Osmotic stress-mediated activation of RET kinases involves intracellular disulfide-bonded dimer formation.
We showed that osmotic stress induces activation of c-RET and second-set activation of constitutively activated RET-MEN2B. A few percentage of RET proteins normally formed disulfide-bonded dimers in the cell, and osmotic stress promoted formation of these dimers. The disulfide-bonded dimers displayed higher levels of autophosphorylation and catalytic activity per molecule than did monomers. Osmotic stress also promoted activation and disulfide-bonded dimerization of the extracellular domain-depleted mutant RET (RET-PTC-1), suggesting that the target amino acid(s) for dimerization is located intracellularly rather than in the cysteine-rich region of the extracellular domain. In the mutant c-RET and RET-PTC-1 in which Cys987 of c-RET or Cys376 of RET-PTC-1 was replaced with Ala, the levels of intrinsic kinase activity were greatly reduced and barely increased in response to osmotic stress. Correspondingly, the Cys376-defective RET-PTC-1 did not form any demonstrable levels of dimers even after exposure to osmotic stress. In contrast, another RET-PTC-1 mutant that had a replacement of Cys365 with Ala mostly behaved like parental RET-PTC-1. These results suggest that Cys987 of c-RET or Cys376 of RET-PTC-1 plays a crucial role in maintenance and promotion of dimerization and activation of the RET kinases.
Takeda
K
K
Department of Immunology, Nagoya University Graduate School of Medicine, Aichi, Japan.
Kato
M
M
Wu
J
J
Iwashita
T
T
Suzuki
H
H
Takahashi
M
M
Nakashima
I
I
D016475:3T3 Cells; D000818:Animals; D003545:Cysteine / Q000737:chemistry; D019281:Dimerization; D004220:Disulfides / Q000737:chemistry; D029721:Drosophila Proteins; D004789:Enzyme Activation; D015151:Immunoblotting; D051379:Mice; D009154:Mutation; D009997:Osmotic Pressure; D017434:Protein Structure, Tertiary; D011518:Proto-Oncogene Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D051096:Proto-Oncogene Proteins c-ret; D020794:Receptor Protein-Tyrosine Kinases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D012462:Saline Solution, Hypertonic
D004220:Disulfides; D029721:Drosophila Proteins; D011518:Proto-Oncogene Proteins; D012462:Saline Solution, Hypertonic; D051096:Proto-Oncogene Proteins c-ret; D020794:Receptor Protein-Tyrosine Kinases; C416649:Ret protein, Drosophila; C494999:Ret protein, mouse; D003545:Cysteine