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Proteolytic processing of Stat6 signaling in mast cells as a negative regulatory mechanism.

Suzuki K, Nakajima H, Kagami S, Suto A, Ikeda K, Hirose K, Hiwasa T, Takeda K, Saito Y, Akira S, Iwamoto I - J. Exp. Med. (2002)

Bottom Line: Accumulating evidence has shown the importance of Stat6-mediated signaling in allergic diseases.When Stat6 is activated by interleukin (IL)-4 and translocated to the nucleus, Stat6 is cleaved by a nucleus-associated protease in mast cells.The cleaved 65-kD Stat6 lacks the COOH-terminal transactivation domain and functions as a dominant-negative molecule to Stat6-mediated transcription.

View Article: PubMed Central - PubMed

Affiliation: Department of Internal Medicine II, Graduate School of Medicine, Chiba University, Chiba 260-8670, Japan.

ABSTRACT
Accumulating evidence has shown the importance of Stat6-mediated signaling in allergic diseases. In this study, we show a novel regulatory mechanism of Stat6-mediated signaling in mast cells. When Stat6 is activated by interleukin (IL)-4 and translocated to the nucleus, Stat6 is cleaved by a nucleus-associated protease in mast cells. The cleaved 65-kD Stat6 lacks the COOH-terminal transactivation domain and functions as a dominant-negative molecule to Stat6-mediated transcription. The retrovirus-mediated expression of cleavage-resistant Stat6 mutants prolongs the nuclear accumulation of Stat6 upon IL-4 stimulation and enhances IL-4-induced gene expression and growth inhibition in mast cells. These results indicate that the proteolytic processing of Stat6 functions as a lineage-specific negative regulator of Stat6-dependent signaling in mast cells, and thus suggest that it may account for the limited role of Stat6 in IL-4 signaling in mast cells.

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Stat6 protease activity is localized in the nucleus. (A) Subfraction of cell extracts (nuclear or cytoplasmic) was prepared from Stat6−/− BMMCs. Where indicated, 1% NP-40 or 420 mM NaCl was added to the cytoplasmic fraction. These extracts were then incubated with transfected Stat6 at 37°C for 20 min and subjected to Western blotting with anti-Stat6 (M200) antibody. (B) Cell extracts from IL-4–stimulated or –unstimulated WT splenocytes were incubated with Stat6−/− BMMC extract and blotted with anti–phospho-Stat6 antibody (top) or anti-Stat6 (M200) antibody (bottom). (C) Subfraction of cell extracts was prepared from either IL-4–stimulated or –unstimulated WT BMMCs and blotted with anti–phospho-Stat6 antibody (top) or anti-Stat6 (M200) antibody (bottom). As a control, cell extracts from IL-4–stimulated or –unstimulated WT splenocytes were blotted with these antibodies.
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fig2: Stat6 protease activity is localized in the nucleus. (A) Subfraction of cell extracts (nuclear or cytoplasmic) was prepared from Stat6−/− BMMCs. Where indicated, 1% NP-40 or 420 mM NaCl was added to the cytoplasmic fraction. These extracts were then incubated with transfected Stat6 at 37°C for 20 min and subjected to Western blotting with anti-Stat6 (M200) antibody. (B) Cell extracts from IL-4–stimulated or –unstimulated WT splenocytes were incubated with Stat6−/− BMMC extract and blotted with anti–phospho-Stat6 antibody (top) or anti-Stat6 (M200) antibody (bottom). (C) Subfraction of cell extracts was prepared from either IL-4–stimulated or –unstimulated WT BMMCs and blotted with anti–phospho-Stat6 antibody (top) or anti-Stat6 (M200) antibody (bottom). As a control, cell extracts from IL-4–stimulated or –unstimulated WT splenocytes were blotted with these antibodies.

Mentions: Next, we examined the subcellular localization of Stat6 protease activity in BMMCs. Cell extracts were prepared from the cytoplasmic or nuclear fraction of Stat6−/− BMMCs and then incubated with 94-kD Stat6. Interestingly, 94-kD Stat6 was cleaved to 65-kD Stat6 by the incubation with nuclear extract but not with the cytoplasmic extract from Stat6−/− BMMCs (Fig. 2 A). To exclude the possibility that the protease is normally in a protected cellular compartment that is detergent or high salt soluble, we added NP-40 or NaCl to the cytoplasmic fraction to the levels that we used for whole cell or nuclear extract preparation (1% NP-40 or 420 mM NaCl), and then examined the Stat6 protease activity. However, there was still no detectable Stat6 protease activity in the cytoplasmic fraction of BMMCs (Fig. 2 A). These results indicate that Stat6 protease activity is localized in the nucleus.


Proteolytic processing of Stat6 signaling in mast cells as a negative regulatory mechanism.

Suzuki K, Nakajima H, Kagami S, Suto A, Ikeda K, Hirose K, Hiwasa T, Takeda K, Saito Y, Akira S, Iwamoto I - J. Exp. Med. (2002)

Stat6 protease activity is localized in the nucleus. (A) Subfraction of cell extracts (nuclear or cytoplasmic) was prepared from Stat6−/− BMMCs. Where indicated, 1% NP-40 or 420 mM NaCl was added to the cytoplasmic fraction. These extracts were then incubated with transfected Stat6 at 37°C for 20 min and subjected to Western blotting with anti-Stat6 (M200) antibody. (B) Cell extracts from IL-4–stimulated or –unstimulated WT splenocytes were incubated with Stat6−/− BMMC extract and blotted with anti–phospho-Stat6 antibody (top) or anti-Stat6 (M200) antibody (bottom). (C) Subfraction of cell extracts was prepared from either IL-4–stimulated or –unstimulated WT BMMCs and blotted with anti–phospho-Stat6 antibody (top) or anti-Stat6 (M200) antibody (bottom). As a control, cell extracts from IL-4–stimulated or –unstimulated WT splenocytes were blotted with these antibodies.
© Copyright Policy
Related In: Results  -  Collection

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fig2: Stat6 protease activity is localized in the nucleus. (A) Subfraction of cell extracts (nuclear or cytoplasmic) was prepared from Stat6−/− BMMCs. Where indicated, 1% NP-40 or 420 mM NaCl was added to the cytoplasmic fraction. These extracts were then incubated with transfected Stat6 at 37°C for 20 min and subjected to Western blotting with anti-Stat6 (M200) antibody. (B) Cell extracts from IL-4–stimulated or –unstimulated WT splenocytes were incubated with Stat6−/− BMMC extract and blotted with anti–phospho-Stat6 antibody (top) or anti-Stat6 (M200) antibody (bottom). (C) Subfraction of cell extracts was prepared from either IL-4–stimulated or –unstimulated WT BMMCs and blotted with anti–phospho-Stat6 antibody (top) or anti-Stat6 (M200) antibody (bottom). As a control, cell extracts from IL-4–stimulated or –unstimulated WT splenocytes were blotted with these antibodies.
Mentions: Next, we examined the subcellular localization of Stat6 protease activity in BMMCs. Cell extracts were prepared from the cytoplasmic or nuclear fraction of Stat6−/− BMMCs and then incubated with 94-kD Stat6. Interestingly, 94-kD Stat6 was cleaved to 65-kD Stat6 by the incubation with nuclear extract but not with the cytoplasmic extract from Stat6−/− BMMCs (Fig. 2 A). To exclude the possibility that the protease is normally in a protected cellular compartment that is detergent or high salt soluble, we added NP-40 or NaCl to the cytoplasmic fraction to the levels that we used for whole cell or nuclear extract preparation (1% NP-40 or 420 mM NaCl), and then examined the Stat6 protease activity. However, there was still no detectable Stat6 protease activity in the cytoplasmic fraction of BMMCs (Fig. 2 A). These results indicate that Stat6 protease activity is localized in the nucleus.

Bottom Line: Accumulating evidence has shown the importance of Stat6-mediated signaling in allergic diseases.When Stat6 is activated by interleukin (IL)-4 and translocated to the nucleus, Stat6 is cleaved by a nucleus-associated protease in mast cells.The cleaved 65-kD Stat6 lacks the COOH-terminal transactivation domain and functions as a dominant-negative molecule to Stat6-mediated transcription.

View Article: PubMed Central - PubMed

Affiliation: Department of Internal Medicine II, Graduate School of Medicine, Chiba University, Chiba 260-8670, Japan.

ABSTRACT
Accumulating evidence has shown the importance of Stat6-mediated signaling in allergic diseases. In this study, we show a novel regulatory mechanism of Stat6-mediated signaling in mast cells. When Stat6 is activated by interleukin (IL)-4 and translocated to the nucleus, Stat6 is cleaved by a nucleus-associated protease in mast cells. The cleaved 65-kD Stat6 lacks the COOH-terminal transactivation domain and functions as a dominant-negative molecule to Stat6-mediated transcription. The retrovirus-mediated expression of cleavage-resistant Stat6 mutants prolongs the nuclear accumulation of Stat6 upon IL-4 stimulation and enhances IL-4-induced gene expression and growth inhibition in mast cells. These results indicate that the proteolytic processing of Stat6 functions as a lineage-specific negative regulator of Stat6-dependent signaling in mast cells, and thus suggest that it may account for the limited role of Stat6 in IL-4 signaling in mast cells.

Show MeSH