IKK phosphorylates p65 at S468 in transactivaton domain 2

Department of Medicine, Columbia University, College of Physicians and Surgeons, New York, New York, USA.
The FASEB Journal (Impact Factor: 5.04). 11/2005; 19(12):1758-60. DOI: 10.1096/fj.05-3736fje
Source: PubMed


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
  • 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.
    Full-text · Article · May 2013 · PLoS ONE
  • Source
    • "degradation (Hacker and Karin, 2006). In addition, while both kinases phosphorylate p65 at Ser 536 to enhance NF-kB transactivation potential, IKKb can also target Ser 468 of p65 to further stimulate NF-kB activity (Schwabe and Sakurai, 2005). IKKa on the other hand, phosphorylates p100 to induce partial proteolysis and activation of the alternative NF-kB pathway (see below), but can also function independently of NF-kB by acting in the nucleus to phosphorylate transcriptional cofactors CBP and SMRT, as well as DNA histone protein H3 (Anest et al., 2003; Hoberg et al., 2004; Huang et al., 2007). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Muscle development, growth, and maintenance require an intricate and timely series of events initiated through a multitude of signaling pathways. The very nature of skeletal muscle requires tremendous plasticity to accommodate the need for anabolism or catabolism, and deregulation of these processes may be a tipping point in the development or progression of various skeletal muscle disorders. Among the relevant signaling pathways, NF-κB has emerged as a critical factor involved in various facets of muscle homeostasis. In this review, we summarize the NF-κB signaling pathway and provide a fresh perspective into the regulation and function of this transcription factor, underlying both the physiological and pathophysiological states of skeletal muscle.
    Full-text · Article · Jan 2011 · Current Topics in Developmental Biology
  • 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.
    Full-text · Article · Feb 2010 · Molecular cell
Show more