Molecular mechanism of TNF receptor superfamily-mediated NF-κB activation

TNF受体超家族介导NF-κB激活的分子机制

• 批准号: 13670323
• 负责人: NAKANO Hiroyasu
• 金额: $ 2.18万
• 依托单位: Juntendo University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13670323/
• 关键词: NF-κB; P65; Knockout mice; TNF; apoptosis; IL-6; phosphorylation; IκB; NF-KB; IKB

NF-κB-dependent gene expression is controlled at several levels, including regulation of nuclear translocation and its transcriptional activity. Phosphorylation of several serine residues especially in the transactivation (TA) domain of p65 NF-κB subunit has been suggested to be important for its transcriptional activity. However, the responsible phosphorylation site of p65 remains controversial. To investigate the biological significance of phosphorylation and to determine the critical phosphorylation sites of p65, we reconstituted murine embryonic fibroblasts (MEFs) from p65^<-/-> mice with various serine to alanine (SA) substituted mutants of p65. Unexpectedly, mutants in the TA domain, including S529A, S536A, and S529A/S536A, completely rescued the defect of p65^<-/-> MEFs as assessed by tumor necrosis factor (TNF)- or interleukin-1(IL-1)-induced IL-6 production and protection from TNF-induced cell death. On the other hand, S276A mutant had an impaired ability to rescue these responses. Moreover, TNF-induced phosphorylation of p65 was severely impaired in S276A mutant. However, nuclear translocation or DNA binding of NF-κB in response to TNF and IL-1 was not impaired in S276A mutant. These results indicated that S276 is the major phosphorylation site of p65 and its phosphorylation is essential for p65-dependent cellular responses

NF-κ B依赖的基因表达在多个水平上受到控制，包括核转位及其转录活性的调节。p65 NF-κ B亚基转录激活区（TA）的丝氨酸残基磷酸化是其转录活性的重要因素。然而，p65的磷酸化位点仍然存在争议。为了研究磷酸化的生物学意义并确定p65的关键磷酸化位点，我们用p65的各种丝氨酸到丙氨酸（SA）取代的突变体重建来自p65 ^<-/->小鼠的鼠胚胎成纤维细胞（MEF）。出乎意料的是，TA结构域中的突变体，包括S529 A、S536 A和S529 A/S536 A，完全挽救了p65 ^<-/-> MEFs的缺陷，如通过肿瘤坏死因子（TNF）或白细胞介素-1（IL-1）诱导的IL-6产生和对TNF诱导的细胞死亡的保护所评估的。另一方面，S276A突变体具有受损的拯救这些反应的能力。此外，TNF诱导的p65磷酸化在S276A突变体中严重受损。然而，在S276 A突变体中，NF-κ B响应TNF和IL-1的核转位或DNA结合未受到损害。这些结果表明S276是p65的主要磷酸化位点，其磷酸化对于p65依赖性细胞反应至关重要

项目成果

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Okazaki, T.: "Phosphorylation of serine 276 is essential for p65 NF-κB subunit-dependent cellular responses"Biochem Biophys Res Commun. 300. 807-812 (2003)

Okazaki, T.：“丝氨酸 276 的磷酸化对于 p65 NF-κB 亚基依赖性细胞反应至关重要”Biochem Biophys Res Commun. 300. 807-812 (2003)

Okazaki, T., S. Sakon, T. Sasazuki, H. Sakurai, T. Doi, H. Yagita, K. Okumura, and H. Nakano: "Phosphorylation ofserine 276 is essential for p65 NF-κB subunit-dependent cellular responses"Biochem Biophys Res Commun. 300. 807-812 (2003)

Okazaki, T.、S. Sakon、T. Sasazuki、H. Sakurai、T. Doi、H. Yagita、K. Okumura 和 H. Nakano：“丝氨酸 276 的磷酸化对于 p65 NF-κB 亚基依赖性细胞至关重要回应“Biochem Biophys Res Commun. 300. 807-812 (2003)

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