Mechanism of activation of the ret proto-oncogene by multiple endocrine neoplasia 2A mutations
Mechanism of activation of the ret proto-oncogene by multiple endocrine neoplasia 2A mutations
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DOI：
10.1128/mcb.15.3.1613
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发表时间：
1995-03
影响因子：
5.3
通讯作者：
N. Asai;T. Iwashita;Mutsushi Matsuyama;M. Takahashi
中科院分区：
文献类型：
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作者：
N. Asai;T. Iwashita;Mutsushi Matsuyama;M. Takahashi
通过转染NIH 3 T3细胞研究了具有多发性内分泌瘤（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型c-ret基因以高效率诱导转化。170-kDa的Ret蛋白存在于转化细胞的细胞表面上的酪氨酸高度磷酸化，并形成二硫键连接的同源二聚体。该结果表明MEN 2A突变诱导细胞表面上的c-Ret蛋白的配体非依赖性二聚化，导致其内在酪氨酸激酶的活化。除了MEN 2A突变外，我们还在钙粘蛋白样结构域的推定Ca（2+）结合位点引入了突变（密码子300 [D300 K]处天冬酰胺酸的赖氨酸）。当c-ret cDNA与MEN 2A和D300 K突变转染到NIH 3 T3细胞，转化活性急剧下降。Western印迹（免疫印迹）分析显示，很少的170-kDa的Ret蛋白与D300 K突变表达的转染子，而保留在内质网中的150-kDa的Ret蛋白的表达不受影响。这一结果也证明了Ret蛋白转运至质膜是其转化活性所必需的。
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.