Regulation of PP2A, PP4, and PP6 holoenzyme assembly by carboxyl-terminal methylation

Scott P Lyons,Mark E Adamo,Elora C Greiner,Arminja N Kettenbach,Lauren E Cressey

doi:10.1038/s41598-021-02456-z

Scott P Lyons, Mark E Adamo + Show 3 more

Open Access

https://doi.org/10.1038/s41598-021-02456-z

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Abstract

The family of Phosphoprotein Phosphatases (PPPs) is responsible for most cellular serine and threonine dephosphorylation. PPPs achieve substrate specificity and selectivity by forming multimeric holoenzymes. PPP holoenzyme assembly is tightly controlled, and changes in the cellular repertoire of PPPs are linked to human disease, including cancer and neurodegeneration. For PP2A, PP4, and PP6, holoenzyme formation is in part regulated by carboxyl (C)-terminal methyl-esterification (often referred to as “methylation”). Here, we use mass spectrometry-based proteomics, methylation-ablating mutations, and genome editing to elucidate the role of C-terminal methylation on PP2A, PP4, and PP6 holoenzyme assembly. We find that the catalytic subunits of PP2A, PP4, and PP6 are frequently methylated in cancer cells and that deletion of the C-terminal leucine faithfully recapitulates loss of methylation. We observe that loss of PP2A methylation consistently reduced B55, B56, and B72 regulatory subunit binding in cancer and non-transformed cell lines. However, Striatin subunit binding is only affected in non-transformed cells. For PP4, we find that PP4R1 and PP4R3β bind in a methylation-dependent manner. Intriguingly, loss of methylation does not affect PP6 holoenzymes. Our analyses demonstrate in an unbiased, comprehensive, and isoform-specific manner the crucial regulatory function of endogenous PPP methylation in transformed and non-transformed cell lines.

Highlights

Holoenzyme assembly is, in part, regulated by post-translational modifications (PTMs) of the catalytic subunits[15,16]
We previously described a chemical proteomics approach to enrich, identify, and quantify endogenous PPPs from cells and tissues that we refer to as Phosphatase Inhibitor Beads combined with Mass Spectrometry (PIB-MS)[32]
To investigate C-terminal methylation of endogenous PP2Acα/β, PP4c, PP6c subunits, we enriched PPPs from the human cancer cell lines SW1088, C32, U-87, MCF-7, CAL-51 using PIBs, digested the isolated proteins into peptides, and analyzed them by liquid-chromatography coupled with tandem mass spectrometry (LC–MS/MS) (Fig. 1B)

Summary

Introduction

Holoenzyme assembly is, in part, regulated by post-translational modifications (PTMs) of the catalytic subunits[15,16]. The deletion of the C-terminal leucine (ΔL309 mutation) of PP2A is frequently used as a mimetic of the unmethylated form This mutation reduces the interaction of PP2Acα with B55, B56, and B72 subunits, while binding of the striatin family is increased[16,27]. For PP4c, loss of methylation reduces PP4R1 binding, while PP6 holoenzyme components bind PP6c independent of m ethylation[21] These results demonstrate the important role of C-terminal methylation in PP2A and PP4 holoenzyme assembly. They qualitatively establish the ability of regulatory subunits to bind to either methylated or unmethylated PP2Acα/β. We generalize the role of C-terminal methylation in holoenzymes assembly across different transformed or non-transformed cell lines

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