Journal article
Reduction of protein phosphatase 2A (PP2A) complexity reveals cellular functions and dephosphorylation motifs of the PP2A/B′δ holoenzyme
The Journal of biological chemistry, Vol.295(17), pp.5654-5668
04/24/2020
DOI: 10.1074/jbc.RA119.011270
PMID: 32156701
Abstract
Protein phosphatase 2A (PP2A) is a large enzyme family responsible for most cellular Ser/Thr dephosphorylation events. PP2A substrate specificity, localization, and regulation by second messengers rely on more than a dozen regulatory subunits (including B/R2, B′/R5, and B″/R3), which form the PP2A heterotrimeric holoenzyme by associating with a dimer comprising scaffolding (A) and catalytic (C) subunits. Because of partial redundancy and high endogenous expression of PP2A holoenzymes, traditional approaches of overexpressing, knocking down, or knocking out PP2A regulatory subunits have yielded only limited insights into their biological roles and substrates. To this end, here we sought to reduce the complexity of cellular PP2A holoenzymes. We used tetracycline-inducible expression of pairs of scaffolding and regulatory subunits with complementary charge-reversal substitutions in their interaction interfaces. For each of the three regulatory subunit families, we engineered A/B charge–swap variants that could bind to one another, but not to endogenous A and B subunits. Because endogenous Aα was targeted by a co-induced shRNA, endogenous B subunits were rapidly degraded, resulting in expression of predominantly a single PP2A heterotrimer composed of the A/B charge–swap pair and the endogenous catalytic subunit. Using B′δ/PPP2R5D, we show that PP2A complexity reduction, but not PP2A overexpression, reveals a role of this holoenzyme in suppression of extracellular signal–regulated kinase signaling and protein kinase A substrate dephosphorylation. When combined with global phosphoproteomics, the PP2A/B′δ reduction approach identified consensus dephosphorylation motifs in its substrates and suggested that residues surrounding the phosphorylation site play roles in PP2A substrate specificity.
Details
- Title: Subtitle
- Reduction of protein phosphatase 2A (PP2A) complexity reveals cellular functions and dephosphorylation motifs of the PP2A/B′δ holoenzyme
- Creators
- Chian Ju Jong - Department of Neuroscience and Pharmacology and Iowa Neuroscience Institute, University of Iowa, Iowa City, Iowa 52242Ronald A Merrill - Department of Neuroscience and Pharmacology and Iowa Neuroscience Institute, University of Iowa, Iowa City, Iowa 52242Emily M Wilkerson - Michael Hooker Proteomics Facility, University of North Carolina, Chapel Hill, North Carolina 27516Laura E Herring - Michael Hooker Proteomics Facility, University of North Carolina, Chapel Hill, North Carolina 27516Lee M Graves - Michael Hooker Proteomics Facility, University of North Carolina, Chapel Hill, North Carolina 27516Stefan Strack - Department of Neuroscience and Pharmacology and Iowa Neuroscience Institute, University of Iowa, Iowa City, Iowa 52242
- Resource Type
- Journal article
- Publication Details
- The Journal of biological chemistry, Vol.295(17), pp.5654-5668
- DOI
- 10.1074/jbc.RA119.011270
- PMID
- 32156701
- NLM abbreviation
- J Biol Chem
- ISSN
- 0021-9258
- eISSN
- 1083-351X
- Publisher
- Elsevier Inc
- Grant note
- DOI: 10.13039/100000002, name: HHS | National Institutes of Health, award: NS056244, NS087908; name: Jordan's Guardian Angels; DOI: 10.13039/100001024, name: Roy J. Carver Charitable Trust, award: 17–4885
- Language
- English
- Date published
- 04/24/2020
- Academic Unit
- Molecular Physiology and Biophysics; Pathology; Iowa Neuroscience Institute; Fraternal Order of Eagles Diabetes Research Center; Neuroscience and Pharmacology
- Record Identifier
- 9984071794602771
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