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

转录因子NF-κB存在于与抑制性I-κB蛋白结合的静息细胞的胞浆中，在许多免疫和炎症相关基因的诱导表达中起关键作用。用特定的诱导剂，如肿瘤坏死因子α或脂多糖刺激，激活IκB激酶(IKK)复合体，使IκB磷酸化，触发其被蛋白酶体降解，并允许游离的NF-κB移位到细胞核并激活基因表达。最近的证据支持核因子-κB p65亚基的磷酸化在决定核因子-κB转录能力中的作用。一些研究为系统研究不同的激酶影响核因子-κB转录活性的机制提供了基础。我们已经证明，位于Ser276的pkA磷酸化p65亚基的催化亚基在体外正向调节核因子-κB的转录活性。为了解决p65被PKA磷酸化的生理作用，我们产生了敲入小鼠，它们在Ser 276替换为Ala(S276A)。S276A转基因小鼠由于未能将新生淋巴管从血管中分离出来，在ED18.5左右死亡。此外，一些S276A胚胎表现出眼睛缺陷。它已经积累了IKKβ直接磷酸化P65亚基在Ser534处诱导NF-κB的转录活性。为了解决Ser534的重要性，我们还一直在创造新的敲入小鼠，它们在Ser534替换为Ala(S534A)。获得3个阳性ES克隆，野生型等位基因与敲入等位基因有同源重组。

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.