The tumour suppressor protein p53 responds to oncogenic stress by inducing cell cycle arrest or apoptosis of potentially malignant cells, thereby preventing the malignant transformation (1). p53 is mutated in around 50% of human cancers. In the other 50% of tumours, where wild-type p53 is intact, the regulation of the p53 pathway is abnormal. A key question in cancer research is how the selection for the p53 response is being made. ASPP2 is a pro-apoptotic protein that stimulates the p53-mediated apoptotic response (2,3). It was previously identified in its shorter forms as 53BP2 and Bbp. ASPP2 is a 1128 amino acids protein, consisting of several structural and functional domains. The C-terminal ankyrin repeats and SH3 domain mediate its binding to p53 (4), as well as to numerous other proteins involved in apoptosis such as NF-kB and Bcl-2. However, the interactions of ASPP2 with other proteins have not been yet characterized structurally and biophysically, and the role of these proteins in regulation of the ASPP2 function is yet unknown. In addition, ASPP2 contains a proline-rich domain between residues 693-918, whose structure and function are unclear. We have used a combination of peptide array screening with biochemical and biophysical methods to gain insight into the protein interaction network of ASPP2 and the roles of its domains in mediating these interactions. To achieve this, we expressed three recombinant ASPP2 fragments: The Ank-SH3 domains only, the proline-rich domain only and a construct containing all three domains. To identify the ASPP2-binding sites in its target proteins, we designed peptide arrays consisting of overlapping peptides derived from a series of ASPP2 binding proteins. Screening of the ASPP2 constructs for binding the peptide arrays identified the precise binding sites in its target proteins, and revealed that only the Ank-SH3 domains, but not the proine-rich domain of ASPP2, mediate protein-protein interactions. Comparison of the ASPP2 binding sites in the three anti-apoptotic Bcl family members Bcl2, Bcl-XL and Bcl-W revealed that all of them bind ASPP2 via their N-terminal BH4 domain, which is essential for their anti-apoptotic activity. We propose that ASPP2 might promote apoptosis by inhibition of this anti-apoptotic BH4 domain. Next, we set to study the structure and function of the proline rich domain of ASPP2. Using biophysical methods including circular dichroism, analytical gel filtration and analytical ultracentrifugation we showed that the proline-rich domain of ASPP2 belongs to the family of natively unfolded proteins (5). SH3 domains are known to bind proline-rich ligands, so we tested whether such an interaction exists between the proline-rich and SH3 domains of ASPP2. Indeed, ASPP2 proline-rich domain bound the Ank-SH3 domains with micromolar affinity as shown by fluorescence spectroscopy and pull-down assays. Using peptide arrays we identified the precise Ank-SH3 binding sites in the proline-rich domain of ASPP2 (5). In summary, our results show that only the Ank-SH3 domains of ASPP2, but not the proline-rich domain, mediate the intermolecular interactions of ASPP2 with its partner proteins. Instead, the proline-rich domain makes an intramolecular interaction with the Ank-SH3 domain of ASPP2. Natively unfolded protein domains usually mediate interactions with numerous partner proteins. The proline rich domain of ASPP2 is a unique case, where such an unstructured domain mediates an intramolecular interaction, while the structured Ank-SH3 mediates the known intermolecular protein-protein interactions (5). Based on our findings, we propose a model for regulation of ASPP2 and of p53-mediated apoptosis. According to our model, the intramolecular interaction between the ASPP2 domains has an important role in regulating the intermolecular interactions of ASPP2 with its partner proteins. When the proline-rich domain is bound to the Ank-SH3 domain, it masks it and makes it unable to bind intermolecularly to its partner proteins. Following a yet unknown regulation mechanism, the intramolecular domain-domain interaction is released and the Ank-SH3 domains become available to bind their target proteins such as p53 or the Bcl family proteins, and promote apoptosis (5).