Inhibition of cell growth by conditional expression of kpm, a human homologue of Drosophila warts/lats tumor suppressor
Inhibition of cell growth by conditional expression of kpm, a human homologue of Drosophila warts/lats tumor suppressor
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DOI：
10.1074/jbc.m211974200
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发表时间：
2003-05-16
影响因子：
4.8
通讯作者：
Hori, T
中科院分区：
文献类型：
--
作者：
Kamikubo, Y;Takaori-Kondo, A;Hori, T
kpm是一种人丝氨酸/苏氨酸激酶，其与果蝇肿瘤抑制因子疣/阔肌及其哺乳动物同源物LATS 1同源。为了确定kpm的生物学功能，我们在HeLa Tet-Off细胞中在四环素响应启动子下产生了野生型kpm（kpm-wt）、kpm（kpm-kd）的激酶死亡突变体和荧光素酶的稳定转染子。Western blot分析表明，高水平的表达kpm-wt以及kpm-kd与表观质量为150 kDa的强力霉素去除后诱导。台盼蓝拒染法和MTT法检测发现，kpm-wt的诱导表达导致活细胞数明显下降，而kpm-kd或荧光素酶的诱导表达则无明显影响。然后我们分析了诱导kpm表达后的细胞周期进程和凋亡。2-3在去除强力霉素后30天，细胞经历G（2）/M停滞，通过碘化丙啶掺入和MPM-2反应性的流式细胞术分析证实。体外激酶活性测定结果表明，kpm-wt的诱导可导致Cdc 2-cyclin B复合物激酶活性的下调，并伴随着Cdc 2过度磷酸化形式的增加和Cdc 25 C磷酸化状态的改变。此外，DAPI染色和TUNEL检测均显示，随着kpm表达的诱导，凋亡细胞的比例增加。综上所述，这些结果表明，kpm通过其激酶活性诱导G（2）/M阻滞和凋亡性细胞死亡来负调节细胞生长。
kpm is a human serine/threonine kinase that is homologous to Drosophila tumor suppressor warts/lats and its mammalian homologue LATS1. In order to define the biological function of kpm, we generated stable transfectants of wild-type kpm (kpm-wt), a kinase-dead mutant of kpm (kpm-kd), and luciferase in HeLa Tet-Off cells under the tetracycline-responsive promoter. Western blot analysis showed that high levels of expression of kpm-wt as well as kpm-kd with an apparent mass of 150 kDa were induced after the removal of doxycycline. Induction of kpm-wt expression resulted in a marked decline in viable cell number measured by both trypan blue dye exclusion and MTT assay, whereas that of kpm-kd or luciferase had no effect. We then analyzed the cell cycle progression and apoptosis upon induction of kpm expression. 2-3 days after removal of doxycycline, cells underwent G(2)/M arrest, demonstrated by flow cytometric analysis of propidium iodide incorporation and MPM-2 reactivity. In vitro kinase assay showed that induction of kpm-wt led to down-regulation of kinase activity of the Cdc2-cyclin B complex, which was accompanied by an increase in the hyperphosphorylated form of Cdc2 and a change of phosphorylation status of Cdc25C. Furthermore, both DAPI staining and TUNEL assay showed that the proportion of apoptotic cells increased as kpm expression was induced. Taken together, these results indicate that kpm negatively regulates cell growth by inducing G(2)/M arrest and apoptotic cell death through its kinase activity.