G-actin provides substrate-specificity to eukaryotic initiation factor 2α holophosphatases

RESEARCH ARTICLE elifesciences.org G-actin provides substrate-specificity to eukaryotic initiation factor 2α holophosphatases Ruming Chen1, Cláudia Rato1†, Yahui Yan1†, Ana Crespillo-Casado1, Hanna J Clarke1, Heather P Harding1, Stefan J Marciniak1*, Randy J Read1*, David Ron1,2,3* 1 Cambridge Institute for Medical Research, University of Cambridge, Cambridge, United Kingdom; 2Wellcome Trust MRC Institute of Metabolic Science, University of Cambridge, Cambridge, United Kingdom; 3NIHR Cambridge, Biomedical Research Centre, University of Cambridge, Cambridge, United Kingdom *For correspondence: sjm20@ cam.ac.uk (SJM); (RJR); (DR) Abstract Dephosphorylation of eukaryotic translation initiation factor 2a (eIF2a) restores protein synthesis at the waning of stress responses and requires a PP1 catalytic subunit and a regulatory subunit, PPP1R15A/GADD34 or PPP1R15B/CReP. Surprisingly, PPP1R15-PP1 binary complexes reconstituted in vitro lacked substrate selectivity. However, selectivity was restored by crude cell lysate or purified G-actin, which joined PPP1R15-PP1 to form a stable ternary complex. In crystal structures of the non-selective PPP1R15B-PP1G complex, the functional core of PPP1R15 made multiple surface contacts with PP1G, but at a distance from the active site, whereas in the substrateselective ternary complex, actin contributes to one face of a platform encompassing the active site. Computational docking of the N-terminal lobe of eIF2a at this platform placed phosphorylated serine 51 near the active site. Mutagenesis of predicted surface-contacting residues enfeebled dephosphorylation, suggesting that avidity for the substrate plays an important role in imparting specificity on the PPP1R15B-PP1G-actin ternary complex. DOI: 10.7554/eLife.04871.001 † These authors contributed equally to this work Competing interests: See page 25 Funding: See page 25 Received: 23 September 2014 Accepted: 12 March 2015 Published: 16 March 2015 Reviewing editor: W James Nelson, Stanford University, United States Copyright Chen et al. This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited. Introduction Reversible phosphorylation of the alpha subunit of translation initiation factor 2 (eIF2a) is pivotal to control of global rates of protein synthesis and to modulating mRNA-specific translation in eukaryotes (Sonenberg and Hinnebusch, 2009). Phosphorylated eIF2 inhibits its guanine nucleotide exchange factor, eIF2B, attenuating the translation of most mRNA, whilst the translation of a small subset of mRNAs, with special 5′ untranslated regions, is increased (Hinnebusch, 2005). As the latter encode potent transcription factors, such as GCN4 in yeasts and ATF4 in animals, eIF2a phosphorylation activates gene expression programs with broad physiological ramifications: the integrated stress response (ISR) in mammals and its yeast counterpart, the general control response (Harding et al., 2003). Four kinases are known to couple diverse upstream signals to eIF2a phosphorylation (Ron and Harding, 2007). PERK restrains protein synthesis in response to unfolded proteins in the endoplasmic reticulum. HRI accomplishes the same in response to heme restriction in developing erythroid precursors, whereas PKR is activated by double-stranded RNA to curtail viral protein synthesis in infected cells. The oldest eIF2a kinase, GCN2, is activated by uncharged tRNAs to restore amino acid balance by ISR activation. In animal cells, eIF2a phosphorylation is reversed by cellular phosphatase complexes consisting of a protein phosphatase 1 catalytic subunit (PP1) and a substrate-specific regulatory subunit. Two such Chen et al. eLife 2015;4:e04871. DOI: 10.7554/eLife.04871 1 of 28 Research article Biochemistry | Biophysics and structural biology eLife digest For a cell to build a protein, it must first copy the instructions contained within a gene. A complex molecular machine called a ribosome then reads these instructions and translates them into a protein. This translation process involves a number of steps. Proteins called eukaryotic translation initiation factors (or eIFs for short) coordinate the first step in the process, which is known as ‘initiation’. The eIFs also provide the cell with ways to control how quickly it makes proteins. For example, when a cell is stressed, either by starvation or toxins, it adds a phosphate group onto part of an eIF protein, called eIF2α. This modification makes this eIF protein less able to initiate translation, and so the cell builds fewer proteins and conserves more of its resources during times of stress. Once the stressful conditions are over, the phosphate group is removed from eIF2α by an enzyme called a phosphatase. This phosphatase contains two subunits: one that recognizes eIF2α and another that removes the phosphate group. However, experiments that attempted to recreate this phosphatase activity using just these two subunits in a test tube failed to generate a working enzyme that specifically targeted the phosphate group of eIF2α. This suggests that in cells this enzyme contains an additional unknown subunit. Now, Chen et al. (and Chambers, Dalton et al.) report the identity of a ‘missing’ third subunit as a protein known as globular-actin or G-actin. First, Chen et al. looked at the three-dimensional structure of a two-subunit complex formed from the previously known subunits of the phosphatase enzyme, and confirmed that it could remove phosphate groups from a range of proteins and not just eIF2α. However, when a mixture of other proteins taken from mouse cells was added to this two-subunit complex, the complex could specifically remove the phosphate group on the eIF2α protein. Further experiments revealed that G-actin was the protein in the mixture that, when added to the two-subunit complex, made it specifically target the eIF2α protein. Chen et al. then used a combination of biochemical and structural biology techniques to investigate the phosphatase activity of the three-subunit complex. These findings suggest a plausible molecular mechanism by which the three-subunit complex becomes selective for its target, but further refinements to the structural work will be needed to critically test these suggestions. DOI: 10.7554/eLife.04871.002 regulatory subunits have been identified in mammals: PPP1R15A (known as GADD34) is encoded by an ISR-inducible gene (Novoa et al., 2001; Ma and Hendershot, 2003), whereas PPP1R15B (known as CReP) is constitutively present (Jousse et al., 2003). Cells lacking PPP1R15A are impaired in recovery of protein synthesis during resolution of the stress response (Novoa et al., 2003; Marciniak et al., 2004), whereas elimination of PPP1R15B results in developmental impairment and perinatal lethality of mice. Importantly, inactivation of both PPP1R15 isoforms is lethal to cells (Tsayt (...truncated)