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Pré-Publication, Document De Travail Année : 2022

A CDK-mediated phosphorylation switch of disordered protein condensation

Guido Van Mierlo
Michiel Vermeulen
Albert Heck
Austin Haider
  • Fonction : Auteur
Kingshuk Ghosh
  • Fonction : Auteur
Pau Bernadó
  • Fonction : Auteur
Puck Knipscheer

Résumé

Abstract Cell cycle transitions arise from collective changes in protein phosphorylation states triggered by cyclin-dependent kinases (CDKs), but conceptual and mechanistic explanations for the abrupt cellular reorganisation that occurs upon mitotic entry are lacking. Specific interactions between distinct CDK-cyclin complexes and sequence motifs encoded in substrates might result in highly ordered phosphorylation1, while bistability in the mitotic CDK1 control network can trigger switch-like phosphorylation2. Yet the dynamics of mitotic phosphorylation has not been demonstrated in vivo, and the roles of most cell cycle-regulated phosphorylations are unclear. Here, we show evidence that switch-like phosphorylation of intrinsically disordered proteins (IDPs) by CDKs contributes to mitotic cellular reorganisation by controlling protein-protein interactions and phase separation. We studied protein phosphorylation in single Xenopus embryos throughout synchronous cell cycles, performed parallel assignment of cell cycle phases using egg extracts, and analysed dynamics of mitotic phosphorylation using quantitative targeted phosphoproteomics. This provided a high-resolution map of dynamic phosphosites from the egg to the 16-cell embryo and showed that mitotic phosphorylation occurs on entire protein complexes involved in diverse subcellular processes and is switch-like in vivo. Most cell cycle-regulated phosphosites occurred in CDK consensus motifs and located to intrinsically disordered regions. We found that substrates of CDKs and other cell cycle kinases are significantly more disordered than phosphoproteins in general, a principle conserved from yeast to humans, while around half are components of membraneless organelles (MLOs), whose assembly is thought to involve phase separation. Analytical modelling predicts modulation of homotypic IDP interactions by CDK-mediated phosphorylation, which was confirmed by biophysical and biochemical analysis of a model IDP, Ki-67. These results highlight the dynamic control of intrinsic disorder as a conserved hallmark of the cell cycle and suggest a mechanism for CDK-mediated mitotic cellular reorganisation.
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Dates et versions

hal-03845313 , version 1 (09-11-2022)

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Juan Valverde, Geronimo Dubra, Henk W.P. van Den Toorn, Guido Van Mierlo, Michiel Vermeulen, et al.. A CDK-mediated phosphorylation switch of disordered protein condensation. 2022. ⟨hal-03845313⟩
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