Phosphorylation of the FOXP2 forkhead domain: the effect on structure and DNA binding using phosphomimetics
Blane, Ashleigh Anne
Transcription factors are proteins that are involved in the regulation of gene expression and are responsible for the tight control of transcription allowing a cell to react to changes in its environment. Transcription factors are thus highly regulated by a variety of mechanisms which include phosphorylation. Forkhead box P2 (FOXP2) is a transcription factor expressed in multiple tissues during embryonic development. FOXP2 like other FOX proteins contains a DNA binding domain known as the forkhead domain (FHD). The effect of phosphorylation of serine 557 in the FHD on the structure and DNA binding was done using a glutamate mutant (to mimic phosphorylation) and an alanine mutant (as a control). Structural characterisation was performed using size exclusion chromatography (SEC), intrinsic fluorescence and far-UV circular dichroism. The effect of phosphorylation on DNA binding was observed using electrophoretic mobility shift assay (EMSA) and isothermal titration calorimetry (ITC). Far-UV circular dichroism and intrinsic fluorescence of the mutants and wild type did not reveal any significant secondary or tertiary structural changes. SEC however revealed a decrease in dimerisation propensity in the Ser557 mutants when compared the wild type (WT). EMSA revealed that DNA binding of S557E is only observed at protein concentrations 40 times in excess of the DNA. DNA binding of the WT and S557A mutants is observed at 5 times and 20 times excess protein respectively. However, using ITC no DNA binding is observed for either S557E or S557A FOXP2 FHD. Thus, it is possible that phosphorylation of serine 557 in the FOXP2 FHD could be a mechanism for inactivation of FOXP2.
A dissertation submitted in fulfilment of the requirements for the degree of Master of Science to the Faculty of Science, University of the Witwatersrand, Johannesburg, 2017
Blane, Ashleigh Anne (2017) Phosphorylation of the FOXP2 forkhead domain: the effect on structure and DNA binding using phosphomimetics, University of the Witwatersrand, Johannesburg, http://hdl.handle.net/10539/23562