Structural and Functional Analysis of the GADD34:PP1 eIF2α Phosphatase

Abstract

Summary The attenuation of protein synthesis via the phosphorylation of eIF2α is a major stress response of all eukaryotic cells. The Growth Arrest and DNA-damaged protein 34 (GADD34) bound to the serine/threonine protein phosphatase 1 (PP1) is the necessary eIF2α phosphatase complex that returns mammalian cells to normal protein synthesis following stress. The molecular basis by which GADD34 recruits PP1 and its substrate eIF2α are not fully understood, hindering our understanding of the remarkable selectivity of the GADD34:PP1 phosphatase for eIF2α. Here we report detailed structural and functional analyses of the GADD34:PP1 holoenzyme and its recruitment of eIF2α. The data highlight independent interactions of PP1 and eIF2α with GADD34, demonstrating that GADD34 functions as a scaffold both in vitro and in cells. This work greatly enhances our molecular understanding of a major cellular eIF2α phosphatase and establishes the foundation for future translational work.

PPP1R15A-mediated dephosphorylation of eIF2α is unaffected by Sephin1 or Guanabenz

“…The latter values agreed with our previous measurements of G-actin’s stimulation of enzymatic activity (in an assay using the murine PPP1R15A) (Chen et al, 2015), whereas the EC 50 of human PPP1R15A was within an order of magnitude of the affinity of human PPP1R15A for PP1, as measured by isothermal titration calorimetry (Choy et al, 2015) (see below).

10.7554/eLife.26109.004Figure 2.eIF2α-P dephosphorylation kinetics as a function of human PPP1R15A 533-624 and G-actin concentration.( A ) Schema of the human PPP1R15A 533-624 construct used. The C-terminal Maltose Binding Protein (MBP) component, which stabilizes the fusion protein, is noted.

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“…The biotinylated PPP1R15A 533-624 ligand showed a robust 1:1 bimolecular interaction, with pure PP1, yielding a k off  = 0.21 ± 0.01 min −1 and a K d  = 20 ± 0.61 nM (Figure 5B). The higher affinity of PPP1R15 for PP1 observed here, compared to isothermal titration calorimetry (ITC) measurements of Choy et al (2015) ( K d  = 62 ± 14 nM) might reflect the contribution of contacts made by residues C-terminal to PPP1R15A L567 , which are present in the construct used here, but absent from the one used in the ITC measurements (Choy et al, 2015). Cooperativity provided by G-actin (present in the enzymatic assay, but absent from the BLI experiment) and steric hindrance from probe components might have contributed to the 3–5 fold lower value of the PPP1R15A EC 50 for eIF2α-P dephosphorylation in the enzymatic assay (7 nM, Figure 2B) compared to the K d observed by BLI.

10.7554/eLife.26109.007Figure 5.Affinity of the components of the tripartite holophosphatase for one another analysed by Bio-Layer Interferometry (BLI).( A ) Schema of the biotinylated human PPP1R15A 533-624 immobilized onto the BLI biosensor tip.

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Mouse Rif1 is a regulatory subunit of protein phosphatase 1 (PP1)

“…Binding curves were fitted using the simple Langmuir binding model 1:1 with K D  = 9.7 ± 1.7 nM. The nanomolar affinity of Rif1 CRI to PP1 is close to the highest values reported for PP1 interactors PNUTS (K D  = 8.7 nM) 40 and spinophilin (K D  = 9.3 nM) 25 , while the determined K D values for NIPP (73 nM) 41 and GADD34 (62 nM) 42 were significantly weaker.

Figure 3Rif1 CRI mediates high-affinity binding to PP1. ( A ) ITC of PP1 and Rif1 CRI was used to quantify the affinity of the interaction (K D  = 22.8 ± 4.2 nM).

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GADD34 Function in Protein Trafficking Promotes Adaptation to Hyperosmotic Stress in Human Corneal Cells

“…3E), with Sal003 having a more prominent effect. Sephin 1 is a very selective GADD34/PP1 inhibitor (Carrara et al, 2017; Das et al, 2015); in contrast, Sal003 has an additional inhibitory effect on PP1 when, instead of GADD34, it contains the constitutively-expressed regulatory subunit CReP (Choy et al, 2015). We next tested if the effects of GADD34/PP1 on Golgi fragmentation involve the well-known eIF2α-P-mediated responses in ISR.…”