Phosphatase specificity principles uncovered by MRBLE:Dephos and global substrate identification.
Date
2023-12-06ICR Author
Author
Hein, JB
Nguyen, HT
Garvanska, DH
Nasa, I
Kruse, T
Feng, Y
Lopez Mendez, B
Davey, N
Kettenbach, AN
Fordyce, PM
Nilsson, J
Type
Journal Article
Metadata
Show full item recordAbstract
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.
Collections
Subject
MRBLE-Pep
MRBLE:Dephos
mitotic exit
protein phosphatase
substrates
Phosphorylation
Protein Phosphatase 2
Mitosis
Signal Transduction
Substrate Specificity
Research team
Short Linear Motif
Language
eng
Date accepted
2023-10-16
License start date
2023-12-06
Citation
Molecular Systems Biology, 2023, 19 (12), pp. e11782 -
Publisher
SPRINGERNATURE