Accéder au contenu
Merck

A substrate-trapping strategy for protein phosphatase PP1 holoenzymes using hypoactive subunit fusions.

The Journal of biological chemistry (2018-08-18)
Dan Wu, Veerle De Wever, Rita Derua, Claudia Winkler, Monique Beullens, Aleyde Van Eynde, Mathieu Bollen
RÉSUMÉ

The protein Ser/Thr phosphatase PP1 catalyzes an important fraction of protein dephosphorylation events and forms highly specific holoenzymes through an association with regulatory interactors of protein phosphatase one (RIPPOs). The functional characterization of individual PP1 holoenzymes is hampered by the lack of straightforward strategies for substrate mapping. Because efficient substrate recruitment often involves binding to both PP1 and its associated RIPPO, here we examined whether PP1-RIPPO fusions can be used to trap substrates for further analysis. Fusions of an hypoactive point mutant of PP1 and either of four tested RIPPOs accumulated in HEK293T cells with their associated substrates and were co-immunoprecipitated for subsequent identification of the substrates by immunoblotting or MS analysis. Hypoactive fusions were also used to study RIPPOs themselves as substrates for associated PP1. In contrast, substrate trapping was barely detected with active PP1-RIPPO fusions or with nonfused PP1 or RIPPO subunits. Our results suggest that hypoactive fusions of PP1 subunits represent an easy-to-use tool for substrate identification of individual holoenzymes.

MATÉRIAUX
Référence du produit
Marque
Description du produit

Sigma-Aldrich
Anti-Histone H3 antibody produced in rabbit, IgG fraction of antiserum, buffered aqueous solution
Sigma-Aldrich
Anticorps anti-phospho-Ser/Thr-Pro MPM-2, clone MPM-2, Upstate®, from mouse
Sigma-Aldrich
Anticorps anti-phospho-histone H3 (Thr3), Upstate®, from rabbit
Sigma-Aldrich
Anti-PPP1R8 antibody produced in rabbit, Prestige Antibodies® Powered by Atlas Antibodies, affinity isolated antibody, buffered aqueous glycerol solution, Ab3