Asai N, Iwashita T, Matsuyama M, Takahashi M
Department of Pathology, Nagoya University School of Medicine, Japan.
Mol Cell Biol. 1995 Mar;15(3):1613-9. doi: 10.1128/MCB.15.3.1613.
Transforming activity of the c-ret proto-oncogene with multiple endocrine neoplasia (MEN) 2A mutations was investigated by transfection of NIH 3T3 cells. Mutant c-ret genes driven by the simian virus 40 or cytomegalovirus promoter induced transformation with high efficiencies. The 170-kDa Ret protein present on the cell surface of transformed cells was highly phosphorylated on tyrosine and formed disulfide-linked homodimers. This result indicated that MEN 2A mutations induced ligand-independent dimerization of the c-Ret protein on the cell surface, leading to activation of its intrinsic tyrosine kinase. In addition to the MEN 2A mutations, we further introduced a mutation (lysine for asparaginic acid at codon 300 [D300K]) in a putative Ca(2+)-binding site of the cadherin-like domain. When c-ret cDNA with both MEN 2A and D300K mutations was transfected into NIH 3T3 cells, transforming activity drastically decreased. Western blot (immunoblot) analysis revealed that very little of the 170-kDa Ret protein with the D300K mutation was expressed in transfectants while expression of the 150-kDa Ret protein retained in the endoplasmic reticulum was not affected. This result also demonstrated that transport of the Ret protein to the plasma membrane is required for its transforming activity.
通过转染NIH 3T3细胞，研究了具有多发性内分泌腺瘤（MEN）2A突变的c-ret原癌基因的转化活性。由猿猴病毒40或巨细胞病毒启动子驱动的突变c-ret基因能高效诱导细胞转化。转化细胞表面存在的170 kDa Ret蛋白在酪氨酸上高度磷酸化，并形成二硫键连接的同型二聚体。这一结果表明，MEN 2A突变诱导细胞表面c-Ret蛋白发生非配体依赖性二聚化，从而激活其内在酪氨酸激酶。除了MEN 2A突变外，我们还在钙黏蛋白样结构域的一个假定钙结合位点引入了一个突变（第300位密码子天冬酰胺突变为赖氨酸[D300K]）。当将同时具有MEN 2A和D300K突变的c-ret cDNA转染到NIH 3T3细胞中时，转化活性急剧下降。蛋白质免疫印迹分析显示，转染细胞中几乎不表达带有D300K突变的170 kDa Ret蛋白，而保留在内质网中的150 kDa Ret蛋白的表达不受影响。这一结果还表明，Ret蛋白转运到质膜对于其转化活性是必需的。
