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

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.

ΔN-ASPP2, a novel isoform of the ASPP2 tumor suppressor, promotes cellular survival

“…Thus, we could hypothesize that ΔN-ASPP2 also binds p53 to prevent it from occupying p53 target gene promoters or to compete with ASPP2 for p53 binding [4]. Thus ΔN-ASPP2 may also directly bind ASPP2 to inhibit its function as would be predicted by structural studies [34,35]. Intriguingly, we observed that expression of ΔN-ASPP2 appears to suppress expression of ASPP2 (Fig.…”

Structure of the Helicobacter pylori CagA oncoprotein bound to the human tumor suppressor ASPP2

“…Although most of the ASPP2-binding proteins that are critical for its proapoptotic function (p53, NF-κB, Bcl-2) interact with the ankyrin-SH3 domain of ASPP2, the plasma membrane localization and the prosenescence activity of ASPP2 depend on cellular partners that bind ASPP2 at its N-terminal region (41)(42)(43)(44). It was shown (41,45) that peptides generated from the proline-rich domain (e.g., 693-746) could bind to the ankyrin-SH3 domain of ASPP2 and compete for binding with NF-κB or p53. This result raised the possibility that an autoinhibitory intramolecular mechanism exists to regulate associations with ankyrin-SH3 domain.…”

Protein Regulation by Intrinsically Disordered Regions: A Role for Subdomains in the IDR of the HIV‐1 Rev Protein

“…[22] Disordered regions are particularly subjecttopost-translational modifications, [23][24][25] and many contain sites for phosphorylation. [26,27] HIV-1 Rev is as mall, 116-residue protein, whiche nables packing of the HIV RNA into the emerging virions. [26,27] HIV-1 Rev is as mall, 116-residue protein, whiche nables packing of the HIV RNA into the emerging virions.…”