Phosphorylation of Re1A/p65 on serine 536 defines an IκBα- independent NF-κB pathway

The association of the NF-κB p65/p50 dimer with IκBα plays a pivotal role in regulating its nuclear translocation and gene transcription. In addition, serine phosphorylation at various sites of the p65 subunit has been shown to be important in initiating transcription. Here we demonstrate that the regulation of nuclear translocation of p65 phosphorylated at serine 536 is not dependent on IκBα. Stimulation of either Jurkat or normal human T cells resulted in the nuclear translocation of phospho-p65 (Ser⁵³⁶). In addition, the phospho-p65 (Ser⁵³⁶) was not associated with either IκBα or p50, and the nuclear translocation of phospho-p65 (Ser⁵³⁶), but not total p65, was unaffected by the proteosome inhibitor MG-132, which blocks IκB protein degradation and prevents p65/p50 dimer nuclear translocation. Accordingly, the co-expression of a dominant negative mutant of IκBα blocked the transcriptional activity mediated by wild type but not the dominant positive p65 mutant (S536D). Furthermore, the transfection of the S536D form of p65 led to the induction of interleukin-8 transcription following stimulation, whereas the S536A form, which cannot be phosphorylated at this site, did not. Together, the findings suggest that p65 phosphorylated on serine 536 is not associated with or regulated by IκBα, that it has a distinct set of target genes, and that it may represent a noncanonical NF-κB pathway that is independent of IκBα regulation.