analyzed. RET-MEN2B mutant proteins were co-immunoprecipitated with all mutant Dok1 proteins at similar levels ( Fig. 4B and data not shown), although tyrosine phosphorylation of Dok1-6F and Dok1-10F was significantly reduced (Fig. 4B). The association of Nck with Dok1 was almost abolished by the Y361F single mutation as well as 6F and 10F mutations including Y361F (Fig. 4B). Because the other nine single tyrosine mutations did not affect the association of both proteins ( Fig. 4B and data not shown), this indicated that tyrosine 361 in mouse Dok1 represents a binding site for Nck.
The association of Ras-GAP with Dok1 was not affected by each single tyrosine mutation or 3F mutation (Fig. 4B and data not shown). Its association was significantly reduced by the 4F mutation and almost abolished by the 6F or 10F mutation (Fig.  4B), suggesting that six tyrosine residues (tyrosines 295, 314, 361, 376, 397, and 408) with the YXXP motif contribute to the association of mouse Dok1 to Ras-GAP.
Regulation of Ras/Erk Activation in the RET Signaling Pathway by Dok1-To investigate the role of Dok1 in the RET signaling pathway, we performed the luciferase reporter-gene assay. We made a reporter construct containing SRE and herpes simplex virus thymidine kinase (TK) minimal promoter immediately upstream of the luciferase gene (Fig. 5A). When the expression plasmid containing the RET-MEN2B gene was cotransfected into SK-N-MC cells with the reporter construct, ϳ2-fold increase of the luciferase activity was observed (Fig.  5C). Co-expression of Dok1-WT or Dok1-Y361F reduced the RET-MEN2B-induced luciferase activity by 50 -60%, whereas expression of Dok1-6F, which cannot bind RAS-GAP, increased its activity by ϳ50% (Fig. 5C). In addition, the RET-MEN2B-induced luciferase activity was decreased to the basal level by expression of dominant-negative Ras (RasN17) and enhanced by expression of Shc, binding of which to RET is responsible for activation of Ras/Erk and PI3-K/Akt signaling pathways (Fig. 5C). Similar effects of Dok1 mutants were observed for GDNF-induced SRE-TK promoter activity in SK-N-MC(RET) cells (Fig. 5D). Because SRE represents a binding site for Elk that is phosphorylated and activated by Erk, these results suggest that Dok1 negatively regulates the Ras/Erk pathway activated by RET via the binding to Ras-GAP.
To see whether Dok1 expression also affects the Ras activa- (dominant negative Ras), or with the empty plasmid (control). Luciferase activity was measured as described previously (32) and normalized by the activities of the Renilla luciferase derived from phRL-TK. Results represent averages from at least three independent experiments, and bars indicate the standard error. D, SK-N-MC expressing wild-type RET were transfected with the SRE-reporter construct and phRL-TK together with the expression plasmids carrying Dok1-WT, Dok1-Y361F, Dok1-6F, or Shc. Twenty-four hours after transfection, the cells were stimulated with GDNF (100 ng/ml) for 24 h and luciferase assays were performed. E, SK-N-MC cells were transiently transfected with pFR-Luc, phRL-TK, and pcDNA-GAL4DB-c-Jun constructs shown in B and pCMV-RET(MEN2B) together with the expression plasmids carrying Dok1-WT, Dok1-Y361F, Dok1-6F, or Shc, or with the empty plasmid (control). F, SK-N-MC(RET) cells were transiently transfected pFR-Luc, phRL-TK, and pcDNAGAL4DB-c-Jun together with the expression plasmids carrying Dok1-WT, Dok1-Y361F, Dok1-6F, or Shc, or with the empty plasmid (control) and luciferase activities were analyzed as described in D.
tion, we compared the amount of GTP-bound Ras among the transfectants. The lysates from GDNF-treated or untreated SK-N-MC(RET) cells transfected with each Dok1 construct were pulled-down with the GST fusion proteins containing the Ras binding domain of c-Raf (designated c-RafRBD). As shown in Fig. 6C, the amount of GTP-bound Ras significantly decreased in the transfectant expressing Dok1-WT or Dok1-Y361F but not in the transfectant expressing Dok1-6F, suggesting that Ras-GAP binding to Dok1 impairs the Ras activation.
Regulation of JNK/Jun Activation in the RET Signaling Pathway by Dok1-We finally investigated the role of Dok1 in the JNK pathway. A construct containing the GAL4-DNA binding domain/c-Jun fusion gene was generated (Fig. 5B) and co-transfected into SK-N-MC cells with the luciferase reportergene plasmid (pFR-Luc in Fig. 5B) and each expression plasmid. Expression of RET-MEN2B in SK-N-MC cells resulted in ϳ2.5-fold increase of the c-Jun-mediated luciferase activity as compared with a control (Fig. 5E). Co-expression of RET-MEN2B and wild-type Dok1 further enhanced the c-Jun-mediated gene expression (ϳ3.5-fold). On the other hand, co-expres-sion of RET-MEN2B and Dok1-Y361F or Dok1-6F decreased the luciferase activity by ϳ25% as compared with that induced by RET-MEN2B expression only (Fig 5E). Similar results were obtained in GDNF-stimulated SK-N-MC(RET) cells that were transfected with each Dok1 construct (Fig. 5F). Shc expression appeared to slightly increase RET-MEN2B and GDNF-induced c-Jun transcriptional activity (Fig. 5, E and F).
In contrast to the effect of Dok1 on Erk activation, overexpression of Dok1-WT enhanced the levels of JNK and c-Jun phosphorylation in NIH-RET(MEN2B) cells and SK-N-MC(RET) cells stimulated with GDNF (Fig. 6, A and B). In addition, expression of Dok1-3F that can bind Nck also increased the JNK and c-Jun phosphorylation (data not shown), whereas expression of Dok1-Y361F or Dok1-6F attenuated their phosphorylation (Fig. 6, A and B). These findings suggested that Nck binding to tyrosine 361 in Dok1 plays a role in the JNK/Jun signaling activated by RET.
Moreover, the level of JNK phosphorylation in NIH-RET(MEN2B) cells were significantly higher than that in NIH-RET(MEN2A) cells, whereas the level of Erk1/2 phosphorylation was comparable between these cells (Fig. 6A). Akt and c-Jun phosphorylation also increased in NIH-RET(MEN2B) cells. Thus, higher activities of JNK, c-Jun, and Akt may be responsible for biological properties of the RET-MEN 2B mutant protein.

DISCUSSION
Dok1 was first identified as a 62-kDa tyrosine-phosphorylated protein associated with Ras-GAP in fibroblasts transfected with v-Src gene (41). To date, five genes belonging to the Dok family (Dok1-5) have been cloned, and all of them were composed of amino-terminal PH domain, PTB domain, and COOH-terminal tail (22,(42)(43)(44)(45). Dok1 is one of the major tyrosine-phosphorylated proteins in v-Src and v-Abl transformed cells and is also rapidly phosphorylated by activation of receptor tyrosine kinases in a variety of cell systems (35, 36, 39, 46 -53). Grimm et al. (22) have recently reported that the Dok proteins directly associate with tyrosine 1062 of RET and could be its substrates. In the current study, we have further extended our understanding of the role of Dok1 in the RET signaling pathway through a combination of in vitro biochemical and cellular approaches.
First, we showed that the PTB domain of Dok1 was required for the binding to tyrosine 1062 in RET. This is consistent with the finding that the sequence (IENKL) amino-terminal to tyrosine 1062 in RET matches the consensus sequence (NXLpY) for binding of the Dok1 PTB domain (37). We and other investigators previously reported that tyrosine 1062 also represents a binding site for the PTB domain of Shc, SNT/Frs2, and IRS-1 (18,19,23,(25)(26)(27), although the amino acid sequences of the Shc PTB domain are not homologous to those of Dok, IRS-1, and Frs2/SNT PTB domains. It is interesting that a single phosphorylated tyrosine residue (tyrosine 1062) in RET can bind the PTB domains of multiple adaptor proteins, leading to the activation of Ras/Erk, PI3-K/Akt, p38MAPK, JNK, and Erk5 signaling pathways (Fig. 7) (30 -33). Similarly, it was reported that tyrosine 1148 in the carboxyl-terminal tail of epidermal growth factor receptor was a binding site for both Shc and Dok-R (Dok-2) PTB domains (54) and tyrosine 499 in the juxtamembrane region of TrkA was a binding site for both Shc and SNT/Frs2 PTB domains (55).
Our previous studies demonstrated that phosphorylation of tyrosine 1062 in RET is crucial for the transforming activity of RET with all forms of MEN2A or MEN2B mutations (19,56). In addition, a couple of Hirschsprung mutations found in the COOH-terminal sequence of RET such as the L1061P and M1064T mutations impaired the binding of docking proteins to tyrosine 1062 (13,57), thus resulting in a defect of RET-mediated signaling. Because leucine 1061 at the Ϫ1 position relative to tyrosine 1062 in RET is included in the consensus sequence for binding of the Dok1 PTB domain (37), it is interesting to speculate that a defect of Dok1-mediated signaling may play an important role in the development of Hirschsprung disease.
Shc and Frs2/SNT are tyrosine-phosphorylated in cells expressing active RET kinase and these phosphorylations establish the binding sites for Grb2, leading to activation of the Ras/Erk signaling pathway (23). Tyrosine phosphorylation of Dok1 results in its binding to the SH2 domain of Ras-GAP and Nck (38). In this study, we showed that phosphorylation of six tyrosine residues with the YXXP motif on mouse Dok1 is necessary for interaction with Ras-GAP in vitro and in vivo. Kashige et al. (46) reported that mutations at five tyrosine residues on human DOK1, including tyrosines 296, 315, 362, 398, and 409, are required for the association with Ras-GAP in vitro, basically consistent with our data. We further demonstrated that overexpression of Dok1 in NIH-RET(MEN2B) or SK-N-MC(RET) cells impairs the Ras/Erk activation induced by active RET, but expression of Dok1-6F mutant that is not able to bind Ras-GAP does not. These results supported the view that Dok1 is a negative regulator for the Ras/Erk signaling pathway activated by RET (Fig. 7) as observed for other signaling systems (52,58). However, Noguchi et al. (48) showed that Dok1 failed to inhibit Erk activation in Chinese hamster ovary cells in response to insulin. This discrepancy may be the result of differences of the ligand-receptor systems used or the experimental conditions. Phosphorylation of tyrosine 361 on mouse Dok1 is required for the association of Dok1 with Nck. In addition, this association appeared to lead to the activation of JNK signaling pathway (Fig. 7). Nck is a ubiquitously expressed protein composed entirely of a single SH2 domain and three SH3 domains. A number of proteins have been shown to bind to the SH3 domains of Nck, including the p21-activated proteins (PAKs), NIK, Wiskott-Aldrich syndrome protein, and PRK2 (59,60). PAKs bind the active forms of Cdc42 and Rac1 (61,62) and are involved in the regulation of actin rearrangements that could associate with cell motility (48). Moreover, several studies demonstrated that the membrane-associated Nck in turn causes, apparently via NIK or PAK1, activation of the JNK/stressactivated protein kinase pathway (63-68). EphB1 receptor with a mutation at tyrosine 594 failed to bind Nck and activate the JNK pathway or transmit attachment signals (65). Becker et al. (63) showed that Nck-NIK complex associated with tyrosine-phosphorylated Dok1 in both EphB1-and EphB2-stimulated cells, and that kinase-defective NIK and Nck bindingdefective NIK blocked ephrinB1-and ephrin B2-induced JNK activation and P19 cell attachment to fibrinogen matrix. Thus, it is possible that Nck-NIK complex formation may also be involved in the activation of RET-mediated JNK signaling pathway.
The MEN2B mutation resulted in increased activity toward the optimal peptide substrates of Src and Abl, and altered the pattern of RET autophosphorylation as well as phosphorylated substrates in NIH 3T3 cells (16,69). The level of Dok1 phosphorylation and its interaction with RET in the MEN2B transfectant were significantly higher than those in the MEN2A transfectant. In the previous study, we also showed that the levels of Gab1 phosphorylation and PI3-K and Akt activation in the MEN2B transfectant were higher than those in the MEN2A transfectant (38). Salvatore et al. (70) reported that the level of phosphorylation of tyrosine 1062 in RET is increased in PC12 cells expressing RET-MEN2B, resulting in enhancement of activation of downstream signaling pathways. Here we demonstrated that the levels of Dok1 and JNK phosphorylation in the MEN2B transfectant are significantly increased compared with those in the MEN2A transfectant, whereas Erk phosphorylation was comparable between both transfectants. The activation of Ras-GAP via Dok1 binding to tyrosine 1062 may decrease the Erk phosphorylation in the MEN2B transfectant to the level in the MEN2A transfectant. The Jun/AP1 complexes induced by JNK activation have been shown to be necessary for cell cycle progression in several systems as well as cellular transformation by a variety of oncogenes, including Src, Ras, and Raf (71). In addition, it was reported that the JNK signaling pathway could be associated with metastatic ability of RET-MEN2B mutant cells (72). Our findings thus suggested that higher levels of the JNK/Jun activation via Dok1 phosphorylation by RET-MEN2B may be responsible for aggressive properties of medullary thyroid carcinoma developed in MEN 2B.