Session: Current Topics in Biological Chemistry:

The ETS transcription factor ERG is a master regulator of multiple vascular processes, including homeostasis, angiogenesis, inflammation and vascular development. ERG is constitutively expressed in vascular endothelial cells (EC); the pathological dysregulation of ERG has been linked to inflammatory disorders and diseases associated with abnormal vascular homeostasis. In non-EC, the aberrant ectopic expression of ERG can result in the pathogenesis of multiple cancers, including prostate cancer. ERG activity can be regulated through ERK2-dependent phosphorylation of serines 96 and 215. Little is known about the molecular mechanisms underlying the regulation of ERG activity through phosphorylation.
We combine in silico Molecular Dynamics (MD) and in vitro assays to characterise the molecular mechanisms underlying the phosphorylation-dependent regulation of ERG’s transcriptional activity.
No full-length crystal structure of ERG has been obtained. Thus, a model of the structure of ERG was generated using MD relying on the available structural information for two conserved domains. This model was then used to show that upon serine 215 and 96 phosphorylation ERG structure underwent conformational changes in the C- and N- termini respectively. To investigate the functional relevance of these changes, we carried out in vitro Luciferase promoter reporter assays using ERG phosphorylation mutants. An electrophoretic mobility shift assay (EMSA) was performed to study the effect of phosphorylation on the binding of ERG to a target DNA sequence. We showed that S215 phosphorylation increased the DNA binding ability of ERG. This finding goes in line with the in silico observed structural change in the C-terminus where the DNA binding domain is located. Ongoing in vitro studies are characterising the effect of ERG phosphorylation on the molecular interactions of ERG with protein binding partners.
Understanding how the dynamic regulation of ERG’s protein structure is linked to its functionality can provide knowledge on how to design therapeutics that can impair or mimic ERG activity in order to tackle diseases associated with abnormal vascular homeostasis.

Graeme Birdsey

Appearances