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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 STAT2 proteins harbor conserved domains and sequences. (A) The NCBI conserved domains database (CDD) and ClustalW alignment showed presence of conserved domains; N-terminal domain (ND), Coiled-coil, DNA binding domain (DBD), SH2 homology domain (SH2) and transcription activation domain (TAD). ClustalW alignment revealed high aa identity between the three identified salmon STAT2 sequences (STAT2a KJ155789, STAT2b KJ155790 and STAT2c FJ173070) and the highest divergence between STAT2c and STAT2a/STAT2b was observed in their Coiled-coil domain (aa 144–199/207). A conserved tyrosine residue (Y) which is a target of phosphorylation by the JAKs is marked with *. DNAB domain of STAT possess conserved aa essential for nuclear localization (marked yellow with †) and nuclear export (marked green with ‡). (B) DNAB domain of Atlantic salmon IRF9 has high sequence identity with IRF9 DNAB domains from other species. ClustalW alignment using the NCBI CDD of the deduced aa sequences of IRF9 DNAB domain from Atlantic salmon (NP_001167190), human (NP_006075), cow (AAI02048), mouse (AAH05435), rat (NP_001012041 XP_224190) and zebrafish (AAH81591). Asterisk (*): indicate conserved tryptophan (W) residues. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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f0005: Salmon STAT2 proteins harbor conserved domains and sequences. (A) The NCBI conserved domains database (CDD) and ClustalW alignment showed presence of conserved domains; N-terminal domain (ND), Coiled-coil, DNA binding domain (DBD), SH2 homology domain (SH2) and transcription activation domain (TAD). ClustalW alignment revealed high aa identity between the three identified salmon STAT2 sequences (STAT2a KJ155789, STAT2b KJ155790 and STAT2c FJ173070) and the highest divergence between STAT2c and STAT2a/STAT2b was observed in their Coiled-coil domain (aa 144–199/207). A conserved tyrosine residue (Y) which is a target of phosphorylation by the JAKs is marked with *. DNAB domain of STAT possess conserved aa essential for nuclear localization (marked yellow with †) and nuclear export (marked green with ‡). (B) DNAB domain of Atlantic salmon IRF9 has high sequence identity with IRF9 DNAB domains from other species. ClustalW alignment using the NCBI CDD of the deduced aa sequences of IRF9 DNAB domain from Atlantic salmon (NP_001167190), human (NP_006075), cow (AAI02048), mouse (AAH05435), rat (NP_001012041 XP_224190) and zebrafish (AAH81591). Asterisk (*): indicate conserved tryptophan (W) residues. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Mentions: STATs have a conserved architecture (Fig. 1) and consist of an N-terminal domain that makes it possible for dimerized STAT to polymerize and bind cooperatively to DNA [19,20], a coiled-coil domain that facilitates interactions to other proteins, followed by a DNA-binding domain (DNAB), and a linker domain that is important for transcription [21,22]. The C-terminal part has a Src-homology 2 (SH2) domain that interacts with receptors [8]. The latter includes a tyrosine phosphorylation site, and the SH2 domain is the domain essential for dimerization. Finally the STATs have a transcriptional activation domain (TAD) which mediates their transcriptional activity [23]. IRF9 is a member of interferon regulatory factor (IRF) family and has an established role in type I IFN responses. This molecule too has a conserved structure which is comprised of a well-defined DNAB in addition to an IRF-association domain (IAD) [24]. The DNAB domain is highly conserved across different species and includes 5 conserved tryptophan residues, while the IAD is much more diverse and works as regulatory domain known to associate with distinct transcription factors [25].


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 STAT2 proteins harbor conserved domains and sequences. (A) The NCBI conserved domains database (CDD) and ClustalW alignment showed presence of conserved domains; N-terminal domain (ND), Coiled-coil, DNA binding domain (DBD), SH2 homology domain (SH2) and transcription activation domain (TAD). ClustalW alignment revealed high aa identity between the three identified salmon STAT2 sequences (STAT2a KJ155789, STAT2b KJ155790 and STAT2c FJ173070) and the highest divergence between STAT2c and STAT2a/STAT2b was observed in their Coiled-coil domain (aa 144–199/207). A conserved tyrosine residue (Y) which is a target of phosphorylation by the JAKs is marked with *. DNAB domain of STAT possess conserved aa essential for nuclear localization (marked yellow with †) and nuclear export (marked green with ‡). (B) DNAB domain of Atlantic salmon IRF9 has high sequence identity with IRF9 DNAB domains from other species. ClustalW alignment using the NCBI CDD of the deduced aa sequences of IRF9 DNAB domain from Atlantic salmon (NP_001167190), human (NP_006075), cow (AAI02048), mouse (AAH05435), rat (NP_001012041 XP_224190) and zebrafish (AAH81591). Asterisk (*): indicate conserved tryptophan (W) residues. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
© Copyright Policy - CC BY
Related In: Results  -  Collection

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

f0005: Salmon STAT2 proteins harbor conserved domains and sequences. (A) The NCBI conserved domains database (CDD) and ClustalW alignment showed presence of conserved domains; N-terminal domain (ND), Coiled-coil, DNA binding domain (DBD), SH2 homology domain (SH2) and transcription activation domain (TAD). ClustalW alignment revealed high aa identity between the three identified salmon STAT2 sequences (STAT2a KJ155789, STAT2b KJ155790 and STAT2c FJ173070) and the highest divergence between STAT2c and STAT2a/STAT2b was observed in their Coiled-coil domain (aa 144–199/207). A conserved tyrosine residue (Y) which is a target of phosphorylation by the JAKs is marked with *. DNAB domain of STAT possess conserved aa essential for nuclear localization (marked yellow with †) and nuclear export (marked green with ‡). (B) DNAB domain of Atlantic salmon IRF9 has high sequence identity with IRF9 DNAB domains from other species. ClustalW alignment using the NCBI CDD of the deduced aa sequences of IRF9 DNAB domain from Atlantic salmon (NP_001167190), human (NP_006075), cow (AAI02048), mouse (AAH05435), rat (NP_001012041 XP_224190) and zebrafish (AAH81591). Asterisk (*): indicate conserved tryptophan (W) residues. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Mentions: STATs have a conserved architecture (Fig. 1) and consist of an N-terminal domain that makes it possible for dimerized STAT to polymerize and bind cooperatively to DNA [19,20], a coiled-coil domain that facilitates interactions to other proteins, followed by a DNA-binding domain (DNAB), and a linker domain that is important for transcription [21,22]. The C-terminal part has a Src-homology 2 (SH2) domain that interacts with receptors [8]. The latter includes a tyrosine phosphorylation site, and the SH2 domain is the domain essential for dimerization. Finally the STATs have a transcriptional activation domain (TAD) which mediates their transcriptional activity [23]. IRF9 is a member of interferon regulatory factor (IRF) family and has an established role in type I IFN responses. This molecule too has a conserved structure which is comprised of a well-defined DNAB in addition to an IRF-association domain (IAD) [24]. The DNAB domain is highly conserved across different species and includes 5 conserved tryptophan residues, while the IAD is much more diverse and works as regulatory domain known to associate with distinct transcription factors [25].

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