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

TL;DR

Researchers developed a new method to identify phosphatase targets on a large scale, revealing how PP2A regulates stress granule formation.

Contribution

A novel in vitro phosphatase assay was developed to screen whole-proteome phosphatase-substrate interactions under native conditions.

Findings

The in vitro phosphatase assay identified thousands of potential target sites for PP1 and PP2A.

PP2A complexes were found to target phosphosites on the protein translation machinery.

PPP2R5E/B56ε-containing PP2A complexes negatively regulate stress granule formation.

Abstract

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 the role of PP2A in regulating stress granule assembly.

•Whole-proteome in vitro phosphatase assay supports phosphatase-target site screening•In vitro and in vivo phosphoproteomics shortlist numerous bona fide PP2A target sites•PP2A complexes target phosphosites on protein translation machinery•PPP2R5E/B56ε-containing complexes negatively regulate stress granule formation

Whole-proteome in vitro phosphatase assay supports phosphatase-target site screening

In vitro and in vivo phosphoproteomics shortlist numerous bona fide PP2A target sites

PP2A complexes target phosphosites on protein translation machinery

PPP2R5E/B56ε-containing complexes negatively regulate stress granule formation

In vitro assays remain the gold standard for proving direct phosphatase-substrate interactions. Performing such assays on proteome scale under native conditions—i.e., retaining the enzymatic activities of endogenous protein complexes—is challenging, as primary and secondary effects are difficult to discriminate. We developed an on-bead in vitro phosphatase assay that supports the inactivation of endogenous phosphatases and the analysis of exogenous phosphatase-substrate interactions on proteome scale. By coupling this assay with quantitative phosphoproteomics, we screen for phosphatase-substrate interactions, identifying bona fide phosphatase target sites in high throughput.

Brunner et al. developed an on-bead in vitro phosphatase screen that uses whole-proteome lysates as input. In combination with in vivo phosphoproteomics, they outline the PP2A target repertoire. Focusing on PPP2R5E/B56ε-containing PP2A complexes, they highlight that PP2A negatively affects stress granule formation.

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