Inducible Phosphorylation of NF-κB p65 at Serine 468 by T Cell Costimulation Is Mediated by IKKϵ

Abstract

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...

The protein kinase IKKε can inhibit HCV expression independently of IFN and its own expression is downregulated in HCV-infected livers

“…With regards to NF-B, IKK⑀ was recently shown to phosphorylate p65 at two of its serine residues (468 and 536) and to control its nuclear import. 28 In summary, the transcriptome profile of IKK⑀ confirms the induction of already described genes and allows depiction of new IKK⑀-induced genes. In particular, these data point out that IRF3-induced genes and genes related to the antiviral action of IFN represent an important fraction of all the genes strongly induced by IKK⑀ ( Table 2).…”

“…25 In addition to phosphorylating IRF3 and inducing IFN, IKK⑀ is also known to induce genes through the NF-B and c/EBP␦ transcription factors. [26][27][28] Therefore, to have exhaustive information on the involvement of IKK⑀ in an antiviral action against HCV, we have analyzed its transcriptome profile after provoking its overexpression by transfection in cells expressing an HCV replicon. This revealed several genes involved in translational control, genes involved in apoptosis, and genes of the ISGylation pathway as well as genes from the chemokine family.…”

Nuclear Accumulation of cRel following C-Terminal phosphorylation by TBK1/IKKε

“…During the course of this study, Mattioli et al (57), reported that IKK was involved in the phosphorylation of RelA/p65 at Ser 468 in response to T cell costimulation and demonstrated that this phosphorylation primarily affected RelA/p65-dependent transactivation. Thus, the function of IKK -mediated phosphorylation of RelA/p65 appears distinct from the function described here for cRel.…”

Zac1 is a histone acetylation-regulated NF-κB suppressor that mediates histone deacetylase inhibitor-induced apoptosis

“…The phosphorylation levels of these two sites are dramatically increased upon stimulation of NF-kB activity. 29,30 Moreover, studies show that tumor cell apoptosis induced by anticancer reagents is associated with decreased p65 phosphorylation at Ser536 residue, [31][32][33] suggesting that p65 Ser536 phosphorylation is important for maintaining cancer cell survival and can be inhibited by anticancer reagents. In this study we showed that not only phosphorylation of Ser536 but also phosphorylation of Ser468 is downregulated during HDAC inhibitor-induced apoptosis of EC cells.…”