Unravelling gene expression for binding proteins
Serum response factor (SRF) regulates the expression of immediate early genes, genes controlling cell adhesion and migration and the expression of many muscle-specific genes, which are a major SRF target. Exclusive binding of MAL and MKL2 co-activators enables differential regulation of expression of SRF action. The activity of MAL and MKL2 is controlled through regulated interaction with G-actin, which is a subunit of microfilaments that are found in the cytoskeleton. It has been shown that depletion of G-actin results in the accumulation of MAL in the nucleus, whereas binding inhibits MAL nuclear import and transcriptional activity. The 'Cell Biology of SRF cofactors' (SRF cofactors) project aimed to enhance understanding of how G-actin binding controls all these events. The original objectives were to develop a system to track MAL in real time and study the interactions between MAL and actin and between MAL and SRF in live cells. MAL senses the G-actin levels in the cell with a so-called RPEL domain (a G-actin binding element). Studies revealed that RPEL1 and RPEL2 peptides bind to actin for the most part through hydrophobic interactions. To test the validity of results obtained, project partners developed disruption mutations to interfere with MAL-actin binding. Providing support for the original model, results showed that all mutations caused at least partial MAL-deregulation. SRF cofactors researchers used Forster resonance energy transfer (FRET) to monitor MAL-actin interaction during signalling. In fixed cells, FRET was observed between the two proteins MAL-GFP and mCherry-actin. Since MAL is implicated in cell movement and metastasis, the project's results encourage further efforts to gain more knowledge about MAL-actin interaction. This pathway could well prove useful in developing new cancer therapies.
