Leucine carboxyl methyltransferase-1 is necessary for normal progression through mitosis in mammalian cells
Leucine carboxyl methyltransferase-1 is necessary for normal progression through mitosis in mammalian cells
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DOI：
10.1074/jbc.m704861200
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发表时间：
2007-10-19
影响因子：
4.8
通讯作者：
Pallas, David C.
中科院分区：
文献类型：
--
作者：
Lee, Jocelyn A.;Pallas, David C.
蛋白磷酸酶2A（PP2A）是一种多功能磷酸酶，在许多细胞过程中发挥重要作用，包括细胞周期和细胞凋亡的调控。由于PP2A参与众多不同的过程，它受到非共价和共价机制的高度调控，这些机制仍在被确定。在这项研究中，我们研究了亮氨酸羧基甲基转移酶 - 1（LCMT - 1）对PP2A甲基化和细胞功能的重要性。我们发现，通过小发夹RNA降低LCMT - 1蛋白水平会使HeLa细胞中PP2A甲基化降低多达70%，这表明LCMT - 1是主要的哺乳动物PP2A甲基转移酶。此外，LCMT - 1的敲低减少了含有Bα调节亚基的PP2A异源三聚体的形成，并且在一部分细胞中诱导了细胞凋亡，其特征为半胱天冬酶激活、核浓缩/碎片化和膜起泡。PP2A Bα调节亚基的敲低诱导了相似程度的细胞凋亡，这表明LCMT - 1部分通过破坏PP2A（BαAC）异源三聚体的形成来诱导细胞凋亡。用一种泛半胱天冬酶抑制剂处理可部分挽救因LCMT - 1或Bα敲低而诱导的细胞凋亡。LCMT - 1敲低细胞和Bα敲低细胞对纺锤体靶向药物诺考达唑更敏感，这表明LCMT - 1和Bα对纺锤体检查点很重要。用胸苷处理LCMT - 1和Bα敲低细胞可显著降低细胞死亡，大概是通过阻断有丝分裂进程。与这些结果一致的是，在小鼠中LCMT - 1的纯合基因捕获敲除导致胚胎致死。总之，我们的结果表明LCMT - 1对有丝分裂的正常进展和细胞存活很重要，并且对小鼠的胚胎发育至关重要。
Protein phosphatase 2A (PP2A) is a multifunctional phosphatase that plays important roles in many cellular processes including regulation of cell cycle and apoptosis. Because PP2A is involved in so many diverse processes, it is highly regulated by both non-covalent and covalent mechanisms that are still being defined. In this study we have investigated the importance of leucine carboxyl methyltransferase-1 (LCMT-1) for PP2A methylation and cell function. We show that reduction of LCMT-1 protein levels by small hairpin RNAs causes up to a 70% reduction in PP2A methylation in HeLa cells, indicating that LCMT-1 is the major mammalian PP2A methyltransferase. In addition, LCMT-1 knockdown reduced the formation of PP2A heterotrimers containing the B alpha regulatory subunit and, in a subset of the cells, induced apoptosis, characterized by caspase activation, nuclear condensation/fragmentation, and membrane blebbing. Knockdown of the PP2A B alpha regulatory subunit induced a similar amount of apoptosis, suggesting that LCMT-1 induces apoptosis in part by disrupting the formation of PP2A(B alpha AC) heterotrimers. Treatment with a pan-caspase inhibitor partially rescued cells from apoptosis induced by LCMT-1 or B alpha knockdown. LCMT-1 knockdown cells and B alpha knockdown cells were more sensitive to the spindle-targeting drug nocodazole, suggesting that LCMT-1 and B alpha are important for spindle checkpoint. Treatment of LCMT-1 and B alpha knockdown cells with thymidine dramatically reduced cell death, presumably by blocking progression through mitosis. Consistent with these results, homozygous gene trap knock-out of LCMT-1 in mice resulted in embryonic lethality. Collectively, our results indicate that LCMT-1 is important for normal progression through mitosis and cell survival and is essential for embryonic development in mice.