biostudies-literaturePadi SKRNINDS NIH HHSNIH HHSNIGMS NIH HHS1373-1382https://www.ebi.ac.uk/biostudies/studies/S-EPMC12350174biostudies-literatureUnknown32(8)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 dephosphorNature structural & molecular biologyCryo-EM structures of PP2A:B55 with p107 and Eya3 define substrate recruitment.PMC12350174R24 GM154192S10 OD032156U24 GM129539R01 NS124666R01 GM144483R01 GM144379R24 GM154185Padi SKRPeti WGodek RJPage RfalseCryo-EM structures of PP2A:B55 with p107 and Eya3 define substrate recruitment.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 dephosphor2025-01-01T00:00:00Z2025 Aug2026-07-15T10:49:39.526Z2026-07-04T03:11:57.229ZS-EPMC123501744024714710.1038/s41594-025-01535-3