Kato M, Iwashita T, Takeda K, Akhand A A, Liu W, Yoshihara M, Asai N, Suzuki H, Takahashi M, Nakashima I
Department of Immunology, Nagoya University School of Medicine, Nagoya, Aichi 466-8550, Japan.
Mol Biol Cell. 2000 Jan;11(1):93-101. doi: 10.1091/mbc.11.1.93.
The c-RET proto-oncogene encodes a receptor-type tyrosine kinase, and its mutations in the germ line are responsible for the inheritance of multiple endocrine neoplasia type 2A (MEN2A) and 2B (MEN2B). Ret kinases are constitutively activated as a result of MEN2A mutations (Ret-MEN2A) or MEN2B mutations (Ret-MEN2B). Here we demonstrate that UV light (UV) irradiation induces superactivation of the constitutively activated Ret-MEN2A and Ret-MEN2B as well as activation of c-Ret. Before UV irradiation, small percentages of c-Ret (3-4%) and Ret-MEN2B (1-2%) and large percentages of Ret-MEN2A (30-40%) were dimerized through disulfide bonds. These dimerized Ret proteins were preferentially autophosphorylated, suggesting a close relation between up-regulated kinase activity and disulfide bond-mediated dimerization of Ret proteins. We found that UV irradiation promotes the disulfide bond-mediated dimerization of the Ret proteins, in close association with activation and superactivation of Ret kinases. UV irradiation also induced dimerization and activation of the extracellular domain-deleted mutant Ret (Ret-PTC-1). Interestingly, the levels of basic kinase activity and dimerization of Ret-PTC-1-C376A, in which cysteine 376 in the tyrosine kinase domain of Ret-PTC-1 was replaced by alanine, were low and were not increased by UV irradiation. These results suggest that Ret-PTC-1 cysteine 376 is one of possibly multiple critical target amino acids of UV for Ret kinase activation. Overexpression of Cu/Zn superoxide dismutase in cells as a result of gene transfection prevented both the UV-mediated promotion of dimerization and the superactivation of Ret-MEN2A kinase. These results suggest that the UV-induced free radicals in cells attack intracellular domains of Ret to dimerize the kinase proteins for superactivation.
c-RET原癌基因编码一种受体型酪氨酸激酶，其种系突变导致2A型多发性内分泌肿瘤（MEN2A）和2B型多发性内分泌肿瘤（MEN2B）的遗传。由于MEN2A突变（Ret-MEN2A）或MEN2B突变（Ret-MEN2B），Ret激酶被组成性激活。在此我们证明，紫外线（UV）照射可诱导组成性激活的Ret-MEN2A和Ret-MEN2B的超激活以及c-Ret的激活。在紫外线照射前，小比例的c-Ret（3-4%）和Ret-MEN2B（1-2%）以及大比例的Ret-MEN2A（30-40%）通过二硫键二聚化。这些二聚化的Ret蛋白优先发生自磷酸化，提示Ret蛋白激酶活性上调与二硫键介导的二聚化之间存在密切关系。我们发现，紫外线照射促进Ret蛋白的二硫键介导的二聚化，与Ret激酶的激活和超激活密切相关。紫外线照射还诱导了细胞外结构域缺失突变体Ret（Ret-PTC-1）的二聚化和激活。有趣的是，Ret-PTC-1-C376A（其中Ret-PTC-1酪氨酸激酶结构域中的半胱氨酸376被丙氨酸取代）的基础激酶活性和二聚化水平较低，且不会因紫外线照射而增加。这些结果表明，Ret-PTC-1半胱氨酸376是紫外线激活Ret激酶可能的多个关键靶氨基酸之一。基因转染导致细胞中铜/锌超氧化物歧化酶的过表达可阻止紫外线介导的二聚化促进作用和Ret-MEN2A激酶超激活。这些结果表明，细胞中紫外线诱导的自由基攻击Ret的细胞内结构域，使激酶蛋白二聚化以实现超激活。
