Regulation of ASPP2 Interaction with p53 Core Domain by an Intramolecular Autoinhibitory Mechanism

Shahar Rotem-Bamberger,Assaf Friedler,Chen Katz,Vladimir N Uversky

doi:10.1371/journal.pone.0058470

Shahar Rotem-Bamberger, Assaf Friedler + Show 2 more

Open Access

https://doi.org/10.1371/journal.pone.0058470

Copy DOI

Journal: PloS one	Publication Date: Mar 5, 2013
Citations: 51	License type: CC BY 4.0

Affiliation: Hebrew University of Jerusalem

Abstract

ASPP2 is a key protein in regulating apoptosis both in p53-dependent and-independent pathways. The C-terminal part of ASPP2 contains four ankyrin repeats and an SH3 domain (Ank-SH3) that mediate the interactions of ASPP2 with apoptosis related proteins such as p53, Bcl-2 and the p65 subunit of NFκB. p53 core domain (p53CD) binds the n-src loop and the RT loop of ASPP2 SH3. ASPP2 contains a disordered proline rich domain (ASPP2 Pro) that forms an intramolecular autoinhibitory interaction with the Ank-SH3 domains. Here we show how this intramolecular interaction affects the intermolecular interactions of ASPP2 with p53, Bcl-2 and NFkB. We used biophysical methods to obtain better understanding of the relationship between ASPP2 and its partners for getting a comprehensive view on ASPP2 pathways. Fluorescence anisotropy competition experiments revealed that both ASPP2 Pro and p53CD competed for binding the n-src loop of the ASPP2 SH3, indicating regulation of p53CD binding to this loop by ASPP2 Pro. Peptides derived from the ASPP2-binding interface of Bcl-2 did not compete with p53CD or NFkB peptides for binding the ASPP2 n-src loop. However, p53CD displaced the NFκB peptide (residues 303–332) from its complex with ASPP2 Ank-SH3, indicating that NFκB 303–332 and p53CD bind a partly overlapping site in ASPP2 SH3, mostly in the RT loop. These results are in agreement with previous docking studies, which showed that ASPP2 Ank-SH3 binds Bcl-2 and NFκB mostly via distinct sites from p53. However they show some overlap between the binding sites of p53CD and NFkB in ASPP2 Ank-SH3. Our results provide experimental evidence that the intramolecular interaction in ASPP2 regulates its binding to p53CD and that ASPP2 Ank-SH3 binds Bcl-2 and NFκB via distinct sites.

Highlights

ASPP2 is a key protein in regulating apoptosis both in p53-dependent and-independent pathways
We have previously demonstrated an intramolecular interaction between ASPP2 Pro and ASPP2 Ank-SH3: ASPP2 Pro binds the first ankyrin repeat and the n-src loop of the SH3 domain (Figure 1D) [13]
We used fluorescence anisotropy competition experiments to reveal the effect of the intramolecular interaction in ASPP2 on its binding to p53 core domain (p53CD)

Summary

Introduction

P53 is one of the major tumor suppressor proteins in the cell. It is a transcription factor that induces growth arrest or apoptosis in response to cellular stress [1,2]. A nine-residue peptide derived from ASPP2 n-src loop (residues 1089–1097), termed CDB3, restored the specific DNA binding activity of the highly destabilized p53CD mutant I195T to the levels similar to the wild-type level [27] This peptide slowed down the unfolding of p53CD at 37uC in a concentration-dependent manner by maintaining the mutant protein in a folded conformation and preventing its aggregation, allowing it enough time to reach the nucleus and bind its sequence-specific target DNA or the p53 binding proteins that stabilize it [27]. They provide experimental evidence that the intramolecular domaindomain interaction in ASPP2 regulates its binding to p53CD and that ASPP2 Ank-SH3 binds Bcl-2 and NFkB via distinct sites

Materials and Methods

Results

Discussion