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Structural and functional characterization of salmon STAT1, STAT2 and IRF9 homologs sheds light on interferon signaling in teleosts.

Sobhkhez M, Skjesol A, Thomassen E, Tollersrud LG, Iliev DB, Sun B, Robertsen B, Jørgensen JB - FEBS Open Bio (2014)

Bottom Line: Overexpression of STAT2a or STAT2b together with STAT1a activated a GAS-containing reporter gene construct in IFNγ-stimulated cells.The highest induction of GAS promoter activation was found in IFNγ-stimulated cells transfected with IRF9 alone.Taken together, these data suggest that salmon STAT2 and IRF9 may have a role in IFNγ-induced signaling and promote the expression of GAS-driven genes in bony fish.

View Article: PubMed Central - PubMed

Affiliation: The Norwegian College of Fishery Science, UiT The Arctic University of Norway, N-9037 Tromsø, Norway.

ABSTRACT
Mammalian IRF9 and STAT2, together with STAT1, form the ISGF3 transcription factor complex, which is critical for type I interferon (IFN)-induced signaling, while IFNγ stimulation is mediated by homodimeric STAT1 protein. Teleost fish are known to possess most JAK and STAT family members, however, description of their functional activity in lower vertebrates is still scarce. In the present study we have identified two different STAT2 homologs and one IRF9 homolog from Atlantic salmon (Salmo salar). Both proteins have domain-like structures with functional motifs that are similar to higher vertebrates, suggesting that they are orthologs to mammalian STAT2 and IRF9. The two identified salmon STAT2s, named STAT2a and STAT2b, showed high sequence identity but were divergent in their transactivation domain (TAD). Like STAT1, ectopically expressed STAT2a and b were shown to be tyrosine phosphorylated by type I IFNs and, interestingly, also by IFNγ. Microscopy analyses demonstrated that STAT2 co-localized with STAT1a in the cytoplasm of unstimulated cells, while IFNa1 and IFNγ stimulation seemed to favor their nuclear localization. Overexpression of STAT2a or STAT2b together with STAT1a activated a GAS-containing reporter gene construct in IFNγ-stimulated cells. The highest induction of GAS promoter activation was found in IFNγ-stimulated cells transfected with IRF9 alone. Taken together, these data suggest that salmon STAT2 and IRF9 may have a role in IFNγ-induced signaling and promote the expression of GAS-driven genes in bony fish. Since mammalian STAT2 is primarily an ISGF3 component and not involved in IFNγ signaling, our finding features a novel role for STAT2 in fish.

No MeSH data available.


Related in: MedlinePlus

A phylogenetic tree of STAT2 protein sequences in Atlantic salmon and selected vertebrate species. The phylogenetic tree was constructed by the neighbor-joining method in the MEGA 5 program and the bootstrap values were calculated with 500 replicates. STAT2 from following species are included: Cow, goldfish, zebrafish, human, mouse, rat, wild boar, western clawed frog, and the three STAT2 isoforms of Atlantic salmon. GenBank accession numbers of the sequences are listed in Table 1.
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f0010: A phylogenetic tree of STAT2 protein sequences in Atlantic salmon and selected vertebrate species. The phylogenetic tree was constructed by the neighbor-joining method in the MEGA 5 program and the bootstrap values were calculated with 500 replicates. STAT2 from following species are included: Cow, goldfish, zebrafish, human, mouse, rat, wild boar, western clawed frog, and the three STAT2 isoforms of Atlantic salmon. GenBank accession numbers of the sequences are listed in Table 1.

Mentions: Sequencing and analysis of the cloned PCR products obtained by the STAT2 primers (Table 1) revealed two fragments with open readings frames of 2445 bp (STAT2are, GenBank accession no. KJ155789) and 2418 bp (STAT2b, GenBank accession no. KJ155790) encoding for putative polypeptides of 813 amino acid (aa) and 805 aa, respectively. Alignment of aa sequences (ClustalW2) showed that STAT2a and STAT2b had a 96% aa identity to each other and their best match (92% and 88% aa identity, respectively) was to an earlier reported Atlantic salmon STAT2 variant (FJ173070) [35]. We propose that the previously published salmon STAT2 (FJ173070) to be referred to as salmon STAT2c. Alignment of the STAT2a and STAT2b with STAT2-sequences from other species showed 57% and 58% aa identities with zebrafish STAT2, while their identities to different mammalian STAT2s were close to 40% (Table 3). In the phylogenetic tree presented in Fig. 2, STAT2a and STAT2b are placed in the same clad and grouped with other fish species.


Structural and functional characterization of salmon STAT1, STAT2 and IRF9 homologs sheds light on interferon signaling in teleosts.

Sobhkhez M, Skjesol A, Thomassen E, Tollersrud LG, Iliev DB, Sun B, Robertsen B, Jørgensen JB - FEBS Open Bio (2014)

A phylogenetic tree of STAT2 protein sequences in Atlantic salmon and selected vertebrate species. The phylogenetic tree was constructed by the neighbor-joining method in the MEGA 5 program and the bootstrap values were calculated with 500 replicates. STAT2 from following species are included: Cow, goldfish, zebrafish, human, mouse, rat, wild boar, western clawed frog, and the three STAT2 isoforms of Atlantic salmon. GenBank accession numbers of the sequences are listed in Table 1.
© Copyright Policy - CC BY
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4215117&req=5

f0010: A phylogenetic tree of STAT2 protein sequences in Atlantic salmon and selected vertebrate species. The phylogenetic tree was constructed by the neighbor-joining method in the MEGA 5 program and the bootstrap values were calculated with 500 replicates. STAT2 from following species are included: Cow, goldfish, zebrafish, human, mouse, rat, wild boar, western clawed frog, and the three STAT2 isoforms of Atlantic salmon. GenBank accession numbers of the sequences are listed in Table 1.
Mentions: Sequencing and analysis of the cloned PCR products obtained by the STAT2 primers (Table 1) revealed two fragments with open readings frames of 2445 bp (STAT2are, GenBank accession no. KJ155789) and 2418 bp (STAT2b, GenBank accession no. KJ155790) encoding for putative polypeptides of 813 amino acid (aa) and 805 aa, respectively. Alignment of aa sequences (ClustalW2) showed that STAT2a and STAT2b had a 96% aa identity to each other and their best match (92% and 88% aa identity, respectively) was to an earlier reported Atlantic salmon STAT2 variant (FJ173070) [35]. We propose that the previously published salmon STAT2 (FJ173070) to be referred to as salmon STAT2c. Alignment of the STAT2a and STAT2b with STAT2-sequences from other species showed 57% and 58% aa identities with zebrafish STAT2, while their identities to different mammalian STAT2s were close to 40% (Table 3). In the phylogenetic tree presented in Fig. 2, STAT2a and STAT2b are placed in the same clad and grouped with other fish species.

Bottom Line: Overexpression of STAT2a or STAT2b together with STAT1a activated a GAS-containing reporter gene construct in IFNγ-stimulated cells.The highest induction of GAS promoter activation was found in IFNγ-stimulated cells transfected with IRF9 alone.Taken together, these data suggest that salmon STAT2 and IRF9 may have a role in IFNγ-induced signaling and promote the expression of GAS-driven genes in bony fish.

View Article: PubMed Central - PubMed

Affiliation: The Norwegian College of Fishery Science, UiT The Arctic University of Norway, N-9037 Tromsø, Norway.

ABSTRACT
Mammalian IRF9 and STAT2, together with STAT1, form the ISGF3 transcription factor complex, which is critical for type I interferon (IFN)-induced signaling, while IFNγ stimulation is mediated by homodimeric STAT1 protein. Teleost fish are known to possess most JAK and STAT family members, however, description of their functional activity in lower vertebrates is still scarce. In the present study we have identified two different STAT2 homologs and one IRF9 homolog from Atlantic salmon (Salmo salar). Both proteins have domain-like structures with functional motifs that are similar to higher vertebrates, suggesting that they are orthologs to mammalian STAT2 and IRF9. The two identified salmon STAT2s, named STAT2a and STAT2b, showed high sequence identity but were divergent in their transactivation domain (TAD). Like STAT1, ectopically expressed STAT2a and b were shown to be tyrosine phosphorylated by type I IFNs and, interestingly, also by IFNγ. Microscopy analyses demonstrated that STAT2 co-localized with STAT1a in the cytoplasm of unstimulated cells, while IFNa1 and IFNγ stimulation seemed to favor their nuclear localization. Overexpression of STAT2a or STAT2b together with STAT1a activated a GAS-containing reporter gene construct in IFNγ-stimulated cells. The highest induction of GAS promoter activation was found in IFNγ-stimulated cells transfected with IRF9 alone. Taken together, these data suggest that salmon STAT2 and IRF9 may have a role in IFNγ-induced signaling and promote the expression of GAS-driven genes in bony fish. Since mammalian STAT2 is primarily an ISGF3 component and not involved in IFNγ signaling, our finding features a novel role for STAT2 in fish.

No MeSH data available.


Related in: MedlinePlus