Suppression of MEK/ERK Signaling Pathway Enhances Cisplatin-induced NF-κB Activation by Protein Phosphatase 4-mediated NF-κB p65 Thr Dephosphorylation

Pei Yen Yeh, Kun-Huei Yeh, Shuang-En Chuang, Ying Chyi Song, Ann-Lii Cheng
2004 Journal of Biological Chemistry  
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-B activation. However, results of immunoblotting analysis showed that neither cisplatin nor MEK/ERK inhibitors induced marked IB␣ degradation, suggesting that suppression of the MEK/ERK signaling pathway may enhance
more » ... ced NF-B 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-B led us to examine the phosphorylation status of NF-B p65. Coincident with activation of NF-B, cisplatin induced Ser phosphorylation but decreased Thr phosphorylation of NF-B p65. Suppression of the MEK/ ERK pathway further enhanced cisplatin-induced Thr dephosphorylation but did not affect cisplatin-induced Ser phosphorylation of NF-B 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-B p65 to nearly undetectable levels, and both basal and cisplatin-induced NF-B activities were higher than those in parental cells. By contrast, cisplatin, either alone or with MEK/ERK inhibitors, induced little NF-B activation in antisense PP4-transfected cells. Coprecipitated complex kinase assay revealed a fragment of NF-B 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.
doi:10.1074/jbc.m402362200 pmid:15073167 fatcat:bg5pre4xtbdqvil4lxbs54vbpe