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

(1)We在RET-PTC-1（RET激酶的胞外结构域缺失突变体）模型中，研究了蛋白酪氨酸激酶（PTKs）的氧化还原相关调节的靶氨基酸。我们制备了突变型RET-PTC-1 cDNA，其中在激酶结构域（RET-PTC-1的Cys 376）中高度保守的半胱氨酸变为丙氨酸、甘氨酸、丝氨酸或赖氨酸，并将其引入NIIH 3 T3细胞中以检测激酶的催化活性。本研究结果证实了半胱氨酸在激酶催化活性的启动中起着至关重要的作用。对Swiss-Prot数据库中人PTKs氨基酸序列的分析表明，81个PTKs中除1个外，其余均含有MXXCW基序中的半胱氨酸。另一方面，通过使用质谱法，我们正式鉴定了Tyr 806、Tyr 809、Tyr 900。激酶结构域中的Tyr 905和Tyr 98 l作为自磷酸化位点。通过使用由北里大学的H.Umeyama开发的计算机建模软件程序，我们确定了c-RET的激酶结构域的三级结构，并提出了一个假设的观点，即MXXCW基序中的半胱氨酸作为全局开关，而激酶结构域中的酪氨酸作为自磷酸化位点一起作为激酶激活的局部开关。（2）亚砷酸钠可诱导细胞凋亡，并与细胞膜筏功能相关的活性氧（ROS）产生有关。我们还发现，当暴露于T细胞时，4-羟基壬烯醛（HNE）通过半胱天冬酶依赖性增加PP 2A活性（使Akt去磷酸化）下调Akt的活性。(3)We已经建立并分析了基因敲除小鼠，其中介导氧化应激相关信号的MKK-6有缺陷，并获得了表明氧化还原相关信号参与胸腺细胞特定亚群的阴性选择步骤和胸腺中T细胞的终末分化步骤的数据。

(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.