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1.
Neurochem Int ; 120: 213-223, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30196145

RESUMO

Brain specific kinases (BRSKs) are serine/threonine kinases, preferentially expressed in the brain after Embryonic Day 12. Although BRSKs are crucial neuronal development factors and regulation of their enzymatic activity has been widely explored, little is known of their transcriptional regulation. In this work, we show that Neuronal Growth Factor (NGF) increased the expression of Brsk1 in PC12 cells. Furthermore, during neuronal differentiation, Brsk1 mRNA increased through a MAPK-dependent Sp1 activation. To gain further insight into this regulation, we analyzed the transcriptional activity of the Brsk1 promoter in PC12 cells treated with NGF. Initially, we defined the minimal promoter region (-342 to +125 bp) responsive to NGF treatment. This region had multiple Sp1 binding sites, one of which was within a CpG island. In vitro binding assays showed that NGF-induced differentiation increased Sp1 binding to this site and that DNA methylation inhibited Sp1 binding. In vitro methylation of the Brsk1 promoter reduced its transcriptional activity and impaired the NGF effect. To evaluate the participation of DNA methyltransferases in Brsk1 gene regulation, the 5'Aza-dC inhibitor was used. 5'Aza-dC acted synergistically with NGF to promote Brsk1 promoter activity. Accordingly, DNMT3B overexpression abolished the response of the Brsk1 promoter to NGF. Surprisingly, we found Dnmt3b to be a direct target of NGF regulation, via the MAPK pathway. In conclusion, our results provide evidence of a novel mechanism of Brsk1 transcriptional regulation changing the promoter's methylation status, which was incited by the NGF-induced neuronal differentiation process.


Assuntos
Encéfalo/efeitos dos fármacos , Fator de Crescimento Neural/farmacologia , Proteínas Quinases/metabolismo , Fator de Transcrição Sp1/efeitos dos fármacos , Animais , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Regulação da Expressão Gênica/efeitos dos fármacos , Metilação/efeitos dos fármacos , Neurogênese/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Células PC12 , Regiões Promotoras Genéticas/genética , Proteínas Quinases/genética , Ratos , Fator de Transcrição Sp1/fisiologia
2.
Mol Cell Biol ; 33(8): 1671-86, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23428871

RESUMO

The mitogen-activated protein (MAP) kinase extracellular signal-regulated kinase 5 (ERK5) plays a crucial role in cell proliferation, regulating gene transcription. ERK5 has a unique C-terminal tail which contains a transcriptional activation domain, and activates transcription by phosphorylating transcription factors and acting itself as a transcriptional coactivator. However, the molecular mechanisms that regulate its nucleocytoplasmatic traffic are unknown. We have used tandem affinity purification to identify proteins that interact with ERK5. We show that ERK5 interacts with the Hsp90-Cdc37 chaperone in resting cells, and that inhibition of Hsp90 or Cdc37 results in ERK5 ubiquitylation and proteasomal degradation. Interestingly, activation of cellular ERK5 induces Hsp90 dissociation from the ERK5-Cdc37 complex, leading to ERK5 nuclear translocation and activation of transcription, by a mechanism which requires the autophosphorylation at its C-terminal tail. Consequently, active ERK5 is no longer sensitive to Hsp90 or Cdc37 inhibitors. Cdc37 overexpression also induces Hsp90 dissociation and the nuclear translocation of a kinase-inactive form of ERK5 which retains transcriptional activity. This is the first example showing that ERK5 transcriptional activity does not require kinase activity. Since Cdc37 cooperates with ERK5 to promote cell proliferation, Cdc37 overexpression (as happens in some cancers) might represent a new, noncanonical mechanism by which ERK5 regulates tumor proliferation.


Assuntos
Transporte Ativo do Núcleo Celular , Proteínas de Ciclo Celular/metabolismo , Núcleo Celular/metabolismo , Chaperoninas/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Proteína Quinase 7 Ativada por Mitógeno/metabolismo , Animais , Proteínas de Ciclo Celular/biossíntese , Proteínas de Ciclo Celular/genética , Linhagem Celular , Proliferação de Células , Chaperoninas/biossíntese , Chaperoninas/genética , Células HEK293 , Células HeLa , Humanos , Camundongos , Proteína Quinase 7 Ativada por Mitógeno/genética , Fosforilação , Interferência de RNA , RNA Interferente Pequeno , Transdução de Sinais , Transcrição Gênica , Ativação Transcricional , Ubiquitinação
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