Suppression of MEK/ERK signaling pathway enhances cisplatin-induced NF-kappaB activation by protein phosphatase 4-mediated NF-kappaB p65 Thr dephosphorylation.
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.
Yeh
Pei Yen
PY
Cancer Research Center, College of Medicine, National Taiwan University, Republic of China.
Yeh
Kun-Huei
KH
Chuang
Shuang-En
SE
Song
Ying Chyi
YC
Cheng
Ann-Lii
AL
D000970:Antineoplastic Agents / Q000494:pharmacology; D002460:Cell Line; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000378:metabolism; D002945:Cisplatin / Q000494:pharmacology*; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000378:metabolism; D005419:Flavonoids / Q000494:pharmacology; D006801:Humans; D015151:Immunoblotting; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D020929:Mitogen-Activated Protein Kinase Kinases / Q000378:metabolism*; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D016328:NF-kappa B / Q000378:metabolism*; D010749:Phosphoprotein Phosphatases / Q000378:metabolism*; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D011485:Protein Binding; D012694:Serine / Q000737:chemistry; D015398:Signal Transduction; D013912:Threonine / Q000737:chemistry*; D051996:Transcription Factor RelA; D014162:Transfection
D000970:Antineoplastic Agents; D004791:Enzyme Inhibitors; D005419:Flavonoids; D016328:NF-kappa B; D051996:Transcription Factor RelA; D013912:Threonine; D012694:Serine; D019950:Mitogen-Activated Protein Kinase 1; D020928:Mitogen-Activated Protein Kinases; D020929:Mitogen-Activated Protein Kinase Kinases; D010749:Phosphoprotein Phosphatases; C080406:protein phosphatase 4; D002945:Cisplatin; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one