A substrate-trapping strategy for protein phosphatase PP1 holoenzymes using hypoactive subunit fusions
| Autor: | Dan Wu, Veerle De Wever, Rita Derua, Aleyde Van Eynde, Mathieu Bollen, Monique Beullens, Claudia Winkler |
|---|---|
| Rok vydání: | 2018 |
| Předmět: |
0301 basic medicine
substrate specificity MYPT1 Biochemistry environment and public health Substrate Specificity Holoenzymes Protein Phosphatase 1 Chlorocebus aethiops CATALYTIC SUBUNIT Phosphorylation SPECIFICITY chemistry.chemical_classification phosphoprotein phosphatase 1 (PP1) Methods and Resources Cell biology PHOSPHORYLATION SITES COS Cells RepoMan REPO-MAN Signal transduction Life Sciences & Biomedicine signal transduction Protein Binding Biochemistry & Molecular Biology animal structures substrate mapping Protein subunit Phosphatase macromolecular substances phosphatase 03 medical and health sciences NUCLEAR INHIBITOR SPLICING FACTORS PNUTS KINASE Animals Humans Immunoprecipitation REGULATORY SUBUNIT Molecular Biology Protein dephosphorylation Cell Nucleus Science & Technology Binding Sites IDENTIFICATION HEK 293 cells fungi Substrate (chemistry) Cell Biology Receptors Neuropeptide Y enzyme enzymes and coenzymes (carbohydrates) 030104 developmental biology Enzyme HEK293 Cells chemistry NIPP1 |
| Zdroj: | The Journal of biological chemistry. 293(39) |
| ISSN: | 1083-351X |
| Popis: | 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. ispartof: JOURNAL OF BIOLOGICAL CHEMISTRY vol:293 issue:39 pages:15152-15162 ispartof: location:United States status: published |
| Databáze: | OpenAIRE |
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