Phosphorylation of Schizosaccharomyces pombe Dss1 mediates direct binding to the ubiquitin-ligase Dma1 in vitro

Nina L. Jacobsen, Magnus Bloch, Peter S. Millard, Sarah F. Ruidiaz, Jonas D. Elsborg, Wouter Boomsma, Ruth Hendus-Altenburger, Rasmus Hartmann-Petersen, Birthe B. Kragelund*

*Corresponding author af dette arbejde

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Abstract

Intrinsically disordered proteins (IDPs) are often multifunctional and frequently posttranslationally modified. Deleted in split hand/split foot 1 (Dss1—Sem1 in budding yeast) is a highly multifunctional IDP associated with a range of protein complexes. However, it remains unknown if the different functions relate to different modified states. In this work, we show that Schizosaccharomyces pombe Dss1 is a substrate for casein kinase 2 in vitro, and we identify three phosphorylated threonines in its linker region separating two known disordered ubiquitin-binding motifs. Phosphorylations of the threonines had no effect on ubiquitin-binding but caused a slight destabilization of the C-terminal α-helix and mediated a direct interaction with the forkhead-associated (FHA) domain of the RING-FHA E3-ubiquitin ligase defective in mitosis 1 (Dma1). The phosphorylation sites are not conserved and are absent in human Dss1. Sequence analyses revealed that the Txx(E/D) motif, which is important for phosphorylation and Dma1 binding, is not linked to certain branches of the evolutionary tree. Instead, we find that the motif appears randomly, supporting the mechanism of ex nihilo evolution of novel motifs. In support of this, other threonine-based motifs, although frequent, are nonconserved in the linker, pointing to additional functions connected to this region. We suggest that Dss1 acts as an adaptor protein that docks to Dma1 via the phosphorylated FHA-binding motifs, while the C-terminal α-helix is free to bind mitotic septins, thereby stabilizing the complex. The presence of Txx(D/E) motifs in the disordered regions of certain septin subunits may be of further relevance to the formation and stabilization of these complexes.
OriginalsprogEngelsk
Artikelnummere4733
TidsskriftProtein Science
Vol/bind32
Udgave nummer9
Antal sider16
ISSN0961-8368
DOI
StatusUdgivet - 2023

Bibliografisk note

Funding Information:
The authors thank Signe A. Sjørup and Anne‐Marie Lauridsen for expert technical assistance and Dr Andreas Prestel for NMR support. This work was made possible by the Novo Nordisk Foundation Challenge grant REPIN— (#NNF18OC0033926 to BBK and RHP). The authors thank Villum Fonden and the Novo Nordisk Foundation for support for NMR infrastructure. NMR data were in part recorded at cOpenNMR—an infrastructure facility funded by the Novo Nordisk Foundation (#NNF18OC0032996). The work was also supported by the Novo Nordisk Foundation through the MLLS Center for Basic Machine Learning Research in Life Science (#NNF20OC0062606). rethinking protein interactions

Funding Information:
The authors thank Signe A. Sjørup and Anne-Marie Lauridsen for expert technical assistance and Dr Andreas Prestel for NMR support. This work was made possible by the Novo Nordisk Foundation Challenge grant REPIN—rethinking protein interactions (#NNF18OC0033926 to BBK and RHP). The authors thank Villum Fonden and the Novo Nordisk Foundation for support for NMR infrastructure. NMR data were in part recorded at cOpenNMR—an infrastructure facility funded by the Novo Nordisk Foundation (#NNF18OC0032996). The work was also supported by the Novo Nordisk Foundation through the MLLS Center for Basic Machine Learning Research in Life Science (#NNF20OC0062606).

Publisher Copyright:
© 2023 The Authors. Protein Science published by Wiley Periodicals LLC on behalf of The Protein Society.

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