Lee K K, Ohyama T, Yajima N, Tsubuki S, Yonehara S
Institute for Virus Research, Kyoto University, Kyoto 606-8507, Japan.
J Biol Chem. 2001 Jun 1;276(22):19276-85. doi: 10.1074/jbc.M005109200. Epub 2001 Mar 7.
The human serine/threonine kinase, mammalian STE20-like kinase (MST), is considerably homologous to the budding yeast kinases, SPS1 and STE20, throughout their kinase domains. The cellular function and physiological activation mechanism of MST is unknown except for the proteolytic cleavage-induced activation in apoptosis. In this study, we show that MST1 and MST2 are direct substrates of caspase-3 both in vivo and in vitro. cDNA cloning of MST homologues in mouse and nematode shows that caspase-cleaved sequences are evolutionarily conserved. Human MST1 has two caspase-cleavable sites, which generate biochemically distinct catalytic fragments. Staurosporine activates MST either caspase-dependently or independently, whereas Fas ligation activates it only caspase-dependently. Immunohistochemical analysis reveals that MST is localized in the cytoplasm. During Fas-mediated apoptosis, cleaved MST translocates into the nucleus before nuclear fragmentation is initiated, suggesting it functions in the nucleus. Transiently expressed MST1 induces striking morphological changes characteristic of apoptosis in both nucleus and cytoplasm, which is independent of caspase activation. Furthermore, when stably expressed in HeLa cells, MST highly sensitizes the cells to death receptor-mediated apoptosis by accelerating caspase-3 activation. These findings suggest that MST1 and MST2 play a role in apoptosis both upstream and downstream of caspase activation.
人类丝氨酸/苏氨酸激酶，即哺乳动物STE20样激酶（MST），在其激酶结构域与芽殖酵母激酶SPS1和STE20具有相当高的同源性。除了在凋亡过程中由蛋白水解切割诱导的激活外，MST的细胞功能和生理激活机制尚不清楚。在本研究中，我们表明MST1和MST2在体内和体外都是caspase-3的直接底物。小鼠和线虫中MST同源物的cDNA克隆表明，caspase切割序列在进化上是保守的。人类MST1有两个可被caspase切割的位点，可产生生化性质不同的催化片段。星形孢菌素以caspase依赖性或非依赖性方式激活MST，而Fas连接仅以caspase依赖性方式激活它。免疫组织化学分析显示，MST定位于细胞质中。在Fas介导的凋亡过程中，切割后的MST在核碎裂开始之前转位到细胞核中，表明它在细胞核中发挥作用。瞬时表达的MST1在细胞核和细胞质中均诱导出明显的凋亡特征性形态变化，这与caspase激活无关。此外，当在HeLa细胞中稳定表达时，MST通过加速caspase-3激活使细胞对死亡受体介导的凋亡高度敏感。这些发现表明，MST1和MST2在caspase激活的上游和下游的凋亡过程中均发挥作用。
