Molecular basis for substrate specificity of the Phactr1/PP1 phosphatase holoenzyme
- Francis Crick Institute, United Kingdom
Abstract
PPP-family phosphatases such as PP1 have little intrinsic specificity. Cofactors can target PP1 to substrates or subcellular locations, but it remains unclear how they might confer sequence-specificity on PP1. The cytoskeletal regulator Phactr1 is a neuronally-enriched PP1 cofactor that is controlled by G-actin. Structural analysis showed that Phactr1 binding remodels PP1's hydrophobic groove, creating a new composite surface adjacent to the catalytic site. Using phosphoproteomics, we identified mouse fibroblast and neuronal Phactr1/PP1 substrates, which include cytoskeletal components and regulators. We determined high-resolution structures of Phactr1/PP1 bound to the dephosphorylated forms of its substrates IRSp53 and spectrin aII. Inversion of the phosphate in these holoenzyme-product complexes supports the proposed PPP-family catalytic mechanism. Substrate sequences C-terminal to the dephosphorylation site make intimate contacts with the composite Phactr1/PP1 surface, which are required for efficient dephosphorylation. Sequence specificity explains why Phactr1/PP1 exhibits orders-of-magnitude enhanced reactivity towards its substrates, compared to apo-PP1 or other PP1 holoenzymes.
Data availability
Diffraction data have been deposited in PDB under the accession code 6ZEE, 6ZEF, 6ZEG, 6ZEH, 6ZEI, 6ZEJ.The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the PRIDE partner repository with the dataset identifiers PXD019977 and PXD019882.All data generated or analysed during this study are included in the manuscript and supporting files.
Article and author information
Author details
Roman O Fedoryshchak
Magdalena Přechová
Funding
H2020 European Research Council (268690)
- Richard Treisman
Cancer Research UK (FC001-190)
- Sila Ultanir
- Richard Treisman
Medical Research Council (FC001-190)
- Sila Ultanir
- Richard Treisman
Medical Research Council (FC001-190)
- Sila Ultanir
- Richard Treisman
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Ethics
Animal experimentation: Animal experimentation complied with all ethical regulations and was carried out under the UK Home Office Project licence P7C307997 in the Crick Biological Research Facilities.
Copyright
© 2020, Fedoryshchak et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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Molecular basis for substrate specificity of the Phactr1/PP1 phosphatase holoenzyme
eLife 9:e61509.
https://doi.org/10.7554/eLife.61509
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