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Co-immunoprecipitation Assay for Studying Functional Interactions Between Receptors and Enzymes
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Co-immunoprecipitation Assay for Studying Functional Interactions Between Receptors and Enzymes

Published on: September 28, 2018

Role of Dok1 in cell signaling mediated by RET tyrosine kinase

Hideki Murakami1, Yumiko Yamamura, Yohei Shimono

  • 1Department of Pathology, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan.

The Journal of Biological Chemistry
|June 28, 2002
PubMed

Insights

Dok1 acts as a docking protein for RET tyrosine kinase, influencing signaling pathways. Its interaction with RET, particularly MEN 2B mutations, affects Ras/Erk and JNK/c-Jun activation, potentially driving medullary thyroid carcinoma.

Area of Science:

  • Molecular Biology
  • Cell Signaling
  • Oncology

Background:

  • RET tyrosine kinase mutations are implicated in Multiple Endocrine Neoplasia (MEN) syndromes, particularly MEN 2A and MEN 2B.
  • Dok1 is a docking protein involved in various signaling pathways, but its specific role in RET-mediated signaling, especially in MEN 2B, requires elucidation.

Purpose of the Study:

  • To identify docking proteins interacting with RET tyrosine kinase.
  • To investigate the role of Dok1 in RET-mediated signaling pathways, including Ras/Erk and JNK/c-Jun.
  • To determine the functional consequences of Dok1-RET interactions in the context of MEN 2B mutations.

Main Methods:

  • Yeast two-hybrid screening to identify Dok1 as a RET-binding protein.
  • Site-directed mutagenesis to define specific tyrosine residues in Dok1 involved in Nck and Ras-GTPase-activating protein (Ras-GAP) binding.
  • Expression of wild-type and mutant Dok1 in cells expressing wild-type RET or RET-MEN2B to analyze downstream signaling activation (Ras/Erk, JNK/c-Jun).

Main Results:

  • Dok1 binds to RET, with stronger binding and phosphorylation observed with RET-MEN2B mutations compared to RET-MEN2A.
  • Tyrosine 361 of Dok1 is crucial for Nck binding and subsequent JNK/c-Jun activation, while other tyrosines (295, 314, 361, 376, 397, 408) are involved in Ras-GAP binding and Ras/Erk pathway inhibition.
  • Dok1 overexpression, particularly variants maintaining Ras-GAP binding, suppresses Ras/Erk activation, whereas Dok1's interaction with Nck is necessary for JNK/c-Jun activation.

Conclusions:

  • Dok1 is a key docking protein mediating RET signaling, with distinct roles for different tyrosine residues.
  • The Nck-Dok1 interaction, facilitated by tyrosine 361, is essential for JNK/c-Jun activation by RET-MEN2B.
  • Enhanced JNK phosphorylation in RET-MEN2B expressing cells suggests that Nck-mediated Dok1 activation contributes to the aggressive phenotype of medullary thyroid carcinoma in MEN 2B.

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