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

The Journal of biological chemistry 2018 Sep 28

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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. © 2018 Wu et al.

Citation

Dan Wu, Veerle De Wever, Rita Derua, Claudia Winkler, Monique Beullens, Aleyde Van Eynde, Mathieu Bollen. A substrate-trapping strategy for protein phosphatase PP1 holoenzymes using hypoactive subunit fusions. The Journal of biological chemistry. 2018 Sep 28;293(39):15152-15162