Suppression of MEK/ERK signaling pathway enhances cisplatin-induced NF-kappaB activation by protein phosphatase 4-mediated NF-kappaB p65 Thr dephosphorylation.
Suppression of MEK/ERK signaling pathway enhances cisplatin-induced NF-kappaB activation by protein phosphatase 4-mediated NF-kappaB p65 Thr dephosphorylation.
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发表时间：
2004
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通讯作者：
P. Yeh;K. Yeh;S. Chuang;Y. Song;A. Cheng
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作者：
P. Yeh;K. Yeh;S. Chuang;Y. Song;A. Cheng
我们以前报道过抑制MEK/ERK通路会增加SiHa细胞的耐药性。在这项研究中，我们进一步描述了这种现象的潜在机制。用MEK/ERK抑制剂预处理SiHa细胞增强顺铂诱导的NF-κ B活化。然而，免疫印迹分析的结果表明，顺铂和MEK/ERK抑制剂均未诱导显著的IkappaB α降解，这表明MEK/ERK信号通路的抑制可能通过常规途径以外的机制增强顺铂诱导的NF-κ B活化。蛋白磷酸酶4（PP4），一种核丝氨酸/苏氨酸磷酸酶，直接与NF-κ B相互作用并激活NF-κ B的先前发现使我们检查NF-κ B p65的磷酸化状态。与NF-κ B的活化一致，顺铂诱导NF-κ B p65的Ser磷酸化，但降低Thr磷酸化。MEK/ERK通路的抑制进一步增强顺铂诱导的Thr去磷酸化，但不影响顺铂诱导的NF-κ B p65的Ser磷酸化。此外，与Thr去磷酸化平行，顺铂处理的细胞中核PP4的蛋白水平增加，并且通过抑制MEK/ERK途径进一步增加。SiHa细胞转染有义或反义PP4基因。PP4过表达细胞显示NF-κ B p65的Thr磷酸化降低到几乎检测不到的水平，并且基础和顺铂诱导的NF-κ B活性均高于亲本细胞。相比之下，顺铂，无论是单独或与MEK/ERK抑制剂，诱导很少的NF-κ B激活的反义PP4转染细胞。共沉淀复合物激酶测定揭示了NF-κ B p65的片段（氨基酸279 - 444）含有直接与PP4相互作用的潜在磷酸化位点。进一步的定点突变研究表明，Thr（435）是主要的磷酸化位点。
We previously reported that suppression of the MEK/ERK pathway increases drug resistance of SiHa cells. In this study, we further characterized the underlying mechanism of this phenomenon. Pretreatment of SiHa cells with MEK/ERK inhibitor enhanced cisplatin-induced NF-kappaB activation. However, results of immunoblotting analysis showed that neither cisplatin nor MEK/ERK inhibitors induced marked IkappaBalpha degradation, suggesting that suppression of the MEK/ERK signaling pathway may enhance cisplatin-induced NF-kappaB activation via mechanisms other than the conventional pathway. Previous findings that protein phosphatase 4 (PP4), a nuclear serine/threonine phosphatase, directly interacts with and activates NF-kappaB led us to examine the phosphorylation status of NF-kappaB p65. Coincident with activation of NF-kappaB, cisplatin induced Ser phosphorylation but decreased Thr phosphorylation of NF-kappaB p65. Suppression of the MEK/ERK pathway further enhanced cisplatin-induced Thr dephosphorylation but did not affect cisplatin-induced Ser phosphorylation of NF-kappaB p65. Further, in parallel with Thr dephosphorylation, the protein level of nuclear PP4 was increased in cisplatin-treated cells and was further increased by suppression of the MEK/ERK pathway. SiHa cells were then transfected by a sense or an antisense PP4 gene. PP4-overexpressing cells showed a decrease in Thr phosphorylation of NF-kappaB p65 to nearly undetectable levels, and both basal and cisplatin-induced NF-kappaB activities were higher than those in parental cells. By contrast, cisplatin, either alone or with MEK/ERK inhibitors, induced little NF-kappaB activation in antisense PP4-transfected cells. Coprecipitated complex kinase assay revealed a fragment of NF-kappaB p65 (amino acids 279-444) to contain potential phosphorylation sites that directly interact with PP4. Further studies by site-directed mutagenesis suggested that Thr(435) was the major phosphorylation site.