Iwashita T, Asai N, Murakami H, Matsuyama M, Takahashi M
Department of Pathology, Nagoya University School of Medicine, Japan.
Oncogene. 1996 Feb 1;12(3):481-7.
The c-ret proto-oncogene with multiple endocrine neoplasia (MEN) 2A or 2B mutation can transform NIH3T3 cells with high efficiencies as a consequence of its constitutive activation. The MEN2A mutation induces ligand-independent homodimerization of the Ret protein on the cell surface while the MEN2B mutation appears to alter the catalytic activity without dimerization. In the present study, we investigated the role of tyrosine residues present in the kinase domain for the transforming activity of the mutant Ret proteins. Substitution of phenylalanine for tyrosine 905 (Y905F) that corresponds to tyrosine 416 of the Src protein abolished the transforming activity of Ret with the MEN2A mutation (MEN2A-Ret) but not with the MEN2B mutation (MEN2B-Ret). On the other hand, the transforming activity of MEN2B-Ret but not MEN2A-Ret significantly decreased by changing tyrosine 864 or 952 to phenylalanine. In addition, double mutations of these tyrosines (Y864/952F) completely abolished the activity of MEN2B-Ret. The Y905F and Y864/952F mutations resulted in severe impairment of the kinase activity of MEN2A-Ret and MEN2B-Ret, respectively. These results thus indicated that tyrosine residues essential for the transforming activity are different between MEN2A-Ret and MEN2B-Ret.
携带多发性内分泌肿瘤（MEN）2A或2B突变的c-ret原癌基因由于其组成性激活，能够高效转化NIH3T3细胞。MEN2A突变诱导细胞表面Ret蛋白的配体非依赖性同源二聚化，而MEN2B突变似乎在不发生二聚化的情况下改变催化活性。在本研究中，我们研究了激酶结构域中酪氨酸残基对突变型Ret蛋白转化活性的作用。将对应于Src蛋白酪氨酸416的酪氨酸905（Y905F）替换为苯丙氨酸消除了携带MEN2A突变的Ret（MEN2A-Ret）的转化活性，但未消除携带MEN2B突变的Ret（MEN2B-Ret）的转化活性。另一方面，将酪氨酸864或952替换为苯丙氨酸会使MEN2B-Ret而非MEN2A-Ret的转化活性显著降低。此外，这些酪氨酸的双突变（Y864/952F）完全消除了MEN2B-Ret的活性。Y905F和Y864/952F突变分别导致MEN2A-Ret和MEN2B-Ret的激酶活性严重受损。因此，这些结果表明，MEN2A-Ret和MEN2B-Ret中转化活性所必需的酪氨酸残基不同。
