JCJC SVSE 8 - JCJC - SVSE 8 - Biochimie, biologie moléculaire et structurale

An Integrated Structural Biology Approach to the study of oncogenic deltaNp73-alpha stabilization induced by the IKK-beta kinase – IKKp73

Structure-function studies of the IKKß/Dp73 complex involved in silencing of p53-regulated gene expression

The IKKß kinase phosphorylates the oncogenic deltaNp73alpha isoform (a member of the p53 tumor suppressor family and named Dp73 from here on) at Ser422. This phosphorylation event promotes the formation of a tight complex between IKKß and Dp73, which associates to the promoters of p53-regulated genes leading to silencing of gene expression. The project aims at characterizing the IKKß/Dp73 complex using an integrated Structural Biology approach.

Structure-function characterization of the IKKß/Dp73 complex implicated in oncogenic repression of p53-regulated gene expression: main objectives.

The inhibitor of kB kinase (IKK) is a master regulator of inflammatory, immune and apoptotic responses. It was recently shown that IKKß mediates nuclear accumulation of Dp73, an oncogenic isoform of p73 that lacks the N-terminal transactivation domain. Interestingly, nuclear accumulation of Dp73 proceeds via IKKß phosphorylation at Ser422 of Dp73. Following this phosphorylation event, IKKß and Dp73 form a tight complex and bind to the promoters of p53-regulated genes as part of large transcriptional complexes, which additionally comprise the enhancer of zeste homolog 2 (EZH2) and the DNA methyltransferase 1 (DNMT1). The assembly of these complexes result in histone H3 and promoter DNA methylation, thereby leading to the repression of gene expression. The project aims at characterizing the IKKß/Dp73 interaction using an integrated Structural Biology approach. The following questions are addressed: <br />• What are the surface regions of IKKß and Dp73 implicated in the interaction? What is the overall structure of the complex?<br />• Ser422 phosphorylation appears to enhance the affinity of the IKKß/Dp73 interaction. What is the mechanism governing this affinity enhancement?<br />• How does the interaction of IKKß with Dp73 differ from the interactions of IKKß with other substrates ( IkB and p53)? <br />• How does IKKß modulate recruitment of Dp73 to DNA regions?<br />Knowledge of binding affinity and kinetics, conformational changes and molecular interfaces will enable design of functional assays that will help to elucidate the role of IKKß/Dp73 in silencing of p53-regulated gene expression. Both IKKß and Dp73 have been found to be upregulated in many human malignancies. For this reason IKKß and Dp73 represent potentially important therapeutic targets.

The project makes use of a variety of methods that are summarized below.

Recombinant protein expression. IKKß and Dp73 recombinant proteins are produced by baculovirus expression in insect cells and bacterial expression, respectively. Cell-free protein expression will be performed in case selective isotope labeling for NMR chemical shift perturbation (CSP) analysis will be necessary.

Binding assays. The IKKß/Dp73 interaction is probed by pulldown assays using purified proteins (see below in results section) and Surface Plasmon Resonance (SPR, experiments in course).

Kinase activity assays. In vivo 32P labeling experiments using an anti phospho-422S Dp73 antibody will be performed to assess kinase specificity. Kinetic parameters will be obtained by performing an in vitro kinase assay that makes use of recombinant proteins and the constitutively active IKKß kinase.

X-ray crystallography. X-ray crystallography is our method of choice for high resolution structure determination of the IKKß/Dp73 complex.

NMR spectroscopy. NMR is a versatile technique that will be used in this project to (i) assess protein folding (thereby allowing construct optimization for structural studies); (ii) probe conformational changes triggered by mutations (in particular the phosphomimic S422E mutation) (iii) detect interactions by CSP; (iv) probe protein dynamics by relaxation experiments; (v) map interfaces by CSP. This latter task requires isotope labeling of samples.

In vivo protein-DNA binding assays. Chromatin immunoprecipitation (ChIP) experiments followed by real-time PCR will be performed to analyze the recruitment of IKKß and Dp73 to p53-regulated promoters.

In vivo transcription assays. p53-null Saos-2 cells will be transfected with DNA plasmids coding for IKKß, Dp73 and p53 proteins. At 24 hours post-transfection cell extracts will be analyzed for endogenous p21 protein levels by immunoblotting.

The proposed research plan was initially divided into three parts. Today we have met most of the objectives of part I and we are currently working on those of part II.

Production of recombinant proteins
IKKß. We based our design of IKKß constructs on the recently published x-ray structures of a large dimeric fragment (residues 1-669) of human IKKß encompassing KD, ULD and SDD domains. We expressed IKKß(1-669) in insect cells using the baculovirus system. After optimization of lysis buffer conditions, soluble IKKß(1-669) was recovered and purified by Ni2+ affinity followed by gel filtration chromatography. IKKß(1-669) eluted from the gel filtration column as a dimer (Fig. 1A).
Dp73. We designed a panel of deletion constructs of Dp73. Each construct has been produced in duplicate, either in the wild-type version or containing the phospho-mimic S422E mutation. Whereas all constructs could be expressed in soluble form as C-terminal fusions of the Maltose Binding Protein (MBP), only a subset of them could be expressed when fused to GST and His6 tags (Fig. 1B).

Interaction studies
Next, we performed pulldown experiments employing MBP-Dp73 constructs and purified IKKß(1-669). We observed interactions for most of the Dp73 constructs, including those encompassing the phospho-mimic S422E mutation (Fig. 1C). These results show that: (i) IKKß and Dp73 establish direct interactions with each other and do not need other cellular mediators to form a complex; (ii) the IKKß/Dp73 complex does not dissociate after Ser422 phosphorylation and this is in agreement with findings reported by our collaborators.

Current work. We are currently performing SPR experiments that will give more quantitative information on the affinities and binding kinetics of the IKKß interactions towards the different Dp73 constructs.
In addition, we are preparing samples of isotopically labeled Dp73 constructs for NMR analysis. The purpose of this analysis is to assess folding of the constructs and to probe putative conformational changes triggered by the phosphomimic S422E mutation.

Future work. In the next coming months, in collaboration with R. Accardi and M. Tommasino (IARC Lyon), we will perform kinase activity experiments to probe IKKß kinase specificity towards the different Dp73 constructs. The ensemble of the binding and kinase activity data will guide construct choice for structural studies.
We plan to start crystallization trials on the IKKß/Dp73 complex during the fall. If well-diffracting crystals will be raised, we will proceed towards x-ray structure determination of the complex. In the contrary case, we will turn to NMR CSP analysis for the mapping of the complex interfaces.
As soon as information on the residues implicated in the IKKß/Dp73 interaction becomes available, we will design interface mutants that will be tested in a number of activity and functional assays. These mutants will serve as tools to further investigate the role of the IKKß/Dp73 interaction in modulating recognition of DNA promoter regions and in the inhibition of p53 gene expression. In addition, mutants of IKKß will be tested for kinase activity towards the other two substrates of IKKß, i.e. IkB and p53, thereby enabling us to obtain a global view on IKKß function.

The results here summarized will be described in a publication.

The inhibitor of kB kinase (IKK) is a regulator of inflammatory, immune and apoptotic responses, best known for its role in the activation of nuclear factor kB (NF-kB). This Ser/Thr kinase is an enzymatic complex consisting of two catalytic and one regulatory subunits. The catalytic IKKß subunit accounts for nearly all kinase activity implicated in the activation of NF-kB.
It has been proposed that IKK acts as a bridge between inflammation and cancer by operating in a number of pathways that promote tumorigenesis. A recent investigation has shown that IKKß regulates the stability of oncogenic deltaNp73-alpha. This latter protein is an isoform of p73 (a member of the tumor suppressor p53 protein family) that lacks the N-terminal transactivation domain. It exerts anti-apoptotic functions and promotes cellular transformation by competing with full-length p53 proteins for binding at p53 regulatory elements within DNA promoter regions. Massimo Tommasino and Rosita Accardi, who participate to this project, have discovered that IKKß phosphorylation at Ser422 of deltaNp73-alpha leads to the stabilization and a strong nuclear accumulation of deltaNp73-alpha and that this results in the inhibition of the expression of p53-regulated genes.
Intriguingly, the interaction between IKKß and deltaNp73-alpha appears to be enhanced by Ser422 phosphorylation. The proposed project aims at characterizing the IKKß/deltaNp73-alpha interaction using an integrated Structural Biology approach, which encompasses x-ray crystallography, NMR spectroscopy and Surface Plasmon Resonance coupled with a number of in vitro and in vivo activity assays and functional studies. Structural studies will provide three-dimensional high-resolution structures or models of IKKß/deltaNp73-alpha complexes, which will be complemented with binding kinetics and kinase activity data. Together structural and activity data will enable to elucidate the mechanisms governing this interaction. Subsequently, specific mutants of IKKß and deltaNp73-alpha will be designed based on knowledge of the interaction mechanisms. These mutants will be tested in a number functional assays in order to better define the role of the IKKß/deltaNp73-alpha interaction in nuclear accumulation of deltaNp73-alpha and subsequent inhibition of the expression of p53-regulated genes.
Both IKKß and deltaNp73-alpha have been found to be upregulated in many human malignancies, including breast, prostate, liver, lung and thyroid cancers. For this reason IKKß and deltaNp73-alpha represent potentially important therapeutic targets. We believe that the present project has the potential to unveil important mechanisms regulating cancer and therefore to be of high impact for public heath.

Project coordinator

Madame Katia ZANIER (Centre National de la Recherche Scientifique-Biotechnologie et Signalisation Cellulaire) – zanier@unistra.fr

The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.


CNRS-BSC Centre National de la Recherche Scientifique-Biotechnologie et Signalisation Cellulaire

Help of the ANR 249,999 euros
Beginning and duration of the scientific project: December 2013 - 42 Months

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