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

Content Provider	Journal of Biological Chemistry (JBC)
Author	Wu, Dan Wever, Veerle De Derua, Rita Winkler, Claudia Beullens, Monique Eynde, Aleyde Van Bollen, Mathieu
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.
Related Links	http://www.jbc.org/content/293/39/15152.abstract
Ending Page	15162
Starting Page	15152
Page Count	11
File Format	HTM / HTML PDF
ISSN	00219258
Journal	Journal of Biological Chemistry (JBC)
Issue Number	39
Volume Number	293
DOI	10.1074/jbc.RA118.004132
e-ISSN	1083351X
Language	English
Publisher	American Society for Biochemistry and Molecular Biology
Publisher Date	2018-09-28
Access Restriction	Open
Subject Keyword	Phosphoprotein phosphatase 1 (PP1) Enzyme Substrate specificity Signal transduction Phosphatase MYPT1 NIPP1 PNUTS RepoMan Substrate mapping Enzymology
Content Type	Text
Resource Type	Article
Subject	Cell Biology Biochemistry Molecular Biology

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