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

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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.