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Suppressor of Cytokine Signaling 2 Negatively Regulates NK Cell Differentiation by Inhibiting JAK2 Activity

View Article: PubMed Central - PubMed

ABSTRACT

Suppressor of cytokine signaling (SOCS) proteins are negative regulators of cytokine responses. Although recent reports have shown regulatory roles for SOCS proteins in innate and adaptive immunity, their roles in natural killer (NK) cell development are largely unknown. Here, we show that SOCS2 is involved in NK cell development. SOCS2−/− mice showed a high frequency of NK cells in the bone marrow and spleen. Knockdown of SOCS2 was associated with enhanced differentiation of NK cells in vitro, and the transplantation of hematopoietic stem cells (HSCs) into congenic mice resulted in enhanced differentiation in SOCS2−/− HSCs. We found that SOCS2 could inhibit Janus kinase 2 (JAK2) activity and JAK2-STAT5 signaling pathways via direct interaction with JAK2. Furthermore, SOCS2−/− mice showed a reduction in lung metastases and an increase in survival following melanoma challenge. Overall, our findings suggest that SOCS2 negatively regulates the development of NK cells by inhibiting JAK2 activity via direct interaction.

No MeSH data available.


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The physical interaction between SOCS2 and JAK2 in NK cells.(A) PLA images for physical interaction between SOCS2 and JAK2. Splenic NK cells was treated with IL-15 (30 ng/ml) for 12 hours. Each red spot represents a SOCS2-JAK2 interaction. Nuclei were stained with DAPI. Scale Bar: 20 um. (B) Interaction of JAK2 with SOCS2 following IL-15 treatment. NK-92 cells were treated with IL-15 (30 ng/ml) for 12 hours. Cell lysates were immunoprecipitated with JAK2 antibodies. Data are representative of two independent experiments. (C,D) Identification of the interaction domains between JAK2 and SOCS2. 293 T cells were transiently transfected with the indicated plasmids and then cell lysates were subjected to a pull-down assay with glutathione-Sepharose beads. Data are representative of three independent experiments.
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f6: The physical interaction between SOCS2 and JAK2 in NK cells.(A) PLA images for physical interaction between SOCS2 and JAK2. Splenic NK cells was treated with IL-15 (30 ng/ml) for 12 hours. Each red spot represents a SOCS2-JAK2 interaction. Nuclei were stained with DAPI. Scale Bar: 20 um. (B) Interaction of JAK2 with SOCS2 following IL-15 treatment. NK-92 cells were treated with IL-15 (30 ng/ml) for 12 hours. Cell lysates were immunoprecipitated with JAK2 antibodies. Data are representative of two independent experiments. (C,D) Identification of the interaction domains between JAK2 and SOCS2. 293 T cells were transiently transfected with the indicated plasmids and then cell lysates were subjected to a pull-down assay with glutathione-Sepharose beads. Data are representative of three independent experiments.

Mentions: SOCS proteins are known to interact with phosphorylated tyrosine motifs in target proteins via an SH2 domain2334. Hence, we hypothesized that SOCS2 might be downregulating a target protein involved in IL-15–induced NK cell differentiation and that this regulation might be recovered by the loss of SOCS2. To verify the endogenous interaction between SOCS2 and JAK2, we examined the interaction among primary NK cells using PLA. In the primary NK cells treated with IL-15 for SOCS2 and JAK2 activation, we identified the interaction between SOCS2 and JAK2 (Fig. 6A). Furthermore, the endogenous interaction between SOCS2 and JAK2 was confirmed in NK-92 cells using co-immunoprecipitation (IP) assays (Fig. 6B). To characterize the domain necessary for the SOCS2-JAK2 interaction, we investigated the binding of SOCS2 to four domains of JAK211. We found that the N-terminal FERM domain of JAK2 is important for their interaction (Fig. 6C). Next, we determined which region of SOCS2 interacted with JAK2. JAK2 precipitated full-length SOCS2 and a SOCS box deletion mutant (GST-SOCS2-∆SOCS) but not a SH2 domain-deletion mutant (GST-SOCS2-∆SH2) (Fig. 6D). Overall, these data suggest that SOCS2 is capable of regulating JAK2-STAT5 signaling pathway via direct interaction.


Suppressor of Cytokine Signaling 2 Negatively Regulates NK Cell Differentiation by Inhibiting JAK2 Activity
The physical interaction between SOCS2 and JAK2 in NK cells.(A) PLA images for physical interaction between SOCS2 and JAK2. Splenic NK cells was treated with IL-15 (30 ng/ml) for 12 hours. Each red spot represents a SOCS2-JAK2 interaction. Nuclei were stained with DAPI. Scale Bar: 20 um. (B) Interaction of JAK2 with SOCS2 following IL-15 treatment. NK-92 cells were treated with IL-15 (30 ng/ml) for 12 hours. Cell lysates were immunoprecipitated with JAK2 antibodies. Data are representative of two independent experiments. (C,D) Identification of the interaction domains between JAK2 and SOCS2. 293 T cells were transiently transfected with the indicated plasmids and then cell lysates were subjected to a pull-down assay with glutathione-Sepharose beads. Data are representative of three independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5382670&req=5

f6: The physical interaction between SOCS2 and JAK2 in NK cells.(A) PLA images for physical interaction between SOCS2 and JAK2. Splenic NK cells was treated with IL-15 (30 ng/ml) for 12 hours. Each red spot represents a SOCS2-JAK2 interaction. Nuclei were stained with DAPI. Scale Bar: 20 um. (B) Interaction of JAK2 with SOCS2 following IL-15 treatment. NK-92 cells were treated with IL-15 (30 ng/ml) for 12 hours. Cell lysates were immunoprecipitated with JAK2 antibodies. Data are representative of two independent experiments. (C,D) Identification of the interaction domains between JAK2 and SOCS2. 293 T cells were transiently transfected with the indicated plasmids and then cell lysates were subjected to a pull-down assay with glutathione-Sepharose beads. Data are representative of three independent experiments.
Mentions: SOCS proteins are known to interact with phosphorylated tyrosine motifs in target proteins via an SH2 domain2334. Hence, we hypothesized that SOCS2 might be downregulating a target protein involved in IL-15–induced NK cell differentiation and that this regulation might be recovered by the loss of SOCS2. To verify the endogenous interaction between SOCS2 and JAK2, we examined the interaction among primary NK cells using PLA. In the primary NK cells treated with IL-15 for SOCS2 and JAK2 activation, we identified the interaction between SOCS2 and JAK2 (Fig. 6A). Furthermore, the endogenous interaction between SOCS2 and JAK2 was confirmed in NK-92 cells using co-immunoprecipitation (IP) assays (Fig. 6B). To characterize the domain necessary for the SOCS2-JAK2 interaction, we investigated the binding of SOCS2 to four domains of JAK211. We found that the N-terminal FERM domain of JAK2 is important for their interaction (Fig. 6C). Next, we determined which region of SOCS2 interacted with JAK2. JAK2 precipitated full-length SOCS2 and a SOCS box deletion mutant (GST-SOCS2-∆SOCS) but not a SH2 domain-deletion mutant (GST-SOCS2-∆SH2) (Fig. 6D). Overall, these data suggest that SOCS2 is capable of regulating JAK2-STAT5 signaling pathway via direct interaction.

View Article: PubMed Central - PubMed

ABSTRACT

Suppressor of cytokine signaling (SOCS) proteins are negative regulators of cytokine responses. Although recent reports have shown regulatory roles for SOCS proteins in innate and adaptive immunity, their roles in natural killer (NK) cell development are largely unknown. Here, we show that SOCS2 is involved in NK cell development. SOCS2−/− mice showed a high frequency of NK cells in the bone marrow and spleen. Knockdown of SOCS2 was associated with enhanced differentiation of NK cells in vitro, and the transplantation of hematopoietic stem cells (HSCs) into congenic mice resulted in enhanced differentiation in SOCS2−/− HSCs. We found that SOCS2 could inhibit Janus kinase 2 (JAK2) activity and JAK2-STAT5 signaling pathways via direct interaction with JAK2. Furthermore, SOCS2−/− mice showed a reduction in lung metastases and an increase in survival following melanoma challenge. Overall, our findings suggest that SOCS2 negatively regulates the development of NK cells by inhibiting JAK2 activity via direct interaction.

No MeSH data available.


Related in: MedlinePlus