Chan, JJ; (2013) Comparative Analysis of Interactions of the RASSF Family Mediated by the Ras-association (RA) and SARAH domains. Doctoral thesis (PhD), UCL (University College London).

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Abstract

Members of the RASSF family (RASSF1-10) have been identified as candidate tumour suppressors that are frequently downregulated by promoter hypermethylation in cancers. These adaptor proteins carry a common Ras-association (RA) and SARAH domain (RASSF1-6) that can potentially bind Ras oncoproteins and mediate protein-protein interactions with other SARAH domain proteins (e.g. MST kinase). However, there is a notable lack of comparative characterisation of the RASSF family, as well as of molecular and structural information that facilitate their tumour suppressive functions. As part of our comparative analysis, we modelled the RA and SARAH domains of the RASSF members based on existing structures and predicted their potential interactions and the key residues involved. These in silico predictions were compared to in vitro studies and intracellular binding assays using Förster Resonance Energy Transfer (FRET). Several SARAH domain mutants were also investigated for their effects on RASSF interactions. Furthermore, we compared the interactions of the RASSF family with several key proteins involved in death and NFκB signalling. Our biochemical data show a diversity of interactions within the RASSF family RA domain, whereas interactions between RASSF and MST correlate with the presence of the SARAH domain, which is supported by the FRET experiments. Mutations of specific non-polar residues in the dimerisation interface of the SARAH domain also prove detrimental to the interaction between selected RASSF members and MST. Moreover, we observed stimulation-dependent interactions between specific RASSF members and MOAP1, TNF-R1, DAPK and TBK1. These results suggest that different members, despite shared general architecture, may have distinct binding properties, but ultimately could share overlapping functions. Current data also support an interaction model where RASSF serves as an adaptor for the assembly of multiple protein complexes and further functional interactions, involving MST kinases and other interacting partners, which could be regulated by Ras.

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