Background Human antigen R (HuR) is a ubiquitously expressed member of the ELAV family, and has relatively high cytoplasmic abundance in lung tissue regenerating after injury. In this study, we investigated whether ...Background Human antigen R (HuR) is a ubiquitously expressed member of the ELAV family, and has relatively high cytoplasmic abundance in lung tissue regenerating after injury. In this study, we investigated whether mitogen-activated protein kinase (MAPK)-activated protein kinase 2 (MK2) and HuR participate in the tumor necrosis factor (TNF)-induced expression of interleukin-6 (IL-6). Methods Human pulmonary microvascular endothelial cells were treated with TNF following short interfering RNAmediated knockdown of MK2 or HuR. Cell supernatants were collected to detect the mRNA and protein expression of IL-6 at different time points, The expression and half-life of IL-6 mRNA were then determined in cells that had been treated with actinomycin D. Finally, after knockdown of MK2, the cytoplasmic expression of HuR protein was analyzed using Western blotting. Results MK2 or HuR knockdown decreased both the mRNA and protein expression of IL-6 in TNF-stimulated cells. In MK2 knockdown cells, the half-life of IL-6 mRNA was reduced to 36 minutes, compared with 67 minutes in the control group. In HuR knockdown cells, the half-life of IL-6 mRNA decreased from 62 minutes to 24 minutes. Further analysis revealed that knockdown of MK2 resulted in reduced HuR protein expression in the cytoplasm. Conclusions MK2 regulates the TNF-induced expression of IL-6 by influencing the cytoplasmic levels of HuR.展开更多
AIM:To identify novel substrates for the mitogen-activated protein kinase-activated protein kinase 5(MK5).METHODS:Yeast two-hybrid screening with MK5 as bait was used to identify novel possible interaction partners.Th...AIM:To identify novel substrates for the mitogen-activated protein kinase-activated protein kinase 5(MK5).METHODS:Yeast two-hybrid screening with MK5 as bait was used to identify novel possible interaction partners.The binding of putative partner was further examined by glutathione S-transferase(GST) pull-down,co-immunoprecipitation and fluorescence resonance energy transfer(FRET) analysis.In vitro kinase and peptide array assays were used to map MK5 phosphoacceptor sites on the new partner.Confocal microscopy was performed to study the subcellular localization of MK5 and its partners.RESULTS:Septin 8 was identified as a novel interaction partner for MK5 by yeast two-hybrid screening.This interaction was confirmed by GST pull-down,coimmunoprecipitation and FRET analysis.Septin 5,which can form a complex with septin 8,did not interact with MK5.Serine residues 242 and 271 on septin 8 were identified as in vitro MK5 phosphorylation sites.MK5 and septin 8 co-localized in the perinuclear area and in cell protrusions.Moreover,both proteins co-localized with vesicle marker synaptophysin.展开更多
基金This study was supported by the National Natural Science Foundation of China (No. 81270138), the Natural Science Foundation of Jiangsu Province (No. BK2011657 and No. BK20130402), and the Medical Technology Innovation Foundation of Nanjing Military Command (No. CWS 12J008).
文摘Background Human antigen R (HuR) is a ubiquitously expressed member of the ELAV family, and has relatively high cytoplasmic abundance in lung tissue regenerating after injury. In this study, we investigated whether mitogen-activated protein kinase (MAPK)-activated protein kinase 2 (MK2) and HuR participate in the tumor necrosis factor (TNF)-induced expression of interleukin-6 (IL-6). Methods Human pulmonary microvascular endothelial cells were treated with TNF following short interfering RNAmediated knockdown of MK2 or HuR. Cell supernatants were collected to detect the mRNA and protein expression of IL-6 at different time points, The expression and half-life of IL-6 mRNA were then determined in cells that had been treated with actinomycin D. Finally, after knockdown of MK2, the cytoplasmic expression of HuR protein was analyzed using Western blotting. Results MK2 or HuR knockdown decreased both the mRNA and protein expression of IL-6 in TNF-stimulated cells. In MK2 knockdown cells, the half-life of IL-6 mRNA was reduced to 36 minutes, compared with 67 minutes in the control group. In HuR knockdown cells, the half-life of IL-6 mRNA decreased from 62 minutes to 24 minutes. Further analysis revealed that knockdown of MK2 resulted in reduced HuR protein expression in the cytoplasm. Conclusions MK2 regulates the TNF-induced expression of IL-6 by influencing the cytoplasmic levels of HuR.
基金Supported by Grants from the Norwegian Cancer Society(A5313) to Shiryaev A
文摘AIM:To identify novel substrates for the mitogen-activated protein kinase-activated protein kinase 5(MK5).METHODS:Yeast two-hybrid screening with MK5 as bait was used to identify novel possible interaction partners.The binding of putative partner was further examined by glutathione S-transferase(GST) pull-down,co-immunoprecipitation and fluorescence resonance energy transfer(FRET) analysis.In vitro kinase and peptide array assays were used to map MK5 phosphoacceptor sites on the new partner.Confocal microscopy was performed to study the subcellular localization of MK5 and its partners.RESULTS:Septin 8 was identified as a novel interaction partner for MK5 by yeast two-hybrid screening.This interaction was confirmed by GST pull-down,coimmunoprecipitation and FRET analysis.Septin 5,which can form a complex with septin 8,did not interact with MK5.Serine residues 242 and 271 on septin 8 were identified as in vitro MK5 phosphorylation sites.MK5 and septin 8 co-localized in the perinuclear area and in cell protrusions.Moreover,both proteins co-localized with vesicle marker synaptophysin.