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Baculovirus protein PK2 subverts eIF2? kinase function by mimicry of its kinase domain C-lobe.

ABSTRACT: Phosphorylation of eukaryotic translation initiation factor 2? (eIF2?) by eIF2? family kinases is a conserved mechanism to limit protein synthesis under specific stress conditions. The baculovirus-encoded protein PK2 inhibits eIF2? family kinases in vivo, thereby increasing viral fitness. However, the precise mechanism by which PK2 inhibits eIF2? kinase function remains an enigma. Here, we probed the mechanism by which PK2 inhibits the model eIF2? kinase human RNA-dependent protein kinase (PKR) as well as native insect eIF2? kinases. Although PK2 structurally mimics the C-lobe of a protein kinase domain and possesses the required docking infrastructure to bind eIF2?, we show that PK2 directly binds the kinase domain of PKR (PKR(KD)) but not eIF2?. The PKR(KD)-PK2 interaction requires a 22-residue N-terminal extension preceding the globular PK2 body that we term the "eIF2? 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 eIF2? 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 eIF2? kinase.

SUBMITTER: Li JJ

PROVIDER: S-EPMC4538665 | biostudies-literature | 2015 Aug

REPOSITORIES: biostudies-literature

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Baculovirus protein PK2 subverts eIF2α kinase function by mimicry of its kinase domain C-lobe.

Li John J JJ Cao Chune C Fixsen Sarah M SM Young Janet M JM Ono Chikako C Bando Hisanori H Elde Nels C NC Katsuma Susumu S Dever Thomas E TE Sicheri Frank F

Proceedings of the National Academy of Sciences of the United States of America 20150727 32

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) ...[more]

PMID: 26216977

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Project description:Enteric bacterial pathogens cause food borne disease, which constitutes an enormous economic and health burden. Enterohemorrhagic Escherichia coli (EHEC) causes a severe bloody diarrhea following transmission to humans through various means, including contaminated beef and vegetable products, water, or through contact with animals. EHEC also causes a potentially fatal kidney disease (hemolytic uremic syndrome) for which there is no effective treatment or prophylaxis. EHEC and other enteric pathogens (e.g., enteropathogenic E. coli (EPEC), Salmonella, Shigella, Yersinia) utilize a type III secretion system (T3SS) to inject virulence proteins (effectors) into host cells. While it is known that T3SS effectors subvert host cell function to promote diarrheal disease and bacterial transmission, in many cases, the mechanisms by which these effectors bind to host proteins and disrupt the normal function of intestinal epithelial cells have not been completely characterized. In this study, we present evidence that the E. coli O157:H7 nleH1 and nleH2 genes encode T3SS effectors that bind to the human ribosomal protein S3 (RPS3), a subunit of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kappaB) transcriptional complexes. NleH1 and NleH2 co-localized with RPS3 in the cytoplasm, but not in cell nuclei. The N-terminal region of both NleH1 and NleH2 was required for binding to the N-terminus of RPS3. NleH1 and NleH2 are autophosphorylated Ser/Thr protein kinases, but their binding to RPS3 is independent of kinase activity. NleH1, but not NleH2, reduced the nuclear abundance of RPS3 without altering the p50 or p65 NF-kappaB subunits or affecting the phosphorylation state or abundance of the inhibitory NF-kappaB chaperone IkappaBalpha NleH1 repressed the transcription of a RPS3/NF-kappaB-dependent reporter plasmid, but did not inhibit the transcription of RPS3-independent reporters. In contrast, NleH2 stimulated RPS3-dependent transcription, as well as an AP-1-dependent reporter. We identified a region of NleH1 (N40-K45) that is at least partially responsible for the inhibitory activity of NleH1 toward RPS3. Deleting nleH1 from E. coli O157:H7 produced a hypervirulent phenotype in a gnotobiotic piglet model of Shiga toxin-producing E. coli infection. We suggest that NleH may disrupt host innate immune responses by binding to a cofactor of host transcriptional complexes.

Dataset Information

Baculovirus protein PK2 subverts eIF2? kinase function by mimicry of its kinase domain C-lobe.

Publications

Baculovirus protein PK2 subverts eIF2α kinase function by mimicry of its kinase domain C-lobe.

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