title: Baculovirus protein PK2 subverts eIF2α kinase function by mimicry of its kinase domain C-lobe.
abstract: Phosphorylation of eukaryotic translation initiation factor 2α (eIF2α) by eIF2α family kinases is a conserved mechanism to limit protein synthesis under specific stress conditions. The baculovirus-encoded protein PK2 inhibits eIF2α family kinases in vivo, thereby increasing viral fitness. However, the precise mechanism by which PK2 inhibits eIF2α kinase function remains an enigma. Here, we probed the mechanism by which PK2 inhibits the model eIF2α kinase human RNA-dependent protein kinase (PKR) as well as native insect eIF2α kinases. Although PK2 structurally mimics the C-lobe of a protein kinase domain and possesses the required docking infrastructure to bind eIF2α, we show that PK2 directly binds the kinase domain of PKR (PKR(KD)) but not eIF2α. The PKR(KD)-PK2 interaction requires a 22-residue N-terminal extension preceding the globular PK2 body that we term the "eIF2α kinase C-lobe mimic" (EKCM) domain. The functional insufficiency of the N-terminal extension of PK2 implicates a role for the adjacent EKCM domain in binding and inhibiting PKR. Using a genetic screen in yeast, we isolated PK2-activating mutations that cluster to a surface of the EKCM domain that in bona fide protein kinases forms the catalytic cleft through sandwiching interactions with a kinase N-lobe. Interaction assays revealed that PK2 associates with the N- but not the C-lobe of PKR(KD). We propose an inhibitory model whereby PK2 engages the N-lobe of an eIF2α kinase domain to create a nonfunctional pseudokinase domain complex, possibly through a lobe-swapping mechanism. Finally, we show that PK2 enhances baculovirus fitness in insect hosts by targeting the endogenous insect heme-regulated inhibitor (HRI)-like eIF2α kinase.
authors.affiliation: Program in Systems Biology, Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, ON M5G 1X5, Canada; Department of Molecular Genetics, University of Toronto, Toronto, ON M5S 1A8, Canada;
authors.forename: John J
authors.initials: JJ
authors.lastname: Li
authors.affiliation: Laboratory of Gene Regulation and Development, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892;
authors.forename: Chune
authors.initials: C
authors.lastname: Cao
authors.affiliation: Department of Human Genetics, University of Utah School of Medicine, Salt Lake City, UT 84112;
authors.forename: Sarah M
authors.initials: SM
authors.lastname: Fixsen
authors.affiliation: Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, WA 98109;
authors.forename: Janet M
authors.initials: JM
authors.lastname: Young
authors.affiliation: Laboratory of Applied Molecular Entomology, Division of Agrobiology, Graduate School of Agriculture, Hokkaido University, Sapporo 060-8589, Japan; katsuma@ss.ab.a.u-tokyo.ac.jp tdever@nih.gov sicheri@lunenfeld.ca.
authors.forename: Chikako
authors.initials: C
authors.lastname: Ono
authors.affiliation: Laboratory of Applied Molecular Entomology, Division of Agrobiology, Graduate School of Agriculture, Hokkaido University, Sapporo 060-8589, Japan;
authors.forename: Hisanori
authors.initials: H
authors.lastname: Bando
authors.affiliation: Department of Human Genetics, University of Utah School of Medicine, Salt Lake City, UT 84112;
authors.forename: Nels C
authors.initials: NC
authors.lastname: Elde
authors.affiliation: Department of Agricultural and Environmental Biology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo 113-8657, Japan; katsuma@ss.ab.a.u-tokyo.ac.jp tdever@nih.gov sicheri@lunenfeld.ca.
authors.forename: Susumu
authors.initials: S
authors.lastname: Katsuma
authors.affiliation: Laboratory of Gene Regulation and Development, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892; katsuma@ss.ab.a.u-tokyo.ac.jp tdever@nih.gov sicheri@lunenfeld.ca.
authors.forename: Thomas E
authors.initials: TE
authors.lastname: Dever
authors.affiliation: Program in Systems Biology, Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, ON M5G 1X5, Canada; Department of Molecular Genetics, University of Toronto, Toronto, ON M5S 1A8, Canada; Department of Biochemistry, University of Toronto, Toronto, Ontario M5S 1A8, Canada katsuma@ss.ab.a.u-tokyo.ac.jp tdever@nih.gov sicheri@lunenfeld.ca.
authors.forename: Frank
authors.initials: F
authors.lastname: Sicheri
mesh_terms: D000818:Animals; D016367:Baculoviridae / Q000502:physiology; D012831:Bombyx / Q000821:virology; D002460:Cell Line; D004252:DNA Mutational Analysis; D008958:Models, Molecular; D018716:Molecular Mimicry; D009154:Mutation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship; D014764:Viral Proteins / Q000737:chemistry* / Q000378:metabolism*; D019892:eIF-2 Kinase / Q000378:metabolism*
chemical_list: D014764:Viral Proteins; C111768:baculovirus PK2; D019892:eIF-2 Kinase
keywords: HRI; PKR; eIF2α kinase inhibition; lobe-swap; viral mimicry