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

STAR Protoc. 2023 Apr 18;4(2):102148. doi: 10.1016/j.xpro.2023.102148. Online ahead of print.

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.

Keywords: Cell Culture; Chemistry; Molecular Biology; Protein Biochemistry; Protein Expression and Purification; Signal Transduction.