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. 2017 Sep;24(9):708-716.
doi: 10.1038/nsmb.3443. Epub 2017 Jul 31.

Decoding the selectivity of eIF2α holophosphatases and PPP1R15A inhibitors

Affiliations

Decoding the selectivity of eIF2α holophosphatases and PPP1R15A inhibitors

Marta Carrara et al. Nat Struct Mol Biol. 2017 Sep.

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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Conflict of interest statement

Competing financial 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.

Figures

Figure 1
Figure 1. Reconstitution of functional eIF2α holophosphatases with recombinant proteins.
(a) Immunoblots showing P-eIF2α and eIF2α following a dephosphorylation reaction of 1 μM P-eIF2α by recombinant PP1 (1 μM) in the presence or absence of 1 μM of recombinant R15A, R15B or R3A. (b,c) Immunoblots of P-eIF2α and eIF2α following a dephosphorylation reaction of 1 μM P-eIF2α by PP1 (1 μM) in the presence or absence of (b) R15A and its inhibitors Guanabenz or Sephin1 and (c) calyculin A. (d) A titration curve of P-eIF2α (1 μM) dephosphorylation by increasing PP1 concentrations. A representative immunoblot corresponding to this titration is shown in Supplementary Figure 2b. Data are means ± s.e.m. (n = 3 independent experiments). (eg) Immunoblots of P-eIF2α and eIF2α following a dephosphorylation reaction of 1 μM P-eIF2α by PP1 (10 nM) in the presence or absence of 1 μM (e) R15A, (f) R15B, or (g) R3A. All dephosphorylation reactions were carried out at 30 °C for 16 h. For all experiments, data shown is representative of one of three independent experiments. Uncropped images for blots shown are available in Supplementary Data Set 1.
Figure 2
Figure 2. Defining the R15 domains required for PP1 binding and eIF2α holophosphatase activity.
(a,b) Schematics of the proteins studied here: (a) R15A and (b) R15B. Amino acid residues delimiting the amino-terminal and carboxy-terminal regions and the presence of an amino-terminal MBP-tag and carboxy-terminal His6-tag are shown. The location of the PP1-binding region is indicated. (c,d) Thermophoresis binding curves of labeled PP1 binding to titrations of unlabeled (c) R15A (amino acids 325–636), R15AN (amino acids 325–512), R15AC (amino acids 513–636) or (d) R15B (amino acids 340–698), R15BN (amino acids 340–635), R15BC (amino acids 636–698). Dissociation constants (KD) are means ± s.e.m. (n = 3 independent experiments). Thermophoresis raw data are available in Supplementary Table 2. (e,f) Immunoblots of P-eIF2α and eIF2α following a dephosphorylation reaction by PP1 (10 nM) in the presence or absence of (e) R15A, R15AN, R15AC or (f) R15B, R15BN, R15BC. Dephosphorylation reactions were carried out at 30 °C for 16 h. Data shown are representative immunoblots of three independent experiments; uncropped images for blots shown are in Supplementary Data Set 1.
Figure 3
Figure 3. Functional R15 holoenzymes have a higher affinity for their substrate than PP1.
(a) Thermophoresis binding curve of labeled P-eIF2α binding to titrations of unlabeled PP1D95A. KD is the mean ± s.e.m. (n = 3; biological replicates). (bd) Thermophoresis-binding curves of labeled P-eIF2α binding to titrations of unlabeled (b) R15A, R15AN, R15AC (c) R15B, R15BN, R15BC, or (d) R3A. KD are means ± s.e.m. (n = 3 independent experiments). Thermophoresis raw data are available in Supplementary Table 2. (e) KD of labeled P-eIF2α to titrations of unlabeled PP1D95A, in the presence of saturating, and unlabeled, functional R15 (R15A or R15B), their nonfunctional carboxy-terminal fragments (R15AC or R15BC), their amino-terminal fragments (R15AN or R15BN), or R3A. The value of P-eIF2α binding to PP1D95A corresponds to that shown in a. KD values are means ± s.e.m. (n = 3 independent experiments). Details in Supplementary Table 1. Statistical significances, relative to PP1D95A binding to P-eIF2α, are shown. **P ≤ 0.01, ***P ≤ 0.001, n.s., not significant (one-way ANOVA).
Figure 4
Figure 4. R15A inhibitors alter the protease sensitivity of R15A and selectively decrease substrate binding to R15A.
(a) Binding of R15A to biotinylated Guanabenz and biotinylated Sephin1 immobilized on streptavidin beads. Immunoblots of input and bound samples, probed with α-MBP (to reveal R15s) are shown. Bound samples (lane 3) were eluted with an excess of Guanabenz (top) or Sephin1 (bottom). Data shown are representative of three independent experiments. (b) The chemical structures of Guanabenz, Sephin1 and C3. (c,d) Coomassie-stained gels showing limited trypsin digestion of (c) R15A and (d) R15B in the presence or absence of Guanabenz, Sephin1 or C3. Trypsin digestions were carried out using 2.5 nM of trypsin, and reactions were allowed to proceed for 0 h, 30 min, 1 h, 2 h and 3 h (left to right lanes of each gel, respectively) at 22 °C and terminated by the addition of 4% SDS Laemmli sample buffer. Data shown are representative of three independent experiments. (eh) Binding of P-eIF2α to MBP-tagged R15s immobilized on magnetic amylose beads in the presence or absence of (e) Guanabenz or (g) Sephin1. Immunoblots of input and bound samples, probed with α-MBP (to reveal R15s) or α-eIF2α antibodies are shown. Data shown are representative of three independent experiments. (f,h) Levels of eIF2α bound to R15s in the presence or absence of (f) Guanabenz or (h) Sephin1. eIF2α immunoblots of three independent pull down experiments, as shown in e and g were quantified and normalized independently for R15A and R15B against DMSO control samples (lanes 10 and 13, for R15A and R15B, respectively, in e and g). Means ± s.e.m. (n = 3 independent experiments) are shown. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, n.s., not significant (two-way ANOVA). Uncropped images of blots shown are in Supplementary Data Set 1.
Figure 5
Figure 5. An activity assay with functional recombinant R15 holoenzymes recapitulates selective inhibition of R15A by Guanabenz and Sephin1.
(a,b) Immunoblots of P-eIF2α and eIF2α following a dephosphorylation reaction of 1 μM P-eIF2α by R15A–PP1 and R15B–PP1 holoenzymes in the presence or absence of (a) Guanabenz or (b) Sephin1. (c) Immunoblots of P-eIF2α and eIF2α following a dephosphorylation reaction of 1 μM P-eIF2α by R15AN–R15BC–PP1 and R15BN–R15AC–PP1 chimeric holoenzymes in the presence or absence of Guanabenz or Sephin1. (d) Immunoblots of P-eIF2α and eIF2α following a dephosphorylation reaction of 1 μM P-eIF2α by R15A–PP1 and R15B–PP1 holoenzymes in the presence or absence of C3. All dephosphorylation reactions were carried out using R15 at 50 nM and PP1 at 10 nM at 30 °C for 16 h. For all experiments, data shown are representative of three independent experiments. P-eIF2α levels were quantified and normalized against levels in lane 2 (P-eIF2α alone) and expressed as percentage relative to levels in lane 2. Uncropped images of blots shown are in Supplementary Data Set 1.

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