title: 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.
authors.affiliation: Institute of Chemistry, The Hebrew University of Jerusalem, Givat Ram, Jerusalem, Israel.
authors.forename: Shahar
authors.initials: S
authors.lastname: Rotem-Bamberger
authors.forename: Chen
authors.initials: C
authors.lastname: Katz
authors.forename: Assaf
authors.initials: A
authors.lastname: Friedler
mesh_terms: D051017:Apoptosis Regulatory Proteins / Q000378:metabolism*; D001665:Binding Sites; D005454:Fluorescence Polarization; D005786:Gene Expression Regulation; D006801:Humans; D008856:Microscopy, Fluorescence; D008958:Models, Molecular; D016328:NF-kappa B / Q000378:metabolism; D010455:Peptides / Q000378:metabolism; D011392:Proline / Q000378:metabolism; D017434:Protein Structure, Tertiary; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism*; D018909:src Homology Domains
chemical_list: D051017:Apoptosis Regulatory Proteins; D016328:NF-kappa B; D010455:Peptides; D019253:Proto-Oncogene Proteins c-bcl-2; C495901:TP53 protein, human; C090468:TP53BP2 protein, human; D016159:Tumor Suppressor Protein p53; D011392:Proline