Watabe M, Kakeya H, Onose R, Osada H
Antibiotics Laboratory, RIKEN, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan.
J Biol Chem. 2000 Mar 24;275(12):8766-71. doi: 10.1074/jbc.275.12.8766.
We found that antitumor drugs such as cytotrienin A, camptothecin, taxol, and 5-fluorouracil induced the activation of a 36-kDa protein kinase (p36 myelin basic protein (MBP) kinase) during apoptosis in human promyelocytic leukemia HL-60 cells. This p36 MBP kinase, which phosphorylates MBP in an in-gel kinase assay, results from the caspase-3-mediated proteolytic cleavage of MST/Krs protein, a mammalian Ste20-like serine/threonine kinase. Herein the correlation between cytotrienin A-induced apoptosis and the activation of MST/Krs proteins was examined in human tumor cell lines, including leukemia-, lung-, epidermoid-, cervix-, stomach-, and brain-derived cell lines. In cytotrienin A-sensitive cell lines, we observed a strong activation of p36 MBP kinase by cleavage of the C-terminal regulatory domain of full-length MST/Krs proteins by caspase-3. When the kinase-inactive mutant form of MST/Krs protein was overexpressed in cytotrienin A-sensitive HL-60 cells, the cytotrienin A-induced apoptosis was partially inhibited. Because cytotrienin A also activated c-Jun N-terminal kinase, we examined the effect of the expression of dominant negative c-Jun on cytotrienin A-induced apoptosis. The expression of dominant negative c-Jun also partially inhibited cytotrienin A-induced apoptosis. Furthermore, coexpression of kinase-inactive MST/Krs protein and dominant negative c-Jun completely suppressed cytotrienin A-induced apoptosis. These findings suggest that the proteolytic activation of MST/Krs and c-Jun N-terminal kinase activation are involved in cytotrienin A-induced apoptosis in human tumor cell lines.
我们发现，诸如细胞三烯素A、喜树碱、紫杉醇和5-氟尿嘧啶等抗肿瘤药物在人早幼粒细胞白血病HL-60细胞凋亡过程中可诱导一种36 kDa蛋白激酶（p36髓鞘碱性蛋白（MBP）激酶）的激活。这种在凝胶内激酶分析中使MBP磷酸化的p36 MBP激酶，是由caspase-3介导的MST/Krs蛋白（一种哺乳动物Ste20样丝氨酸/苏氨酸激酶）的蛋白水解切割产生的。在此，我们在包括白血病、肺癌、表皮样癌、宫颈癌、胃癌和脑源性细胞系在内的人肿瘤细胞系中研究了细胞三烯素A诱导的凋亡与MST/Krs蛋白激活之间的相关性。在对细胞三烯素A敏感的细胞系中，我们观察到全长MST/Krs蛋白的C末端调节结构域被caspase-3切割后，p36 MBP激酶有强烈激活。当激酶失活的MST/Krs蛋白突变体形式在对细胞三烯素A敏感的HL-60细胞中过表达时，细胞三烯素A诱导的凋亡被部分抑制。由于细胞三烯素A也激活c-Jun氨基末端激酶，我们研究了显性负性c-Jun的表达对细胞三烯素A诱导凋亡的影响。显性负性c-Jun的表达也部分抑制了细胞三烯素A诱导的凋亡。此外，激酶失活的MST/Krs蛋白与显性负性c-Jun的共表达完全抑制了细胞三烯素A诱导的凋亡。这些发现表明，MST/Krs的蛋白水解激活和c-Jun氨基末端激酶激活参与了细胞三烯素A诱导的人肿瘤细胞系凋亡。
