Analysis of stable protein methylation in cultured cells.
Analysis of stable protein methylation in cultured cells.
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DOI：
10.1016/0003-9861(92)90369-8
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发表时间：
1992-02
影响因子：
3.9
通讯作者：
J. Najbauer;B. A. Johnson;D. Aswad
中科院分区：
文献类型：
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作者：
J. Najbauer;B. A. Johnson;D. Aswad
最近证明，蛋白质N-甲基转移酶的底物（J. Najbauer和D. W. Aswad，1990，J.Biol.Chem.265，12，717 - 12，721）和蛋白质羧基甲基转移酶（J. Najbauer，B. A.约翰逊和D. W. Aswad，1991，Anal. 197，412-420）在甲基化抑制剂腺苷二醛存在下培养大鼠PC 12细胞时积累。在本报告中，我们进一步描述了PC 12细胞和其他两种广泛使用的细胞类型中的这种现象。腺苷二醛被发现增加人类皮肤成纤维细胞和小鼠Sp2/0骨髓瘤细胞中蛋白质的甲基接受能力。然而，在未处理的细胞中的甲基掺入的水平和抑制剂处理所提供的刺激量在这些细胞中比在PC 12细胞中低得多。所有三种细胞系在17-21 kDa处积累甲基受体。PC 12细胞和成纤维细胞也表现出在33- 38-kDa区域中的三种明显相似的蛋白质的刺激，在该区域中预期有几种参与RNA加工的精氨酸甲基化蛋白质。对PC 12细胞提取物甲基化的最佳条件进行了表征，包括pH值、甲基化时间和S-[甲基-3H]腺苷-L-甲硫氨酸浓度。腺苷二醛处理后短至2 h，检测到甲基掺入增加，并且随着处理时间延长至72 h，大多数底物的甲基化继续增加。积累的动力学因基质而异。来自在S-[甲基-3H]腺苷-1-甲硫氨酸存在下孵育的未处理的PC 12细胞的提取物的二维凝胶的荧光图显示了与处理的细胞相似的甲基掺入模式，但是需要更长的暴露时间（例如，35天vs 7天）。这些发现表明，抑制剂治疗主要通过抑制“正常”细胞蛋白质阵列的翻译后或共翻译甲基化来发挥作用。
It was demonstrated recently that substrates for proteinN-methyltransferases (J. Najbauer and D. W. Aswad, 1990,J. Biol. Chem.265, 12,717–12,721) and protein carboxyl methyltransferases (J. Najbauer, B. A. Johnson, and D. W. Aswad, 1991,Anal. Biochem.197, 412–420) accumulate when rat PC12 cells are cultured in the presence of the methylation inhibitor, adenosine dialdehyde. In the present report, we have further characterized this phenomenon in PC12 cells and in two other, widely used cell types. Adenosine dialdehyde was found to increase the methyl-accepting capacity of proteins in human skin fibroblasts and mouse Sp2/0 myeloma cells. However, both the level of methyl incorporation in untreated cells and the amount of stimulation afforded by inhibitor treatment were substantially lower in these cells than in PC12 cells. All three cell lines accumulated methyl acceptor(s) at 17–21 kDa. The PC12 cells and the fibroblasts also exhibited stimulation of three apparently similar proteins in the 33- to 38-kDa region, where several arginine-methylated proteins involved in RNA processing would be expected. The optimal conditions for methylation of PC12 cell extracts with regard to pH, time of methylation, andS-[methyl-3H]adenosyl-l-methionine concentration were characterized. Increased methyl incorporation was detected after adenosine dialdehyde treatments as short as 2 h, and methylation of most substrates continued to increase as the time of treatment was extended to 72 h. The kinetics of accumulation varied from substrate to substrate. Fluorograms of two-dimensional gels of extracts from untreated PC12 cells incubated in the presence ofS-[methyl-3H] adenosyl-l-methionine revealed patterns of methyl incorporation similar to those of treated cells, but longer exposure times were necessary (e.g., 35 days vs 7 days). These findings suggest that the inhibitor treatment works mainly by inhibiting the post- or cotranslational methylation of a “normal” array of cellular proteins.