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Regulation of NF-kappaB transactivation by modulatory phosphorylations of the DNA-binding subunits during T cell activation

English title Regulation of NF-kappaB transactivation by modulatory phosphorylations of the DNA-binding subunits during T cell activation
Applicant Schmitz M. Lienhard
Number 107498
Funding scheme Project funding (Div. I-III)
Research institution Biochemisches Institut
Institution of higher education University of Berne - BE
Main discipline Biochemistry
Start/End 01.05.2005 - 31.03.2007
Approved amount 223'278.00
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All Disciplines (2)

Immunology, Immunopathology

Keywords (3)

transactivation; NF-kB; phosphorylation

Lay Summary (English)

Lay summary
Using a combination of peptide arrays with in vitro kinase assays we identify serine 468 as a novel phosphorylation site of p65 NF-kB. Follow-up experiments allowed the identification of two kinases which are able to phosphorylate p65 serine 468. Basal phosphorylation is mediated by GSK3b. Serine 468 lies within a GSK-3b consensus site, and recombinant GSK-3b specifically phosphorylates a GST-p65-(354-551) fusion protein at Serine 468 in vitro. In intact cells, phosphorylation of endogenous Ser(468) of p65 is induced by the PP1/PP2A phosphatase inhibitor calyculin A and this effect is inhibited by the GSK-3b inhibitor LiCl. Reconstitution of p65-deficient cells with a p65 protein where serine 468 was mutated to alanine revealed a negative regulatory role of serine 468 for NF-kB activation. Collectively our results suggest that a GSK-3b-PP1-dependent mechanism regulates phosphorylation of p65 NF-kB at Serine 468 in unstimulated cells and thereby controls the basal activity of NF-kB. Further work allowed the identification of IKKe as a p65 kinase mediating inducible ser 536 and ser 468 phosphorylation. Downregulation of IKKepsilon levels by small interfering RNA does not affect inducible phosphorylation of Serine 536 but largely prevents Ser468 phosphorylation induced by T cell costimulation. Serine 536-phosphorylated p65 is found predominantly in the cytosol. In contrast, the Serine 468 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 Serine 468 serves to enhance p65-dependent transactivation. These results also provide a mechanistic link that helps to explain the relevance of IKKe for the expression of a subset of NF-kB target genes without affecting cytosolic IkBa degradation.
Direct link to Lay Summary Last update: 21.02.2013

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Associated projects

Number Title Start Funding scheme
66794 Regulation of NF-kappa B transactivation by modulatory phospho- rylations of the DNA-binding subunits during T-cell activation 01.05.2002 Project funding (Div. I-III)