A Novel NF- B Pathway Involving IKK and p65/RelA Ser-536 Phosphorylation Results in p53 Inhibition in the Absence of NF- B Transcriptional Activity*

Nuclear factor B (NFB) plays an important role in regulating cellular transformation and apoptosis. The human T-cell lymphotropic virus type I protein, Tax, which is critical for viral transformation, modulates the transcription of several cellular genes through activation of NFB. We have demonstrated previously that Tax inhibits p53 activity through the p65/RelA subunit of NFB. We now present evidence that suggests that the upstream kinase IKK plays an important role in Tax-induced p53 inhibition through phosphorylation of p65/RelA at Ser-536. First, mouse embryo fibroblast (MEF) IKK / cells did not support Tax-mediated p53 inhibition, whereas MEFs lacking IKK allowed Tax inhibition of p53. Second, transfection of IKK wild type (WT), but not a kinase-dead mutant, into IKK / cells rescued p53 inhibition by Tax. Third, the IKK -specific inhibitor SC-514 decreased the ability of Tax to inhibit p53. Fourth, we show that phosphorylation of p65/RelA at Ser-536 is important for Tax inhibition of p53 using MEF p65/RelA / cells transfected with p65/RelA WT or mutant plasmids. Moreover, Tax induced p65/RelA Ser536 phosphorylation in WT or IKK / cells but failed to induce the phosphorylation of p65/RelA Ser-536 in IKK / cells, suggesting a link between IKK and p65/ RelA phosphorylation. Consistent with this observation, blocking IKK kinase activity by SC-514 decreases the phosphorylation of p65/RelA at Ser-536 in the presence of Tax in human T-cell lymphotropic virus type I-transformed cells. Finally, the ability of Tax to inhibit p53 is distinguished from the NFB transcription activation pathway. Our work, therefore, describes a novel TaxNFB p65/RelA pathway that functions to inhibit p53 but does not require NFB transcription activity.