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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

Atlantic salmon STAT1a, STAT2a and STAT2b are tyrosine phosphorylated upon IFN stimulation. (A) CHSE-214 cells were transfected with plasmids expressing GFP-tagged STAT1a and either were left unstimulated or were stimulated with 200 U/ml IFNa1, b and c and 500 ng/ml IFNγ for 30 min before harvest. (B) CHSE-214 cells were transfected with GFP-tagged STAT2a and STAT2b. The cells were either left unstimulated or they were stimulated with 200 U/ml IFNa1, and 500 ng/ml IFNγ for 30 min before harvest. IP was performed with anti-GFP antibody and tyrosine-phosphorylated proteins were detected by Western blot using anti-phosphotyrosine antibody. The membranes were stripped and re-probed with anti-GFP antibody to detect the total amount of immunoprecipitated proteins. These experiments were repeated 3 times with the same result (results not shown).
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f0020: Atlantic salmon STAT1a, STAT2a and STAT2b are tyrosine phosphorylated upon IFN stimulation. (A) CHSE-214 cells were transfected with plasmids expressing GFP-tagged STAT1a and either were left unstimulated or were stimulated with 200 U/ml IFNa1, b and c and 500 ng/ml IFNγ for 30 min before harvest. (B) CHSE-214 cells were transfected with GFP-tagged STAT2a and STAT2b. The cells were either left unstimulated or they were stimulated with 200 U/ml IFNa1, and 500 ng/ml IFNγ for 30 min before harvest. IP was performed with anti-GFP antibody and tyrosine-phosphorylated proteins were detected by Western blot using anti-phosphotyrosine antibody. The membranes were stripped and re-probed with anti-GFP antibody to detect the total amount of immunoprecipitated proteins. These experiments were repeated 3 times with the same result (results not shown).

Mentions: Tyrosine phosphorylation is a key step in STAT-mediated IFN signal transduction and both salmon STAT1a [33] and the two salmon STAT2 isoforms identified in this study possess phosphorylation sites that are conserved between salmon and human. The conventional view is that type I IFN signals through STAT1/STAT2 heterodimers while IFNγ signals through STAT1 homodimers. In Atlantic salmon 4 different type I IFN subtypes are identified, IFNa, b, c and d, and recent studies have revealed distinct roles for the different subtypes [41]. In an earlier study we have shown that endogenous STAT1a is phosphorylated upon stimulation with IFNa1 and IFNγ [33]. Here, we extended the studies and tested tyrosine phosphorylation of overexpressed STAT1a, when stimulated with recombinant IFNa1, IFNb, IFNc and IFNγ in CHSE-214 cells. IFNa1 and IFNγ were also tested for their ability to phosphorylate both STAT2a and STAT2b. The GFP-tagged STAT expressing plasmids were transfected in CHSE-214 and transfectants were stimulated with IFNs. The transfectants were harvested after 30 min of stimulation with the IFNs and anti-GFP antibody was used to immune precipitate the various overexpressed GFP-tagged STAT proteins. The results were visualized by Western blotting. As shown in Fig. 4A STAT1a is phosphorylated when stimulated with IFNa1, IFNc and IFNγ, while no phosphorylation was detected for cells stimulated with IFNb. Phosphorylated STAT2a and b were detected after 30 min stimulation by IFNa1 and also, very interestingly, by IFNγ (Fig. 4B). The phosphorylation was not as strong as observed for STAT1a, but still higher when compared to the unstimulated control.


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)

Atlantic salmon STAT1a, STAT2a and STAT2b are tyrosine phosphorylated upon IFN stimulation. (A) CHSE-214 cells were transfected with plasmids expressing GFP-tagged STAT1a and either were left unstimulated or were stimulated with 200 U/ml IFNa1, b and c and 500 ng/ml IFNγ for 30 min before harvest. (B) CHSE-214 cells were transfected with GFP-tagged STAT2a and STAT2b. The cells were either left unstimulated or they were stimulated with 200 U/ml IFNa1, and 500 ng/ml IFNγ for 30 min before harvest. IP was performed with anti-GFP antibody and tyrosine-phosphorylated proteins were detected by Western blot using anti-phosphotyrosine antibody. The membranes were stripped and re-probed with anti-GFP antibody to detect the total amount of immunoprecipitated proteins. These experiments were repeated 3 times with the same result (results not shown).
© Copyright Policy - CC BY
Related In: Results  -  Collection

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f0020: Atlantic salmon STAT1a, STAT2a and STAT2b are tyrosine phosphorylated upon IFN stimulation. (A) CHSE-214 cells were transfected with plasmids expressing GFP-tagged STAT1a and either were left unstimulated or were stimulated with 200 U/ml IFNa1, b and c and 500 ng/ml IFNγ for 30 min before harvest. (B) CHSE-214 cells were transfected with GFP-tagged STAT2a and STAT2b. The cells were either left unstimulated or they were stimulated with 200 U/ml IFNa1, and 500 ng/ml IFNγ for 30 min before harvest. IP was performed with anti-GFP antibody and tyrosine-phosphorylated proteins were detected by Western blot using anti-phosphotyrosine antibody. The membranes were stripped and re-probed with anti-GFP antibody to detect the total amount of immunoprecipitated proteins. These experiments were repeated 3 times with the same result (results not shown).
Mentions: Tyrosine phosphorylation is a key step in STAT-mediated IFN signal transduction and both salmon STAT1a [33] and the two salmon STAT2 isoforms identified in this study possess phosphorylation sites that are conserved between salmon and human. The conventional view is that type I IFN signals through STAT1/STAT2 heterodimers while IFNγ signals through STAT1 homodimers. In Atlantic salmon 4 different type I IFN subtypes are identified, IFNa, b, c and d, and recent studies have revealed distinct roles for the different subtypes [41]. In an earlier study we have shown that endogenous STAT1a is phosphorylated upon stimulation with IFNa1 and IFNγ [33]. Here, we extended the studies and tested tyrosine phosphorylation of overexpressed STAT1a, when stimulated with recombinant IFNa1, IFNb, IFNc and IFNγ in CHSE-214 cells. IFNa1 and IFNγ were also tested for their ability to phosphorylate both STAT2a and STAT2b. The GFP-tagged STAT expressing plasmids were transfected in CHSE-214 and transfectants were stimulated with IFNs. The transfectants were harvested after 30 min of stimulation with the IFNs and anti-GFP antibody was used to immune precipitate the various overexpressed GFP-tagged STAT proteins. The results were visualized by Western blotting. As shown in Fig. 4A STAT1a is phosphorylated when stimulated with IFNa1, IFNc and IFNγ, while no phosphorylation was detected for cells stimulated with IFNb. Phosphorylated STAT2a and b were detected after 30 min stimulation by IFNa1 and also, very interestingly, by IFNγ (Fig. 4B). The phosphorylation was not as strong as observed for STAT1a, but still higher when compared to the unstimulated control.

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