Investigation of a novel protein-protein interaction involving the hdm2 oncoprotein

Abstract

The p53 tumor suppressor protein has a critical role in the cellular response to stress, and when activated, functions to transactivate genes which induce apoptosis or growth arrest.  The hdm2 oncoprotein is the most significant regulator of p53 activity, and it achieves this partly through inhibition of p53 transactivating ability.  Hdm2 also interacts with other non-p53 proteins and through its many protein-protein interactions, influences diverse cellular pathways.  Work by our group previously identified a putative protein-protein interaction between hdm2 and the redox-sensitive transcriptional co-repressor CtBP2.  Based on this observation, the objectives of the current study were:  (i) To verify that hdm2 and CtBP2 interact, (ii) To characterise the physical nature of the interaction, (iii) to determine the functional consequences of the interaction and how it may be regulated.

The interaction between hdm2 and CtBP2 was verified using in vitro and in vivo protein binding assays.  Deletion mutants were used to identify the interacting domains in the proteins, and the effect of the interaction on p53 activity assessed using reporter gene assays.

Hdm2 participates in a novel protein-protein interaction with CtBP2 both in vitro and in vivo.  The acidic domain of hdm2 and the N-terminus of CtBP2 are necessary and sufficient for this interaction.  CtBP proteins undergo an NADH-induced conformational change, which we show results in a loss of its hdm2 binding ability.  This negative regulation is dependent on the conserved NADH-binding GXGXXG motif in CtBP2.

The recruitment of CtBP2 by hdm2 results in a promoter selective repression of p53-depdnent transcription. Furthermore, hypoxia-mimicking conditions which increase intracellular NADH levels abolish this repression, thus enhancing p53 activity.

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