Study on redox-linked regulatory mechanism of intracellular signal transduction for differentiation and death of T lymphocytes

T淋巴细胞分化和死亡的细胞内信号转导氧化还原调控机制研究

基本信息

批准号：
14390026
负责人：
NAKASHIMA Izumi
金额：
$ 5.63万
依托单位：
Nagoya University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2002
资助国家：
日本
起止时间：
2002 至 2003
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-14390026/
关键词：
Tyrosine kinase redox RET T-lymphocyte reft apoptosis MKK6--mouse negative selection チロシンキナーゼ

项目摘要

(1)We surveyed target amino acid(s) of redox-linked regulation of protein tyrosine kinases (PTKs) in a model of RET-PTC-1,an extracellular domain-deleted mutant of RET kinase. We prepared mutant RET-PTC-1 cDNA in which the highly conserved cysteine in the kinase domain (Cys376 of RET-PTC-1) to alanine, glycine, serine or lysine and introduced it into NIIH3T3 cells for examination of the catalytic activity of the kinase. The result of this study confirmed that the cysteine plays a crucial role in the initiation of the catalytic activity of the kinase. Analyses of database of Swiss-Prot on amino acid sequences of human PTKs demonstrated that all but one of 81 PTKs had the cysteine in the MXXCW motif. On the other hand, by use of massspectrometry we formally identified Tyr806,Tyr809,Tyr900. Tyr905 and Tyr98l in the kinase domain as autophosphorylation sites. By using a computer modeling software program developed by H.Umeyama, Kitasato University we determined the tertiary structure of the kinase domain of c-RET and proposed a hypothetical view that the cysteine in the MXXCW motif works as a global switch whereas the tyrosines in the kinase domain as autophosphorylation sites work together as a local switch for activation of the kinase.(2)Arsenite was shown to transduce a signal for induction of apoptotic cell death and this signal was found to involve production of reactive oxygen species (ROS) in a manner dependent on the membrane raft function. We also showed that 4-hydroxynonenal (HNE), when exposed to T cells, downreuglates the activity of Akt through a caspase-dependent increase in the PP2A activity that dephosphorylates Akt.(3)We have established and analysed a gene-knockout mice in which the oxidative stress-linked signal-mediating MKK-6 was defective and obtained data suggesting that a redox-linked signal is involved in the step of negative selection of a specific subpopulation of thymocytes and in the step of terminal differentiation of T cells in the thymus.

（1）我们调查了在RET-PTC-1的模型（一种RET-PTC-1）模型中，氧化还原连接的蛋白酪氨酸激酶（PTK）的靶氨基酸是一种RET激酶的细胞外结构域删除的突变体。我们准备了突变ret-PTC-1 cDNA，其中激酶结构域（RET-PTC-1的Cys376）中高度保守的半胱氨酸将其放到丙氨酸，甘氨酸，丝氨酸或赖氨酸中，并将其引入NIIH3T3细胞中，以检查激酶的催化活性。这项研究的结果证实，半胱氨酸在激酶的催化活性的开始中起着至关重要的作用。瑞士 - 普罗特数据库对人类PTK的氨基酸序列的分析表明，除81个PTK中的一个外，所有其他人都在MXXCW基序中具有半胱氨酸。另一方面，通过使用质谱法，我们正式确定了Tyr806，Tyr809，Tyr900。激酶结构域中的Tyr905和Tyr98L作为自磷酸化位点。 By using a computer modeling software program developed by H.Umeyama, Kitasato University we determined the tertiary structure of the kinase domain of c-RET and proposed a hypothetical view that the cysteine in the MXXCW motif works as a global switch whereas the tyrosines in the kinase domain as autophosphorylation sites work together as a local switch for activation of the kinase.(2)Arsenite was shown to transduce诱导凋亡细胞死亡的信号和该信号以依赖于膜筏功能的方式涉及活性氧（ROS）的产生。我们还表明，当暴露于T细胞时，4-羟基苯烯（HNE）通过caspase依赖性的PP2A活性增加了Akt的活性，使Akt降低了Akt。氧化还原连接的信号参与了胸腺细胞特定亚群的阴性选择以及胸腺中T细胞末端分化的步骤。