PP2A/B55α substrate recruitment as defined by the retinoblastoma-related protein p107

  1. Holly Fowle
  2. Ziran Zhao
  3. Qifang Xu
  4. Jason S Wasserman
  5. Xinru Wang
  6. Mary Adeyemi
  7. Felicity Feiser
  8. Alison N Kurimchak
  9. Diba Atar
  10. Brennan C McEwan
  11. Arminja N Kettenbach
  12. Rebecca Page
  13. Wolfgang Peti
  14. Roland L Dunbrack Jr.
  15. Xavier Graña  Is a corresponding author
  1. Temple University Lewis Katz School of Medicine, United States
  2. Fox Chase Cancer Center, United States
  3. University of Arizona, United States
  4. Hitchcock Medical Center at Dartmouth, United States
  5. UConn Health, United States

Abstract

Protein phosphorylation is a reversible post-translation modification essential in cell signaling. This study addresses a long-standing question as to how the most abundant serine/threonine Protein Phosphatase 2 (PP2A) holoenzyme, PP2A/B55α, specifically recognizes substrates and presents them to the enzyme active site. Here, we show how the PP2A regulatory subunit B55α recruits p107, a pRB-related tumor suppressor and B55α substrate. Using molecular and cellular approaches, we identified a conserved region 1 (R1, residues 615-626) encompassing the strongest p107 binding site. This enabled us to identify an 'HxRVxxV619-625' short linear motif (SLiM) in p107 as necessary for B55α binding and dephosphorylation of the proximal pSer-615 in vitro and in cells. Numerous B55α/PP2A substrates, including TAU, contain a related SLiM C-terminal from a proximal phosphosite, 'p[ST]-P-x(4,10)-[RK]-V-x-x-[VI]-R'. Mutation of conserved SLiM residues in TAU dramatically inhibits dephosphorylation by PP2A/B55α, validating its generality. A data-guided computational model details the interaction of residues from the conserved p107 SLiM, the B55α groove, and phosphosite presentation. Altogether these data provide key insights into PP2A/B55α mechanisms of substrate recruitment and active site engagement, and also facilitate identification and validation of new substrates, a key step towards understanding PP2A/B55α role in multiple cellular processes.

Data availability

Raw MS data for the the data depicted in Figure 6B are available at MassIVEhttps://massive.ucsd.edu/ProteoSAFe/dataset.jsp?task=9c21e08f6a524d7097e8bd45f0d2f375PXD028612.All NMR chemical shifts (Figure 1E-F) have been deposited in the BioMagResBank (BMRB: 28091).Source code folder (PeptideDock_sourceCode) for Figure 7 is a C# project, including retrieval of peptide structures from PDB and other sources such as PISCES, and calculation of distances and data analyses. https://github.com/DunbrackLab/PP2A_PeptideDock.All other data generated or analysed during this study are included in the manuscript and supporting files. Source Data files have been provided.

The following data sets were generated
The following previously published data sets were used

Article and author information

Author details

  1. Holly Fowle

    Fels Institute for Cancer Research and Molecular Biology, Temple University Lewis Katz School of Medicine, Philadelphia, United States
    Competing interests
    The authors declare that no competing interests exist.
  2. Ziran Zhao

    Fels Institute for Cancer Research and Molecular Biology, Temple University Lewis Katz School of Medicine, Philadelphia, United States
    Competing interests
    The authors declare that no competing interests exist.
  3. Qifang Xu

    Institute for Cancer Research, Fox Chase Cancer Center, Philadelphia, United States
    Competing interests
    The authors declare that no competing interests exist.
  4. Jason S Wasserman

    Fels Institute for Cancer Research and Molecular Biology, Temple University Lewis Katz School of Medicine, Philadelphia, United States
    Competing interests
    The authors declare that no competing interests exist.
  5. Xinru Wang

    Department of Chemistry and Biochemistry, University of Arizona, Tucson, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-5994-707X
  6. Mary Adeyemi

    Fels Institute for Cancer Research and Molecular Biology, Temple University Lewis Katz School of Medicine, Philadelphia, United States
    Competing interests
    The authors declare that no competing interests exist.
  7. Felicity Feiser

    Fels Institute for Cancer Research and Molecular Biology, Temple University Lewis Katz School of Medicine, Philadelphia, United States
    Competing interests
    The authors declare that no competing interests exist.
  8. Alison N Kurimchak

    Fels Institute for Cancer Research and Molecular Biology, Temple University Lewis Katz School of Medicine, Philadelphia, United States
    Competing interests
    The authors declare that no competing interests exist.
  9. Diba Atar

    Fels Institute for Cancer Research and Molecular Biology, Temple University Lewis Katz School of Medicine, Philadelphia, United States
    Competing interests
    The authors declare that no competing interests exist.
  10. Brennan C McEwan

    Department of Biochemistry and Cell Biology, Hitchcock Medical Center at Dartmouth, Lebanon, United States
    Competing interests
    The authors declare that no competing interests exist.
  11. Arminja N Kettenbach

    Department of Biochemistry and Cell Biology, Hitchcock Medical Center at Dartmouth, Lebanon, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-3979-4576
  12. Rebecca Page

    Department Cell Biology,, UConn Health, Farmington, United States
    Competing interests
    The authors declare that no competing interests exist.
  13. Wolfgang Peti

    6Department Molecular Biology and Biophysics, UConn Health, Farmington, United States
    Competing interests
    The authors declare that no competing interests exist.
  14. Roland L Dunbrack Jr.

    Institute for Cancer Research, Fox Chase Cancer Center, Philadelphia, United States
    Competing interests
    The authors declare that no competing interests exist.
  15. Xavier Graña

    Fels Institute for Cancer Research and Molecular Biology, Temple University Lewis Katz School of Medicine, Philadelphia, United States
    For correspondence
    xgrana@temple.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-7134-0473

Funding

National Institute of General Medical Sciences (R01 GM117437)

  • Xavier Graña

National Cancer Institute (R03 CA216134-01)

  • Xavier Graña

WW Smith charitable Trust Award (no reference number)

  • Xavier Graña

National Cancer Institute (P30 CA006927)

  • Roland L Dunbrack Jr.
  • Xavier Graña

National Cancer Institute (U54 CA221704)

  • Holly Fowle
  • Ziran Zhao

National Institute of General Medical Sciences (R01GM134683)

  • Wolfgang Peti

National Institute of Neurological Disorders and Stroke (R01NS091336)

  • Wolfgang Peti

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Copyright

© 2021, Fowle et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

Metrics

  • 2,298
    views
  • 307
    downloads
  • 24
    citations

Views, downloads and citations are aggregated across all versions of this paper published by eLife.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  1. Holly Fowle
  2. Ziran Zhao
  3. Qifang Xu
  4. Jason S Wasserman
  5. Xinru Wang
  6. Mary Adeyemi
  7. Felicity Feiser
  8. Alison N Kurimchak
  9. Diba Atar
  10. Brennan C McEwan
  11. Arminja N Kettenbach
  12. Rebecca Page
  13. Wolfgang Peti
  14. Roland L Dunbrack Jr.
  15. Xavier Graña
(2021)
PP2A/B55α substrate recruitment as defined by the retinoblastoma-related protein p107
eLife 10:e63181.
https://doi.org/10.7554/eLife.63181

Share this article

https://doi.org/10.7554/eLife.63181

Further reading

    1. Biochemistry and Chemical Biology
    Jianheng Fox Liu, Ben R Hawley ... Samie R Jaffrey
    Tools and Resources

    N 6,2’-O-dimethyladenosine (m6Am) is a modified nucleotide located at the first transcribed position in mRNA and snRNA that is essential for diverse physiological processes. m6Am mapping methods assume each gene uses a single start nucleotide. However, gene transcription usually involves multiple start sites, generating numerous 5’ isoforms. Thus, gene-level annotations cannot capture the diversity of m6Am modification in the transcriptome. Here, we describe CROWN-seq, which simultaneously identifies transcription-start nucleotides and quantifies m6Am stoichiometry for each 5’ isoform that initiates with adenosine. Using CROWN-seq, we map the m6Am landscape in nine human cell lines. Our findings reveal that m6Am is nearly always a high stoichiometry modification, with only a small subset of cellular mRNAs showing lower m6Am stoichiometry. We find that m6Am is associated with increased transcript expression and provide evidence that m6Am may be linked to transcription initiation associated with specific promoter sequences and initiation mechanisms. These data suggest a potential new function for m6Am in influencing transcription.

    1. Biochemistry and Chemical Biology
    2. Microbiology and Infectious Disease
    Eva Herdering, Tristan Reif-Trauttmansdorff ... Ruth Anne Schmitz
    Research Article

    Glutamine synthetases (GS) are central enzymes essential for the nitrogen metabolism across all domains of life. Consequently, they have been extensively studied for more than half a century. Based on the ATP-dependent ammonium assimilation generating glutamine, GS expression and activity are strictly regulated in all organisms. In the methanogenic archaeon Methanosarcina mazei, it has been shown that the metabolite 2-oxoglutarate (2-OG) directly induces the GS activity. Besides, modulation of the activity by interaction with small proteins (GlnK1 and sP26) has been reported. Here, we show that the strong activation of M. mazei GS (GlnA1) by 2-OG is based on the 2-OG dependent dodecamer assembly of GlnA1 by using mass photometry (MP) and single particle cryo-electron microscopy (cryo-EM) analysis of purified strep-tagged GlnA1. The dodecamer assembly from dimers occurred without any detectable intermediate oligomeric state and was not affected in the presence of GlnK1. The 2.39 Å cryo-EM structure of the dodecameric complex in the presence of 12.5 mM 2-OG demonstrated that 2-OG is binding between two monomers. Thereby, 2-OG appears to induce the dodecameric assembly in a cooperative way. Furthermore, the active site is primed by an allosteric interaction cascade caused by 2-OG-binding towards an adaption of an open active state conformation. In the presence of additional glutamine, strong feedback inhibition of GS activity was observed. Since glutamine dependent disassembly of the dodecamer was excluded by MP, feedback inhibition most likely relies on the binding of glutamine to the catalytic site. Based on our findings, we propose that under nitrogen limitation the induction of M. mazei GS into a catalytically active dodecamer is not affected by GlnK1 and crucially depends on the presence of 2-OG.