Protocol to assess substrate dephosphorylation by serine/threonine phosphoprotein phosphatases in vitro

Jason S. Wasserman; Felicity Feiser; Seren Palacio; Kishan Patel; Joy Gonzalez; Holly Fowle; Xavier Graña

doi:10.1016/j.xpro.2023.102148

Protocol to assess substrate dephosphorylation by serine/threonine phosphoprotein phosphatases in vitro

Jason S. Wasserman, Felicity Feiser, Seren Palacio, Kishan Patel, Joy Gonzalez, Holly Fowle, Xavier Graña

Temple University

Research output: Contribution to journal › Article › peer-review

1 Scopus citations

Abstract

Serine/threonine protein phosphatase 2 (PP2A) forms heterotrimeric holoenzymes, where a scaffold subunit bridges the PP2A catalytic subunit to a B regulatory subunit, e.g., B55α. The PP2A/B55α holoenzyme plays key roles in signaling and cell-cycle control targeting multiple substrates. Here, we describe semiquantitative approaches to determine PP2A/B55α substrate specificity. Parts I and II detail approaches to assess PP2A/B55α-mediated dephosphorylation of immobilized substrate peptide variants. Parts III and IV detail methods to assess PP2A/B55α-substrate-binding specificity. These approaches are adaptable to other serine/threonine phosphatases. For complete details on the use and execution of this protocol, please refer to Fowle et al.¹

Original language	English
Article number	102148
Pages (from-to)	102148
Journal	STAR Protocols
Volume	4
Issue number	2
Early online date	Mar 18 2023
DOIs	https://doi.org/10.1016/j.xpro.2023.102148
State	Published - Jun 16 2023

Keywords

Cell Culture
Chemistry
Molecular Biology
Protein Biochemistry
Protein Expression and Purification
Signal Transduction

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10.1016/j.xpro.2023.102148

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title = "Protocol to assess substrate dephosphorylation by serine/threonine phosphoprotein phosphatases in vitro",

abstract = "Serine/threonine protein phosphatase 2 (PP2A) forms heterotrimeric holoenzymes, where a scaffold subunit bridges the PP2A catalytic subunit to a B regulatory subunit, e.g., B55α. The PP2A/B55α holoenzyme plays key roles in signaling and cell-cycle control targeting multiple substrates. Here, we describe semiquantitative approaches to determine PP2A/B55α substrate specificity. Parts I and II detail approaches to assess PP2A/B55α-mediated dephosphorylation of immobilized substrate peptide variants. Parts III and IV detail methods to assess PP2A/B55α-substrate-binding specificity. These approaches are adaptable to other serine/threonine phosphatases. For complete details on the use and execution of this protocol, please refer to Fowle et al.1",

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AU - Wasserman, Jason S.

AU - Feiser, Felicity

AU - Palacio, Seren

AU - Patel, Kishan

AU - Gonzalez, Joy

AU - Fowle, Holly

AU - Graña, Xavier

PY - 2023/6/16

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AB - Serine/threonine protein phosphatase 2 (PP2A) forms heterotrimeric holoenzymes, where a scaffold subunit bridges the PP2A catalytic subunit to a B regulatory subunit, e.g., B55α. The PP2A/B55α holoenzyme plays key roles in signaling and cell-cycle control targeting multiple substrates. Here, we describe semiquantitative approaches to determine PP2A/B55α substrate specificity. Parts I and II detail approaches to assess PP2A/B55α-mediated dephosphorylation of immobilized substrate peptide variants. Parts III and IV detail methods to assess PP2A/B55α-substrate-binding specificity. These approaches are adaptable to other serine/threonine phosphatases. For complete details on the use and execution of this protocol, please refer to Fowle et al.1

KW - Cell Culture

KW - Chemistry

KW - Molecular Biology

KW - Protein Biochemistry

KW - Protein Expression and Purification

KW - Signal Transduction

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ER -

Protocol to assess substrate dephosphorylation by serine/threonine phosphoprotein phosphatases in vitro

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