Quantitative determination of Rap 1 activation in cyclic nucleotide-treated HL-60 leukemic cells: lack of Rap 1 activation in variant cells.
Quantitative determination of Rap 1 activation in cyclic nucleotide-treated HL-60 leukemic cells: lack of Rap 1 activation in variant cells.
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环核苷酸处理的 HL-60 白血病细胞中 Rap 1 活化的定量测定：变异细胞中缺乏 Rap 1 活化。
DOI：
10.1038/sj.onc.1203741
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发表时间：
2000
期刊：
影响因子：
--
通讯作者：
Boss,GR
中科院分区：
文献类型：
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作者：
vonLintig,FC;Pilz,RB;Boss,GR
我们先前已经分离了对cGMP诱导的分化有抗性的变体HL-60细胞，并且显示它们缺乏Rap 1A的蛋白水解切割和/或羧基甲基化（J. Biol. Chem. 269，32155-32161，1994和Oncogene 17，2211-2233，1998）。我们现在已经开发了一种基于酶的方法来评估Rap 1激活，该方法是定量的，并提供了处于活性GTP结合状态的Rap分子百分比的测量。使用这种方法，我们表明，cAMP和cGMP类似物激活RAP 1在父母的HL-60细胞，但不是在变异细胞和H-89，cAMP依赖性蛋白激酶抑制剂，cAMP诱导的RAP 1激活在父母的细胞没有影响。因此，cAMP激活HL-60细胞中的Rap 1可能是通过cAMP调节的鸟嘌呤核苷酸交换因子（cAMP-GEF），并且由于cAMP在变体细胞中不激活Rap 1，数据表明Rap 1的完全翻译后加工对于Rap 1的cAMP-GEF激活是必需的。以前没有发现Rap 1被cGMP类似物激活，这表明NO/cGMP信号转导途径和Rap 1信号传导之间可能存在串扰。
We have previously isolated variant HL-60 cells that are resistant to cGMP-induced differentiation and showed that they are deficient in proteolytic cleavage and/or carboxyl methylation of Rap 1A (J. Biol. Chem. 269, 32155–32161, 1994 and Oncogene 17, 2211–2233, 1998). We have now developed an enzyme-based method for assessing Rap 1 activation which is quantitative and provides a measurement of the per cent of Rap molecules in the active GTP-bound state. Using this method, we show that cAMP and cGMP analogs activate Rap 1 in parental HL-60 cells but not in the variant cells and that H-89, a cAMP-dependent protein kinase inhibitor, has no effect on cAMP-induced Rap 1 activation in parental cells. Thus, cAMP activation of Rap 1 in HL-60 cells is likely through a cAMP-regulated guanine nucleotide exchange factor (cAMP-GEF) and since cAMP does not activate Rap 1 in the variant cells, the data suggest that full post-translational processing of Rap 1 is necessary for cAMP-GEF activation of Rap 1. Activation of Rap 1 by cGMP analogs has not been previously found and suggests possible cross-talk between the NO/cGMP signal transduction pathway and Rap 1 signaling.