Regulation of the NF-kappaB p65 subunit by phosphorylation and ubiquitination

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GengHui-2008-05-29.pdf (2.5 MB)

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2008

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DOI:
http://dx.doi.org/10.22029/jlupub-10093

Abstract

The inducible transcription factor NF-kappaB regulates a wide variety of target genes and plays a key role in many biological processes. While all NF-kappaB activation pathways share a critical step involving proteasome-mediated degradation of inhibitory proteins and the release of DNA-binding subunits, NF-kappaB itself is also regulated by post-translational modification of the DNA-binding subunits. This second level of regulation is required to determine the specificity and to control the amplitude as well as duration of the transcriptional response. This study identified IKKepsilon as a novel p65 kinase mediating inducible phosphorylation at Ser 468 and also Ser 536 in response to T cell costimulation. In costimulated T cells, multiple experimental approaches revealed an important role of IKKepsilon for p65 phosphorylation at Ser 468, whereas Ser 536 phosphorylation also occurred in the absence of this kinase. These results also provide a mechanistic clue that helps to explain the relevance of IKKepsilon for the expression of a subset of NF-kappaB target genes without affecting IKK activity. The functional role of these phosphorylation sites was tested in reconstitution experiments. Gene arrays and real-time PCR analyses showed that the effect of Ser 468 phosphorylation depends on the individual target gene. While some genes show strict dependency on the integrity of this phosphorylation site, other genes are expressed at even higher rates when the phosporylatable serine is replaced by an alanine. The latter finding can be explained by an increased stability of the mutated p65 protein, which is largely refractory to ubiquitination and proteasome-dependent elimination. Further experiments showed that TNF-induced p65 phosphorylation at Ser 468 controls its ability to associate with COMMD1 and Cullin-2, components of a multisubunit ubiquitin ligase complex. These proteins in turn mediate p65 ubiquitination and allow for proteasome-dependent degradation of this transcription factor. ChIP assays revealed that phosphorylation of p65 at Ser 468 leads to ubiquitin/proteasome-dependent removal of chromatin-bound p65, thus contributing to the selective termination of late NF-kappaB-dependent gene expression.

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