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

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journal contribution
posted on 05.11.2020, 13:17 by Roman O Fedoryshchak, Magdalena Přechová, Abbey M Butler, Rebecca Lee, Nicola O'Reilly, Helen R Flynn, Ambrosius P Snijders, Noreen Eder, Sila Ultanir, Stephane Mouilleron, Richard Treisman
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.


Crick (Grant ID: 10201, Grant title: Ultanir FC001201) Crick (Grant ID: 10190, Grant title: Treisman FC001190) Crick (Grant ID: 10013, Grant title: O'Reilly FC001999) Crick (Grant ID: 10011, Grant title: Snijders FC001999)