Suppression of MEK/ERK signaling pathway enhances cisplatin-induced NF-κB activation by protein phosphatase 4-mediated NF-κB p 65 Thr dephosphorylation

Abstract

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-κB activation. However, results of immunoblotting analysis showed that neither cisplatin nor MEK/ERK inhibitors induced marked IκBα degradation, suggesting that suppression of the MEK/ERK: signaling pathway may enhance cisplatin-induced NF-κB 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-κB led us to examine the phosphorylation status of NF-κB p65. Coincident with activation of NF-κB, cisplatin induced Ser phosphorylation but decreased Thr phosphorylation of NF-κB p65. Suppression of the MEK/ERK pathway further enhanced cisplatin-induced Thr dephosphorylation but did not affect cisplatin-induced Ser phosphorylation of NF-κB 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. Silla cells were then transfected by a sense or an antisense PP4 gene. PP4-overexpressing cells showed a decrease in Thr phosphorylation of NF-κB p65 to nearly undetectable levels, and both basal and cisplatin-induced NF-κB activities were higher than those in parental cells. By contrast, cisplatin, either alone or with MEK/ERK inhibitors, induced little NF-κB activation in antisense PP4-transfected cells. Coprecipitated complex kinase assay revealed a fragment of NF-κB p65 (amino acids 279-444) to contain potential phosphorylation sites that directly interact with PP4. Further studies by site-directed mutagenesis suggested that Thr485 was the major phosphorylation site.