RESUMO
The signal pathway by which 14-3-3epsilon inhibits cell migration induced by MAPK-activated protein kinase 5 (MK5) was investigated in cultured HeLa cells. Both in vivo and in vitro analyses have revealed that 14-3-3epsilon interacts with MK5. 14-3-3epsilon bound to MK5 inhibits the phosphorylation of HSP27, a known substrate of MK5. Disturbance of actin cytoskeleton organization by 14-3-3epsilon was shown in transfected cells transiently expressing 14-3-3epsilon as well as established cells stably expressing 14-3-3epsilon. Moreover, overexpression of 14-3-3epsilon resulted in the inhibition of cell migration induced by MK5 overexpression or TNFalpha treatment. Our results suggest that 14-3-3epsilon bound to MK5 inhibits cell migration by inhibiting the phosphorylation of HSP27 whose phosphorylation regulates F-actin polymerization, actin cytoskeleton organization and subsequent actinfilament dynamics.
Assuntos
Proteínas 14-3-3/metabolismo , Actinas/química , Actinas/metabolismo , Movimento Celular , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Movimento Celular/efeitos dos fármacos , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/metabolismo , Proteínas de Choque Térmico HSP27 , Células HeLa , Proteínas de Choque Térmico/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Modelos Biológicos , Chaperonas Moleculares , Proteínas de Neoplasias/metabolismo , Fosforilação/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
We propose a biochemical mechanism by which Daxx modulates NF-kappaB transcriptional activity. Both chromatin immunoprecipitation (ChIP) assay and electrophoretic mobility shift assay (EMSA) have confirmed Daxx-mediated repression of transcriptional competence of NF-kappaB in HeLa cells. Overexpression of Daxx repressed the expression of NF-kappaB-regulated genes such as I kappa B alpha and IL8. Co-immunoprecipitation assay revealed the existence of intermolecular association between endogenous Daxx and p65 subunit of NF-kappaB stimulated by TNFalpha. Here, we suggest that Daxx-mediated repression of NF-kappaB transactivation correlates with the inhibition of p65 acetylation by Daxx. Based on the finding that the Daxx binding N-terminal side of p65 includes the major sites of acetylation mediated by p300/CBP, we further propose that the physical interaction between Daxx and p65 provides a functional framework for the inhibition of p65 acetylation by p300/CBP and subsequent repression of NF-kappaB transcriptional activity.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Nucleares/metabolismo , Fator de Transcrição RelA/antagonistas & inibidores , Fator de Transcrição RelA/genética , Ativação Transcricional/genética , Acetilação , Núcleo Celular/metabolismo , Proteínas Correpressoras , Proteína p300 Associada a E1A/metabolismo , Células HeLa , Humanos , Chaperonas Moleculares , Fosforilação , Ligação Proteica , Transporte Proteico , Frações Subcelulares/metabolismo , Fator de Transcrição RelA/metabolismoRESUMO
The proapoptotic function of SPOP protein was investigated in HeLa cells. HeLa cells underwent apoptosis by the overexpression of SPOP. Studies using SPOP deletion mutants suggest that BTB/POZ domain of SPOP protein is important for the induction of apoptosis in transfected cells. This study first proposes the proapoptotic aspect of the BTB/POZ domain of SPOP protein based on the finding that cells expressing the C-terminal fragment of SPOP containing the BTB/POZ domain underwent apoptosis.