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

Abstract

The phosphoprotein phosphatase (PPP) family of ser/thr phosphatases are responsible for the majority of all ser/thr dephosphorylation in cells. However, unlike their kinase counterpart, they do not achieve specificity via phosphosite recognition sequences, but instead bind substrates and regulators using PPP-specific short linear and/or helical motifs (SLiMs, SHelMs). Protein phosphatase 2A (PP2A) is a highly conserved PPP that regulates cell signaling and is a tumor suppressor. Here, we investigate the mechanisms of substrate and regulator recruitment to the PP2A:B55 holoenzyme to define how substrates and regulators engage B55 and understand, in turn, how these interactions direct phosphosite dephosphorylation. Our cryo-EM structures of PP2A:B55 bound to p107 (substrate) and Eya3 (regulator), coupled with biochemical, biophysical and cell biology assays, show that while B55 associates using a common set of interaction pockets, the mechanisms of substrate and regulator binding can differ substantially. This shows that B55-mediated substrate recruitment is distinct from that observed for PP2A:B56 and other PPPs. It also allowed us to identify the core B55 recruitment motif in Eya3 proteins, a sequence we show is conserved amongst the Eya family. Finally, using NMR-based dephosphorylation assays, we also showed how B55 recruitment directs PP2A:B55 fidelity, via the selective dephosphorylation of specific phosphosites. Because of the key regulatory functions of PP2A:B55 in 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.