Phosphatase specificity principles uncovered by MRBLE:Dephos and global substrate identification.
MRBLE-Pep
MRBLE:Dephos
mitotic exit
protein phosphatase
substrates
Journal
Molecular systems biology
ISSN: 1744-4292
Titre abrégé: Mol Syst Biol
Pays: England
ID NLM: 101235389
Informations de publication
Date de publication:
06 Dec 2023
06 Dec 2023
Historique:
revised:
14
10
2023
received:
21
05
2023
accepted:
16
10
2023
medline:
7
12
2023
pubmed:
2
11
2023
entrez:
2
11
2023
Statut:
ppublish
Résumé
Phosphoprotein phosphatases (PPPs) regulate major signaling pathways, but the determinants of phosphatase specificity are poorly understood. This is because methods to investigate this at scale are lacking. Here, we develop a novel in vitro assay, MRBLE:Dephos, that allows multiplexing of dephosphorylation reactions to determine phosphatase preferences. Using MRBLE:Dephos, we establish amino acid preferences of the residues surrounding the dephosphorylation site for PP1 and PP2A-B55, which reveals common and unique preferences. To compare the MRBLE:Dephos results to cellular substrates, we focused on mitotic exit that requires extensive dephosphorylation by PP1 and PP2A-B55. We use specific inhibition of PP1 and PP2A-B55 in mitotic exit lysates coupled with phosphoproteomics to identify more than 2,000 regulated sites. Importantly, the sites dephosphorylated during mitotic exit reveal key signatures that are consistent with MRBLE:Dephos. Furthermore, integration of our phosphoproteomic data with mitotic interactomes of PP1 and PP2A-B55 provides insight into how binding of phosphatases to substrates shapes dephosphorylation. Collectively, we develop novel approaches to investigate protein phosphatases that provide insight into mitotic exit regulation.
Identifiants
pubmed: 37916966
doi: 10.15252/msb.202311782
pmc: PMC10698503
doi:
Substances chimiques
Protein Phosphatase 2
EC 3.1.3.16
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
e11782Subventions
Organisme : NIGMS NIH HHS
ID : DP2 GM123641
Pays : United States
Organisme : NCI NIH HHS
ID : P30 CA023108
Pays : United States
Organisme : NIGMS NIH HHS
ID : R35 GM119455
Pays : United States
Informations de copyright
© 2023 The Authors. Published under the terms of the CC BY 4.0 license.
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