Proceedings of The Physiological Society

Europhysiology 2018 (London, UK) (2018) Proc Physiol Soc 41, PCA343

Poster Communications


B. Griesler1, S. Ruhs1, N. Strätz1, C. Grossmann1

1. Julius-Bernstein-Institute of Physiology, Halle (Saale), Germany.

The mineralocorticoid receptor (MR) is a ligand-dependent transcription factor. Upon binding of aldosterone the MR translocates into the nucleus, binds to GRE elements and promotes the transcription of its target genes. Clinical trials have identified the MR as a mediator of cardiovascular disease (CVD). The mechanisms that trigger the switch of the MR from a regulator of water-electrolyte homeostasis to a mediator of pathological changes remain unclear. As the shift towards pathological MR effects is not caused by elevated blood pressure or aldosterone excess, an inappropriate activation of the MR by factors in the micro-milieu has been suggested. There are indications that post-translational modifications of the receptor can modulate its activity. A potential kinase for the MR is the ubiquitously expressed casein kinase 1 (CK1). Our aim is to investigate the interaction between CK1 and MR as a mechanism for mediating pathophysiological MR effects. To examine if CK1 affects genomic MR activity, GRE reporter assays were performed in HEK cells transfected with MR after incubation with the pharmacological CK1-inhibitor D4476 (10 µM). We found that CK1 inhibition leads to a reduction in basal and in aldosterone-induced genomic MR-activity (41% and 35% reduction N=4, n=8-12; p<0.05). This effect was confirmed with another CK1-inhibitor PF670462 (5µM) (41.7% and 56.8% reduction ; N=3, n=7-9; p<0.05).To test if CK1 modulates MR function through direct phosphorylation we performed peptide microarrays and correlated the results with in silico predictions using NetPhos 2.0. Altogether 5 potentially CK1-dependently modified amino acids (S170, S468; S501; T800; S936) were identified and analyzed further. To narrow down the search for relevant sites, we examined the effects of D4476 on a truncated MR construct missing the regulatory N-terminal domain (CDEF-MR). We observed a clear reduction in reporter activity in stimulated as well as in unstimulated cells (19.4% and 45.7% reduction; N=3, n=7-9; p<0.05). Furthermore we performed reporter assays overexpressing the glucocorticoid receptor, a close MR relative. Under D4476-treatment the GR shows the same tendencies as the MR with reduced basal as well as dexamethasone-induced genomic activity (45% and 36% reduction; N=2, n=4-6; p<0.05 respectively). Our results implicate, that CK1 can modulate the genomic activity of the MR and GR and that this interaction does not require the N-terminal A/B domain. Results from in silico prediction and peptide array experiments suggest that CK1 can phosphorylate the MR directly. Whether the main regulatory effects are exerted by direct phosphorylation of the receptors and/or by phosphorylation of e.g. the translocation machinery remains a subject of future investigations.

Where applicable, experiments conform with Society ethical requirements