Protein phosphorylation is a crucial regulatory mechanism in cells, with protein kinases – the enzymes that catalyze phosphorylation – being among the most significant drug targets. DarkPhospho will investigate the “dark matter of the phosphoproteome”: the
process of multiphosphorylation of dense serine-threonine (S/T) clusters. The S/T cluster-containing proteins are common in key biological pathways, including Wnt signalling and the circadian clock, both regulated by casein kinase 1 (CK1). Despite its biological
importance, the kinetic mechanism of multiphosphorylation – and how it depends on the sequence and spatial arrangement of S/T sites – remains poorly understood.
DarkPhospho aims to elucidate this mechanism using a panel of CK1 enzymes and their substrates as a model system and experimental toolkit. The project will take a multidisciplinary approach, comprising: (1) biophysical analysis of multiphosphorylation
using nuclear magnetic resonance (NMR) and mass spectrometry (MS); (2) development of mathematical models describing the dynamics of multiphosphorylation; and (3) assessment of biological relevance of multiphosphorylation focusing on protein liquidliquid
phase separation (via confocal microscopy) and Wnt signalling activity (via dual luciferase reporter assay).
This project challenges the traditional view of protein phosphorylation as a simple on/off switch. Its findings will contribute to the rational design of CK1 inhibitors with potential clinical applications in cancer therapy. The fellow will receive training in NMR and protein purification, and will also develop transferable skills aligned with the long-term goal of establishing an independent research career.

Sustainable Development Goals

Masaryk University is committed to the UN Sustainable Development Goals, which aim to improve the conditions and quality of life on our planet by 2030.