Signalling and Transcription Laboratory, The Francis Crick Institute, London, United Kingdom; Structural Biology Science Technology Platform, The Francis Crick Institute, London, United Kingdom
Rebecca Lee
Signalling and Transcription Laboratory, The Francis Crick Institute, London, United Kingdom; Structural Biology Science Technology Platform, The Francis Crick Institute, London, United Kingdom
Proteomics Science Technology Platform, The Francis Crick Institute, London, United Kingdom
Noreen Eder
Proteomics Science Technology Platform, The Francis Crick Institute, London, United Kingdom; Kinases and Brain Development Laboratory The Francis Crick Institute, London, United Kingdom
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.
