Kato M, Iwashita T, Akhand A A, Liu W, Takeda K, Takeuchi K, Yoshihara M, Hossain K, Wu J, Du J, Oh C, Kawamoto Y, Suzuki H, Takahashi M, Nakashima I
Department of Immunology, Nagoya University Graduate School of Medicine, Nagoya, Aichi 466-8550, Japan.
Antioxid Redox Signal. 2000 Winter;2(4):841-9. doi: 10.1089/ars.2000.2.4-841.
The catalytic activities of Ret tyrosine kinases as the products of oncogene RET with multiple endocrine neoplasia type 2A (Ret-MEN2A) or 2B (Ret-MEN2B) mutations and the hybrid gene from c-RET and RFP (Rfp-Ret) were higher than those of c-Ret. We demonstrated that ultraviolet light (UV) irradiation induced activation of c-Ret and superactivation of genetically mutated, and thereby constitutively activated, Ret-MEN2A, Ret-MEN2B, and Rfp-Ret. We found that small proportions of c-Ret and Ret-MEN2B and a large proportion of MEN2A were dimerized due to disulfide bonds and that high kinase activity resided in these fractions. The UV-induced activation of c-Ret and superactivation of Ret-MEN2A and Ret-MEN2B were then shown to be closely associated with promotion of the disulfide bond-mediated dimerization of the Ret proteins. Furthermore, we showed that a large proportion of Rfp-Ret was dimerized or polymerized and that almost all kinase activities resided in the highly polymerized but not dimerized fraction. The UV-induced superactivation of Rfp-Ret was also found to be closely associated with promotion of polymerization but not with dimerization of Rfp-Ret. Further experiments revealed that UV induced intracellular dimerization and activation of the extracellular domain-deleted mutant Ret (Ret-PTC-1). Most importantly, the levels of basal kinase activity and dimerization of Ret-TPC-1-C376A, in which cysteine 376 in the tyrosine kinase domain of Ret-TPC-1 was replaced with alanine, were low and were not increased by UV irradiation. These results suggest that the cysteine at this position works as the primary target of dimerization of Ret proteins inside the cell for both the maintenance of the basal kinase activity and its promotion by UV, possibly in co-operation with the cysteine(s) in the extracellular domain of Ret-MEN2A and Rfp-Ret, which is the target of dimerization and polymerization outside the cell. The potential biological significance of the UV-mediated superactivation of mutant Ret through the newly proposed mechanism in oncogenesis is discussed.
作为致癌基因RET的产物，具有2A型多发性内分泌肿瘤（Ret-MEN2A）或2B型（Ret-MEN2B）突变的Ret酪氨酸激酶以及来自c-RET和RFP的杂交基因（Rfp-Ret）的催化活性高于c-Ret。我们证明，紫外线（UV）照射可诱导c-Ret激活以及基因发生突变从而组成性激活的Ret-MEN2A、Ret-MEN2B和Rfp-Ret的超激活。我们发现，小部分的c-Ret和Ret-MEN2B以及大部分的MEN2A因二硫键而二聚化，且高激酶活性存在于这些组分中。随后表明，UV诱导的c-Ret激活以及Ret-MEN2A和Ret-MEN2B的超激活与促进Ret蛋白的二硫键介导的二聚化密切相关。此外，我们表明大部分Rfp-Ret二聚化或聚合，且几乎所有激酶活性存在于高度聚合而非二聚化的组分中。还发现UV诱导的Rfp-Ret超激活与促进聚合密切相关，而与Rfp-Ret的二聚化无关。进一步实验表明，UV诱导细胞内缺失细胞外结构域的突变体Ret（Ret-PTC-1）二聚化并激活。最重要的是，Ret-TPC-1-C376A（其中Ret-TPC-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，且未因UV照射而增加。这些结果表明，该位置的半胱氨酸作为细胞内Ret蛋白二聚化的主要靶点，对于维持基础激酶活性以及UV对其的促进作用可能是必需的，这可能与Ret-MEN2A和Rfp-Ret细胞外结构域中的半胱氨酸协同作用，后者是细胞外二聚化和聚合的靶点。讨论了通过新提出的机制在肿瘤发生中UV介导的突变体Ret超激活的潜在生物学意义。
