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A Substrate-Trapping Strategy for Protein Phosphatase PP1 Holoenzymes Using Hypoactive Subunit Fusions

A Substrate-Trapping Strategy for Protein Phosphatase PP1 Holoenzymes Using Hypoactive Subunit Fusions

Dan Wu, Veerle De Wever, Rita Derua, Claudia Winkler, Monique Beullens, Aleyde Van Eynde, Mathieu Bollen

J Biol Chem. 2018 Sep 28;293(39):15152-15162.

PMID: 30115685

Abstract:

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.

Chemicals Related in the Paper:

Catalog Number	Product Name	Structure	CAS Number	Price
AP172889268-A	PP1		172889-26-8	Price

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