Combination of <i>in vivo</i> and <i>in vitro</i> phosphoproteomics determines the PP2A target repertoire on proteome scale

Dengjel, Joern

doi:10.1016/j.crmeth.2025.101084

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Combination of in vivo and in vitro phosphoproteomics determines the PP2A target repertoire on proteome scale

DOKPE

Dengjel, Joern ORCID University of Fribourg

2025

Published in:

Cell Reports Methods. - US: Elsevier BV. - 2025, p. 1-18

English Dynamics of protein phosphorylation are regulated by the interplay of kinases and phosphatases. Current mass spectrometry-based phosphoproteomic approaches are extremely powerful in identifying and quantifying tens of thousands of phosphosites in single biological samples. However, whereas the mapping of phosphosites is successfully automated supporting high sample throughput, the characterization of responsible kinases and phosphatases still largely depends on laborious protein biochemical assays. To show direct (de)phosphorylation events in vitro kinase or phosphatase assays using single substrates or peptide arrays are often used. Here we describe the development of an in vitro phosphatase assay using whole proteome under native conditions as input. We employ this approach to study the PP1 and PP2A target repertoire, characterizing thousands of potential target sites. Focusing on PPP2R5E/B56ε-containing complexes, we combine in vitro with in vivo phosphoproteomics to characterize bona fide target sites, which highlight PP2A`s role in regulating stress granule assembly.

Faculty

Faculté des sciences et de médecine

Department

Département de Biologie

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