Molecular basis for substrate specificity of the Phactr1/PP1 phosphatase holoenzyme

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 controlle...

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Published in:eLife 2020-09, Vol.9
Main Authors: Fedoryshchak, Roman O, Přechová, Magdalena, Butler, Abbey M, Lee, Rebecca, O'Reilly, Nicola, Flynn, Helen R, Snijders, Ambrosius P, Eder, Noreen, Ultanir, Sila, Mouilleron, Stephane, Treisman, Richard
Format: Article
Language:English
Subjects:
actin
Animals
Biochemistry and Chemical Biology
Catalytic Domain
Crystallization
cytoskeleton
Cytoskeleton - metabolism
Holoenzymes - chemistry
Holoenzymes - metabolism
IRSp53
Mice
Microfilament Proteins - chemistry
Microfilament Proteins - metabolism
Nerve Tissue Proteins - metabolism
Phactr1
Phosphatases
Phosphates
Phosphates - metabolism
Protein Conformation
Protein Phosphatase 1
Proteins
RPEL
Spectrin - metabolism
Structural Biology and Molecular Biophysics
Substrate Specificity
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Summary: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 αII. 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.
ISSN:2050-084X
2050-084X
DOI:10.7554/eLife.61509