Role of Dok1 in Cell Signaling Mediated by RET Tyrosine Kinase

Abstract

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. 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. glial cell line-derived neurotrophic factor multiple endocrine neoplasia familial medullary thyroid carcinoma papillary thyroid carcinoma medullary thyroid carcinoma Ras-GTPase-activating protein Jun amino-terminal kinase phosphotyrosine-binding pleckstrin homology serum response element glial cell line-derived neurotrophic factor family receptor phosphatidylinositol 3-kinase phenylmethylsulfonyl fluoride insulin-like receptor substrate-1 COOH-terminal tail p21-activated protein cytomegalovirus thymidine kinase DNA binding domain Nck-interacting kinase RET is a tyrosine kinase receptor that plays an important role in the development of the enteric nervous system and the kidney (1Takahashi M. Cytokine Growth Factor Rev. 2001; 12: 361-373Crossref PubMed Scopus (363) Google Scholar, 2Manieá S. Santoro M. Fusco A. Billaud M. Trends Genet. 2001; 17: 580-589Abstract Full Text Full Text PDF PubMed Scopus (242) Google Scholar). It has been demonstrated that members of the glial cell line-derived neurotrophic factor (GDNF)1family 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 α 1–4 (GFRα 1–4) (1Takahashi M. Cytokine Growth Factor Rev. 2001; 12: 361-373Crossref PubMed Scopus (363) Google Scholar, 2Manieá S. Santoro M. Fusco A. Billaud M. Trends Genet. 2001; 17: 580-589Abstract Full Text Full Text PDF PubMed Scopus (242) Google Scholar). GDNF, neurturin, artemin, and persephin use GFRα 1, GFRα 2, GFRα 3, and GFRα 4 as the preferred receptors, respectively, and play specific roles in vivo through these preferred ligand-receptor complex formation. For example, Gdnf- orGfrα1-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 (3Schuchardt A. D'Agati V. Larsson B.L. Costantini F. Pachnis V. Nature. 1994; 367: 380-383Crossref PubMed Scopus (1416) Google Scholar, 4Sánchez M.P. Silos-Santiago I. Friseán J., He, B. Lira S.A. Barbacid M. Nature. 1996; 382: 70-73Crossref PubMed Scopus (1041) Google Scholar, 5Pichel J.G. Shen L. Sheng H.Z. Granholm A.-C. Drago J. Grinberg A. Lee E.J. Huang S.P. Saarma M. Hoffer B.J. Sariola H. Westphal H. Nature. 1996; 382: 73-76Crossref PubMed Scopus (1003) Google Scholar, 6Moore M.W. Klein R.D. Fariñas I. Sauer H. Armanini M. Phillips H. Reichardt L.F. Ryan A.M. Carver-Moore K. 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Oncogene. 2000; 19: 5590-5597Crossref PubMed Scopus (244) Google Scholar). Loss-of-function mutations of RET lead to the development of Hirschsprung’s disease (13Geneste O. Bidaud C., De Vita G. Hofstra R.M. Tartare D.S. Buys C.H. Lenoir G.M. Santoro M. Billaud M. Hum. Mol. Genet. 1999; 8: 1989-1999Crossref PubMed Scopus (53) Google Scholar, 14Iwashita T. Kurokawa K. Qiao S. Murakami H. Asai N. Hashimoto M. Watanabe T. Kawai K. Ichihara M. Takahashi M. Gastroenterology. 2001; 121: 24-33Abstract Full Text PDF PubMed Scopus (60) Google Scholar), 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 (12Jhiang S.M. Oncogene. 2000; 19: 5590-5597Crossref PubMed Scopus (244) Google Scholar, 15Asai N. Iwashita T. Matsuyama M. Takahashi M. Mol. Cell. Biol. 1995; 15: 1613-1619Crossref PubMed Google Scholar, 16Santoro M. 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Oncogene. 2000; 19: PubMed Scopus Google Scholar), in activation of the Ras/Erk and the and are through tyrosine is In this to the activation of through RET we to the that with phosphorylated RET by a yeast two-hybrid We identified Dok1, the phosphotyrosine-binding domain of was responsible for the binding to tyrosine in RET in response to 361 in mouse Dok1 bound the Nck adaptor and six tyrosines with the bound to the JNK and c-Jun activation and of the In we enhanced JNK activation in the cells expressing RET-MEN2B that may a high level of Dok1 the domain of human RET with a was by and to the the DNA binding domain in the The was into the of with two-hybrid human The and with to the and to be for and to the to yeast with or the domain of human in and identified by the characterized by and for homology by the human RET was introduced into the mouse wild-type Dok1 mutant by with of in the The introduced into the the Dok1 with the Dok1 mutations introduced into DNA by the site-directed mutagenesis to the of the The of by DNA mouse and human cell in with and cell with the wild-type RET or N. 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Oncogene. 1999; PubMed Scopus Google Scholar). to to the that The cells expressing wild-type RET and mutant RET in with of and and the cell by The with of or for 4 and with The bound to by in by and with or For serum response element cells in to The cells with of of the of and of mutant Dok1 or by to the of the The of by including the The cells for and for the and the for the a the by RET-MEN2B in the cells The cells with of of of DNA binding of and of Dok1 or by The cells and for the the effect of GDNF the cells with of of and of For of the cells with of of of and of Dok1 or GDNF was for and with the the was to The including the domain of in as and to the of in the or cells expressing RET for in and and the 4 for The was to of bound to and 4 for with The in and in The of was by with We a yeast two-hybrid to the that with the domain of RET in a human to the domain with or a mutation was in to the DNA binding domain of factor in the as a with and showed that in the yeast by the the level of tyrosine phosphorylation of the protein was than that of the protein of human in the and with identified of of the human and of the the human and RET by a a protein was in as a protein. The protein was with the of cells expressing human RET with or The showed that the protein with as well as RET-MEN2B not In the human cells the wild-type was for the the protein with RET phosphorylated by GDNF Dok1 of an amino-terminal pleckstrin homology a and a COOH-terminal tail tyrosine phosphorylation N. D. A. D. R. B. B. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar, Y. D. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). domain of Dok1 the with we the domain domain COOH-terminal tail and or and COOH-terminal tail in 2B, the the domain to strongly the RET-MEN2B protein. In was that and in the Dok1 domain are for its with Y. D. D. J. 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Biol. 2001; PubMed Scopus Google Scholar), we replaced tyrosine with phenylalanine the cells expressing or RET-MEN2B with the mutation for of RET with Dok1 was the of tyrosine for Dok1 Dok1 more strongly with RET-MEN2B than we the mouse into and with showed that the of RET-MEN2B protein was than that of protein the or Dok1 was into of RET with was On the other hand, wild-type Dok1 and with RET similar that the domain is for the Dok1 binding to RET in the cells We demonstrated that phosphorylated Dok1 with and Nck in cells expressing and RET-MEN2B Iwashita T. Asai N. Y. Y. Kawai K. Takahashi M. 1999; PubMed Scopus Google Scholar). In binding with a has that the domain of Nck to a that the the of to L.C. M. T. J. PubMed Scopus Google Scholar, M. G. T. F. T. S. H. B. L.C. Cell. 12: Scholar). The is tyrosines 295, 314, 361, 376, 397, and 408 in mouse Dok1, and the is tyrosine the binding site of mouse Dok1 with Nck and we replaced tyrosine 295, 314, 361, 376, 397, and in mouse Dok1 with phenylalanine and mutant genes with tyrosine In we Dok1 with tyrosine mutations and 4 tyrosine mutations and tyrosine mutations and or tyrosine mutations and mutant genes into and the association of RET with mutant Dok1 protein was RET-MEN2B mutant with mutant Dok1 similar and not tyrosine phosphorylation of and was reduced The association of Nck with Dok1 was by the Y361F mutation as well as and mutations including Y361F Because the other tyrosine mutations not the association of and not this that tyrosine 361 in mouse Dok1 represents a binding site for The association of with Dok1 was not by tyrosine mutation or mutation and not association was reduced by the mutation and by the or mutation that six tyrosine 295, 314, 361, 376, 397, and with the contribute to the association of mouse Dok1 to the role of Dok1 in the RET we the We a and thymidine kinase of the the the RET-MEN2B was into cells with the of the was observed of or Dok1-Y361F reduced the by of Dok1–6F, its by In the was to the level by of and enhanced by of binding of to RET is responsible for activation of Ras/Erk and of Dok1 observed for in cells Because represents a binding site for that is phosphorylated and by these that Dok1 the Ras/Erk by RET via the binding to the role of Dok1 in we Dok1-Y361F, or into or and the of phosphorylation the with the of the overexpression of or Dok1-Y361F the phosphorylation induced by RET-MEN2B or GDNF Dok1 the we the of the The or cells with Dok1 with the the binding domain of (designated in the of in the expressing or Dok1-Y361F but not in the expressing Dok1–6F, that binding to Dok1 the activation. We the role of Dok1 in the JNK the binding was and into cells with the in and of RET-MEN2B in cells in of the as with a of RET-MEN2B and wild-type Dok1 enhanced the On the other hand, of RET-MEN2B and Dok1-Y361F or the by as with that induced by RET-MEN2B in cells that with Dok1 to RET-MEN2B and c-Jun and In to the effect of Dok1 overexpression of enhanced the of JNK and c-Jun phosphorylation in cells and cells with GDNF and In of that Nck the JNK and c-Jun phosphorylation not of Dok1-Y361F or their phosphorylation suggested that Nck binding to tyrosine 361 in Dok1 plays a role in the by the level of JNK phosphorylation in cells than that in the level of phosphorylation was these cells and c-Jun phosphorylation in of and may be responsible for properties of the 2B mutant protein. Dok1 was identified as a protein with in with C. M. F. T. Nature. PubMed Scopus Google Scholar). genes to the family been and of of amino-terminal and COOH-terminal tail J. M. S. T. K. J. 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