TY - JOUR TI - Molecular basis for substrate specificity of the Phactr1/PP1 phosphatase holoenzyme AU - Fedoryshchak, Roman O AU - Přechová, Magdalena AU - Butler, Abbey M AU - Lee, Rebecca AU - O'Reilly, Nicola AU - Flynn, Helen R AU - Snijders, Ambrosius P AU - Eder, Noreen AU - Ultanir, Sila AU - Mouilleron, Stephane AU - Treisman, Richard A2 - Hunter, Tony A2 - Cooper, Jonathan A A2 - Hunter, Tony A2 - Barford, David VL - 9 PY - 2020 DA - 2020/09/25 SP - e61509 C1 - eLife 2020;9:e61509 DO - 10.7554/eLife.61509 UR - https://doi.org/10.7554/eLife.61509 AB - 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. KW - Phactr1 KW - Protein Phosphatase 1 KW - RPEL KW - actin KW - cytoskeleton KW - IRSp53 JF - eLife SN - 2050-084X PB - eLife Sciences Publications, Ltd ER -