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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

Salmon STAT1a is phosphorylated on a single site (Tyr 695) in the SH2-domain in type I IFN-stimulated cells and this phosphorylation requires expression of full-length STAT1a. (A) Constructs used for immunoprecipitations: full-length STAT1a, STAT1a Y695A and 4 different deletion mutants. (B) CHSE-214 cells were transfected with plasmids encoding different GFP-tagged STAT1a, STAT1a deletion mutants or point mutated STAT1a Y695A. The cells were harvested 72 h post transfection, and 30 min prior to harvest they were stimulated with 200 U/ml of IFNa1, b or c. Cell extracts were immunoprecipitated with anti-GFP antibody and tyrosine-phosphorylation was detected by immunoblotting using α-phospho-tyrosine antibody. The membrane was stripped and re-probed with α-GFP to detect the total amount of immunoprecipitated proteins.
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f0025: Salmon STAT1a is phosphorylated on a single site (Tyr 695) in the SH2-domain in type I IFN-stimulated cells and this phosphorylation requires expression of full-length STAT1a. (A) Constructs used for immunoprecipitations: full-length STAT1a, STAT1a Y695A and 4 different deletion mutants. (B) CHSE-214 cells were transfected with plasmids encoding different GFP-tagged STAT1a, STAT1a deletion mutants or point mutated STAT1a Y695A. The cells were harvested 72 h post transfection, and 30 min prior to harvest they were stimulated with 200 U/ml of IFNa1, b or c. Cell extracts were immunoprecipitated with anti-GFP antibody and tyrosine-phosphorylation was detected by immunoblotting using α-phospho-tyrosine antibody. The membrane was stripped and re-probed with α-GFP to detect the total amount of immunoprecipitated proteins.

Mentions: Next, we wanted to test the functional importance of the tyrosine 695 in salmon STAT1a [33] and we made a construct where this tyrosine was replaced with alanine (STAT1a Y695A) and tested it together with wild-type STAT1a for the abilities of these two constructs to be phosphorylated upon IFN-treatments. Additionally, different STAT1a deletion constructs (Fig. 5A) were made and also tested in a similar manner.


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)

Salmon STAT1a is phosphorylated on a single site (Tyr 695) in the SH2-domain in type I IFN-stimulated cells and this phosphorylation requires expression of full-length STAT1a. (A) Constructs used for immunoprecipitations: full-length STAT1a, STAT1a Y695A and 4 different deletion mutants. (B) CHSE-214 cells were transfected with plasmids encoding different GFP-tagged STAT1a, STAT1a deletion mutants or point mutated STAT1a Y695A. The cells were harvested 72 h post transfection, and 30 min prior to harvest they were stimulated with 200 U/ml of IFNa1, b or c. Cell extracts were immunoprecipitated with anti-GFP antibody and tyrosine-phosphorylation was detected by immunoblotting using α-phospho-tyrosine antibody. The membrane was stripped and re-probed with α-GFP to detect the total amount of immunoprecipitated proteins.
© Copyright Policy - CC BY
Related In: Results  -  Collection

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

f0025: Salmon STAT1a is phosphorylated on a single site (Tyr 695) in the SH2-domain in type I IFN-stimulated cells and this phosphorylation requires expression of full-length STAT1a. (A) Constructs used for immunoprecipitations: full-length STAT1a, STAT1a Y695A and 4 different deletion mutants. (B) CHSE-214 cells were transfected with plasmids encoding different GFP-tagged STAT1a, STAT1a deletion mutants or point mutated STAT1a Y695A. The cells were harvested 72 h post transfection, and 30 min prior to harvest they were stimulated with 200 U/ml of IFNa1, b or c. Cell extracts were immunoprecipitated with anti-GFP antibody and tyrosine-phosphorylation was detected by immunoblotting using α-phospho-tyrosine antibody. The membrane was stripped and re-probed with α-GFP to detect the total amount of immunoprecipitated proteins.
Mentions: Next, we wanted to test the functional importance of the tyrosine 695 in salmon STAT1a [33] and we made a construct where this tyrosine was replaced with alanine (STAT1a Y695A) and tested it together with wild-type STAT1a for the abilities of these two constructs to be phosphorylated upon IFN-treatments. Additionally, different STAT1a deletion constructs (Fig. 5A) were made and also tested in a similar manner.

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