RESUMO
MEKK2, MEK5, and extracellular signal-regulated kinase 5 (ERK5) are members of a three-kinase cascade for the activation of ERK5. MEK5 is the only MAP2K to express a PB1 domain, and we have shown that it heterodimerizes with the PB1 domain of MEKK2. Here we demonstrate the MEK5 PB1 domain is a scaffold that also binds ERK5, functionally forming a MEKK2-MEK5-ERK5 complex. Reconstitution assays and CFP/YFP imaging (fluorescence resonance energy transfer [FRET]) measuring YFP-MEKK2/CFP-MEK5 and CFP-MEK5/YFP-ERK5 interactions define distinct MEK5 PB1 domain binding sites for MEKK2 and ERK5, with a C-terminal extension of the PB1 domain contributing to ERK5 binding. Stimulus-dependent CFP/YFP FRET in combination with mutational analysis was used to define MEK5 PB1 domain residues critical for the interaction of MEKK2/MEK5 and MEK5/ERK5 required for activation of the ERK5 pathway in living cells. Fusion of the MEK5 PB1 domain to the N terminus of MEK1 confers ERK5 regulation by a MAP2K normally regulating only ERK1/2. The MEK5 PB1 domain confers stringent MAP3K regulation of ERK5 relative to more promiscuous MAP3K control of ERK1/2, JNK, and p38.
Assuntos
MAP Quinase Quinase 5/metabolismo , Proteína Quinase 7 Ativada por Mitógeno/metabolismo , Transdução de Sinais , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Células Cultivadas , Dimerização , Ativação Enzimática , Transferência Ressonante de Energia de Fluorescência , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , MAP Quinase Quinase 5/efeitos dos fármacos , MAP Quinase Quinase 5/genética , MAP Quinase Quinase Quinase 2/genética , MAP Quinase Quinase Quinase 2/metabolismo , Proteína Quinase 7 Ativada por Mitógeno/efeitos dos fármacos , Proteína Quinase 7 Ativada por Mitógeno/genética , Dados de Sequência Molecular , Complexos Multiproteicos , Mutação , Mapeamento de Interação de Proteínas , Estrutura Terciária de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sorbitol/farmacologiaRESUMO
Genistein, an isoflavonoid present in soybeans, exhibits anti-carcinogenic effects. Several studies have shown that genistein inhibits cell proliferation and triggers apoptosis in human breast cancer cells. In this study, we assessed the role of the MEK-ERK cascade in the regulation of genistein-mediated cell apoptosis in MDA-MB-231 cells. The results indicate that genistein, in a concentration-dependent manner, suppresses the protein levels of MEK5, total ERK5, and phospho-ERK5, effects that are consistent with inhibition of cell growth and induction of apoptosis. Exposure of these cells to genistein results in a concentration-dependent decrease in NF-kappaB/p65 protein levels and DNA-binding activity of NF-kappaB. Genistein down-regulates Bcl-2 and up-regulates Bax. NF-kappaB binding sites are present in the promoter of Bcl-2, suggesting that genistein might inhibit the expression of Bcl-2 through down-regulation of NF-kappaB. Exposure of MDA-MB-231 cells to genistein results in cleavage of caspase-3 and induction of caspase-3 activity in a concentration-dependent manner. Genistein inhibits NF-kappaB activity via the MEK5/ERK5 pathway; it also inhibits cell growth and induces apoptosis. In conclusion, inhibition of the MEK5/ERK5/NF-kappaB pathway may be an important mechanism by which genistein suppresses cell growth and induces apoptosis.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Genisteína/farmacologia , Proteínas Quinases Ativadas por Mitógeno/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Genisteína/administração & dosagem , Genisteína/isolamento & purificação , Humanos , MAP Quinase Quinase 5/efeitos dos fármacos , MAP Quinase Quinase 5/metabolismo , Proteína Quinase 7 Ativada por Mitógeno/efeitos dos fármacos , Proteína Quinase 7 Ativada por Mitógeno/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , NF-kappa B/efeitos dos fármacos , NF-kappa B/metabolismo , Glycine max/químicaRESUMO
OBJECTIVE: To study the role of ERK5 MAPK signaling transduction pathway in the inhibition of genistein to MDA-MB-231 breast cancer cells. METHODS: The inhibitive effects of genistein on MDA-MB-231 cells were observed with the MTT assay. The cell apoptosis was detected by flow cytometry. The ERK5 MAPK, Bax and Caspase3 protein expressions were detected by using western blot method. RESULTS: genistein inhibited the cell proliferation of MDA-MB-231 cells and induced apoptosis. The protein expression of ERK5 decreased and those of Bax and Caspase3 increased with the dose of genistein. CONCLUSION: Genistein can affect the ERK5 MAPK signaling transduction pathway and induce the expressions of apoptosis related proteins to inhibit the proliferation of MDA-MB-231 cells.