The role of phosphorylation of p65 subunit on transcriptional activity of NF-κB

p65亚基磷酸化对NF-κB转录活性的作用

• 批准号: 16390536
• 负责人: JIMI Eijiro
• 金额: $ 9.28万
• 依托单位: Kyushu Dental College (2005)Fukuoka Dental College (2004)
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2004
• 资助国家: 日本
• 起止时间: 2004 至 2005
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-16390536/
• 关键词: Transcription factor; NF-κB; Phosphorylation

The transcription factor NF-κB, which is critical for inducible expression of many genes involevdinimmunity and inflammation, exists in the cytosol of resting cells bound to inhibitory IκB proteins. Stimulation with specific inducers, such as TNFα or LPS activates an IκB kinase (IKK) complex that phosphorylates IκB, triggering its degradation by the proteasome and allowing free NF-κB to translocate to the nucleus and activate gene expression. Recent evidence supports the role for phosphorylation of NF-κB p65 subunit in determining the transcriptional capacity of NF-κB. Several studies provide the basis for systemic examination of the mechanisms through which distinct kinases influence the transcriptional activity of NF-κB. We have shown that the catalytic subunit of PKA phosphorylatesp65 subunit at Ser 276 positively regulates transcriptional activity of NF-κB in vitro. To address the physiological role of p65 phosphorylation by PKA, we generated knock-in mice, which replace at Ser 276 to Ala (S276A). S276A knock-in mice died around ED18.5 due to a failure to separate emerging lymphatic vessels from blood vessels. Furthermore, some S276A embryos showed eye defect.It has been accumulating that IKKβ directly phosphorylates p65 subunit at Ser 534 to induce transcriptional activity of NF-κB. To address the importance of Ser 534, we have also been generating new knock-in mice, which replace at Ser 534 to Ala (S534A). We have gotten 3 positive ES clones, which have homologous recombination in wild-type allele to knock-in allele.

转录因子NF-κB存在于静息细胞的胞浆中，与抑制性IκB蛋白结合，对免疫和炎症相关基因的诱导表达至关重要。用特异性诱导剂（如TNFα或LPS）刺激可激活IκB激酶（IKK）复合物，使IκB磷酸化，触发其被蛋白酶体降解，并使游离的NF-κB易位至细胞核并激活基因表达。最近的证据支持NF-κB p65亚基的磷酸化在决定NF-κB转录能力中的作用。几项研究为系统研究不同激酶影响NF-κB转录活性的机制提供了基础。我们已经证明PKA磷酸化sp 65亚基Ser 276的催化亚基在体外正调控NF-κB的转录活性。为了解决PKA磷酸化p65的生理作用，我们产生了敲入小鼠，其将Ser 276替换为Ala（S276 A）。S276 A基因敲入小鼠在ED 18.5左右死亡，原因是未能将新生淋巴管与血管分离。IKKβ直接磷酸化p65亚基Ser 534，诱导NF-κB B的转录活性，这是IKKβ在S276 A胚胎中的一个重要作用机制。为了解决Ser 534的重要性，我们还产生了新的敲入小鼠，将Ser 534替换为Ala（S534 A）。获得了3个ES阳性克隆，这些克隆在野生型等位基因到敲入型等位基因之间存在同源重组。