Yeh Pei Yen, Yeh Kun-Huei, Chuang Shuang-En, Song Ying Chyi, Cheng Ann-Lii
Cancer Research Center, College of Medicine, National Taiwan University, Republic of China.
J Biol Chem. 2004 Jun 18;279(25):26143-8. doi: 10.1074/jbc.M402362200. Epub 2004 Apr 8.
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.
我们之前报道过，抑制MEK/ERK通路会增加SiHa细胞的耐药性。在本研究中，我们进一步探究了这一现象的潜在机制。用MEK/ERK抑制剂预处理SiHa细胞可增强顺铂诱导的NF-κB活化。然而，免疫印迹分析结果显示，顺铂和MEK/ERK抑制剂均未诱导明显的IκBα降解，这表明抑制MEK/ERK信号通路可能通过不同于传统途径的机制增强顺铂诱导的NF-κB活化。先前的研究发现，核丝氨酸/苏氨酸磷酸酶蛋白磷酸酶4（PP4）直接与NF-κB相互作用并激活NF-κB，这促使我们检测NF-κB p65的磷酸化状态。与NF-κB活化一致，顺铂诱导NF-κB p65的丝氨酸磷酸化增加，但苏氨酸磷酸化减少。抑制MEK/ERK通路进一步增强了顺铂诱导的苏氨酸去磷酸化，但不影响顺铂诱导的NF-κB p65的丝氨酸磷酸化。此外，与苏氨酸去磷酸化同时发生的是，顺铂处理的细胞中核PP4的蛋白水平升高，并且通过抑制MEK/ERK通路进一步升高。然后用PP4基因的正义或反义序列转染SiHa细胞。过表达PP4的细胞中NF-κB p65的苏氨酸磷酸化降低至几乎检测不到的水平，基础和顺铂诱导的NF-κB活性均高于亲本细胞。相比之下，顺铂单独或与MEK/ERK抑制剂联合使用时，在反义PP4转染的细胞中几乎不诱导NF-κB活化。共沉淀复合激酶分析显示，NF-κB p65的一个片段（氨基酸279 - 444）含有与PP4直接相互作用的潜在磷酸化位点。定点诱变的进一步研究表明，Thr(435)是主要的磷酸化位点。
