Characterization of the function of iASPP as a scaffolding protein for selected proteins involved in inflammation

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The ASPP (apoptosis-stimulating protein of p53) family of proteins is involved in many cellular interactions and is starting to emerge as a major scaffolding hub for numerous proteins involved in cancer biology and inflammation. It consists of the three members ASPP1, ASPP2 and iASPP which are best known for modulating the apoptotic function of p53, thereby directing cell fate decision. While ASPP1 and ASPP2 are supposed to stimulate the pro-apoptotic function of p53, iASPP has the opposing effect and inhibits the p53 mediated transcription of pro-apoptotic genes. All ASPP family members consist of three domains: the proline-rich region, the ankyrin repeats (ARs) and the SH3 domain. Of these the ARs and SH3 domain constitute one structural entity at the very C-terminus (CTD; C-terminal domain). In this grant application we describe experiments to characterize the interaction of iASPP with further interaction partners by a combination of in vitro interaction studies (isothermal titration calorimetry, pull down experiments), structure determination and cell based functional assays (transactivation assays, mass spectrometry). In addition to p53, two more interaction partners of iASPP are partially characterized: NF-kB and PP1. In contrast to p53 which interacts via its Ankyrin repeat and SH3 domains with iASPP, NF-kB and PP1 bind via a poly-proline domain to the SH3 domain. We have determined a consensus peptide sequence for interaction with the iASPP SH3 domain and scanned the KEGG GENES database. This has identified potential binding sites in many different proteins, including the AP1 family, the E3 ligase Itch, the MAPK kinase MKK7, the atypical MAP kinase ERK8 and RASSF5 of the RASSF family. We have started to characterize the binding and the functional consequences of the interaction for the AP1 family showing that iASPP binding has inhibitory effects on the transcriptional activity. Here we propose to extend these investigations and to extend them to mechanistic investigations of the inhibitory effect on NF-kB and characterization of the interaction with Itch.