title: G-actin provides substrate-specificity to eukaryotic initiation factor 2α holophosphatases.
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 substrate-selective 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.
authors.affiliation: Cambridge Institute for Medical Research, University of Cambridge, Cambridge, United Kingdom.
authors.forename: Ruming
authors.initials: R
authors.lastname: Chen
authors.affiliation: Cambridge Institute for Medical Research, University of Cambridge, Cambridge, United Kingdom.
authors.forename: Cláudia
authors.initials: C
authors.lastname: Rato
authors.affiliation: Cambridge Institute for Medical Research, University of Cambridge, Cambridge, United Kingdom.
authors.forename: Yahui
authors.initials: Y
authors.lastname: Yan
authors.affiliation: Cambridge Institute for Medical Research, University of Cambridge, Cambridge, United Kingdom.
authors.forename: Ana
authors.initials: A
authors.lastname: Crespillo-Casado
authors.affiliation: Cambridge Institute for Medical Research, University of Cambridge, Cambridge, United Kingdom.
authors.forename: Hanna J
authors.initials: HJ
authors.lastname: Clarke
authors.affiliation: Cambridge Institute for Medical Research, University of Cambridge, Cambridge, United Kingdom.
authors.forename: Heather P
authors.initials: HP
authors.lastname: Harding
authors.affiliation: Cambridge Institute for Medical Research, University of Cambridge, Cambridge, United Kingdom.
authors.forename: Stefan J
authors.identifier: http://orcid.org/0000-0001-8472-7183
authors.initials: SJ
authors.lastname: Marciniak
authors.affiliation: Cambridge Institute for Medical Research, University of Cambridge, Cambridge, United Kingdom.
authors.forename: Randy J
authors.identifier: http://orcid.org/0000-0001-8273-0047
authors.initials: RJ
authors.lastname: Read
authors.affiliation: Cambridge Institute for Medical Research, University of Cambridge, Cambridge, United Kingdom.
authors.forename: David
authors.identifier: http://orcid.org/0000-0002-3014-5636
authors.initials: D
authors.lastname: Ron
mesh_terms: D000199:Actins / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D020134:Catalytic Domain; D002417:Cattle; D017124:Conserved Sequence; D006224:Cricetinae; D003412:Cricetulus; D015852:Eukaryotic Initiation Factor-2 / Q000378:metabolism*; D006801:Humans; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D010766:Phosphorylation; D054645:Protein Phosphatase 1 / Q000378:metabolism*; D011817:Rabbits; D013379:Substrate Specificity
chemical_list: D000199:Actins; D015852:Eukaryotic Initiation Factor-2; C513256:Ppp1r15a protein, mouse; D054645:Protein Phosphatase 1
keywords: E. coli; biochemistry; biophysics; cell lines; human; mouse; rabbit; structural biology