Evidence of both extra- and intracellular cysteine targets of protein modification for activation of RET kinase.
By use of a specifically sulfhydryl group-reactive chemical, 1,4-butanediyl-bismethanethiosulfonate (BMTS), we studied the localization of oxidative stress-responsive target cysteines for activation of a receptor-type protein tyrosine kinase, c-RET. The chemical, which reacted with RET proteins on the cell surface for sulfhydryl-linked aggregation, induced autophosphorylation and activation of RET kinase. When extracellular domain-deleted RET mutant (RET-PTC-1) cells were exposed to BMTS, neither the molecular status nor the activity of the enzyme was affected, suggesting that the target cysteines of BMTS to which cells were exposed for reaction are located in the cysteine-rich region of the extracellular domain of RET kinase. Despite this result, the exposure of a subcellular form of c-RET or RET-PTC-1 kinase isolated by immunoprecipitation to BMTS did induce activation of the enzyme. These results suggest that cysteines in both the extracellular and the intracellular domains of RET can work as target sites of accessible BMTS and possibly other oxidative elements for structural modification and activation of RET kinase.
Akhand
Anwarul A
AA
Department of Immunology, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya, 466-8550, Japan.
Ikeyama
Takanari
T
Akazawa
Satoru
S
Kato
Masashi
M
Hossain
Khaled
K
Takeda
Kozue
K
Suzuki
Haruhiko
H
Takahashi
Masahide
M
Nakashima
Izumi
I
D016475:3T3 Cells; D000818:Animals; D003545:Cysteine / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D029721:Drosophila Proteins; D004789:Enzyme Activation / Q000187:drug effects; D005110:Extracellular Space / Q000378:metabolism*; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D007424:Intracellular Fluid / Q000378:metabolism*; D051379:Mice; D015514:Oncogene Proteins, Fusion / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D011233:Precipitin Tests; D011485:Protein Binding / Q000187:drug effects; D011499:Protein Processing, Post-Translational / Q000502:physiology*; D011505:Protein-Tyrosine Kinases; D011518:Proto-Oncogene Proteins / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D051096:Proto-Oncogene Proteins c-ret; D020794:Receptor Protein-Tyrosine Kinases / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D013886:Thiosulfonic Acids / Q000494:pharmacology; D014162:Transfection
C454105:1,4-butanediylbismethanethiosulfonate; D029721:Drosophila Proteins; D015514:Oncogene Proteins, Fusion; D011518:Proto-Oncogene Proteins; D013439:Sulfhydryl Reagents; D013886:Thiosulfonic Acids; D011505:Protein-Tyrosine Kinases; D051096:Proto-Oncogene Proteins c-ret; D020794:Receptor Protein-Tyrosine Kinases; C416649:Ret protein, Drosophila; C494999:Ret protein, mouse; C093573:ret-PTC fusion oncoproteins, human; D003545:Cysteine