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Cell Biology of SRF cofactors

Final Report Summary - SRF COFACTORS (Cell biology of SRF cofactors)

Development and function of a multicellular organism requires a complex interplay between cells and a tight control of proliferation and differentiation. Cells respond to extracellular signals with changes in their gene expression pattern, a process which is regulated by transcription factors. SRF (serum response factor) is a transcription factor which regulates many immediate early genes as well as many genes controlling cell adhesion and migration. The third major group of SRF targets are muscle-specific genes.

Differential regulation of SRF-activity is achieved by mutually exclusive binding of specific coactivators. MAL (MRTF-A/MKL1), a coactivator of the myocardin family, links SRF activity to actin. MAL localisation and transcriptional activity is controlled through regulated interaction with monomeric G-actin. Stimulation with serum leads to actin-polymerisation and G-actin depletion. Upon G-actin depletion MAL accumulates in the nucleus, where it activates transcription (Miralles et al. (2003) Cell 113(3): 329-42, reviewed in Posern and Treisman (2006) Trends Cell Biol 16(11): 588-96). Aim of this project was to understand the mechanism by which G-actin binding controls these events.

MAL senses the G-actin levels in the cell with a so-called RPEL domain. It consists of three RPEL motifs, which are connected by linker-sequences (Miralles et al. (2003) Cell 113(3): 329-42). To investigate the nature of the actin-binding to the RPEL domain and the stoichiometry of the complex, we applied x-ray crystallography in collaboration with Sebastian Guettler, Stephane Mouilleron and Neil McDonald. We obtained crystal-structures for the isolated RPEL1 and RPEL2-motif bound to latB-actin. Both RPEL peptides bound to actin mainly through hydrophobic interactions via two alpha-helices and a C-terminal capping region (Mouilleron et al. (2008) EMBO J 27: 3198-3208). To get more insight into the stoichiometry of the actin-MAL complex, we crystallised full RPEL domain bound to latB-actin. We obtained two distinct structures, one with three actins bound to the RPEL domain, one with five actins.

To validate these results in a biological context, we disrupted MAL-actin binding by site-directed mutagenesis of crucial actin binding residues in full-length MAL and assessed their behaviour in a cell based localisation and activation assay. All disrupting mutations caused partial MAL-deregulation, which supports the structural model.

To study changes in the MAL-actin interaction during signalling we planned to use FRET analysis in live cells. FRET (Forster resonance energy transfer) is a photophysical effect, which can be used to monitor transient protein-protein interactions. Fluorescent proteins can be genetically fused to the proteins of interest and be used for analysis of protein-protein interactions in a live cell environment. We were trying to monitor the MAL-actin interactions using MAL-GFP and mcherry-actin as a FRET-pair. In fixed cells we could observe FRET between MAL-GFP and mcherry-actin in serum-starved cells, indicating an interaction between these two proteins. FRET was abolished when the cells were treated with a drug that disrupts MAL-actin binding. However, the effect was very small, which makes this system not applicable for in vivo work. Current work aims to improve the FRET-efficiency. This project is carried out in collaboration with Banafshe Larijani.

MAL is implicated in cell motility and metastasis (Medjikane et al. (2009) Nat Cell Biol 11(3): 257-268), which makes this pathway interesting as a potential target for new cancer therapies. Deeper understanding of the MAL-actin interaction might prove useful in the fight against cancer. From the crystal-structures combined with the site-directed mutagenesis we were able to define crucial actin binding residues in MAL. This might be a first step into new therapeutic avenues.