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

(1)我们在RET激酶胞外结构域缺失突变体RET- ptc -1模型中研究了氧化还原连接蛋白酪氨酸激酶（PTKs）调控的靶氨基酸。我们制备了RET-PTC-1突变体cDNA，其中高度保守的激酶结构域半胱氨酸（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，检测该激酶的催化活性。本研究结果证实了半胱氨酸在启动激酶的催化活性中起着至关重要的作用。通过Swiss-Prot数据库对人类PTKs的氨基酸序列进行分析，发现81个PTKs除1个外，其余均含有MXXCW基序的半胱氨酸。另一方面，通过质谱分析，我们正式鉴定了Tyr806，Tyr809,Tyr900。Tyr905和Tyr98l作为自磷酸化位点。通过使用北上大学H.Umeyama开发的计算机建模软件程序，我们确定了c-RET激酶结构域的三级结构，并提出了一种假设观点，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共同作为激酶激活的局部开关。(2)亚砷酸盐可转导诱导凋亡细胞死亡的信号，该信号涉及活性氧（ROS）的产生，其方式依赖于膜筏功能。我们还发现，当暴露于T细胞时，4-羟基壬烯醛（HNE）通过caspase依赖性的PP2A活性增加来下调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MKK-6缺陷的基因敲除小鼠，获得的数据表明氧化还原相关信号参与了胸腺细胞特定亚群的负选择步骤和胸腺T细胞的终末分化步骤。

(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.