ESTABLISHMENT OF LIPOPOLYSACCHARIDE-DEPENDENT NUCLEAR FACTOR KAPPA-B ACTIVATION IN A CELL-FREE SYSTEM
ESTABLISHMENT OF LIPOPOLYSACCHARIDE-DEPENDENT NUCLEAR FACTOR KAPPA-B ACTIVATION IN A CELL-FREE SYSTEM
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DOI：
10.1074/jbc.270.8.4158
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发表时间：
1995-02-24
影响因子：
4.8
通讯作者：
MATSUSHIMA, K
中科院分区：
文献类型：
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作者：
ISHIKAWA, Y;MUKAIDA, N;MATSUSHIMA, K
核因子-kappaB由p50和p65组成，在静息状态下与细胞质保留蛋白I-kappa B结合，在多种炎症刺激下诱导核转录因子-kappa B与I-kappaB解离和核转位，从而激活一些参与炎症反应的基因，如白介素6、白介素8和肿瘤坏死因子α。为了阐明NE-kappa B激活的确切机制，我们利用从人单核细胞系THP-1中提取的质膜富集物、胞浆和核组分，在无细胞系统中建立了依赖内毒素(LPS)的NF-kappa B激活机制。只有在有ATP存在的情况下，质膜富集组分和胞浆的结合才足以以依赖于内毒素/CD14的方式激活NF-kappa B，这是通过电泳迁移率改变分析中NF-kappa B与IL-8基因kappa B的结合来判断的。内毒素依赖的NP-kappa B激活可被蛋白激酶抑制剂抑制，但丝裂原激活的蛋白激酶底物、cGMP依赖的蛋白激酶、cAMP依赖的蛋白激酶、蛋白激酶C和钙调素依赖的蛋白激酶II抑制作用不明显，提示星状孢子素敏感的蛋白激酶(S)和酪氨酸激酶(S)参与了脂多糖介导的NF-kappa B的激活。此外，在无细胞体系中，内毒素在刺激后5min开始诱导I kappa B-α的磷酸化。此外，除草霉素A和酪氨酸氨基转移酶抑制磷酸化，但不能抑制星形孢子素，这表明这些蛋白激酶抑制剂在不同的信号传递步骤中起作用。在无细胞体系中建立配体依赖的核因子-kappa B激活机制，有助于鉴定参与内毒素介导的核因子-kappaB激活的蛋白激酶(S)及其底物(S)。
Nuclear factor kappa B (NF-kappa B), consisting of p50 and p65, is bound to a cytoplasmic retention protein, I kappa B, in a resting state, and the stimulation of cells with a variety of inflammatory stimuli induces the dissociation of NF-kappa B from I kappa B and the nuclear translocation of NF-kappa B, thereby activating several genes involved in inflammatory responses, such as interleukin (IL)-6, IL-8, and tumor necrosis factor alpha. In order to elucidate the precise mechanism of NE-kappa B activation, we have established Lipopolysaccharide (LPS)-dependent NF-kappa B activation in a cell-free system using plasma membrane-enriched, cytosol, and nuclear fractions extracted from a human monocytic cell Line, THP-1, by disruption with sonication followed by a differential centrifugation. The combination of plasma membrane enriched fraction and cytosol was sufficient to activate NF-kappa B in a LPS/CD14-dependent manner only in the presence of ATP as judged by the binding of NF-kappa B to the IL-8 gene kappa B site on an electrophoretic mobility shift assay. LPS-dependent NP-kappa B activation was inhibited by protein kinase inhibitors, such as staurosporine, herbimycin A, tyrphostin, and genistein, but not mitogen-activated protein kinase substrate, cGMP-dependent protein kinase, cAMP-dependent protein kinase, protein kinase C, and calmodulin-dependent protein kinase II inhibitory peptides, suggesting that staurosporine-sensitive kinase(s) as well as tyrosine kinase(s) are involved in LPS-mediated NF-kappa B activation. In addition, LPS induced the phosphorylation of I kappa B-alpha, starting at 5 min after the stimulation in a cell-free system. Moreover, the phosphorylation was inhibited by herbimycin A and tyrphostin, but not staurosporine, suggesting that these protein kinase inhibitors act at distinct steps of signal transmission. Establishment of ligand-dependent activation of NF-kappa B in a cell-free system will facilitate identification of protein kinase(s) and its substrate(s) involved in LPS-mediated NF-kappa B activation.