Cryo-EM and cellular dissection uncover versatile PME-1 activities in PP2A holoenzyme demethylation and inhibition

Abstract Protein phosphatase 2A (PP2A) methylesterase 1 (PME-1) is cancer-promoting but essential for development, and long-believed to remove carboxymethylation, a central modification, from the common PP2A core enzyme but not diverse holoenzymes that target broad cellular signaling by recognizing specific disordered motifs via regulatory subunits. On the contrary, our biochemical dissection and high-resolution cryo-EM structural analysis of a PP2A-B’ holoenzyme-PME-1 complex reveal that PME-1 disordered motifs, including phosphatase substrate-mimicking motifs, tether to the holoenzyme at remote sites, block its substrate binding, and allow large structural shifts in both holoenzyme and PME-1 to enable multiptle dynamic structured contacts and induce methylesterase activation toward the holoenzyme. PME-1 inhibitor and B’-interface mutations differentially modulate cellular PP2A methylation and allow us to uncover cellular PME-1 functions in AKT-p53 signaling. Our studies demonstrate how dynamic structured cores and disordered motifs create versatile activities and lay a foundation for investigating and targeting multifaceted activities toward broad PP2A complexes in cellular signaling.

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Protein interactomes of protein phosphatase 2A B55 regulatory subunits reveal B55-mediated regulation of replication protein A under replication stress

Scientific Reports ◽

10.1038/s41598-018-21040-6 ◽

2018 ◽

Vol 8 (1) ◽

Cited By ~ 7

Author(s):

Feifei Wang ◽

Songli Zhu ◽

Laura A. Fisher ◽

Weidong Wang ◽

Gregory G. Oakley ◽

...

Keyword(s):

Protein Phosphatase ◽

Protein Phosphatase 2A ◽

Protein A ◽

Replication Stress ◽

Replication Protein A ◽

Replication Protein ◽

Regulatory Subunits ◽

Phosphatase 2A

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Identification of Integrator-PP2A complex (INTAC), an RNA polymerase II phosphatase

Science ◽

10.1126/science.abb5872 ◽

2020 ◽

Vol 370 (6520) ◽

pp. eabb5872

Author(s):

Hai Zheng ◽

Yilun Qi ◽

Shibin Hu ◽

Xuan Cao ◽

Congling Xu ◽

...

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Protein Phosphatase ◽

Rna Cleavage ◽

Rna Transcripts ◽

Core Enzyme ◽

Repeat Domain ◽

Phosphatase 2A ◽

Carboxy Terminal ◽

Resolution Structure

The 14-subunit metazoan-specific Integrator contains an endonuclease that cleaves nascent RNA transcripts. Here, we identified a complex containing Integrator and protein phosphatase 2A core enzyme (PP2A-AC), termed INTAC. The 3.5-angstrom-resolution structure reveals that nine human Integrator subunits and PP2A-AC assemble into a cruciform-shaped central scaffold formed by the backbone and shoulder modules, with the phosphatase and endonuclease modules flanking the opposite sides. As a noncanonical PP2A holoenzyme, the INTAC complex dephosphorylates the carboxy-terminal repeat domain of RNA polymerase II at serine-2, -5, and -7 and thus regulates transcription. Our study extends the function of PP2A to transcriptional regulation and reveals how dual enzymatic activities—RNA cleavage and RNA polymerase II dephosphorylation—are structurally and functionally integrated into the INTAC complex.

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Protein phosphatase 2A: a highly regulated family of serine/threonine phosphatases implicated in cell growth and signalling

Biochemical Journal ◽

10.1042/bj3530417 ◽

2001 ◽

Vol 353 (3) ◽

pp. 417-439 ◽

Cited By ~ 727

Author(s):

Veerle JANSSENS ◽

Jozef GORIS

Keyword(s):

Protein Phosphatase ◽

Cellular Localization ◽

Protein Phosphatase 2A ◽

Viral Proteins ◽

Catalytic Subunit ◽

Post Translational Modification ◽

Regulatory Subunits ◽

Regulatory Ability ◽

Phosphatase 2A ◽

Cycle Regulation

Protein phosphatase 2A (PP2A) comprises a family of serine/threonine phosphatases, minimally containing a well conserved catalytic subunit, the activity of which is highly regulated. Regulation is accomplished mainly by members of a family of regulatory subunits, which determine the substrate specificity, (sub)cellular localization and catalytic activity of the PP2A holoenzymes. Moreover, the catalytic subunit is subject to two types of post-translational modification, phosphorylation and methylation, which are also thought to be important regulatory devices. The regulatory ability of PTPA (PTPase activator), originally identified as a protein stimulating the phosphotyrosine phosphatase activity of PP2A, will also be discussed, alongside the other regulatory inputs. The use of specific PP2A inhibitors and molecular genetics in yeast, Drosophila and mice has revealed roles for PP2A in cell cycle regulation, cell morphology and development. PP2A also plays a prominent role in the regulation of specific signal transduction cascades, as witnessed by its presence in a number of macromolecular signalling modules, where it is often found in association with other phosphatases and kinases. Additionally, PP2A interacts with a substantial number of other cellular and viral proteins, which are PP2A substrates, target PP2A to different subcellular compartments or affect enzyme activity. Finally, the de-regulation of PP2A in some specific pathologies will be touched upon.

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The variable subunit associated with protein phosphatase 2A0 defines a novel multimember family of regulatory subunits

Biochemical Journal ◽

10.1042/bj3170187 ◽

1996 ◽

Vol 317 (1) ◽

pp. 187-194 ◽

Cited By ~ 55

Author(s):

Stanislaw ZOLNIEROWICZ ◽

Christine VAN HOOF ◽

Nataša ANDJELKOVIĆ ◽

Peter CRON ◽

Ilse STEVENS ◽

...

Keyword(s):

Amino Acid ◽

Protein Phosphatase ◽

Amino Acid Sequences ◽

Regulatory Subunits ◽

Consensus Sequences ◽

Reverse Transcription Pcr ◽

Genes Encoding ◽

Phosphatase 2A ◽

Molecular Masses ◽

Specific Mrnas

Two protein phosphatase 2A (PP2A) holoenzymes were isolated from rabbit skeletal muscle containing, in addition to the catalytic and PR65 regulatory subunits, proteins of apparent molecular masses of 61 and 56 kDa respectively. Both holoenzymes displayed low basal phosphorylase phosphatase activity, which could be stimulated by protamine to an extent similar to that of previously characterized PP2A holoenzymes. Protein microsequencing of tryptic peptides derived from the 61 kDa protein, termed PR61, yielded 117 residues of amino acid sequence. Molecular cloning by enrichment of specific mRNAs, followed by reverse transcription–PCR and cDNA library screening, revealed that this protein exists in multiple isoforms encoded by at least three genes, one of which gives rise to several splicing variants. Comparisons of these sequences with the available databases identified one more human gene and predicted another based on a rabbit cDNA-derived sequence, thus bringing the number of genes encoding PR61 family members to five. Peptide sequences derived from PR61 corresponded to the deduced amino acid sequences of either α or β isoforms, indicating that the purified PP2A preparation was a mixture of at least two trimers. In contrast, the 56 kDa subunit (termed PR56) seems to correspond to the ϵ isoform of PR61. Several regulatory subunits of PP2A belonging to the PR61 family contain consensus sequences for nuclear localization and might therefore target PP2A to nuclear substrates.

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