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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

STAT1a interacts with Tyk2 and IRF9, and STAT2a and STAT2b interact with Tyk2, IRF9 and STAT1a. HEK 293 cells co-transfected with plasmids expressing TYK2, IRF9 or STAT1a with FLAG-tag in combination with plasmids expressing either STAT2a or STAT2b with GFP-tag. The study also included plasmids expressing FLAG-tagged Tyk2 or IRF9 in combination with STAT1a with GFP-tag. The IP was performed with anti-FLAG M2 affinity gel and interacting partners were detected by Western blot using antibodies against GFP, membrane was stripped and treated with anti-FLAG antibody. An empty pDEST-3xFlag vector was included as control. All the proteins were shown to be expressed in total lysate.
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f0030: STAT1a interacts with Tyk2 and IRF9, and STAT2a and STAT2b interact with Tyk2, IRF9 and STAT1a. HEK 293 cells co-transfected with plasmids expressing TYK2, IRF9 or STAT1a with FLAG-tag in combination with plasmids expressing either STAT2a or STAT2b with GFP-tag. The study also included plasmids expressing FLAG-tagged Tyk2 or IRF9 in combination with STAT1a with GFP-tag. The IP was performed with anti-FLAG M2 affinity gel and interacting partners were detected by Western blot using antibodies against GFP, membrane was stripped and treated with anti-FLAG antibody. An empty pDEST-3xFlag vector was included as control. All the proteins were shown to be expressed in total lysate.

Mentions: The results presented in Fig. 6B show that while wild-type STAT1a was tyrosine phosphorylated upon IFNa1 and IFNc treatment, the STAT1aY695A mutant showed no phosphorylation in response to the same stimuli. As presented in Fig. 6B, the phosphorylated form of STAT1 (green band) migrated slightly slower than the non-phosphorylated form (red band). These results suggest STAT1a is phosphorylated on a single site (tyrosine 695). No tyrosine phosphorylation was detected on constructs expressing the SH2 domain alone or the SH2 in combination with TAD domain (Fig. 5C), which was surprising since tyrosine 695 is located in the SH2 domain. The same experiment as presented in Fig. 6 was also set up with IFNγ stimulation and gave similar results (results not shown).


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)

STAT1a interacts with Tyk2 and IRF9, and STAT2a and STAT2b interact with Tyk2, IRF9 and STAT1a. HEK 293 cells co-transfected with plasmids expressing TYK2, IRF9 or STAT1a with FLAG-tag in combination with plasmids expressing either STAT2a or STAT2b with GFP-tag. The study also included plasmids expressing FLAG-tagged Tyk2 or IRF9 in combination with STAT1a with GFP-tag. The IP was performed with anti-FLAG M2 affinity gel and interacting partners were detected by Western blot using antibodies against GFP, membrane was stripped and treated with anti-FLAG antibody. An empty pDEST-3xFlag vector was included as control. All the proteins were shown to be expressed in total lysate.
© Copyright Policy - CC BY
Related In: Results  -  Collection

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

f0030: STAT1a interacts with Tyk2 and IRF9, and STAT2a and STAT2b interact with Tyk2, IRF9 and STAT1a. HEK 293 cells co-transfected with plasmids expressing TYK2, IRF9 or STAT1a with FLAG-tag in combination with plasmids expressing either STAT2a or STAT2b with GFP-tag. The study also included plasmids expressing FLAG-tagged Tyk2 or IRF9 in combination with STAT1a with GFP-tag. The IP was performed with anti-FLAG M2 affinity gel and interacting partners were detected by Western blot using antibodies against GFP, membrane was stripped and treated with anti-FLAG antibody. An empty pDEST-3xFlag vector was included as control. All the proteins were shown to be expressed in total lysate.
Mentions: The results presented in Fig. 6B show that while wild-type STAT1a was tyrosine phosphorylated upon IFNa1 and IFNc treatment, the STAT1aY695A mutant showed no phosphorylation in response to the same stimuli. As presented in Fig. 6B, the phosphorylated form of STAT1 (green band) migrated slightly slower than the non-phosphorylated form (red band). These results suggest STAT1a is phosphorylated on a single site (tyrosine 695). No tyrosine phosphorylation was detected on constructs expressing the SH2 domain alone or the SH2 in combination with TAD domain (Fig. 5C), which was surprising since tyrosine 695 is located in the SH2 domain. The same experiment as presented in Fig. 6 was also set up with IFNγ stimulation and gave similar results (results not shown).

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