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.


Available from: Hiroaki Sakurai
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    • "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. "
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    • "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). "
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    • "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). "
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