Cryo-EM structures of PP2A:B55 with p107 and Eya3 define substrate recruitment

Abstract

Phosphoprotein phosphatases (PPPs) achieve specificity by binding substrates and regulators using PPP-specific short motifs. Protein phosphatase 2A (PP2A) is a highly conserved phosphatase that regulates cell signaling and is a tumor suppressor. Here, we use cryo-electron microscopy and nuclear magnetic resonance (NMR) spectroscopy to investigate the mechanisms of human p107 substrate and Eya3 regulator recruitment to the PP2A:B55 holoenzyme. We show that, while they associate with B55 using a common set of interaction pockets, the mechanism of substrate and regulator binding differs and is distinct from that observed for PP2A:B56 and other PPPs. We also identify the core B55 recruitment motif in Eya3 proteins, a sequence conserved amongst the Eya family. Lastly, using NMR-based dephosphorylation assays, we demonstrate how B55 recruitment directs PP2A:B55 fidelity through the selective dephosphorylation of specific phosphosites. As PP2A:B55 orchestrates mitosis and DNA damage repair, these data provide a roadmap for pursuing new avenues to therapeutically target this complex by individually blocking a subset of regulators that use different B55 interaction sites. Padi and Godek et al. solve cryo-electron microscopy structures of protein phosphatase 2A (PP2A):B55 bound to p107 and Eya3, which, together with nuclear magnetic resonance dephosphorylation experiments, show how PP2A:B55 recruits specific substrates and how it achieves efficient dephosphorylation of phosphosites with high cellular fidelity.