Here we identify IKK⑀ as a novel NF-B p65 kinase that mediates inducible phosphorylation of Ser 468 and Ser 536 in response to T cell costimulation. In addition, the kinase activity of IKK⑀ contributes to the control of p65 nuclear uptake. Serines 468 and 536 are evolutionarily conserved, and the surrounding amino acids display sequence homology. Down-regulation of IKK⑀ levels by small interfering RNA does not affect inducible phosphorylation of Ser 536 but largely prevents Ser 468 phosphorylation induced by T cell costimulation. Ser 536 -phosphorylated p65 is found predominantly in the cytosol. In contrast, the Ser 468 phosphorylated form of this transcription factor occurs mainly in the nucleus, suggesting a function for transactivation. Reconstitution of p65 ؊/؊ cells with either wild type p65 or point-mutated p65 variants showed that inducible phosphorylation of Ser 468 serves to enhance p65-dependent transactivation. These results also provide a mechanistic link that helps to explain the relevance of IKK⑀ for the expression of a subset of NF-B target genes without affecting cytosolic IB␣ degradation.The NF-B transcription factor system serves to control the expression of an extraordinarily wide array of genes in response to infections, inflammation, and other harmful situations (1, 2). NF-B target genes (such as immunoreceptors, cytokines, and chemokines) contribute to the innate immune response but also serve to control cell survival and proliferation (3). NF-B is a collective name for homo-or heterodimers composed of five different DNA-binding subunits, with the most frequently detected form being a heterodimer of p50 and p65 (RelA). The p65 subunit contains two strong, acidic transactivation domains called TAD1 3 and TAD2 in its C-terminal portion (4). A large variety of different inducers leads to NF-B activation by activation of numerous cellular and membrane receptors, including toll-like receptors and the T cell receptor. Thus far, three major pathways mediating NF-B activation have been identified, the so-called canonical and noncanonical pathways and the DNA damage-induced NF-B pathway. All NF-B activating events have in common that they lead to the proteasome-dependent generation of DNA-binding dimers (5). NF-B signals activating the canonical pathway funnel into the IKK complex, which is composed of the enzymatically active subunits IKK␣ and IKK and the regulatory subunits IKK␥/NEMO (6, 7) and ELKS (8). IKK-mediated I phosphorylation allows subsequent ubiquitination and proteolytic destruction of this inhibitory protein. This leads to an unmasking of the p65 nuclear localization sequence and results in NF-B nuclear immigration, DNA binding, and gene expression.Once activated, inducible post-translational modifications, including phosphorylation, acetylation, ubiquitination, or prolyl isomerization, allow the regulation of NF-B transcriptional activity (9, 10). Thus far, eight different phosphorylation sites have been mapped for the strongly activating NF-B p65 subunit. Three sites are contain...