MAPキナーゼ阻害剤・活性化剤の探索定
概要
MAPキナーゼは、増殖因子を培養細胞に添加すると活性化するセリン/スレオニン/チロシンキナーゼとして同定されたが、神経分化や免疫細胞の活性化や分泌、最近では海馬における長期増強、さらに微小管ダイナミクスをM期型に変換するなど様々な細胞内シグナル伝達に関与していることがわかっている。MAPキナーゼ阻害剤の開発は、このようなMAPキナーゼの生体における機能を明らかにするうえで有効な道具となりうる。そこで、PC12などの細胞から得たライゼートを用い、MAPキナーゼ阻害剤・活性化剤をスクリーニングする。
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Cylolrienin A activates a kinase with an apparent molecular mass of 36 kDa with kinetics different from JNK/SAPK and p38 MAPK in HL-60 cells.
(A) in-gel kinase activity of cells treated with cytotrienin A. Cells were incubated with cytotrienin A at the concentrations indicated. Cell lysates were eleclrophoresed in 10% SDS-PAGE, and kinase activity was determined by an in-gel kinase assay using MBP as a substrate. (B) kinetics of the activation of p.36 MBP kinase. JNK/SAPK. and p.38 MAPK during apoptosis induced by cytotrienin A. Cells were incubated with 100 ng/ml cytotrienin A for the times indicated, and the activation of the JNK/SAPK and p38 MAPK was analyzed by Western blotting using antibodies that specifically recognize the activated JNK/SAPK and p38 MAPK kinases. The p36 MBP kinase activity was measured as described in A. (C) effect of a specific inhibitor of p38 MAPK, SB203580, on p36 MBP kinase activation. Cells were treated with 100 ng/ml cytotrienin A in the presence of SB203580. After incubating for 2 h, p38 MAPK activation and p36 MBP kinase activity were measured as described in B. (Ref:1)
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References:
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H. Kakeya, R. Onose, H. Osada.
Caspase-mediated activation of a 36-kDa myelin basic protein kinase during anticancer drug-induced apoptosis.
Cancer Res., 58, 4888- 4894 (1998).
