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

Roman O Fedoryshchak,Ambrosius P Snijders,Sila Ultanir,Richard Treisman,Helen R Flynn,Nicola O'Reilly,Noreen Eder,Abbey M Butler,Rebecca Lee,Stephane Mouilleron,Magdalena Přechová

doi:10.7554/elife.61509

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 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.

Highlights

PPP family phosphatases are metalloenzymes that carry out the majority of protein serine/threonine dephosphorylation (Brautigan and Shenolikar, 2018)
As will be described below, the pH8.5 structure is adopted by Phactr1(516580)/PP1a(7-300) when substrates occupy the Phosphatase 1 (PP1) active site; it is supported by Bio-layer interferometry (BLI) data, and is likely to exist at physiological pH
Interaction with Phactr1 confers substrate specificity on PP1 Since the composite hydrophobic surface of the Phactr1/PP1 holoenzyme plays an important role in substrate binding and catalytic efficiency, we investigated to what extent interaction with Phactr1 confers substrate specificity on

Summary

Introduction

PPP family phosphatases are metalloenzymes that carry out the majority of protein serine/threonine dephosphorylation (Brautigan and Shenolikar, 2018). The PP1 catalytic site lies at the intersection of three putative substrate-binding grooves (Egloff et al, 1995; Goldberg et al, 1995), and PIPs can interact both with these grooves and with other PP1 surface features To do this they use a variety of short sequence elements, of which the best understood is the RVxF motif (Choy et al, 2014; Egloff et al, 1997; Hendrickx et al, 2009; Hurley et al, 2007; O'Connell et al, 2012; Ragusa et al, 2010; Terrak et al, 2004). How this affects substrate selection remains unclear, the sequence-specificity of these PIP/PP1 holoenzymes, and how they bind substrates, have not been characterized

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Conclusion