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

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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