Inducible Phosphorylation of NF- B p65 at Serine 468 by T Cell Costimulation Is Mediated by IKK

Hannover Medical School, Hanover, Lower Saxony, Germany
Journal of Biological Chemistry (Impact Factor: 4.57). 04/2006; 281(10):6175-83. DOI: 10.1074/jbc.M508045200
Source: PubMed


Here we identify IKKepsilon as a novel NF-kappaB p65 kinase that mediates inducible phosphorylation of Ser468 and Ser536 in response to T cell costimulation. In addition, the kinase activity of IKKepsilon 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 IKKepsilon levels by small interfering RNA does not affect inducible phosphorylation of Ser536 but largely prevents Ser468 phosphorylation induced by T cell costimulation. Ser536-phosphorylated p65 is found predominantly in the cytosol. In contrast, the Ser468 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 Ser468 serves to enhance p65-dependent transactivation. These results also provide a mechanistic link that helps to explain the relevance of IKKepsilon for the expression of a subset of NF-kappaB target genes without affecting cytosolic IkappaBalpha degradation.

12 Reads
  • Source
    • "Substitution of Ser468 with an alanine impedes this interaction and almost completely prevents COMMD1-mediated RELA ubiquitination and proteasomal degradation. Upon NF-κB activation , RELA S468A substitution results in prolonged binding of RELA to a selective set of NF-κB target genes [49] [50] [51]. Interestingly, after removal of RELA from the promoter site, COMMD1 was detected at the same "
    [Show abstract] [Hide abstract]
    ABSTRACT: NF-κB is an important regulator of immunity and inflammation, and its activation pathway has been studied extensively. The mechanisms that downregulate the activity of NF-κB have also received a lot of attention, particularly since its activity needs to be terminated to prevent chronic inflammation and subsequent tissue damage. The COMMD family has been identified as a new group of proteins involved in NF-κB termination. All ten COMMD members share the structurally conserved carboxy-terminal motif, the COMM domain, and are ubiquitously expressed. They seem to play distinct and non-redundant roles in various physiological processes, including NF-κB signaling. In this review, we describe the mechanisms and proteins involved in the termination of canonical NF-κB signaling, with a specific focus on the role of the COMMD family in the down-modulation of NF-κB.
    Biochimica et Biophysica Acta 09/2013; 1832(12). DOI:10.1016/j.bbadis.2013.09.014 · 4.66 Impact Factor
  • Source
    • "Previous studies have demonstrated that serine phosphorylation of STAT1 is required for its maximum transactivation activity [34]. Also, several serine phosphorylation sites have been identified in NF-κB subunit p65 [35] [36] [37] [38] [39]. Although our preliminary studies suggest these transcription factors are serine phosphorylated by ERKs, we have not examined whether this phosphorylation mediates the ERKs dependent effects on the LCN2 expression. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Lipocalin-2 (LCN2) is induced in conditions of obesity and Type 2 diabetes (T2DM). IFNγ and TNFα induce LCN2 expression in adipocytes in a manner that is dependent on transcription. The effects of these cytokines are additive. IFNγ induced STAT1 and TNFα induced NF-κB play a role in the induction of LCN2. In the LCN2 promoter, one NF-κB binding site and four STAT1 binding sites were identified by in silico and in vitro approaches. MAPK (ERKs 1 and 2) activation was required for the IFNγ and TNFα induction of LCN2 expression, but did not affect the nuclear translocation or DNA binding activity of STAT1 or NF-κB. The NF-κB binding site and the STAT1 binding sites we identified in vitro were confirmed by in vivo studies. Transfection of a LCN2 promoter/luciferase reporter construct confirmed acute activation by IFNγ and TNFα. Our studies identify mechanisms involved in the actions of cytokines secreted from immune cells in adipose tissue that induce LCN2 expression in conditions of obesity and T2DM.
    Molecular Metabolism 08/2013; 2(3):161-70. DOI:10.1016/j.molmet.2013.04.003
  • Source
    • "The plasmids pSG5-SUMO1, pSG5-His-SUMO1 [25] and pSG5-His-SUMO2 were a gift from Stefan Müller. The p(NF-κB)3-luc reporter plasmid [26] was obtained from Lienhard Schmitz. The HA-PPARγ1 K77/365R double mutant was generated by replacing an EcoRV-XbaI fragment of pcDNA3-HA-PPARγ1 K77R with the corresponding fragment from the pcDNA3-HA-PPARγ K365R plasmid. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Peroxisome proliferator-activated receptor gamma (PPARγ) is a ligand-activated nuclear receptor regulating adipogenesis, glucose homeostasis and inflammatory responses. The activity of PPARγ is controlled by post-translational modifications including SUMOylation and phosphorylation that affects its biological and molecular functions. Several important aspects of PPARγ SUMOylation including SUMO isoform-specificity and the impact of ligand binding on SUMOylation remain unresolved or contradictory. Here, we present a comprehensive study of PPARγ1 SUMOylation. We show that PPARγ1 can be modified by SUMO1 and SUMO2. Mutational analyses revealed that SUMOylation occurs exclusively within the N-terminal activation function 1 (AF1) domain predominantly at lysines 33 and 77. Ligand binding to the C-terminal ligand-binding domain (LBD) of PPARγ1 reduces SUMOylation of lysine 33 but not of lysine 77. SUMOylation of lysine 33 and lysine 77 represses basal and ligand-induced activation by PPARγ1. We further show that lysine 365 within the LBD is not a target for SUMOylation as suggested in a previous report, but it is essential for full LBD activity. Our results suggest that PPARγ ligands negatively affect SUMOylation by interdomain communication between the C-terminal LBD and the N-terminal AF1 domain. The ability of the LBD to regulate the AF1 domain may have important implications for the evaluation and mechanism of action of therapeutic ligands that bind PPARγ.
    PLoS ONE 06/2013; 8(6):e66947. DOI:10.1371/journal.pone.0066947 · 3.23 Impact Factor
Show more