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Decoding the selectivity of eIF2α holophosphatases and PPP1R15A inhibitors

Abstract

The reversible phosphorylation of proteins controls most cellular functions. Protein kinases have been popular drug targets, unlike phosphatases, which remain a drug discovery challenge. Guanabenz and Sephin1 are selective inhibitors of the phosphatase regulatory subunit PPP1R15A (R15A) that prolong the benefit of eIF2α phosphorylation, thereby protecting cells from proteostatic defects. In mice, Sephin1 prevents two neurodegenerative diseases, Charcot–Marie–Tooth 1B (CMT-1B) and SOD1-mediated amyotrophic lateral sclerosis (ALS). However, the molecular basis for R15A inhibition is unknown. Here we reconstituted human recombinant eIF2α holophosphatases, R15A–PP1 and R15B–PP1, whose activity depends on both the catalytic subunit PP1 (protein phosphatase 1) and either R15A or R15B. This system enabled the functional characterization of these holophosphatases and revealed that Guanabenz and Sephin1 induced a selective conformational change in R15A, detected by resistance to limited proteolysis. This altered the recruitment of eIF2α, preventing its dephosphorylation. This work demonstrates that regulatory subunits of phosphatases are valid drug targets and provides the molecular rationale to expand this concept to other phosphatases.

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Figure 1: Reconstitution of functional eIF2α holophosphatases with recombinant proteins.
Figure 2: Defining the R15 domains required for PP1 binding and eIF2α holophosphatase activity.
Figure 3: Functional R15 holoenzymes have a higher affinity for their substrate than PP1.
Figure 4: R15A inhibitors alter the protease sensitivity of R15A and selectively decrease substrate binding to R15A.
Figure 5: An activity assay with functional recombinant R15 holoenzymes recapitulates selective inhibition of R15A by Guanabenz and Sephin1.

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Acknowledgements

We thank members of the Bertolotti laboratory for advice and discussions, S. McLaughlin for help with light scattering and M. Goedert and R. Taylor for comments on the manuscript. This work was supported by the Medical Research Council (UK) grant MC_U105185860 and the European Research Council (ERC) under the European Union's Seventh Framework Programme (FP7/2007-2013)/ERC grant 309516. A.B. is an honorary fellow of the Clinical Neurosciences Department of Cambridge University.

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Contributions

M.C. designed, performed and analyzed all experiments, prepared the figures and helped with the manuscript. A.S. discovered compound C3 and performed cytotoxicity experiments. A.B. designed and guided the study and wrote the manuscript.

Corresponding author

Correspondence to Anne Bertolotti.

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Competing interests

M.C. and A.B. are co-inventors on Great Britain patent application 1709927.6 on the activity assays and methods described in this manuscript.

Integrated supplementary information

Supplementary Figure 1 Coomassie-stained gel of purified recombinant proteins

Coomassie-stained gel of ~ 3 μg of purified R15A, R15B, R3A, eIF2α and PP1 proteins. Molecular weight markers, in kilodalton (kDa), are indicated.

Supplementary Figure 2 Reconstitution of functional eIF2α holophosphatases with recombinant proteins

(a,c,d,e,f) Phos-tag gels of experiments shown in Fig 1. Supplementary Figure 2a,c,d,e,f correspond to samples analyzed by immunoblotting shown in Fig 1a,d,e,f,g respectively. Samples were run on 15% Phos-tag gels and visualized by Coomassie staining. (b) Representative immunoblot of P-eIF2α and eIF2α following a dephosphorylation reaction, of 1 μM P-eIF2α, by increasing amounts of PP1 used for the titration curve in Fig. 1d. The concentration of PP1 used is indicated. Dephosphorylation reactions were carried out at 30 °C for 16 h. Representative results of three independent experiments are shown (n = 3; biological replicates).

Supplementary Figure 3 Functional R15-PP1 do not dephosphorylate 33P Phosphorylase a

Phosphorimaging and Coomassie gel of 33P Phosphorylase a following a dephosphorylation reaction, of 1 μM 33P Phosphorylase a, by PP1 (1 μM and 10 nM) in the presence or absence of 1 μM R15A or R15B. All dephosphorylation reactions were carried out at 30 °C for 16 h. Representative results of three independent experiments are shown (n = 3; biological replicates).

Supplementary Figure 4 PP1D95A is catalytically dead

Immunoblot of P-eIF2α and eIF2α following a dephosphorylation reaction, of 1 μM P-eIF2α, by PP1 (10 nM) or PP1D95A (10 nM) in the presence of 1 μM R15A, or R15B. All dephosphorylation reactions were carried out at 30 °C for 16 h. Representative results of three independent experiments are shown (n = 3; biological replicates).

Supplementary Figure 5 Cytoprotection of Tunicamycin-treated cells by Guanabenz, but not by C3

Dose dependent cytoprotection of HeLa cells by Guanabenz, but not the inactive derivative C3, in response to Tunicamycin stress. Vehicle concentrations represent the corresponding amounts of DMSO used in compound treated samples (from 0 to 0.04% highest concentration). Cell viability after 72 h of treatment was monitored using the IncuCyte ZOOM system. Representative results of three independent experiments are shown (n = 3; biological replicates).

Supplementary Figure 6 Limited trypsin digestion of MBP

Coomassie-stained gel showing limited trypsin digestion of MBP in the presence or absence of Guanabenz or Sephin1. Trypsin digestions were carried out using 2.5 nM of trypsin. Reactions were allowed to proceed for 0 h (first lane in each gel), 30 min, 1 h, 2 h or 3 h at 22 °C. Reactions were terminated by addition of 4% SDS Laemmli sample buffer. Representative results of three independent experiments are shown (n = 3; biological replicates).

Supplementary Figure 7 Aggregation of R15A with Salubrinal but not with saturating concentrations of Guanabenz, Sephin1 or C3

Light scattering measurements of 5 μM R15A in the presence of 1 mM Guanabenz, 1 mM Sephin1, 1 mM C3, 50 μM Salubrinal or DMSO vehicle. Absorbance at 380 nm was monitored over 10 min at 20 °C with constant stirring. 100 data points are plotted for each sample.

Supplementary Figure 8 Binding of recombinant PP1 to R15s in the presence or absence of Guanabenz or Sephin1

Binding of recombinant PP1 to MBP-tagged R15s immobilized on magnetic amylose beads (see methods) in the presence or absence of Guanabenz or Sephin1. Immunoblots of input and bound samples, probed with α-MBP (to reveal R15s) or α-PPP1A (to reveal PP1) antibodies are shown. Representative results of three independent experiments are shown (n = 3; biological replicates).

Supplementary Figure 9 Salubrinal inhibits PP1 and causes aggregation of P-eIF2α, in contrast to Sephin1 and Guanabenz that have no effect in these assays

(a-d) Immunoblots of P-eIF2α and eIF2α following a dephosphorylation reactions, of 1 μM P-eIF2α, by increasing amounts of PP1, in the presence of (a) DMSO vehicle control, (b) Guanabenz, (c) Sephin1, or (d) Salubrinal. The concentration of PP1 used is indicated. All dephosphorylation reactions were carried out at 30 °C for 16 h. (e,f) Images of the effect of adding increasing amounts (from 30 μM up to 2 mM) Salubrinal to (e) 1μM P-eIF2α and (f) 1μM PP1. (g,h) Images of the effect of adding 2 mM Guanabenz or Sephin1 to (g) 1 μM P-eIF2α and (h) 1 μM PP1.

Supplementary Figure 10 Time course of P-eIF2α dephosphorylation by PP1 and R15A-PP1 holophosphatase

Phos-tag gel showing P-eIF2α and eIF2α following a dephosphorylation reaction, of 1μM P-eIF2α, using free PP1 (10 nM) or PP1 (10 nM) plus 50 nM R15A. Dephosphorylation reactions were carried out at 30 °C for the time indicated (min (‘) or hours (h)). Note that similar to what has been reported (Crespillo-Casado, A. et al., eLife. 6, e26109, 2017), no dephosphorylation was seen after 20 min in either condition. Representative results of three independent experiments are shown (n = 3; biological replicates).

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–10 and Supplementary Table 1. (PDF 1011 kb)

Life Sciences Reporting Summary (PDF 140 kb)

Supplementary Table 2

Raw values of Figs. 1, 2c, 2d, 3, 4f, 4h, 5, Supplementary Fig 5, Supplementary Fig 7. (XLSX 145 kb)

Supplementary Data Set 1

Uncropped images of all gels. (PDF 8028 kb)

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Carrara, M., Sigurdardottir, A. & Bertolotti, A. Decoding the selectivity of eIF2α holophosphatases and PPP1R15A inhibitors. Nat Struct Mol Biol 24, 708–716 (2017). https://doi.org/10.1038/nsmb.3443

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