Tyrosine kinase activity of the ret proto-oncogene products in vitro.
We investigated tyrosine kinase activity of the ret proto-oncogene products (proto-Ret proteins), using a cell lysate of NB-39-nu neuroblastoma cells. The 150 kDa and 170 kDa proto-Ret proteins immunoprecipitated with antibodies against their carboxy-terminal 20 amino acids were shown to be phosphorylated predominantly on tyrosine residues in immunocomplex kinase assay. The level of tyrosine phosphorylation of the 150 kDa proto-Ret protein was approximately 10-fold higher than that of the 170 kDa proto-Ret protein, although both proteins were expressed at similar levels in neuroblastoma cells. This result was confirmed by using a lysate of SK-N-MC human primitive neuroectodermal tumor cells transfected with the ret proto-oncogene. The kinase activity of proto-Ret proteins was significantly inhibited by antibodies against their kinase domain, indicating that these antibodies recognize crucial epitopes for the enzymatic activity. On the other hand, the proto-Ret proteins were not phosphorylated in vivo in NB-39-nu cells and SK-N-MC transfectants.
Miyazaki
K
K
Department of Pathology, Nagoya University School of Medicine, Japan.
Asai
N
N
Iwashita
T
T
Taniguchi
M
M
Isomura
T
T
Funahashi
H
H
Takagi
H
H
Matsuyama
M
M
Takahashi
M
M
D000596:Amino Acids / Q000032:analysis; D015153:Blotting, Western; D029721:Drosophila Proteins; D006801:Humans; D008970:Molecular Weight; D009447:Neuroblastoma; D010710:Phosphates / Q000378:metabolism; D010750:Phosphoproteins / Q000302:isolation & purification / Q000378:metabolism; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D019000:Phosphotyrosine; D011505:Protein-Tyrosine Kinases / Q000235:genetics / Q000378:metabolism*; D000090063:Proto-Oncogene Mas; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism*; D051096:Proto-Oncogene Proteins c-ret; D011519:Proto-Oncogenes; D020794:Receptor Protein-Tyrosine Kinases; D014407:Tumor Cells, Cultured; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000032:analysis
D000596:Amino Acids; D029721:Drosophila Proteins; C000715470:MAS1 protein, human; D010710:Phosphates; D010750:Phosphoproteins; D010761:Phosphorus Radioisotopes; D000090063:Proto-Oncogene Mas; D011518:Proto-Oncogene Proteins; D019000:Phosphotyrosine; D014443:Tyrosine; D011505:Protein-Tyrosine Kinases; D051096:Proto-Oncogene Proteins c-ret; D020794:Receptor Protein-Tyrosine Kinases; C416649:Ret protein, Drosophila