Nerve growth factor-specific regulation of protein methylation during neuronal differentiation of PC12 cells.
Nerve growth factor-specific regulation of protein methylation during neuronal differentiation of PC12 cells.
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DOI：
10.1083/jcb.138.5.1089
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发表时间：
1997-09-08
期刊：
影响因子：
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通讯作者：
Aletta JM
中科院分区：
文献类型：
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作者：
Cimato TR;Ettinger MJ;Zhou X;Aletta JM
蛋白质甲基化是一种翻译后修饰，可能以与蛋白质磷酸化类似的方式调节信号转导途径。通过用l-[甲基-3H]甲硫氨酸代谢标记PC12细胞蛋白和用l-[甲基-3H]S-腺苷甲硫氨酸体外标记细胞蛋白来检查蛋白质甲基化在NGF信号传导中的作用。 NGF 的作用在 15 分钟内就被检测到。通过一维和二维 SDS-PAGE 解析甲基标记的蛋白质。 NGF 影响几种 68-60-kD 蛋白质 (pI 5.8-6.4) 和 50-kD 蛋白质（等电点 pH 6.7-6.8 和 5.8-6.2）的甲基化。 NGF 诱导的甲基化变化在数小时和 4 天内有所增加。此外，仅在 NGF 处理后才检测到几种特定蛋白质的甲基标记，但在未处理的对照中未检测到。 NGF 对蛋白质甲基化的影响是 NGF 特异性的，因为在 EGF 或胰岛素中没有观察到这种影响。神经突生长需要蛋白质甲基化，这通过两种甲基化抑制剂之一得到证实：二羟基环戊烯基腺嘌呤（DHCA）和同型半胱氨酸。 DHCA 是两者中更有效的，可显着抑制蛋白质甲基化和神经突生长，而不影响细胞生长、NGF 诱导的存活、细胞扁平化或与早期信号事件相关的几种蛋白质磷酸化。 DHCA 的去除会导致几种蛋白质的快速甲基化和同时的神经突生长。结果表明，NGF 调节几种特定蛋白质的甲基化，并且蛋白质甲基化参与 PC12 细胞的神经突生长。
Protein methylation is a posttranslational modification that can potentially regulate signal transduction pathways in a similar manner as protein phosphorylation. The role of protein methylation in NGF signaling was examined by metabolic labeling of PC12 cell proteins with l-[methyl-3H]methionine and by in vitro labeling of cell proteins with l-[methyl-3H]S-adenosylmethionine. Effects of NGF were detected within 15 min. Methyl-labeled proteins were resolved by one and two dimensional SDS-PAGE. NGF affected the methylation of several 68–60-kD proteins (pI 5.8–6.4) and 50-kD proteins (isoelectric point pH 6.7–6.8 and 5.8–6.2). Several NGF-induced changes in methylation increased over several hours and through 4 d. Moreover, methyl labeling of several specific proteins was only detected after NGF treatment, but not in nontreated controls. The effects of NGF on protein methylation were NGF specific since they were not observed with EGF or insulin. A requirement for protein methylation for neurite outgrowth was substantiated with either of two methylation inhibitors: dihydroxycyclopentenyl adenine (DHCA) and homocysteine. DHCA, the more potent of the two, markedly inhibits protein methylation and neurite outgrowth without affecting cell growth, NGF-induced survival, cell flattening, or several protein phosphorylations that are associated with early signaling events. Removal of DHCA leads to rapid protein methylation of several proteins and concurrent neurite outgrowth. The results indicate that NGF regulates the methylation of several specific proteins and that protein methylation is involved in neurite outgrowth from PC12 cells.