Molecular mechanism of PP2A/B55α phosphatase inhibition by IER5.

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Published in final edited form as: Cell Chem Biol. 2025 Apr 9;32(4):631–642.e7. doi: 10.1016/j.chembiol.2025.03.004 Search in PMC Search in PubMed View in NLM Catalog Add to search Molecular Mechanism of PP2A/B55α Phosphatase Inhibition by IER5 Ruili Cao Ruili Cao 1 Department of Biological Chemistry and Molecular Pharmacology, Blavatnik Institute, Harvard Medical School, Boston, MA, USA Find articles by Ruili Cao 1, # , Daniel TD Jones Daniel TD Jones 1 Department of Biological Chemistry and Molecular Pharmacology, Blavatnik Institute, Harvard Medical School, Boston, MA, USA Find articles by Daniel TD Jones 1, # , Li Pan Li Pan 2 Department of Pathology, Brigham and Women’s Hospital, Boston, MA, USA Find articles by Li Pan 2, # , Annie Yang Annie Yang 1 Department of Biological Chemistry and Molecular Pharmacology, Blavatnik Institute, Harvard Medical School, Boston, MA, USA Find articles by Annie Yang 1 , Shumei Wang Shumei Wang 2 Department of Pathology, Brigham and Women’s Hospital, Boston, MA, USA Find articles by Shumei Wang 2 , Sathish K R Padi Sathish K R Padi 3 Department of Molecular Biology and Biophysics, University of Connecticut Health Center, Farmington, CT, USA Find articles by Sathish K R Padi 3 , Shaun Rawson Shaun Rawson 1 Department of Biological Chemistry and Molecular Pharmacology, Blavatnik Institute, Harvard Medical School, Boston, MA, USA Find articles by Shaun Rawson 1 , Jon C Aster Jon C Aster 2 Department of Pathology, Brigham and Women’s Hospital, Boston, MA, USA Find articles by Jon C Aster 2, * , Stephen C Blacklow Stephen C Blacklow 1 Department of Biological Chemistry and Molecular Pharmacology, Blavatnik Institute, Harvard Medical School, Boston, MA, USA 4 Department of Cancer Biology, Dana Farber Cancer Institute, Boston, MA, USA 5 Lead contact Find articles by Stephen C Blacklow 1, 4, 5, * Author information Article notes Copyright and License information 1 Department of Biological Chemistry and Molecular Pharmacology, Blavatnik Institute, Harvard Medical School, Boston, MA, USA 2 Department of Pathology, Brigham and Women’s Hospital, Boston, MA, USA 3 Department of Molecular Biology and Biophysics, University of Connecticut Health Center, Farmington, CT, USA 4 Department of Cancer Biology, Dana Farber Cancer Institute, Boston, MA, USA 5 Lead contact # Equal contributors AUTHOR CONTRIBUTIONS JCA and SCB conceived the project and acquired funding. RC purified PP2A/B55α-IER5 complexes, acquired biochemical data, prepared samples for cryo-EM data collection, and collected cryo-EM images. SKRP provided purified FAM122A protein. DTDJ processed and analyzed cryo-EM data with input from SR and RC, and DTDJ built the structural models with input from SCB and RC. LP performed co-immunoprecipitation studies and KRT1 expression analyses in SC2 cells. DTDJ performed structural bioinformatic studies in this work, and AY and DTDJ performed co-immunoprecipitation studies in HEK293T cells. All authors participated in data analysis and interpretation. SCB, DTDJ, RC, and JCA wrote and edited the manuscript with input from all authors. All authors agreed on the final manuscript. * Correspondence: stephen_blacklow@hms.harvard.edu ; jaster@bwh.harvard.edu Issue date 2025 Apr 17. PMC Copyright notice PMCID: PMC12067903  NIHMSID: NIHMS2073241  PMID: 40209703 The publisher's version of this article is available at Cell Chem Biol Previous version available: This article is based on a previously available preprint posted on bioRxiv on August 26, 2024: " Molecular Mechanism of PP2A/B55α Phosphatase Inhibition by IER5 ". SUMMARY PP2A serine/threonine phosphatases are heterotrimeric complexes that execute many essential physiologic functions. These activities are modulated by additional regulatory proteins, such as ARPP19, FAM122A, and IER5. Here, we report the cryoelectron microscopy structure of a complex of PP2A/B55α with the N-terminal structured region of IER5 (IER5-N50), which occludes a surface on B55α used for substrate recruitment, and show that IER5-N50 inhibits PP2A/B55α catalyzed dephosphorylation of pTau in biochemical assays. Mutations of full-length IER5 that disrupt its PP2A/B55α interface interfere with co-immunoprecipitation of PP2A/B55α. IER5 antagonism of B55α in keratinocytes is required for expression of KRT1 , a differentiation marker. Mini-IER5 composed of IER5-N50 and a nuclear localization sequence restores this activity in IER5 knockout cells. Using structural bioinformatics, we identify homology of IER5-N50 with SERTA domain containing proteins. These studies define the molecular basis of PP2A/B55α nuclear inhibition by IER5 and suggest a roadmap for selective pharmacologic modulation of PP2A/B55α complexes. Graphical Abstract eTOC blurb Cao et al. determined a cryoEM structure of PP2A/B55α bound to the N-terminal portion of the immediate early response protein IER5, which revealed a molecular basis for phosphatase inhibition that was confirmed in functional studies. Structural bioinformatics and follow-up studies also identified SERTAD proteins as PP2A/B55α-binding IER5 homologs. INTRODUCTION PP2A serine/threonine protein phosphatases are assembled from a scaffolding subunit (A, A’), a catalytic subunit (C, C’) and a regulatory subunit derived from one of four different protein subfamilies (B/B55, B’/B56, B’’/PR48-PR70, and B’’’/striatin) 1 . The B55α form of PP2A (PP2A/B55α) has critical roles in cell cycle regulation, mitotic exit, and the DNA damage response 2 – 6 . In addition, the PP2A A-C subcomplex is incorporated into the INTAC submodule of the integrator complex, a transcriptional regulator 7 , 8 . A structure of PP2A/B55α bound to microcystin-LR defined the architecture of the heterotrimer and showed how microcystin-LR inhibits dephosphorylation of pTau by binding to the active site of the catalytic subunit 9 . FAM122A and ARPP19, proteins that regulate cell cycle progression by selectively inhibiting PP2A/B55α 10 – 14 , both engage the heterotrimer using a bipartite binding interface in structures determined by single particle electron cryomicroscopy (cryo-EM), contacting both a B55α surface and the catalytic subunit at the active site 11 . IER5 , a member of the AP1-regulated immediate early response (IER) gene family 15 , encodes a protein of 327 amino acids. IER5 is induced in response to ionizing radiation and is implicated in the cellular response to DNA damaging agents and heat shock 16 – 18 . In squamous cell carcinoma (SCC) cells, IER5 is a direct transcriptional target of activated Notch that is required for induction of a cell differentiation program that arrests cell growth and stimulates expression of KRT1 and other differentiation-associated keratinocyte genes 19 . IER5 executes its cellular function, at least in part, by binding to and modulating the activity of heterotrimeric B55α holoenzyme complexes of PP2A 19 – 22 . One model for IER5 function proposes that it acts to direct selection of PP2A substrates such as S6K and HSF1 for dephosphorylation 22 ; however, in SCC cells, IER5 induction of gene transcription is mediated by suppression of PP2A/B55α activity, suggesting an alternative model where IER5 functions to antagonize PP2A activity against at least a subset of substrates 19 . Here, we report the structure of a PP2A/B55α in complex with the N-terminal domain of IER5 (IER5-N50), thereby uncovering the molecular basis of IER5’s ability to inhibit PP2A/B55α. We show that full length IER5 and IER5-N50 inhibit PP2A/B55α phosphatase activity in a pTau dephosphorylation assay with K i values similar to that of the PP2A/B55α phosphatase inhibitor FAM122A. Notch-induced expression of KRT1 , a marker of keratinocyte differentiation that depends on IER5 antagonism of B55α 19 , is restored in IER5 knockout SCC cells by a mini-IER5 consisting only of IER5-N50 tethered to a nuclear localization sequence, establishing that IER5 acts as an inhibitor of nuclear PP2A/B55α. Lastly, using structural bioinformatics, we identify and validate SERTADs as structural homologs of IER5-N50, demonstrating the existence of a family of proteins that bind PP2A/B55α through the same interface. Our structure of the PP2A/B55α complex with IER5-N50, combined with our functional validation of IER5 as a PP2A/B55α nuclear inhibitor, identifies strategies for selective modulation of PP2A/B55α activity. RESULTS Structure of a PP2A/B55α complex with IER5-N50. Previous work demonstrated that the 50-residue N-terminal domain of IER5 (IER5-N50), predicted to be a helical hairpin by Alphafold2 ( Fig. S1A , B , related to Fig. 1 ), is necessary and sufficient for binding to PP2A/B55α, whereas the C-terminal region (IER5-C) of IER5, predicted to be unstructured ( Fig. S1A , related to Fig. 1 ), does not interact 19 . To elucidate the molecular basis for IER5 recruitment to PP2A/B55α, we expressed and purified a complex of PP2A/B55α with IER5-N50 ( Fig. S1C related to Fig. 1 ) and determined its structure using cryo-EM ( Fig. 1 and Figs. S2 and S3 , related to Fig. 1 ). During data processing, we observed that the complexes existed in monomeric and dimeric assemblies ( Fig. S2 , related to Fig. 1 ). We utilized both assemblies during data processing and determined a final map of the monomeric assembly with a global resolution of 3.27 Å ( Table S1 , related to Fig. 1 ; Figs. 1A , 1B and Fig. S3 , related to Fig. 1 ). In complex with IER5-N50, the curvature of the A subunit of the PP2A heterotrimer is increased compared to that of PP2A-B55α in the structure with bound microcystin-LR (PDB:3DW8 9 , Fig. S4A , related to Fig. 1 ), and similar to that seen in PP2A complexes with FAM122A and ARPP19 11 ( Fig. S4B , related to Fig. 1 ). We thus used the structure of PP2A-B55α in complex with FAM122A (PDB:8SO0 11 ) as an initial model in building the PP2A heterotrimer in the IER5 complex. IER5 residues 3–45 were then built into the unmodelled region of the map adjacent to B55α using an initial model of IER5-N50 derived from Alphafold2 23 ( Fig. S1A , related to Fig. 1 ). In the final refined model ( Fig. 1A ), the RMSD of the PP2A/B55α portion of the model for the IER5 complex has values of 1.4 Å and 1.5 Å when compared to the FAM122A and ARPP19 complexes, respectively ( Fig. S4B ) 11 . Fig. 1. Cryo-EM structure of a PP2A-IER5 complex and interface analysis. Open in a new tab (A) cryo-EM map of the PP2A/B55α-IER5 complex. The scaffolding A subunit is green, the regulatory B55α subunit is purple, the catalytic C subunit is wheat, and IER5 is cyan. (B) cartoon rendering of the modelled structure, with iron and zinc atoms of the catalytic subunit rendered as brown and grey spheres, respectively. (C) Open book view depicting the interface between B55α and IER5 using a surface representation. IER5 is cyan, and B55α is purple, with residues at the contact interface between B55α and IER5 colored pink and dark blue, respectively. (D) Electrostatic surface representation of the complex (center), of B55α alone (left), and IER5 alone (right), colored on a sliding scale from blue to red (blue: basic, red: acidic). The catalytic and scaffolding subunits have been removed for clarity in panels C and D. See also Supplementary Figs. 1 – 4 and Table S1 . IER5-N50 adopts a helical hairpin conformation in the complex, forming an extensive contact interface with B55α to bury a total surface area of 2381 Å 2 ( Fig. 1C ). This site overlaps with the binding interfaces seen in complexes of PP2A/B55α with ARPP19 and FAM122A 11 ( Fig. S4B , related to Fig. 1 ). IER5-N50, however, does not make any contacts with either the scaffolding or catalytic subunit in the complex. There is electrostatic complementarity between the B55α binding surface, which contains acidic patches on its top face, and the binding surface of IER5, which is largely basic ( Fig. 1D ). The contact interface on B55α derives from the exposed face of its beta propeller, which presents a trio of short helices and a series of four extended loops to create a groove with two interaction sites for IER5 ( Fig. 2A ). At the first site, the helices of B55α engage the two helices of IER5 using contacts that are predominantly hydrophobic, with residues F281, I284, Y337, and F343 of B55α packing against I10, I13, L35, V36, and V39 of IER5 ( Fig. 2B ). S14 intramolecularly bridges the two helices of IER5 by reaching within hydrogen bonding distance of the backbone carbonyl of V39. The side chain of Y337 on B55α sits centrally in this surface, with its hydroxyl group approaching within hydrogen bonding distance of the backbone carbonyl of V36. Additionally, there are electrostatic contacts between the side chains of E338 from B55α and R9 of IER5, and between the K17 side chain of IER5 and the backbone carbonyls of Y337 and E338 of B55α. At the N-terminal end of IER5 helix 2, L30 and L34 pack in a hydrophobic cluster with M222, L225, and V228 of B55α ( Fig. 2B , right). H31 and K32 impart a positive electrostatic surface for interaction with the acidic platform of B55α, with the IER5 H31 side chain coming within hydrogen bonding distance of K345 on B55α. Fig. 2. Intermolecular contacts between B55α and IER5 and pTau dephosphorylation assay. Open in a new tab (A) Overview of the IER5 interface with B55α. The B55α regulatory subunit is rendered as a purple cartoon with a transparent surface and IER5-N50 is cyan, with structural elements of the IER5 helix-loop-helix identified with black labels. (B) and (C) Close-up views highlighting intermolecular contacts between B55α and IER5. Side chains of interacting residues are shown as sticks in CPK colors, with IER5 carbon atoms in green and B55α carbon atoms in yellow. Hydrogen bonds are depicted using dashed red lines. (B) Interactions of helices 1 and 2 with B55α. The left side shows IER5 helix 1 in the foreground and the right side shows IER5 helix 2 in the foreground after a 180° rotation. (C) Interactions between the loop of IER5 and B55α. D, Western blot analysis of anti-HA immunoprecipitates from I5 cells expressing wild-type or mutant HA-tagged IER5 and endogenous B55a. EV: empty vector. See also Supplementary Fig. 4 . (E) and (F) anti-pTau (S396) Western blots comparing the pTau phosphatase activity of PP2A in the presence of increasing concentrations (slanted triangle) of added inhibitory protein. (E) Effect of IER5-N50, IER5-N50-K17E, and FAM122A on Tau dephosphorylation. (F) effect of IER5-FL and IER5-FL-K17E on Tau dephosphorylation. (G) Plot of phosphatase activity as a function of inhibitor concentration for the five proteins tested, based on densitometry analysis. Data represent mean ± s.d. of three independent replicates. Data are normalized to the control condition without PP2A/B55α or inhibitor proteins. Data points were fitted using a log(inhibitor) vs. response (variable slope, four parameters) least squares fit model in Prism. The curves were used to estimate the IC50 value for each protein. See also Supplementary Fig. 5 . The second site on B55α is derived from the loops connecting adjacent beta strands of the propeller, which present an acidic surface to the basic loop of IER5 that connects its helices ( Fig. 2C ). This segment of IER5 resembles a Short Linear Motif (SLiM) 24 , with a side chain electrostatic interaction between R25 and E93 of B55α. The R25 side chain is also within hydrogen bond distance of the backbone carbonyls of E91 and I92 on B55α, and the K29 side chain amino group is positioned to form a salt bridge with the carboxyl group of D116 from B55α. H179 of B55α also approaches within hydrogen bonding distance of the K29 amino group. Validation of the binding interface. To test the importance of the hydrophobic and electrostatic surfaces of IER5-N50 in formation of complexes with PP2A/B55α, we introduced single point mutations into full-length IER5 (IER5-FL) at B55α interface residues or at control sites (L15 and N20) distant from the contact interface and tested their effects on B55α co-immunoprecipitation in IER5 knockout cells ( Fig. 2D ). Wild-type IER5, the IER5 L15R mutant, and the N20A mutant all strongly co-immunoprecipitated B55α. In contrast, interface mutations either completely prevented (S14A, K17E, L30E) or greatly reduced (I10A, R25E, K29E, H31A, K32E, L35A, V39A) co-immunoprecipitation of B55α, confirming that the interface seen in the cryo-EM structure is required for binding of IER5-FL to B55α in cells. IER5 inhibits PP2A dephosphorylation of pTau. The region of PP2A/B55α bound by IER5-N50 overlaps with residues required for recruitment of a broad set of substrates to PP2A/B55α 9 , 25 , 26 ( Fig. S4C , related to Fig. 2 ). To investigate the effect of IER5 on PP2A/B55α substrate dephosphorylation, we purified the PP2A/B55α holoenzyme, FLAG-tagged MBP fusions of IER5-N50 and full-length IER5 (IER5-FL), and FLAG-tagged MBP fusions of IER5-N50 and IER5-FL with the K17E mutation ( Fig. S5 , related to Fig. 2 ). Using pTau as a substrate 9 , we compared the inhibitory activity of these proteins with that of FAM122A, a known PP2A/B55α inhibitor 10 , 11 , 14 ( Fig. 2E – G ). Both IER5-FL (IC50, 2.2 μM) and IER5-N50 (IC50, 2.3 μM) MBP fusion proteins inhibited pTau dephosphorylation by PP2A/B55α similarly to each other and to FAM122A (IC50, 3.1 μM) ( Fig. 2E – G ). In contrast, the K17E mutated forms of IER5-FL and IER5-N50 failed to inhibit pTau dephosphorylation ( Fig. 2E – G ), confirming that IER5 inhibition of PP2A/B55α requires complex formation. Determining the minimal molecular requirements of IER5 essential for KRT1 induction. In the SCC cell line SC2, IER5 is required for Notch-dependent induction of KRT1 expression ( Fig. 3A , adapted from 19 ). This activity relies on the function of IER5 as an antagonist of the B55α isoform of PP2A because the requirement for IER5 in KRT1 induction is relieved when B55α is knocked out and restored when wild-type IER5-FL is reintroduced into IER5 knockout, B55α wild-type SC2 cells 19 . Reintroduction of either the L15R or N20A mutant of IER5-FL, neither of which disrupt recovery of B55α in the co-immunoprecipitation assay ( Fig. 2D ), rescues expression of KRT1 in response to Notch activation, whereas IER5-FL variants harboring I10A, S14A, K17E, or L30E mutations that disrupt the binding interface all sharply reduce KRT1 expression, as determined by RT-qPCR ( Fig. 3B ). These data show that mutants that abrogate binding of IER5 to B55α result in IER5 loss-of-function in SC2 cells. Fig. 3. Effects of IER5 mutations and deletions on downstream signaling responses, and generation of a mini-IER5. Open in a new tab (A) Experimental design for controlled activation of Notch1 by GSI washout. The readout for IER5 dependence in keratinocyte differentiation is the induction of KRT1 expression (figure design adapted from ref. 19 ). (B) RT-qPCR analysis of KRT1 RNA abundance measured 72 h after GSI washout in I5 cells expressing wild-type or mutant forms of IER5-FL. Data points are from biological replicates (n = 6). (C, F, I) Schematic representations of IER5 constructs analyzed in panels D-E, G-H, and J-K, respectively. (D, G, J) RT-qPCR analysis of KRT1 RNA abundance measured 72 h after GSI washout in I5 cells expressing the indicated protein constructs. Data points are from biological replicates (n=3). (E, H, K) Western blots showing the amount of expressed protein for the variants tested in panels D, G, and J, respectively. For each data set, transcript abundance was normalized against GAPDH, and error bars represent standard deviations of the mean. One-way analysis of variance (ANOVA) was used with Dunnett’s multiple-comparison post-hoc test to compare the difference between IER5-FL to all other samples individually (** P < 0.01;*** P < 0.001;**** P < 0.0001). EV: empty vector. (L) Schematic representation showing full-length IER5, and two mini-IER5 proteins, one consisting of IER5-N50 fused to its natural NLS, and a second of IER5-N50 fused to the nucleoplasmin NLS. (M) RT-qPCR analysis of KRT1 RNA abundance measured 72 h after GSI washout in I5 cells expressing the indicated protein constructs. Data points are from two biological replicates (n=2). (N) Western blot showing the amount of expressed protein for the variants tested in panel B. Transcript abundance was normalized against GAPDH, and error bars represent standard deviations of the mean. One-way analysis of variance (ANOVA) was used with Dunnett’s multiple-comparison post-hoc test to compare the difference between IER5-FL to all other samples individually (** P < 0.01;*** P < 0.001;**** P < 0.0001). EV: empty vector. We next performed structure-function studies to determine the minimal molecular requirements for IER5 rescue of KRT1 expression ( Fig. 3 ). IER5-N50 did not restore KRT1 expression, showing that PP2A binding activity alone is insufficient for inhibitory function in cells. IER5-C also failed to rescue KRT1 expression ( Fig. 3C – E ). In contrast, serial truncations and internal deletions within the IER5-C region showed that inclusion of just the IER5-N50 region and a C-terminal bipartite nuclear localization sequence 27 was sufficient for induction of KRT1 expression ( Fig. 3F – K ). Our deletion analysis suggested that we could engineer a functional, mini-IER5 by simply fusing the IER5-N50 region to its bipartite NLS (IER5-N50-NLS, residues 217 – 244). Indeed, this mini-IER5 was sufficient to complement KRT1 expression in IER5 knockout cells ( Fig. 3L – N ). To eliminate the unlikely possibility that the natural IER5 NLS provided a second activity required for KRT1 induction, we fused IER5-N50 to the NLS from nucleoplasmin (IER5-N50-NP-NLS) and found that this molecule was also sufficient to complement KRT1 expression. Taken together, mini-IER5 appears sufficient to complement IER5 antagonism of nuclear PP2A/B55α in IER5 knockout SC2 cells. Identification of IER5-N50 homology to SERTA domain containing proteins. We next queried the entire human alphafold-predicted proteome using Foldseek 28 to determine if any other proteins are predicted to contain a helical hairpin domain related to that of IER5-N50. Among the top hits were SRTD2 and CDCA4 (Probability 0.9 and 0.82, respectively) ( Fig. 4A , B ), proteins belonging to a larger family of SERTA domain-containing (SERTAD) proteins previously implicated as PP2A/B55α binders 29 , 30 . Notably, the positions on IER5 that are essential for PP2A/B55α binding are among the most conserved residues in the IER/SERTAD protein superfamily ( Fig. 4B , red dots). Based on structure-based sequence alignment, there are consensus motifs of ([I/L][F/W]XFSFXKF) in helix 1, and ([H/K/R]XXL[L/I]X[S/N]) in helix 2 (with aliphatic and aromatic residues denoted as F or W, respectively; Fig. 4C ). Interestingly, the loop of IER5, which is analogous to a SLiM 24 on other PP2A-binding proteins, is more poorly conserved. Structures of complexes between PP2A/B55α and SERTAD proteins using this helical hairpin in the contact interface are predicted with high-confidence using alphafold multimer 31 , 32 ( Fig. 4D and Table S2 , related to Fig. 4 ). Fig. 4. Homology of a predicted PP2A/B55α binding interface among IER family members and SERTA domain containing proteins. Open in a new tab (A) Searching the human alphafold predicted proteome using IER5 as input in Foldseek 28 led to the identification of CDCA4 and SRTD2 as structural homologues. (A) Multiple sequence alignment focusing on the helix-loop-helix motif of IER5, aligning IER, SRTAD, and CDCA4 proteins. The helix 1 and helix 2 segments of IER5 seen in the structure of the PP2A/B55α complex with IER5-N50 are indicated above the alignment. Red dots indicate sites of mutations in IER5 that interfere with co-immunoprecipitation of B55α. The alignment is shown using the Zappo color scheme for the 20 amino acids: pink, aliphatic; green, hydrophilic; blue, basic; red, acidic; orange, aromatic; yellow, cysteine; magenta, proline or glycine. (A) Helical wheel diagram of IER5 helix 1 and helix 2 (left) and a consensus for helix 1 and 2 based on residue conservation among aligned IER, SRTAD and CDCA4 proteins (aliphatic and aromatic residues are denoted as F or W, respectively). The helical face directed at B55α is marked by a magenta arc. (D) Best scoring structural models for alphafold2-predicted interactions of IER, SERTAD and CDCA4 proteins with PP2A/B55α. IER, SERTAD, and CDCA4 predictions were restricted to the aligned region in (B). B55α is magenta, the PP2A catalytic subunit is wheat, the PP2A scaffolding subunit is green, and the predicted interactor is cyan. See also Table S2 , related to Fig. 4 . (E) Western blot analysis of immunoprecipitants prepared with streptavidin magnetic beads from HEK293T cells expressing wild-type or mutant N-terminus HA-tagged and C-terminus Twin-Strep tagged IER5 family and SERTAD proteins. Both the input and the immunoprecipitants were probed for HA-tagged bait proteins and for endogenous B55α. To test this prediction, we expressed N-terminal HA, C-terminal Twin-Strep tagged wild type and K17E-equivalent mutated forms of other IER and SERTAD proteins in HEK293T cells and performed a co-immunoprecipitation experiment with streptavidin magnetic beads, probing for recovery of B55α by western blotting ( Fig. 4E ). In all cases, except IER3/IEX1 which does not contain a SERTA domain, wild type proteins pulled down B55α, whereas the mutant proteins did not ( Fig. 4E ). For SERTAD2, the K17E equivalent mutation (K47E) partially abrogated B55α recruitment, and combining K47E with a L30E equivalent mutation (L59E) in cis completely blocked B55α interaction. This analysis indicates that IER5 is a member of a broader family of proteins that modulate PP2A/B55α function by binding to B55α using a conserved helical hairpin motif. In a related study, B55α binding proteins identified by mass spectrometry and from the literature were also subjected to Alphafold2 multimer predictions, which were used to predict common structural features of B55α-binding proteins and to create a synthetic, high-affinity protein inhibitor for the predicted B55α docking site 33 . Those studies identified a helical segment as a critical secondary structural element of the predicted B55α-binding interface and are highly complementary to the structural and informatic analysis of IER5-related proteins reported here. DISCUSSION The PP2A phosphatase utilizes a set of regulatory subunits, including B55α, to maintain cellular homeostasis by specifically recruiting substrates for dephosphorylation in response to cellular signaling cues. Modulators of PP2A/B55α activity, such as the inhibitors FAM122A and ARPP19, have overlapping and distinct roles in cell cycle-specific control of PP2A/B55α activity 10 – 14 . In previous work, we showed that IER5 interacts with and is epistatic to B55α in SCC cells, in which markers of differentiation such as KRT1 depend on Notch-induced upregulation of IER5 for expression 19 . The work reported here shows that the N-terminal helical hairpin of IER5 acts as an inhibitor of PP2A/B55α phosphatase activity by occluding the substrate-binding platform of the B55α regulatory subunit. IER5-N50 masks an extensive region on the surface of B55α that faces, but does not directly contact, the catalytic subunit ( Figs. 1 , 2 ). Substrate recruitment to B55α relies primarily on short linear motifs (SLiMs), which may adopt an alpha helical conformation when bound 24 , 25 . Residues on B55α reported to participate in substrate recruitment and function show substantial overlap with the IER5 binding site 9 , 25 , 26 ( Fig. S4C ). In addition, recently reported structures of Eya3 and p107 peptides bound to PP2A/B55α also have footprints that overlap with IER5 34 , consistent with our findings that IER5-FL and IER5-N50 inhibit PP2A/B55α pTau dephosphorylation ( Fig. 2 ). The mode of IER5-N50 binding differs from that of the PP2A inhibitors FAM122A and ARPP19, which both engage the B55α subunit with a short helical motif and contact the catalytic subunit using a discontinuous unstructured segment 11 , 14 ( Fig. S4B ). The more closed conformation of the PP2A-IER5 complex, as compared to the microcystin-LR-inhibited structure 9 , also allows the B55α regulatory subunit to contact the C-terminal tail of the catalytic domain directly ( Fig. S4 , related to Fig. 1 ). IER5 function appears to require both B55α binding and nuclear entry, because IER5 does not restore the expression of KRT1 in IER5 knockout cells unless the N50 module and a nuclear localization sequence 27 are both present ( Fig. 3 ). Others have proposed that IER5 functions as a substrate adaptor, enabling PP2A to dephosphorylate proteins that do not bind B55α directly 22 . Though the findings reported here disfavor this possibility, they do not exclude the possibility that loading of IER5 onto PP2A/B55α complexes could inhibit dephosphorylation of some substrates while promoting dephosphorylation of other proteins. By mining the alphafold predicted human proteome with a structure similarity search 28 , we also found that IER5 is likely representative of a larger protein family that regulates PP2A/B55α by using a helix-loop-helix motif to recognize B55α. Among these helix-loop-helix containing proteins are the SERTADs, for which a functional link to PP2A/B55α has already been established 29 , 30 . Other studies have shown that SERTAD proteins contain potent transcriptional activation domains 35 – 38 , and recent work has also suggested that IER5 has activity as a transcriptional regulator 20 . How the transcriptional regulatory activity of IER5 relates to its activity as a selective inhibitor of nuclear PP2A/B55α complexes and whether this is a property shared with SERTADs and other helix-loop-helix superfamily members should be fertile ground for future studies. The immediate early response genes also include a family member called IER3 (also known as IEX-1), which is closely related to IER5. IER3 does not bind B55α (Figure 6E) and instead inhibits the activity of the PP2A/B56 isoform by promoting the dissociation of B56 from the active enzyme 39 , 40 . Of interest, IER3 also appears to be a direct target of Notch signaling in SCC cells 19 . The induction of different immediate early response proteins by Notch and cell stresses such as radiation-induced DNA damage may serve to induce cell cycle arrest and differentiation of squamous cells through the coordinated inhibition of multiple PP2A holoenzyme species. Lastly, there is interest in development of PP2A modulators for diseases ranging from neurodegeneration to cancer 41 , 42 . The molecularly distinct IER5 contact site identified here could serve as a target surface for developing protein-protein interaction (PPI) inhibitors that selectively block substrate recruitment to PP2A/B55α complexes, or conversely, for molecular glue modulators that can direct dephosphorylation of specific proteins, analogous to protein-degrading IMiDs 43 . LIMITATIONS OF THE STUDY Given that our Cryo-EM structure is of IER5-N50 and not full length IER5 bound to PP2A/B55α, we cannot absolutely exclude the possibility that a segment of IER5-C interacts with the PP2A/B55α at a secondary site. However, IER5-N50 is equipotent to full-length IER5 in inhibiting the dephosphorylation of pTau ( Fig. 2 ), and the function of IER5 in the KRT1 induction assay can be fully restored by fusing IER5-N50 to either a C-terminal IER5 nuclear localization sequence or the heterologous NLS from nucleoplasmin ( Fig. 3 ). Thus, the simplest model to explain these findings is one in which IER5 inhibits PP2A/B55α in the nucleus as a consequence of occlusion of the B55α binding platform, thereby preventing substrate recruitment. However, our work does not formally rule out the possibility that IER5 can function as a substrate adaptor to deliver PP2A/B55α to alternative substrates, nor has this work identified specific substrates of PP2A/B55α that are inhibited by IER5 binding in cells. RESOURCE AVAILABILITY Lead contact Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Stephen C. Blacklow ( stephen_blacklow@hms.harvard.edu ). Materials availability Plasmids and cell lines generated in this study are available upon request from the Lead Contact after completion of a Materials Transfer Agreement. Data and code availability Atomic coordinates and EM map have been deposited in the RCSB Protein Data Bank (PDB) under the accession number 8UO5 and are publicly available as of the date of publication. The accession number is also listed in the key resources table . Original Western Blot images are available for review upon request. This paper does not report original code. Any additional information required to reanalyze the data reported in this work is available from the Lead Contact upon request. Key Resources Table REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies anti-B55α Cell Signaling Technology Cat# 2290S anti-IER5 Sigma Cat# HPA029894 anti-HA Cell Signaling Technology Cat# 3724S anti-HA (HRP conjugate) Cell Signaling Technology Cat# 2999S anti-pTau Thermo Scientific Cat # 44-752G anti-β-actin Sigma Cat# A1978 IRDye 800CW Donkey anti-Rabbit LI-COR Biosciences Cat# 926-32213, RRID:AB_621848 Anti-Mouse IgG, HRP-linked Antibody Cell Signaling Technology Cat# 7076 Anti-Mouse IgG, HRP-linked Antibody Cell Signaling Technology Cat# 7074 Bacterial and virus strains BL21(DE3) New England Biolabs (NEB) Cat# C2527H Stellar competent cells Takara Bio Cat# 636766 Chemicals, peptides, and recombinant proteins FBS GeminiBio Cat# 100-106 Penicillin and streptomycin ThermoFisher Scientific Cat# 15140163 Trypsin/2.21 mM EDTA in HBSS Corning Cat# 25-053-CI DMEM Corning Cat# 10-017-CV DPBS Corning Cat# 21-031-CV NaCl VWR Cat# 0241-10KG Glycerol americanbio Cat# AB00751-04000 HEPES Sigma-Aldrich Cat# H4034-1KG Trizma base Sigma-Aldrich Cat# T1503-5KG Glycine Sigma-Aldrich Cat# G7126-5KG b-Mercaptoethanol Sigma-Aldrich Cat# M6250-250ML SDS Sigma Cat# 75746-1KG Methanol VWR Cat# BDH2018-1GLP Tween-20 Sigma Cat# P7949-500ML DMSO Sigma-Aldrich Cat# D2650 Polyplus-transfection FectoPRO ® VWR Cat#10118-842 Lipofectamine 2000 Transfection Reagent ThermoFisher Cat# 11668019 Valproic acid sodium salt Sigma-Aldrich P4543-100G D-(+)-Glucose solution Sigma-Aldrich G8769-100ML Expi293 expression medium ThermoFisher Cat # A1435103 ESF 921 Insect Cell Culture Medium, Protein-Free Expression Systems Cat # 96001 Strep-Tactin ® XT 4Flow ® high capacity resin NEUROMICS Cat # 2-5030-025 Anti-Flag resin Produced in-house N/A Amylose Resin High Flow NEB Cat# E8022S Compound E EMD Millipore Cat # 565790 Critical commercial assays QIAprep Spin Miniprep Kit Qiagen Cat# 27106 PureLink ™ HiPure Plasmid Filter Maxiprep Kit Invitrogen Cat# K210016 RNeasy Mini Kit Qiagen Cat# 74104 High-Capacity cDNA Reverse Transcription Kit ThermoFisher Cat# 4368814 PowerUp SYBR Green Master Mix for qPCR ThermoFisher Cat# A25742 QuickChange II XL Site-Directed Mutagenesis Kit Agilent Cat# 200521 Pierce HA-Tag Magnetic IP/Co-IP Kit ThermoFisher Cat# 88838 In-Fusion HD Cloning Plus Clontech Cat # 638911 SuperSignal West Dura Extended Duration Substrate ThermoFisher Cat# 34075 Deposited data PP2A/B55α-IER5-N50 complex structure coordinates and EM density map This paper PDB ID: 8UO5 https://www.rcsb.org/structure/8UO5 https://doi.org/10.2210/pdb8uo5/pdb EMDB ID: EMD-42428 Experimental models: cell lines Expi293F cells ThermoFisher Cat # A14527 HEK293T cells ATCC Cat # CRL-3216 Sf9 Expression Systems Cat# 94-001F SC2 cells Pan, L. et al. 19 N/A SC2 IER5 KO cells (I5 cells) Pan, L. et al. 19 N/A Oligonucleotides KRT1 forward 5’-GGACAGCTCCTTAGCATCTTATC-3’ This paper N/A KRT1 reverse 5’-GGAGTTTAAGACCTCTCCACAAA-3’ This paper N/A GAPDH forward 5’-GAAGGTGAAGGTCGGAGTCAAC-3’ This paper N/A GAPDH reverse 5’-TGGAAGATGGTGATGGGATTTC-3’ This paper N/A Recombinant DNA pcDNA3.1/Hygro(+) ThermoFisher Scientific Cat # V87020 pAC8RedNK-Flag-CA Gift from Eric Fischer at the Dana-Farber Cancer Institute N/A pAC8RedNK-B55α Gift from Eric Fischer at the Dana-Farber Cancer Institute N/A pAC8RedNK-His-R1A Gift from Eric Fischer at the Dana-Farber Cancer Institute N/A pAC8RedNK-Strep-IER5-N50 This paper N/A gBlocks Integrated DNA Technologies N/A pcDNA3.1-Flag-MBP-IER5-FL This paper N/A pcDNA3.1-Flag-MBP-IER5-FL (K17E) This paper N/A pcDNA3.1-Flag-MBP-IER5-N50 This paper N/A pcDNA3.1-Flag-MBP-IER5-N50 (K17E) This paper N/A MIEG3-IER5-FH This paper N/A MIEG3-IER5-FH (I10A) This paper N/A MIEG3-IER5-FH (S14A) This paper N/A MIEG3-IER5-FH (L15R) This paper N/A MIEG3-IER5-FH (K17E) This paper N/A MIEG3-IER5-FH (N20A) This paper N/A MIEG3-IER5-FH (R25E) This paper N/A MIEG3-IER5-FH (K29E) This paper N/A MIEG3-IER5-FH (L30E) This paper N/A MIEG3-IER5-FH (H31A) This paper N/A MIEG3-IER5-FH (K32E) This paper N/A MIEG3-IER5-FH (L35A) This paper N/A MIEG3-IER5-FH (V39A) This paper N/A MIEG3-IER5-FH-N50-NLS This paper N/A MIEG3-IER5-FH-N50-Nucleoplasmin NLS This paper N/A pcDNA3.1-HA-SRTD1-TwinStrep This paper N/A pcDNA3.1-HA-SRTD2-TwinStrep This paper N/A pcDNA3.1-HA-SRTD3-TwinStrep This paper N/A pcDNA3.1-HA-SRTD4-TwinStrep This paper N/A pcDNA3.1-HA-IER2-TwinStrep This paper N/A pcDNA3.1-HA-IER5-TwinStrep This paper N/A pcDNA3.1-HA-CDCA4-TwinStrep This paper N/A pcDNA3.1-HA-IEX1-TwinStrep This paper N/A pcDNA3.1-HA-SRTD1-TwinStrep (K52E) This paper N/A pcDNA3.1-HA-SRTD2-TwinStrep (K47E) This paper N/A pcDNA3.1-HA-SRTD2-TwinStrep (L59E) This paper N/A pcDNA3.1-HA-SRTD2-TwinStrep (K47E/L59E) This paper N/A pcDNA3.1-HA-SRTD3-TwinStrep (K40E) This paper N/A pcDNA3.1-HA-SRTD4-TwinStrep (K115E) This paper N/A pcDNA3.1-HA-IER2-TwinStrep (K17E) This paper N/A pcDNA3.1-HA-IER5-TwinStrep (K17E) This paper N/A pcDNA3.1-HA-CDCA4-TwinStrep (K44E) This paper N/A pcDNA3.1-HA-IEX1-TwinStrep This paper N/A Software and algorithms SerialEM Mastronarde 44 https://bio3d.colorado.edu/SerialEM/ SBGrid Consortium Morin et al. 45 https://sbgrid.org cryoSPARC Structura Biotechnology Inc. 46 https://cryosparc.com DeepEMhancer Sanchez-Garcia et al. 47 https://github.com/rsanchezgarc/deepEMhancer/ UCSF Chimera Pettersen et al. 48 https://www.rbvi.ucsf.edu/chimera/ Phenix Adams, P. D. et al. 49 https://sbgrid.org/software/ Coot Emsley, P. & Cowtan, K. 50 https://sbgrid.org/software/ Pymol Schrödinger https://pymol.org/2/ PDB validation server World Wide Protein Data Bank https://www.wwpdb.org/ GraphPad Prism GraphPad RRID:SCR_002798 Other Quantifoil 400 mesh copper grids R 1.2/1.3 Electron Microscopy Sciences Q4100CR1.3 Open in a new tab STAR METHODS EXPERIMENTAL MODEL AND STUDY PARTICIPANT DETAILS Expi293F cells (female) were maintained in suspension at 37 °C in Expi293 expression medium (ThermoFisher) in a 5% CO2, humidified incubator. Human embryonic kidney HEK293T (human, female) were cultured in DMEM (Corning) supplemented with 10% FBS (GeminiBio) at 37°C in a 5% CO2, humidified incubator. Studies with HEK293T cells (female) are IRB exempt. Studies with Expi293F and HER293T cells are IRB exempt. SC2 cells and IER5 knock-out cell line I5 (female) were cultured in keratinocyte medium (300 mL of DMEM, 100 mL of Ham’s F-12 supplemented with 10% FBS, 0.4 mg/mL hydrocortisone, 5 mg/mL insulin, 10 ng/mL EGF, 5 mg/mL transferrin, 8.4 ng/mL cholera toxin, and 13 ng/mL liothyronine) in the presence of GSI (1 μM compound E). All cell lines were periodically tested for mycoplasma by PCR. Adherent cells were detached from plates after a PBS rinse using 0.25% Trypsin/2.21 mM EDTA in HBSS (Corning) for 5–10 min at 37°C unless otherwise specified. Sf9 and Hi5 cells were cultured in ESF 921 Insect Cell Culture medium (Expression Systems). E. coli BL21 (DE3) cells were cultured in LB media. METHOD DETAILS Plasmid construction cDNAs encoding PPP2R1A, PPP2CA, and PPP2R2A (B55α) assembled in a pAC-derived baculovirus expression vector (pAC8RedNK) were gifts from the Fischer lab (Dana Farber Cancer Institute). PPP2R1A was engineered to include an N-terminal His6 tag followed by a Tobacco etch virus (TEV) cleavage site; the cDNA for PPP2R2A (B55α) had no affinity tag; and PPP2CA had an N-terminal Flag tag followed by a TEV cleavage site. The IER5 (1–50) fragment was subcloned into pAC8RedNK with an N-terminal Strep tag II followed by a TEV cleavage site. Both the IER5 (1–50) and full length IER5 were cloned into the pcDNA3.1/hygro(+) vector with an N-terminal Flag-MBP tag followed by a TEV cleavage site. K17E mutations of IER5 proteins were made using site-directed mutagenesis. The full-length Tau protein was cloned into the bacterial expression vector ptd68, incorporating an N-terminal His6-SUMO tag. Insert sequences were confirmed by Sanger sequencing. Protein sequences encoding SRTD1–4, IER2, IER3/IEX1 and CDCA4 were retrieved from Uniprot, and codon optimised for homologous expression by Twist, with exclusion of internal NheI, EcoRI, NotI and XhoI restriction enzyme cutting sites. Expression constructs were engineered to contain an N-terminus HA tag and a C-terminus Twin-Strep tag, flanked at the 5’ end with a NheI site, and the 3’ end site a XhoI site, in the vector backbone of pcDNA3.1/G418(+). The amino acids between the N-terminal HA tag and protein of interest encode “EF” residues, and from the protein of interest to the C-terminus Twin-Strep tag encode “AAA” residues, due to the presence of in-frame EcoRI and NotI sites, respectively. Mutations were introduced by site-directed mutagenesis. Insert sequences were confirmed by whole plasmid sequencing. Protein Expression The PP2A/B55α heterotrimer and the PP2A/B55α IER5-N50 complex were expressed in Hi5 cells concurrently transfecting 1.5 µg of the pAC8RedNK vector and 0.5 µg of linearized baculoviral DNA into 1×10 6 Sf9 cells using 6 µl of FuGene HD (Promega) in ESF 921 Insect Cell Culture medium (Expression Systems). After 5 to 7 days of incubation, the supernatant was collected to harvest the baculovirus. The virus was subsequently amplified over 2 to 3 cycles using Sf9 cells at a concentration of 2×10 6 . The collected viruses were then used to infect Hi5 cells for protein expression. For expression of the PP2A heterotrimer, a viral stoichiometric ratio of 1:1:1 was used for Hi5 cell infection. For the PP2A-IER5 complex, a ratio of 1:1:1:1.5 (IER5) was used. Cells were shaken at 27 °C for 72 hours before harvesting by centrifugation. Cell pellets were collected and stored at −80 °C until purification. Flag-MBP-IER5-N50 and Flag-MBP-IER5-FL protein were expressed in Expi293F cells. Cells were grown in Expi293 media to a density of 3 × 10 6 cells/ml and then transfected with 1.0 mg DNA/L of culture using the FectroPro transfection reagent (Polyplus) at a 1:1 DNA/FectroPro ratio. After 24 hours, 45% D-(+)-Glucose solution (Sigma-Aldrich, 10 mL per L of culture) and 3 mM valproic acid sodium salt (Sigma-Aldrich) were added to the cells to enhance protein expression. The cells were cultured for an additional 24 hours before harvesting by centrifugation. Cell pellets were collected and stored at −80 °C until purification. Recombinant Tau protein was expressed in E. coli BL21 (DE3) cells. Protein expression was induced at a culture optical density (OD) of 0.8 by addition of 0.2 mM isopropyl-1-thio-D-galactopyranoside (IPTG) and the culture was maintained at 16°C overnight. Cell pellets were collected and stored at −80 °C until purification. Recombinant FAM122A was expressed and purified as reported 11 . Protein purification For the PP2A heterotrimer, cells were resuspended in lysis buffer containing 20mM Tris-HCl, pH 7.6, 200mM NaCl, 2mM Tris-(2-carboxyethyl) phosphine (TCEP), 0.1% (v/v) Triton X-100, protease inhibitor cocktail (Sigma) and Benzonase (EMD Millipore). Cells were lysed by sonication and centrifuged at 50,000 g for 1 h. The soluble fraction was passed over an anti-FLAG M2 affinity resin. The resin was washed with 10 column volumes (CVs) of wash buffer (20 mM Tris-HCl, pH 7.6, 200 mM NaCl, 2 mM TCEP), and the protein was then eluted using wash buffer supplemented with 0.2 mg/ml of FLAG peptide. The elution fractions were collected, concentrated, and further purified using size-exclusion chromatography (SEC) on a Superdex S200 10/300 column, which was pre-equilibrated with buffer (20 mM Tris-HCl, pH 7.6, 200 mM NaCl, 2 mM TCEP). Protein purity was assessed by SDS-PAGE using a Coomassie blue stain. Peak fractions were pooled for biochemical studies. For the PP2A/B55-IER5 complex, lysis and ultracentrifugation were performed as above. The affinity purification step was performed using Strep-Tactin XT Superflow resin (IBA), followed by elution with wash buffer supplemented with 50 mM biotin. After elution from the column the fractions were concentrated and further purified using size exclusion chromatography on a Superdex S200 10/300 column. Protein purity was assessed by SDS-PAGE using a Coomassie blue stain. Peak fractions were pooled for biochemical studies and for Cryo-EM data collection. For purification of IER5 proteins, cells were resuspended in lysis buffer containing 20mM Tris-HCl, pH 7.6, 200mM NaCl, 2mM Tris-(2-carboxyethyl) phosphine (TCEP), protease inhibitor cocktail (Sigma) and Benzonase (EMD Millipore). Cells were lysed by sonication and centrifuged at 50,000 g for 1 h. The soluble fraction was passed over amylose resin. The resin was washed with 10 column volumes (CVs) of wash buffer (20 mM Tris-HCl, pH 7.6, 200 mM NaCl, 2 mM TCEP), 50 CVs of wash buffer supplied with 10mM Mgcl2, 5mM ATP, and 5CVs of wash buffer, and the protein was then eluted using wash buffer supplemented with 10 mM maltose. The elution fractions were collected, concentrated, and further purified using size-exclusion chromatography (SEC) on a Superdex S200 10/300 column, which was pre-equilibrated with buffer (20 mM Tris-HCl, pH 7.6, 100 mM NaCl, 2 mM TCEP). Protein purity was assessed by SDS-PAGE using a Coomassie blue stain. Peak fractions were pooled for biochemical studies. For preparation of Tau protein, bacterial cells were resuspended in lysis buffer containing 20 mM Tris-HCl, pH 8, 200 mM NaCl, 2 mM Tris-(2-carboxyethyl) phosphine (TCEP), protease inhibitor cocktail (Sigma), 20 mM Imidazole. Cells were lysed by sonication and centrifuged to remove debris. After centrifugation, the supernatant containing the recombinant His-SUMO-Tau was applied to Ni(NTA) affinity resin, and the resin was washed using lysis buffer. The His-SUMO tag was removed overnight by on-column cleavage with ULP1 protease. Protease-liberated Tau was recovered from the column flow-through and was further purified by size exclusion chromatography using a Superdex 75 10/300 GL (GE Healthcare) column in buffer consisting of 20 mM Tris-HCl pH 8, 200 mM NaCl, and 2mM TCEP. Peak fractions were pooled for phosphorylation using GSK3β. The concentration of purified Tau was determined by UV absorbance at 280 nm. Phosphorylation of Tau protein and purification of pTau Purified Tau was incubated with GSK3β (SinoBiological) in a 100:1 ratio, using a buffer of 25 mM HEPES pH 7.5, containing 100 mM NaCl, 10 mM MgCl 2 , 10 mM ATP, and 2 mM TCEP. The reaction was allowed to proceed for 19 hours at 37 °C. Verification that the reaction had gone to completion was performed by SDS-PAGE with Coomassie blue staining. The pTau was further purified by size exclusion chromatography using a Superdex 75 10/300 GL (GE Healthcare) column in buffer consisting of 20 mM Tris-HCl pH 8, 200 mM NaCl, and 2mM TCEP. The concentration of purified pTau was determined by UV absorbance at 280 nm. Fractions were collected and frozen in −80 prior to use. Cryo-EM grid preparation and data collection Samples were frozen on Cryo-EM grids using a Vitrobot Mark IV (Thermo Fisher Scientific) instrument for vitrification. Freshly purified PP2A-IER5 complex (3.5 µl at a concentration of 2 mg/ml) was deposited onto glow-discharged C-flat holey carbon grids (R1.2/1.3, 400 mesh copper, Electron Microscopy Sciences). These grids were blotted for 6 seconds with a blot force of 15 at 100% humidity and 22 °C before being rapidly submerged into liquid nitrogen-cooled liquid ethane. Images were acquired on a Titan Krios microscope equipped with a BioQuantum K3 Imaging Filter (slit width 25 eV) and a K3 direct electron detector (Gatan), operating at an acceleration voltage of 300 kV. Images were recorded at a defocus range of −0.8 to −2.0 μm with a nominal magnification of 105 kx, resulting in a pixel size of 0.825 Å. Each image was dose fractionated into 51 movie frames with a total exposure time of 2.8 s, resulting in a total dose of ∼53.7 electrons per Å 2 . SerialEM was used for data collection 44 . Structure Determination Data were processed using CryoSPARC 46 unless explicitly stated in the text, as summarized in Fig. S3 . A total of 5,383 micrographs were subjected to patch motion correction and patch CTF estimation. Particles (3.95 million) were identified and extracted using a box size of 360 pixels using the CryoSPARC blob picker. 2D-classification was used to remove poorly classified particles, reducing the total number of particles to 1.17 million. From the projected 2D-class averages, a mixture of monomeric and dimeric PP2A-IER5 assemblies was evident. 100k particles were split and used to generate three preliminary maps representing the different species present in the data. The three maps were subjected to heterogeneous refinement using all identified particles, resulting in three classes designated as monomeric (~47%), dimeric (~25%) and junk (~27%) (e.g., scaffolding subunit only) classes. The monomeric and dimeric classes were then processed independently. Non-uniform refinement, local motion correction, and another round of non-uniform refinement was applied to the monomer class, yielding a map with a nominal resolution of 3.67 Å. The monomer class displayed no clear IER5 density at this stage of processing. Non-uniform refinement, local motion correction, and another round of non-uniform refinement was applied to the dimeric class, resulting in a resolution of 3.67 Å for the dimer class map. Alignment of the dimeric map along an apparent C2 symmetry axis followed by non-uniform refinement under a C2 symmetry constraint improved the map to 3.44 Å resolution. A further round of local motion correction and two rounds of sequential non-uniform refinement produced a map of 3.17 Å resolution. Symmetry expansion was performed along the C2 symmetry axis. Masked local refinement with a soft mask (shown in black outline box named “local refinement mask”) around a single copy from an asymmetric unit produced a map at 3.04 Å resolution. Despite this reasonable global resolution, the map displayed strong directional anisotropy. Masked non-uniform refinement using a single copy of an asymmetric unit of the symmetry expanded dimer map, using the monomeric particles as input, resulted in a 3.82 Å map with improved signal for IER5. We then combined all the aligned monomer and symmetry expanded dimer particles and performed a masked local refinement that resulted in a combined map of 3.09 Å resolution. To resolve conformational heterogeneity in IER5 we generated a mask around IER5 only and performed masked 3D classification without alignment in Relion 51 (K = 4, T = 20). Once particle class convergence was reached, a single class contained the majority particles (60.1 %) and displayed high resolution features. Further 3D classification was ineffective at improving map quality, we therefore utilised CryoSieve 52 with reconstruction in Relion and reduced the particle stack to ~99k particles without impacting the quality of IER5 (nominal resolution of 3.14 Å). We then made a mask on the alternate copy of PP2A/B55α-IER5, attributed by the dimer particles of PP2A/B55, and performed a local refinement on this copy 53 . Using the same mask, we then performed particle subtraction on this copy of PP2A/B55α-IER5 from the map. Using the particle subtracted stack and the alignments from the CryoSieved map reconstruction as input, we performed a local refinement around the previously refined copy of PP2A/B55α-IER5, reaching 3.18 Å resolution. In the previous step we determined the per particle input scale during local refinement, and used the score with the rebalance orientations job in cryosparc, reducing to a final particle stack of 67k particles with a nominal resolution of 3.27 Å. We post-processed the output half maps using DeepEMhancer 47 , and used this map to aid inspection, manual model building, and visual illustration. Model refinement and atomic model building We used the PP2A/B55α-FAM122A structure (PDB 8SO0 11 ) as an initial model for the PP2A/B55α heterotrimer, and an initial model for IER5 as predicted by alphafold 23 . The models were fitted to the map by hand using Chimerax 48 . The model fit to the map was improved using ISOLDE and adaptive distance constraints to maintain local geometries and distances when applicable 54 . The model was refined iteratively using REFMAC Servalcat 55 and Phenix Real-Space Refine 56 . During refinement in REFMAC Servalcat or manual refinement in Coot, positional restraints generated using ProSMART 57 were used. The final models were evaluated using MolProbity 58 in Phenix validation report. Statistics of the map reconstruction and model refinement are presented in Table S1 and taken from the Phenix validation report. Structural biology applications used in this project (except CryoSPARC) were compiled and configured by SBGrid 45 . Molecular graphics and analyses were performed with UCSF ChimeraX 48 (developed by the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco, with support from National Institutes of Health R01-GM129325 and the Office of Cyber Infrastructure and Computational Biology, National Institute of Allergy and Infectious Diseases), or PyMOL (Schrödinger). Enzymatic analysis of dephosphorylation of pTau in vitro PP2A/B55α holoenzyme in enzyme buffer (20 mM Tris pH 7.6, 100 mM NaCl, 2 mM TCEP) was pre-incubated with various concentrations of IER5-N50, IER5-FL, IER5-N50 variants, IER5-FL variants, or FAM122A for 2 h on ice. The reaction was started by adding pTau (final concentration 0.35 μM) to the PP2A/B55α - inhibitor complexes (final concentration of PP2A/B55α holoenzyme, 20 nM) and incubating at 30 °C. After 30 min, the reaction was stopped by adding SDS loading buffer and the samples were loaded onto SDS-PAGE. The phosphorylation status of Tau was examined by western blot using an antibody (Thermo Scientific) that specifically recognizes phosphorylated serine 396 of Tau. The experiments were independently repeated at least 3 times (n ≥ 3). Cell culture Cells were grown under 5% CO2 at 37°C in media supplemented with streptomycin/penicillin. IER5 knock-out cell line I5 was derived from SC2 cells, which are engineered to contain a cDNA encoding a mutated truncated form of NOTCH1, ΔEGF-L1596H, that is regulatable with a γ-secretase inhibitor (GSI) 19 . I5 and its derivatives overexpressing wide-type or mutant IER5 were cultured in keratinocyte medium 59 (300 mL of DMEM, 100 mL of Ham’s F-12 supplemented with 10% FBS, 0.4 mg/mL hydrocortisone, 5 mg/mL insulin, 10 ng/mL EGF, 5 mg/mL transferrin, 8.4 ng/mL cholera toxin, and 13 ng/mL liothyronine) in the presence of GSI (1 μM compound E) to maintain Notch in the off-state. Timed activation of Notch was triggered by GSI washout. Cells were washed twice with media containing 0.1% DMSO instead of GSI and replenished with media containing 0.1% DMSO instead of GSI. In mock washouts, all media used for washing and replenishment contained 1 μM GSI 19 . Expression Constructs, Viral Production and Infection of Cells The expression vector MIEG3-IER5-FH was constructed by inserting human IER5 tagged with HA and 3XFLAG into the MIEG3 vector 19 , which is a murine stem cell virus (MSCV)‐based bicistronic retroviral construct expressing EGFP. MIEG3 expression plasmids containing IER5 and variants with point mutations were generated using a QuickChange II Site-Directed Mutagenesis Kit (Agilent). MIEG3 expression plasmids containing deletional IER5 mutants were constructed by replacing full-length IER5 in MIEG3-IER5-FH with PCR-generated DNA fragments containing mutant forms of IER5. Plasmids IER5-N50-NLS and IER5-N50-Nucleoplasmin NLS were constructed by replacing IER5 in MIEG3-IER5-FH with DNA sequences corresponding to IER5-N50 plus IER5 NLS or Nucleoplasmin NLS, respectively. The amino acid sequences for IER5 NLS and Nucleoplasmin NLS are RKRCAAGVGGGPAGCPAPGSTPLKKPRR and KRPAATKKAGQAKKKK, respectively. Retrovirus was prepared by transfecting Phoenix-gp cells with the MIEG3 vector and expression vectors for Gag/Pol and GALV. Viral supernatant was collected 48 h after transfection, centrifuged, and filtered through a 0.45 µm filter (Corning). For infection of target cells, 1 ml of virus was mixed with cells and protamine sulfate in a 6-well plate, and the plate was then centrifuged at 2,250 rpm for 90 min at room temperature. GFP-expressing cells were then isolated by sorting for GFP positivity using a Sony SH800S flow sorter several days after transduction 19 . Quantitative RT-PCR After 72 hours of GSI washout, cells were resuspended in Trizol (Life Technologies) and total RNA was prepared with RNeasy Mini kit (Qiagen). cDNA was synthesized with High-Capacity cDNA Reverse Transcripition Kit (Applied Biosystems). PCR was performed using the PowerUP SYBR Green Master Mix (Applied Biosystems) with QuantStudio 3 Real-Time PCR System (Applied Biosystems). Primers used for KRT1 and GAPDH are: forward 5’-GGACAGCTCCTTAGCATCTTATC-3’, reverse 5’-GGAGTTTAAGACCTCTCCACAAA-3’, and forward 5’-GAAGGTGAAGGTCGGAGTCAAC-3’, reverse 5’-TGGAAGATGGTGATGGGATTTC-3’, respectively. Gene expression was normalized to GAPDH using the ΔΔCT method. Immunoprecipitation and Western Blotting For immunoprecipitation assays from SC2 cells, cells in 10-cm dishes were washed in cold PBS and lysed in 1 ml of Pierce IP Lysis Buffer (Thermo Scientific) supplemented with protease inhibitors (Sigma). Cell lysates with equal amounts of protein were incubated with 25µl of washed Pierce Anti-HA Magnetic Beads (Thermo Scientific) overnight at 4°C with mixing. The beads were then washed three times with TBS-T and once with ultrapure water. The HA-tagged IER5 and its associated proteins were eluted with Pierce HA peptide (Themo Scientific). The eluates and input proteins were loaded on 3–8% SDS-polyacrylamide gels and resolved by electrophoresis. Following transfer to nitrocellulose membranes, proteins were incubated at 4° C overnight with the following primary antibodies: anti-PP2A B55 (100C1) or anti-HA (C29F4) (both from Cell Signaling Technology); or anti-IER5 (HPA029894) or anti-Flag (F3165) (both from Sigma). Secondary antibody was either goat anti-rabbit (7074) or horse anti-mouse (7076) IgG conjugated with horseradish peroxidase (Cell Signaling Technology). Staining was developed with SuperSignal West Dura Extended Duration Substrate (Thermo Scientific) for 2min at room temperature and documented by exposure to x-ray film. For immunoprecipitation assays for SERTA domain identification (Fig. 6E), HEK293T cells in 10-cm dishes were washed in cold PBS and lysed in 1 mL of Pierce IP Lysis Buffer (ThermoScientific) supplemented with protease inhibitors (Roche), and phosphatase inhibitors (PhosSTOP, Roche). Cell lysates were spun down at 21,000 x g and incubated with 25 uL Pierce Streptavidin Magnetic Beads (ThermoScientific) for 3 hours at 4°C with rotating. The beads were then washed three times using a buffer of 20 mM HEPES pH 7.5, 150 mM NaCl. The bound proteins were eluted with SDS-PAGE loading dye supplemented with protease inhibitors (Roche), and Phosphatase inhibitors (PhosSTOP, Roche). Samples were sonicated using a QSonica instrument prior to loading. The input and eluent proteins were loaded on 4–20% SDS-polyacrylamide gels and resolved by electrophoresis. Following transfer to nitrocellulose membranes, proteins were blocked at 4 °C overnight with non-fat milk. Proteins were incubated at room temperature for one hour with the following primary antibodies: anti-HA HRP conjugate (6E2) and anti-B55α (100C1) (both from Cell Signaling Technology). Secondary antibody was goat anti-rabbit IgG conjugated with IRDye 800CW (Fisher Scientific). Blot was first read out on a LI-COR Odyssey CLx imager, then staining was developed with Western Lightning Plus-ECL Substrate (Revvity) using a BioRad imager. Quantification and Statistical analysis Statistical analysis was performed using GraphPad Prism version 10 (GraphPad). Statistical details are indicated in the Figure Legends. Sample distribution and normality tests were performed for each data set and significance was determined using One-way analysis of variance (ANOVA) with Dunnett’s multiple-comparison post-hoc test. Supplementary Material 1 NIHMS2073241-supplement-1.pdf (19.8MB, pdf) SIGNIFICANCE. IER5 is a member of the AP1-regulated immediate early response gene family that is induced in response to DNA-damaging agents and heat shock, but its cellular function and molecular mechanism of action remains poorly understood. This work makes the fundamental conceptual advance that IER5 acts as a nuclear inhibitor of PP2A/B55α activity and reveals the structural basis for this effect. The cryo-EM structure of IER5-N50 bound to PP2A/B55α, supported by extensive validation of the binding pose, offers a roadmap for development of selective B55α inhibitor or molecular glue design. These studies also demonstrate that an engineered mini-IER5, which contains only IER5 and a nuclear localization sequence, is sufficient to recapitulate the biological activity of IER5 as a PP2A/B55α antagonist in keratinocytes, rescuing Notch-induced expression of a differentiation marker, KRT1 , in IER5 knockout cells. Lastly, this work extends beyond IER5 and its immediate relatives to show that a similar interaction occurs between PP2A/B55α and SERTA domain containing proteins (SERTADs). Together, these findings reveal a molecular basis for modulation of PP2A/B55α activity by IER5 in response to various environmental stressors and uncover biochemical parallels among helical hairpin proteins that regulate the activity and function of PP2A/B55α. HIGHLIGHTS. Cryo-EM structure of an IER5-N50 complex with PP2A/B55α reveals the binding site IER5-N50 inhibits pTau dephosphorylation by PP2A/B55α An engineered mini-IER5 complements biological function A domain homologous to IER5-N50 identified in SERTAD proteins also binds B55α ACKNOWLEDGMENTS We thank the Cryo-EM Center for Structural Biology at Harvard Medical School for help and advice with data collection, and members of the Blacklow lab and Alan Brown for helpful discussions. We thank Ernst Schmid for running and scoring alphafold predictions. This work was supported by NIH awards 1R35 CA220340 (to SCB) and 1R01 CA272484 (to SCB), the Ludwig Center at Harvard (JCA), the Warren Alpert Foundation (to SCB), and the Blavatnik Institute of Harvard Medical School. Footnotes Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. DECLARATION OF INTERESTS SCB is on the board of directors of the non-profit Institute for Protein Innovation and the Revson Foundation, is on the scientific advisory board for and receives funding from Erasca, Inc. for an unrelated project, is an advisor to MPM Capital, and is a consultant for IFM, Scorpion Therapeutics, Odyssey Therapeutics, Droia Ventures, and Ayala Pharmaceuticals for unrelated projects. JCA is a consultant for Ayala Pharmaceuticals, Cellestia, Inc., SpringWorks Therapeutics, and Remix Therapeutics. The other authors declare that they have no competing interests. REFERENCES 1. Amin P, Awal S, Vigneron S, Roque S, Mechali F, Labbé JC, Lorca T, and Castro A (2021). PP2A-B55: substrates and regulators in the control of cellular functions. Oncogene 2021 41:1 41, 1–14. 10.1038/s41388-021-02068-x. [ DOI ] [ PubMed ] [ Google Scholar ] 2. Grallert A, Boke E, Hagting A, Hodgson B, Connolly Y, Griffiths JR, Smith DL, Pines J, and Hagan IM (2015). A PP1/PP2A phosphatase relay controls mitotic progression. Nature 517, 94. 10.1038/NATURE14019. [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 3. Krasinska L, Domingo-Sananes MR, Kapuy O, Parisis N, Harker B, Moorhead G, Rossignol M, Novák B, and Fisher D (2011). Protein phosphatase 2A controls the order and dynamics of cell-cycle transitions. Molecular cell 44, 437–450. 10.1016/J.MOLCEL.2011.10.007. [ DOI ] [ PubMed ] [ Google Scholar ] 4. Swartz SZ, Nguyen HT, McEwan BC, Adamo ME, Cheeseman IM, and Kettenbach AN (2021). Selective dephosphorylation by pp2a-b55 directs the meiosis imeiosis ii transition in oocytes. eLife 10. 10.7554/ELIFE.70588. [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 5. Wurzenberger C, and Gerlich DW (2011). Phosphatases: providing safe passage through mitotic exit. Nature Reviews Molecular Cell Biology 2011 12:8 12, 469–482. 10.1038/nrm3149. [ DOI ] [ PubMed ] [ Google Scholar ] 6. Schmitz MHA, Held M, Janssens V, Hutchins JRA, Hudecz O, Ivanova E, Goris J, Trinkle-Mulcahy L, Lamond AI, Poser I, et al. (2010). Live-cell imaging RNAi screen identifies PP2A–B55α and importin-β1 as key mitotic exit regulators in human cells. Nature Cell Biology 12, 886–893. 10.1038/ncb2092. [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 7. Huang KL, Jee D, Stein CB, Elrod ND, Henriques T, Mascibroda LG, Baillat D, Russell WK, Adelman K, and Wagner EJ (2020). Integrator Recruits Protein Phosphatase 2A to Prevent Pause Release and Facilitate Transcription Termination. Molecular cell 80, 345. 10.1016/J.MOLCEL.2020.08.016. [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 8. Zheng H, Qi Y, Hu S, Cao X, Xu C, Yin Z, Chen X, Li Y, Liu W, Li J, et al. (2020). Identification of Integrator-PP2A complex (INTAC), an RNA polymerase II phosphatase. Science 370. 10.1126/science.abb5872. [ DOI ] [ PubMed ] [ Google Scholar ] 9. Xu Y, Chen Y, Zhang P, Jeffrey PD, and Shi Y (2008). Structure of a Protein Phosphatase 2A Holoenzyme: Insights into B55-Mediated Tau Dephosphorylation. Molecular Cell 31, 873–885. 10.1016/j.molcel.2008.08.006. [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 10. Fan L, Liu M-H, Guo M, Hu C-X, Yan Z-W, Chen J, Chen G-Q, and Huang Y (2016). FAM122A, a new endogenous inhibitor of protein phosphatase 2A. Oncotarget 7, 63887–63900. 10.18632/ONCOTARGET.11698. [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 11. Padi SKR, Vos MR, Godek RJ, Fuller JR, Kruse T, Hein JB, Nilsson J, Kelker MS, Page R, and Peti W (2023). Cryo-EM structures of PP2A:B55–FAM122A and PP2A:B55–ARPP19. Nature 2023 625:7993 625, 195–203. 10.1038/s41586-023-06870-3. [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 12. Gharbi-Ayachi A, Labbé J-C, Burgess A, Vigneron S, Strub J-M, Brioudes E, Van-Dorsselaer A, Castro A, and Lorca T (2010). The Substrate of Greatwall Kinase, Arpp19, Controls Mitosis by Inhibiting Protein Phosphatase 2A. Science 330, 1673–1677. 10.1126/science.1197048. [ DOI ] [ PubMed ] [ Google Scholar ] 13. Mochida S, Maslen SL, Skehel M, and Hunt T (2010). Greatwall Phosphorylates an Inhibitor of Protein Phosphatase 2Α That Is Essential for Mitosis. Science 330, 1670–1673. 10.1126/science.1195689. [ DOI ] [ PubMed ] [ Google Scholar ] 14. Wasserman JS, Faezov B, Patel KR, Kurimchak AM, Palacio SM, Glass DJ, Fowle H, McEwan BC, Xu Q, Zhao Z, et al. (2024). FAM122A ensures cell cycle interphase progression and checkpoint control by inhibiting B55α/PP2A through helical motifs. Nature Communications 2024 15:1 15, 1–20. 10.1038/s41467-024-50015-7. [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 15. Williams M, Lyu MS, Yang YL, Lin Edward P, Dunbrack R, Birren B, Cunningham J, and Hunter K (1999). Ier5, a novel member of the slow-kinetics immediate-early genes. Genomics 55, 327–334. 10.1006/GENO.1998.5679. [ DOI ] [ PubMed ] [ Google Scholar ] 16. Ding K-K, Shang Z-F, Hao C, Xu Q-Z, Shen J-J, Yang C-J, Xie Y-H, Qiao C, Wang Y, Xu L-L, et al. (2009). Induced expression of the IER5 gene by γ-ray irradiation and its involvement in cell cycle checkpoint control and survival. Radiation and Environmental Biophysics 48, 205–213. 10.1007/s00411-009-0213-4. [ DOI ] [ PubMed ] [ Google Scholar ] 17. Ishikawa Y, and Sakurai H (2015). Heat-induced expression of the immediate-early gene IER5 and its involvement in the proliferation of heat-shocked cells. The FEBS Journal 282, 332–340. 10.1111/FEBS.13134. [ DOI ] [ PubMed ] [ Google Scholar ] 18. Kis E, Szatmári T, Keszei M, Farkas R, Ésik O, Lumniczky K, Falus A, and Sáfrány G (2006). Microarray analysis of radiation response genes in primary human fibroblasts. International Journal of Radiation Oncology Biology Physics 66, 1506–1514. 10.1016/j.ijrobp.2006.08.004. [ DOI ] [ PubMed ] [ Google Scholar ] 19. Pan L, Lemieux ME, Thomas T, Rogers JM, Lipper CH, Lee W, Johnson C, Sholl LM, South AP, Marto JA, et al. (2020). IER5, a dna damage response gene, is required for notch-mediated induction of squamous cell differentiation. eLife 9, 1–32. 10.7554/ELIFE.58081. [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 20. Asano Y, Kawase T, Okabe A, Tsutsumi S, Ichikawa H, Tatebe S, Kitabayashi I, Tashiro F, Namiki H, Kondo T, et al. (2016). IER5 generates a novel hypophosphorylated active form of HSF1 and contributes to tumorigenesis. Scientific Reports 2016 6:1 6, 1–19. 10.1038/srep19174. [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 21. Ishikawa Y, Kawabata S, and Sakurai H (2015). HSF1 transcriptional activity is modulated by IER5 and PP2A/B55. FEBS Letters 589, 1150–1155. 10.1016/J.FEBSLET.2015.03.019. [ DOI ] [ PubMed ] [ Google Scholar ] 22. Kawabata S, Ishita Y, Ishikawa Y, and Sakurai H (2015). Immediate-early response 5 (IER5) interacts with protein phosphatase 2A and regulates the phosphorylation of ribosomal protein S6 kinase and heat shock factor 1. FEBS Letters 589, 3679–3685. 10.1016/j.febslet.2015.10.013. [ DOI ] [ PubMed ] [ Google Scholar ] 23. Jumper J, Evans R, Pritzel A, Green T, Figurnov M, Ronneberger O, Tunyasuvunakool K, Bates R, Žídek A, Potapenko A, et al. (2021). Highly accurate protein structure prediction with AlphaFold. Nature 596, 583–589. 10.1038/s41586-021-03819-2. [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 24. Kumar M, Gouw M, Michael S, Sámano-Sánchez H, Pancsa R, Glavina J, Diakogianni A, Valverde JA, Bukirova D, Signalyševa J, et al. (2020). ELM—the eukaryotic linear motif resource in 2020. Nucleic Acids Research 48, D296–D306. 10.1093/NAR/GKZ1030. [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 25. Fowle H, Zhao Z, Xu Q, Wasserman JS, Wang X, Adeyemi M, Feiser F, Kurimchak A, Atar D, McEwan BC, et al. (2021). Pp2a/b55a substrate recruitment as defined by the retinoblastoma-related protein p107. eLife 10, 1–26. 10.7554/eLife.63181. [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 26. Cundell MJ, Hutter LH, Bastos RN, Poser E, Holder J, Mohammed S, Novak B, and Barr FA (2016). A PP2A-B55 recognition signal controls substrate dephosphorylation kinetics during mitotic exit. The Journal of Cell Biology 214, 539. 10.1083/JCB.201606033. [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 27. Yamano S, Kimura M, Chen Y, Imamoto N, and Ohki R (2020). Nuclear import of IER5 is mediated by a classical bipartite nuclear localization signal and is required for HSF1 full activation. Experimental Cell Research 386, 111686. 10.1016/J.YEXCR.2019.111686. [ DOI ] [ PubMed ] [ Google Scholar ] 28. van Kempen M, Kim SS, Tumescheit C, Mirdita M, Lee J, Gilchrist CLM, Söding J, and Steinegger M (2023). Fast and accurate protein structure search with Foldseek. Nature Biotechnology. 10.1038/s41587-023-01773-0. [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 29. Zang ZJ, Gunaratnam L, Cheong JK, Lai LY, Hsiao LL, O’Leary E, Sun X, Salto-Tellez M, Bonventre JV, and Hsu SIH (2009). Identification of PP2A as a novel interactor and regulator of TRIP-Br1. Cellular Signalling 21, 34–42. 10.1016/j.cellsig.2008.09.018. [ DOI ] [ PubMed ] [ Google Scholar ] 30. Yadav L, Tamene F, Göös H, van Drogen A, Katainen R, Aebersold R, Gstaiger M, and Varjosalo M (2017). Systematic Analysis of Human Protein Phosphatase Interactions and Dynamics. Cell Systems 4, 430–444.e5. 10.1016/j.cels.2017.02.011. [ DOI ] [ PubMed ] [ Google Scholar ] 31. Mirdita M, Schütze K, Moriwaki Y, Heo L, Ovchinnikov S, and Steinegger M (2022). ColabFold: making protein folding accessible to all. Nature Methods 19, 679–682. 10.1038/s41592-022-01488-1. [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 32. Evans R, O’Neill M, Pritzel A, Antropova N, Senior A, Green T, Žídek A, Bates R, Blackwell S, Yim J, et al. (2022). Protein complex prediction with AlphaFold-Multimer. bioRxiv. 10.1101/2021.10.04.463034. [ DOI ] [ Google Scholar ] 33. Kruse T, Garvanska DH, Varga JK, Garland W, McEwan BC, Hein JB, Weisser MB, Benavides-Puy I, Chan CB, Sotelo-Parrilla P, et al. (2024). Substrate recognition principles for the PP2A-B55 protein phosphatase. Science Advances 10. 10.1126/sciadv.adp5491. [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 34. Padi SKR, Godek RJ, Peti W, and Page R (2024). Cryo-EM structures of PP2A : B55-Eya3 and PP2A : B55-p107 define PP2A : B55 substrate recruitment. bioRxiv. 10.1101/2024.09.16.613291. [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 35. Hsu SIH, Yang CM, Sim KG, Hentschel DM, O’leary E, and Bonventre JV (2001). TRIP-Br: A novel family of PHD zinc finger- and bromodomain-interacting proteins that regulate the transcriptional activity of E2F-1/DP-1. EMBO Journal 20, 2273–2285. 10.1093/emboj/20.9.2273. [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 36. Cho JM, Song DJ, Bergeron J, Benlimame N, Wold MS, and Alaoui-Jamali MA (2000). RBT1, a novel transcriptional co-activator, binds the second subunit of Replication Protein A. Nucleic Acids Research 28, 3478–3485. 10.1093/nar/28.18.3478. [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 37. Hayashi R, Goto Y, Ikeda R, Yokoyama KK, and Yoshida K (2006). CDCA4 is an E2F transcription factor family-induced nuclear factor that regulates E2F-dependent transcriptional activation and cell proliferation. Journal of Biological Chemistry 281, 35633–35648. 10.1074/jbc.M603800200. [ DOI ] [ PubMed ] [ Google Scholar ] 38. Alerasool N, Leng H, Lin Z-Y, Gingras A-C, and Taipale M (2022). Identification and functional characterization of transcriptional activators in human cells. Molecular Cell 82, 677–695.e7. 10.1016/j.molcel.2021.12.008. [ DOI ] [ PubMed ] [ Google Scholar ] 39. Letourneux C, Rocher G, and Porteu F (2006). B56-containing PP2A dephosphorylate ERK and their activity is controlled by the early gene IEX-1 and ERK. The EMBO journal 25, 727–738. 10.1038/SJ.EMBOJ.7600980. [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 40. Rocher G, Letourneux C, Lenormand P, and Porteu F (2007). Inhibition of B56-containing protein phosphatase 2As by the early response gene IEX-1 leads to control of Akt activity. Journal of Biological Chemistry 282, 5468–5477. 10.1074/jbc.M609712200. [ DOI ] [ PubMed ] [ Google Scholar ] 41. Clark AR, and Ohlmeyer M (2019). Protein phosphatase 2A as a therapeutic target in inflammation and neurodegeneration. Pharmacology & Therapeutics 201, 181. 10.1016/J.PHARMTHERA.2019.05.016. [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 42. Ronk H, Rosenblum JS, Kung T, and Zhuang Z (2022). Targeting PP2A for cancer therapeutic modulation. Cancer Biology & Medicine 19, 1428. 10.20892/J.ISSN.2095-3941.2022.0330. [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 43. Fischer ES, Böhm K, Lydeard JR, Yang H, Stadler MB, Cavadini S, Nagel J, Serluca F, Acker V, Lingaraju GM, et al. (2014). Structure of the DDB1-CRBN E3 ubiquitin ligase in complex with thalidomide. Nature 512, 49. 10.1038/NATURE13527. [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 44. Mastronarde DN (2003). SerialEM: A program for automated tilt series acquisition on Tecnai microscopes using prediction of specimen position. Microscopy and Microanalysis 9, 1182–1183. 10.1017/s1431927603445911. [ DOI ] [ Google Scholar ] 45. Morin A, Eisenbraun B, Key J, Sanschagrin PC, Timony MA, Ottaviano M, and Sliz P (2013). Collaboration gets the most out of software. eLife 2, e01456. 10.7554/eLife.01456. [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 46. Punjani A, Rubinstein JL, Fleet DJ, and Brubaker MA (2017). cryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination. Nature Methods 2017 14:3 14, 290–296. 10.1038/nmeth.4169. [ DOI ] [ PubMed ] [ Google Scholar ] 47. Sanchez-Garcia R, Gomez-Blanco J, Cuervo A, Carazo JM, Sorzano COS, and Vargas J (2021). DeepEMhancer: a deep learning solution for cryo-EM volume post-processing. Communications Biology 2021 4:1 4, 1–8. 10.1038/s42003-021-02399-1. [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 48. Pettersen EF, Goddard TD, Huang CC, Meng EC, Couch GS, Croll TI, Morris JH, and Ferrin TE (2021). UCSF ChimeraX: Structure visualization for researchers, educators, and developers. Protein Science 30, 70–82. 10.1002/pro.3943. [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 49. Adams PD, Afonine PV, Bunkóczi G, Chen VB, Davis IW, Echols N, Headd JJ, Hung LW, Kapral GJ, Grosse-Kunstleve RW, et al. (2010). PHENIX: A comprehensive Python-based system for macromolecular structure solution. Acta Crystallographica Section D: Biological Crystallography 66, 213–221. 10.1107/S0907444909052925. [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 50. Emsley P, Lohkamp B, Scott WG, and Cowtan K (2010). Features and development of Coot. Acta Crystallographica Section D: Biological Crystallography 66, 486–501. 10.1107/S0907444910007493. [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 51. Scheres SHW (2012). RELION: Implementation of a Bayesian approach to cryo-EM structure determination. Journal of Structural Biology 180, 519–530. 10.1016/j.jsb.2012.09.006. [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 52. Zhu J, Zhang Q, Zhang H, Shi Z, Hu M, and Bao C (2023). A minority of final stacks yields superior amplitude in single-particle cryo-EM. Nature Communications 14, 1–11. 10.1038/s41467-023-43555-x. [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 53. Scheres SHW (2016). Processing of Structurally Heterogeneous Cryo-EM Data in RELION. Methods in Enzymology 579, 125–157. 10.1016/BS.MIE.2016.04.012. [ DOI ] [ PubMed ] [ Google Scholar ] 54. Croll TI (2018). ISOLDE : a physically realistic environment for model building into low-resolution electron-density maps. Acta Crystallographica Section D Structural Biology 74, 519–530. 10.1107/S2059798318002425. [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 55. Yamashita K, Palmer CM, Burnley T, and Murshudov GN (2021). Cryo-EM single-particle structure refinement and map calculation using Servalcat. Acta Crystallographica Section D: Structural Biology 77, 1282–1291. 10.1107/S2059798321009475. [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 56. Afonine PV, Poon BK, Read RJ, Sobolev OV, Terwilliger TC, Urzhumtsev A, and Adams PD (2018). Real-space refinement in PHENIX for cryo-EM and crystallography. Acta Crystallographica Section D: Structural Biology 74, 531–544. 10.1107/S2059798318006551. [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 57. Nicholls RA, Long F, and Murshudov GN (2012). Low-resolution refinement tools in REFMAC5. Acta Crystallographica Section D: Biological Crystallography 68, 404. 10.1107/S090744491105606X. [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 58. Williams CJ, Headd JJ, Moriarty NW, Prisant MG, Videau LL, Deis LN, Verma V, Keedy DA, Hintze BJ, Chen VB, et al. (2018). MolProbity: More and better reference data for improved all-atom structure validation. Protein science : a publication of the Protein Society 27, 293–315. 10.1002/pro.3330. [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 59. Purdie KJ, Pourreyron C, and South AP (2011). Isolation and Culture of Squamous Cell Carcinoma Lines. Methods in Molecular Biology 731, 151–159. 10.1007/978-1-61779-080-5_14. [ DOI ] [ PubMed ] [ Google Scholar ] Associated Data This section collects any data citations, data availability statements, or supplementary materials included in this article. Supplementary Materials 1 NIHMS2073241-supplement-1.pdf (19.8MB, pdf) Data Availability Statement Atomic coordinates and EM map have been deposited in the RCSB Protein Data Bank (PDB) under the accession number 8UO5 and are publicly available as of the date of publication. The accession number is also listed in the key resources table . Original Western Blot images are available for review upon request. This paper does not report original code. Any additional information required to reanalyze the data reported in this work is available from the Lead Contact upon request. Key Resources Table REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies anti-B55α Cell Signaling Technology Cat# 2290S anti-IER5 Sigma Cat# HPA029894 anti-HA Cell Signaling Technology Cat# 3724S anti-HA (HRP conjugate) Cell Signaling Technology Cat# 2999S anti-pTau Thermo Scientific Cat # 44-752G anti-β-actin Sigma Cat# A1978 IRDye 800CW Donkey anti-Rabbit LI-COR Biosciences Cat# 926-32213, RRID:AB_621848 Anti-Mouse IgG, HRP-linked Antibody Cell Signaling Technology Cat# 7076 Anti-Mouse IgG, HRP-linked Antibody Cell Signaling Technology Cat# 7074 Bacterial and virus strains BL21(DE3) New England Biolabs (NEB) Cat# C2527H Stellar competent cells Takara Bio Cat# 636766 Chemicals, peptides, and recombinant proteins FBS GeminiBio Cat# 100-106 Penicillin and streptomycin ThermoFisher Scientific Cat# 15140163 Trypsin/2.21 mM EDTA in HBSS Corning Cat# 25-053-CI DMEM Corning Cat# 10-017-CV DPBS Corning Cat# 21-031-CV NaCl VWR Cat# 0241-10KG Glycerol americanbio Cat# AB00751-04000 HEPES Sigma-Aldrich Cat# H4034-1KG Trizma base Sigma-Aldrich Cat# T1503-5KG Glycine Sigma-Aldrich Cat# G7126-5KG b-Mercaptoethanol Sigma-Aldrich Cat# M6250-250ML SDS Sigma Cat# 75746-1KG Methanol VWR Cat# BDH2018-1GLP Tween-20 Sigma Cat# P7949-500ML DMSO Sigma-Aldrich Cat# D2650 Polyplus-transfection FectoPRO ® VWR Cat#10118-842 Lipofectamine 2000 Transfection Reagent ThermoFisher Cat# 11668019 Valproic acid sodium salt Sigma-Aldrich P4543-100G D-(+)-Glucose solution Sigma-Aldrich G8769-100ML Expi293 expression medium ThermoFisher Cat # A1435103 ESF 921 Insect Cell Culture Medium, Protein-Free Expression Systems Cat # 96001 Strep-Tactin ® XT 4Flow ® high capacity resin NEUROMICS Cat # 2-5030-025 Anti-Flag resin Produced in-house N/A Amylose Resin High Flow NEB Cat# E8022S Compound E EMD Millipore Cat # 565790 Critical commercial assays QIAprep Spin Miniprep Kit Qiagen Cat# 27106 PureLink ™ HiPure Plasmid Filter Maxiprep Kit Invitrogen Cat# K210016 RNeasy Mini Kit Qiagen Cat# 74104 High-Capacity cDNA Reverse Transcription Kit ThermoFisher Cat# 4368814 PowerUp SYBR Green Master Mix for qPCR ThermoFisher Cat# A25742 QuickChange II XL Site-Directed Mutagenesis Kit Agilent Cat# 200521 Pierce HA-Tag Magnetic IP/Co-IP Kit ThermoFisher Cat# 88838 In-Fusion HD Cloning Plus Clontech Cat # 638911 SuperSignal West Dura Extended Duration Substrate ThermoFisher Cat# 34075 Deposited data PP2A/B55α-IER5-N50 complex structure coordinates and EM density map This paper PDB ID: 8UO5 https://www.rcsb.org/structure/8UO5 https://doi.org/10.2210/pdb8uo5/pdb EMDB ID: EMD-42428 Experimental models: cell lines Expi293F cells ThermoFisher Cat # A14527 HEK293T cells ATCC Cat # CRL-3216 Sf9 Expression Systems Cat# 94-001F SC2 cells Pan, L. et al. 19 N/A SC2 IER5 KO cells (I5 cells) Pan, L. et al. 19 N/A Oligonucleotides KRT1 forward 5’-GGACAGCTCCTTAGCATCTTATC-3’ This paper N/A KRT1 reverse 5’-GGAGTTTAAGACCTCTCCACAAA-3’ This paper N/A GAPDH forward 5’-GAAGGTGAAGGTCGGAGTCAAC-3’ This paper N/A GAPDH reverse 5’-TGGAAGATGGTGATGGGATTTC-3’ This paper N/A Recombinant DNA pcDNA3.1/Hygro(+) ThermoFisher Scientific Cat # V87020 pAC8RedNK-Flag-CA Gift from Eric Fischer at the Dana-Farber Cancer Institute N/A pAC8RedNK-B55α Gift from Eric Fischer at the Dana-Farber Cancer Institute N/A pAC8RedNK-His-R1A Gift from Eric Fischer at the Dana-Farber Cancer Institute N/A pAC8RedNK-Strep-IER5-N50 This paper N/A gBlocks Integrated DNA Technologies N/A pcDNA3.1-Flag-MBP-IER5-FL This paper N/A pcDNA3.1-Flag-MBP-IER5-FL (K17E) This paper N/A pcDNA3.1-Flag-MBP-IER5-N50 This paper N/A pcDNA3.1-Flag-MBP-IER5-N50 (K17E) This paper N/A MIEG3-IER5-FH This paper N/A MIEG3-IER5-FH (I10A) This paper N/A MIEG3-IER5-FH (S14A) This paper N/A MIEG3-IER5-FH (L15R) This paper N/A MIEG3-IER5-FH (K17E) This paper N/A MIEG3-IER5-FH (N20A) This paper N/A MIEG3-IER5-FH (R25E) This paper N/A MIEG3-IER5-FH (K29E) This paper N/A MIEG3-IER5-FH (L30E) This paper N/A MIEG3-IER5-FH (H31A) This paper N/A MIEG3-IER5-FH (K32E) This paper N/A MIEG3-IER5-FH (L35A) This paper N/A MIEG3-IER5-FH (V39A) This paper N/A MIEG3-IER5-FH-N50-NLS This paper N/A MIEG3-IER5-FH-N50-Nucleoplasmin NLS This paper N/A pcDNA3.1-HA-SRTD1-TwinStrep This paper N/A pcDNA3.1-HA-SRTD2-TwinStrep This paper N/A pcDNA3.1-HA-SRTD3-TwinStrep This paper N/A pcDNA3.1-HA-SRTD4-TwinStrep This paper N/A pcDNA3.1-HA-IER2-TwinStrep This paper N/A pcDNA3.1-HA-IER5-TwinStrep This paper N/A pcDNA3.1-HA-CDCA4-TwinStrep This paper N/A pcDNA3.1-HA-IEX1-TwinStrep This paper N/A pcDNA3.1-HA-SRTD1-TwinStrep (K52E) This paper N/A pcDNA3.1-HA-SRTD2-TwinStrep (K47E) This paper N/A pcDNA3.1-HA-SRTD2-TwinStrep (L59E) This paper N/A pcDNA3.1-HA-SRTD2-TwinStrep (K47E/L59E) This paper N/A pcDNA3.1-HA-SRTD3-TwinStrep (K40E) This paper N/A pcDNA3.1-HA-SRTD4-TwinStrep (K115E) This paper N/A pcDNA3.1-HA-IER2-TwinStrep (K17E) This paper N/A pcDNA3.1-HA-IER5-TwinStrep (K17E) This paper N/A pcDNA3.1-HA-CDCA4-TwinStrep (K44E) This paper N/A pcDNA3.1-HA-IEX1-TwinStrep This paper N/A Software and algorithms SerialEM Mastronarde 44 https://bio3d.colorado.edu/SerialEM/ SBGrid Consortium Morin et al. 45 https://sbgrid.org cryoSPARC Structura Biotechnology Inc. 46 https://cryosparc.com DeepEMhancer Sanchez-Garcia et al. 47 https://github.com/rsanchezgarc/deepEMhancer/ UCSF Chimera Pettersen et al. 48 https://www.rbvi.ucsf.edu/chimera/ Phenix Adams, P. D. et al. 49 https://sbgrid.org/software/ Coot Emsley, P. & Cowtan, K. 50 https://sbgrid.org/software/ Pymol Schrödinger https://pymol.org/2/ PDB validation server World Wide Protein Data Bank https://www.wwpdb.org/ GraphPad Prism GraphPad RRID:SCR_002798 Other Quantifoil 400 mesh copper grids R 1.2/1.3 Electron Microscopy Sciences Q4100CR1.3 Open in a new tab ACTIONS View on publisher site PDF (2.0 MB) Cite Collections Permalink PERMALINK Copy RESOURCES Similar articles Cited by other articles Links to NCBI Databases Cite Copy Download .nbib .nbib Format: AMA APA MLA NLM Add to Collections Create a new collection Add to an existing collection Name your collection * Choose a collection Unable to load your collection due to an error Please try again Add Cancel Follow NCBI NCBI on X (formerly known as Twitter) NCBI on Facebook NCBI on LinkedIn NCBI on GitHub NCBI RSS feed Connect with NLM NLM on X (formerly known as Twitter) NLM on Facebook NLM on YouTube National Library of Medicine 8600 Rockville Pike Bethesda, MD 20894 Web Policies FOIA HHS Vulnerability Disclosure Help Accessibility Careers NLM NIH HHS USA.gov Back to Top

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