Lehmann David M, Galloway Chad A, MacElrevey Celeste, Sowden Mark P, Wedekind Joseph E, Smith Harold C
Environmental Health Sciences Center, Department of Toxicology, University of Rochester, Rochester, NY 14642, USA.
Biochim Biophys Acta. 2007 Mar;1773(3):408-18. doi: 10.1016/j.bbamcr.2006.11.019. Epub 2006 Dec 8.
ApoB mRNA editing involves site-specific deamination of cytidine 6666 producing an in-frame translation stop codon. Editing minimally requires APOBEC-1 and APOBEC-1 complementation factor (ACF). Metabolic stimulation of apoB mRNA editing in hepatocytes is associated with serine phosphorylation of ACF localized to editing competent, nuclear 27S editosomes. We demonstrate that activation of protein kinase C (PKC) stimulated editing and enhanced ACF phosphorylation in rat primary hepatocytes. Conversely, activation of protein kinase A (PKA) had no effect on editing. Recombinant PKC efficiently phosphorylated purified ACF64 protein in vitro, whereas PKA did not. Mutagenesis of predicted PKC phosphorylation sites S154 and S368 to alanine inhibited ethanol-stimulated induction of editing suggesting that these sites function in the metabolic regulation of editing. Consistent with this interpretation, substitution of S154 and S368 with aspartic acid stimulated editing to levels comparable to ethanol treatment in control McArdle RH7777 cells. These data suggest that phosphorylation of ACF by PKC may be a key regulatory mechanism of apoB mRNA editing in rat hepatocytes.
载脂蛋白B（ApoB）信使核糖核酸（mRNA）编辑涉及胞嘧啶6666的位点特异性脱氨，产生一个符合读框的翻译终止密码子。编辑过程至少需要载脂蛋白B mRNA编辑酶催化多肽1（APOBEC-1）和APOBEC-1互补因子（ACF）。肝细胞中apoB mRNA编辑的代谢刺激与定位于具有编辑能力的核27S编辑体的ACF的丝氨酸磷酸化有关。我们证明，蛋白激酶C（PKC）的激活刺激了大鼠原代肝细胞中的编辑并增强了ACF磷酸化。相反，蛋白激酶A（PKA）的激活对编辑没有影响。重组PKC在体外有效地磷酸化了纯化的ACF64蛋白，而PKA则没有。将预测的PKC磷酸化位点丝氨酸154（S154）和丝氨酸368（S368）突变为丙氨酸可抑制乙醇刺激的编辑诱导，表明这些位点在编辑的代谢调节中起作用。与此解释一致，在对照的麦卡德尔RH7777细胞中，用天冬氨酸替代S154和S368可将编辑刺激到与乙醇处理相当的水平。这些数据表明，PKC介导的ACF磷酸化可能是大鼠肝细胞中apoB mRNA编辑的关键调节机制。
