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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)以酪氨酸激酶胞外区缺失突变体RET-PTC-1为模型，研究了蛋白酪氨酸激酶(PTKs)氧化还原调节的靶氨基酸(S)。我们制备了突变的RET-PTC-1基因，将RET-PTC-1结构域中高度保守的半胱氨酸(RET-PTC-1的Cys376)转化为丙氨酸、甘氨酸、丝氨酸或赖氨酸，并将其导入NIIH3T3细胞，检测其催化活性。本研究的结果证实了半胱氨酸在启动该酶的催化活性中起着至关重要的作用。瑞士Prot数据库对人PTK的氨基酸序列分析表明，81个PTK中除1个外，其余均含有MXXCW基序中的半胱氨酸。另一方面，通过质谱分析，我们正式鉴定了Tyr806、Tyr809、Tyr900。酪氨酸酶切域中的905和981为自身磷酸化位点。利用北里大学H.Umeyama开发的计算机模拟软件程序，我们确定了c-RET激动域的三级结构，并提出了一个假说，即MXXCW基序中的半胱氨酸起着全局开关的作用，而激酶结构域中的酪氨酸作为自动磷酸化位点协同作用，起着局部开关的作用。(2)亚砷酸盐被证明可以传递诱导细胞死亡的信号，并发现该信号参与了依赖膜筏功能的活性氧物种(ROS)的产生。我们还发现，当暴露在T细胞中时，4-羟基壬烯醛(HNE)通过caspase依赖的PP2A活性的增加下调Akt的活性，从而使Akt去磷酸化。(3)我们建立并分析了氧化应激相关信号介导的MKK-6缺陷的基因敲除小鼠，并获得了数据表明，氧化还原关联信号参与了特定胸腺细胞亚群的负选择步骤和胸腺T细胞的末端分化步骤。