| Research Abstract |
Among the three MAP kinase cascades, two of them that converge on JNKs and p38 MAP kinases are preferentially activated by cytotoxic stresses such as UV radiation, X-ray, heat shock and osmotic shock, and by proinflammatory cytokines such as tumor necrosis factor (TNF) and interleukin-1. One of the important biological responses mediated through these stress-activated MAP kinase pathways appears to be the decision of cell fate by regulating apoptosis. In this research project, we intended to develop a novel, rapid and sensitive assessment system for various biomaterials by assaying the activation status of the stress-activated MAP kinase pathways. We first established the sensitive assay system for INK and p38 activation, and clearly showed that apoptosis signal^egulating kinase 1 (ASK1), which is known to regulate the JNK and p38 MAP kinase pathways, is required for oxidative stress-nduced activation of JNK and p38. We next established the sensitive assay system for ASK1 activation. We found that phosphorylation of Thr845 in the kinase domain of ASK1 is required for the activation of ASK1, and raised a polyclonal antibody that specifically recognized the activation status of Thr 845 of ASK 1. This anti-phospho-ASKl antibody could detect the activation state of ASK1 with much higher sensitivity than the conventional "in vitro kinase assay". By the various biochemical analyzes using this antibody and the further investigation of ASKl-deficient mice, we have found that ASK1 is necessary for not only oxidative stress- but also ER stress-induced apoptosis, suggesting that the ASK1-MAP kinase pathways is a common signal mediator of various kind of stressors. Thus, accurate and sensitive detection of the activation status of the ASK1-MAP kinase pathways appears to be useful for the development and application of our assessment system for various biomaterials.
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