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Title: Molecular mechanisms of MRTF-A regulation
Author: Pawlowski, R.
Awarding Body: University College London (University of London)
Current Institution: University College London (University of London)
Date of Award: 2011
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Transcription factor SRF (Serum Response Factor) and its coactivators MRTFs (Myocardin-Related Transcription Factors) regulate expression of the target genes in response to changes in actin dynamics. Via its RPEL domain, MRTF-A forms complexes with monomeric, but not F-actin and remains transcriptionaly inactive. In serum-starved cells, MRTF-A continuously shuttles through the nucleus, but as a result of the export rate being much faster than the import, MRTF-A predominantly localises to the cytoplasm. When actin polymerisation is induced (e.g. upon serum stimulation), G-actin concentration drops and MRTF-A cannot be efficiently exported back to the cytoplasm, accumulating in the nucleus. This coincides with MRTFs phosphorylation. Two aims of this thesis were to 1) establish the mechanism of MRTF-A shuttling and 2) the role of its phosphorylation and regulation by ERK and PKC family of enzymes. MRTF-A import to the nucleus utilises Importin β (Impβ) and its adaptor protein Importin α (Impα). Impα binds to a bipartite nuclear localisation signal (NLS) within the RPEL domain of MRTF-A constituted by two basic residues boxes (B2 and B3) separated by an unusually long (30 amino acids) linker. Actin overexpression inhibits MRTF-A nuclear accumulation after serum stimulation, reflecting a direct competition between actin and the Impα/Impβ complex binding to the RPEL domain. The mechanism of MRTF-A export is less clear. Seven putative nuclear export signals (NESs) may contribute to MRTF-A export. It has been suggested that actin is required for Crm1-dependent export. I find, however, that it does not facilitate Crm1 binding in vitro. I show that TPA treatment results in ERK-dependent partial nuclear accumulation of MRTF-A, which may require phosphorylation of Ser98. Concomitantly, TPA treatment inhibits Rho-mediated actin polymerisation in response to serum, preventing efficient nuclear accumulation of MRTF-A. Finally, I show evidence suggesting that MRTF-A phosphorylation at multiple residues correlates with its activation.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available