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

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(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.