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) in Dok1 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.
Murakami
Hideki
H
Department of Pathology, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan.
Yamamura
Yumiko
Y
Shimono
Yohei
Y
Kawai
Kumi
K
Kurokawa
Kei
K
Takahashi
Masahide
M
D016475:3T3 Cells; D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D002460:Cell Line; D004268:DNA-Binding Proteins; D029721:Drosophila Proteins; D004789:Enzyme Activation; D005982:Glutathione Transferase / Q000378:metabolism; D015151:Immunoblotting; D051379:Mice; D048055:Mitogen-Activated Protein Kinase 8; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D008954:Models, Biological; D018814:Multiple Endocrine Neoplasia Type 2b / Q000235:genetics*; D009154:Mutation; D015513:Oncogene Proteins / Q000378:metabolism; D010649:Phenylalanine / Q000378:metabolism; D010750:Phosphoproteins / Q000502:physiology*; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D051096:Proto-Oncogene Proteins c-ret; D016601:RNA-Binding Proteins; D020794:Receptor Protein-Tyrosine Kinases / Q000378:metabolism*; D015398:Signal Transduction; D020798:Two-Hybrid System Techniques; D014443:Tyrosine / Q000378:metabolism; D020703:ras GTPase-Activating Proteins / Q000378:metabolism; D018631:ras Proteins / Q000378:metabolism
D048868:Adaptor Proteins, Signal Transducing; D004268:DNA-Binding Proteins; C489561:Dok1 protein, mouse; D029721:Drosophila Proteins; C074561:GAP-associated protein p62; C078332:Nck protein; D015513:Oncogene Proteins; D010750:Phosphoproteins; D011518:Proto-Oncogene Proteins; D016601:RNA-Binding Proteins; D020703:ras GTPase-Activating Proteins; D014443:Tyrosine; D010649:Phenylalanine; D005982:Glutathione Transferase; D051096:Proto-Oncogene Proteins c-ret; D020794:Receptor Protein-Tyrosine Kinases; C416649:Ret protein, Drosophila; C494999:Ret protein, mouse; D048055:Mitogen-Activated Protein Kinase 8; D020928:Mitogen-Activated Protein Kinases; D018631:ras Proteins