Article

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.

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Available from: Hiroaki Sakurai, Aug 29, 2015
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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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    • "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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    • "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]. "
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