IKKβ phosphorylates p65 at S468 in transactivation domain 2

Department of Medicine, Columbia University, College of Physicians and Surgeons, New York, New York, USA.
The FASEB Journal (Impact Factor: 5.48). 11/2005; 19(12):1758-60. DOI: 10.1096/fj.05-3736fje
Source: PubMed

ABSTRACT The transcription factor nuclear factor-kappa B (NF-kappaB) subunit p65 is phosphorylated by IkappaB kinase (IKK) at S536 in transactivation domain (TAD) 1. In this study, we investigate the presence of IKK sites in TAD2 of p65. Recombinant IKKbeta, but not IKKalpha, phosphorylated a GST-p65 substrate in which TAD1 was deleted. Mutational analysis revealed S468 as the only IKK site in TAD2. S468 phosphorylation occurred rapidly after TNF-alpha and IL-1beta in T cell, B cell, cervix carcinoma, hepatoma, breast cancer, and astrocytoma lines and in primary hepatic stellate cells as well as peripheral blood mononuclear cells. S468-phosphorylated p65 coimmunoprecipitated with IkappaBalpha, indicating that p65 is phosphorylated while bound to IkappaBalpha. Dominant negative IKKbeta or pharmacological IKK inhibition blocked S468 phosphorylation after TNF-alpha or IL-1beta, whereas dominant negative IKKalpha or inhibitors of MEK, p38, JNK, PI-3 kinase, or GSK-3 had no effect. p65S468A-reconstituted p65-/- mouse embryonic fibroblasts (MEFs) showed a small, but significant, elevation of NF-kappaB-driven luciferase activity and RANTES mRNA levels after TNF-alpha and IL-1beta in comparison to wtp65-reconstituted MEFs. p65 nuclear translocation was not altered in p65S468A-expressing MEFs. In conclusion, our results indicate that 1) IKKbeta phosphorylates multiple p65 sites, 2) IKKbeta phosphorylates p65 in an IkappaB-p65 complex, and 3) S468 phosphorylation slightly reduces TNF-alpha- and IL-1beta-induced NF-kappaB activation.

Download full-text


Available from: Hiroaki Sakurai, Aug 29, 2015
  • Source
    • "Phosphorylation of p65 by IKKβ at Ser468 [36] promotes its ubiquitination and degradation when bound to specific gene targets [37], [38], thus playing an important role in the control of NF-κB responses [51]. It was considered that PB1-F2 might influence p65 ubiquitination by favoring the IKKβ-mediated phosphorylation at Ser468 and induce p65 degradation. "
    [Show abstract] [Hide abstract]
    ABSTRACT: PB1-F2, a protein encoded by a second open reading frame of the influenza virus RNA segment 2, has emerged as a modulator of lung inflammatory responses but the molecular mechanisms underlying this are only poorly understood. Here we show that PB1-F2 inhibits the activation of NF-κB dependent signalling pathways in luciferase reporter assays. PB1-F2 proteins from four different viruses interact with IKKβ in yeast two-hybrid assays and by co-immunoprecipitation. PB1-F2 expression did not inhibit IKKβ kinase activity or NF-κB translocation into the nucleus, but NF-κB binding to DNA was severely impaired in PB1-F2 transfected cells as assessed by Electrophoretic Mobility Shift Assay. Neither the N-terminal 57 amino acid truncated forms nor the C-terminus of PB1-F2 were able to inhibit NF-κB dependent signalling, indicating that the full length protein is necessary for the inhibition.
    PLoS ONE 05/2013; 8(5):e63852. DOI:10.1371/journal.pone.0063852 · 3.23 Impact Factor
  • Source
    • "A number of p65 phosphorylation sites have been revealed such as serine 468, which is contained in a C-terminal transactivation domain. Inducible phosphorylation of this site is exerted by IKKb and the noncanonical IKK family member IKK3, also called IKKi (Mattioli et al., 2006; Schwabe and Sakurai, 2005). Besides its function as a NF-kB kinase, IKK3 has been identified as an important mediator of the antiviral interferon response (Chau et al., 2008; Fitzgerald et al., 2003; Matsui et al., 2006; Sharma et al., 2003) and as a breast cancer oncogene (Adli and Baldwin, 2006; Boehm et al., 2007). "
    [Show abstract] [Hide abstract]
    ABSTRACT: The IKK-related kinase IKKepsilon contributes to the antiviral response and can function as an oncogene that is frequently amplified in breast cancer. Here we report on an additional role of IKKepsilon as a mediator protecting from DNA-damage-induced cell death. Genotoxic stress allows for kinase-dependent entry of IKKepsilon into the nucleus, where IKKepsilon-dependent PML phosphorylation is a prerequisite for retention of this kinase in PML nuclear bodies. Within these subnuclear structures IKKepsilon inducibly colocalizes with TOPORS, which functions as a SUMO E3 ligase mediating SUMOylation of IKKepsilon at lysine 231. SUMO modification of IKKepsilon is required to trigger phosphorylation of nuclear substrates including NF-kappaB p65, thereby contributing to the antiapoptotic function of NF-kappaB in response to DNA damage.
    Molecular cell 02/2010; 37(4):503-15. DOI:10.1016/j.molcel.2010.01.018 · 14.46 Impact Factor
  • Source
    • "Reconstituting Rela−/− MEFs with a phospho-mimicking S536D mutant produced enhanced binding to TAFII31 and enhanced transcriptional activity, reciprocating effects of IL-1-induced NF-κB activation [9]. Finally, phosphorylation at Ser468 has been reported to have both inhibitory and activatory effects on transactivation, and reconstituting Rela−/− MEFs with a non-phosphorylatable S468A mutant form of human RelA has been shown to have a negative effect on both IL-1- and TNFα-induced NF-κB transactivation [19]. TNFα-induced Ser468 phosphorylation was also found to regulate ubiquitin-mediated proteasome-dependent removal of chromatin-bound RelA on certain NF-κB gene targets [13]. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Phosphorylation of the RelA (p65) NF-kappaB (nuclear factor kappaB) subunit has been previously shown to modulate its ability to induce or repress transcription. In the present study we have investigated the consequences of Thr435 phosphorylation within the C-terminal transactivation domain of RelA. We confirm that Thr435 is phosphorylated in cells and is induced by TNFalpha (tumour necrosis factor alpha) treatment. Mutational analysis of this site revealed gene-specific effects on transcription, with a T435D phosphomimetic mutant significantly enhancing Cxcl2 (CXC chemokine ligand 2) mRNA levels in reconstituted Rela-/- mouse embryonic fibroblasts. Chromatin immunoprecipitation analysis revealed that this mutation results in enhanced levels of histone acetylation associated with decreased recruitment of HDAC1 (histone deacetylase 1). Moreover, mutation of this site disrupted RelA interaction with HDAC1 in vitro. Thr435 phosphorylation of promoter-bound RelA was also detected at NF-kappaB target genes following TNFalpha treatment in wild-type mouse embryonic fibroblasts. Phosphorylation at this site therefore provides an additional mechanism through which the specificity of NF-kappaB transcriptional activity can be modulated in cells.
    Biochemical Journal 12/2009; 426(3):345-54. DOI:10.1042/BJ20091630 · 4.78 Impact Factor
Show more