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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: 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.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.

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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A 65-kD isoform of Stat6 is produced by proteolytic processing. (A) Cell extracts from WT splenocytes were incubated with cell extracts of BMMCs from Stat6−/− mice at 37°C for 20 min and analyzed by Western blotting with anti-Stat6 (M200) antibody (top) or anti-Stat6 (M20) antibody (bottom). As controls, cell extracts from WT BMMCs and Stat6−/− BMMCs were blotted with anti-Stat6 antibodies. Representative blots from four independent experiments are shown. (B) COS7 cells were transfected with Stat6 expression vector and their cell extracts were used as a source of Stat6 protein. Transfected Stat6 was incubated with cell extracts of thymocytes, splenocytes, or BMMCs from Stat6−/− mice at 37°C for 20 min and analyzed by Western blotting with anti-Stat6 (M200) antibody. A representative blot from four independent experiments is shown.
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fig1: A 65-kD isoform of Stat6 is produced by proteolytic processing. (A) Cell extracts from WT splenocytes were incubated with cell extracts of BMMCs from Stat6−/− mice at 37°C for 20 min and analyzed by Western blotting with anti-Stat6 (M200) antibody (top) or anti-Stat6 (M20) antibody (bottom). As controls, cell extracts from WT BMMCs and Stat6−/− BMMCs were blotted with anti-Stat6 antibodies. Representative blots from four independent experiments are shown. (B) COS7 cells were transfected with Stat6 expression vector and their cell extracts were used as a source of Stat6 protein. Transfected Stat6 was incubated with cell extracts of thymocytes, splenocytes, or BMMCs from Stat6−/− mice at 37°C for 20 min and analyzed by Western blotting with anti-Stat6 (M200) antibody. A representative blot from four independent experiments is shown.

Mentions: In previous reports, we and others have shown that a 65-kD isoform of Stat6 (65-kD Stat6) is expressed in BMMCs (13, 14). The 65-kD Stat6 in BMMCs is detected by anti-Stat6 (M200) antibody, which recognizes the middle portion of Stat6 (aa 280–480), but not by anti-Stat6 (M20) antibody, which recognizes the COOH terminus of Stat6 (13, 14). In addition, when BMMCs are stimulated with IL-4, the phosphorylated form of Stat6 is detected at 65 kD by anti-phospho Stat6 antibody, which recognizes the tyrosine residue at aa 641 (Y641) of Stat6 (13). These findings indicate that the 65-kD Stat6 lacks the COOH terminus but contains the Y641, which is essential for the homodimerization of Stat6 (3). To determine whether the 65-kD Stat6 is a product of protein processing, we first performed the coincubation assay in which the conventional 94-kD Stat6 from splenocytes was incubated with cell extracts of BMMCs and analyzed for the size of Stat6 protein by anti-Stat6 Western blotting. To eliminate the influence of endogenous Stat6 expression in BMMCs, we prepared whole cell extracts from BMMCs in Stat6−/− mice (Stat6−/− BMMCs) as a possible source of the protease(s). Interestingly, when conventional Stat6 (94-kD Stat6) was incubated with Stat6−/− BMMC extract, the 94-kD Stat6 was cleaved to 65 kD (Fig. 1 A, compare lanes 3 and 4). The cleaved Stat6 was detected by anti-Stat6 (M200) antibody (Fig. 1 A, top) but not by anti-Stat6 (M20) antibody (Fig. 1 A, bottom), suggesting that the cleaved Stat6 also lacks the COOH terminus. These results indicate that the 65-kD Stat6 is produced by the cleavage of the 94-kD Stat6 in BMMCs.


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)

A 65-kD isoform of Stat6 is produced by proteolytic processing. (A) Cell extracts from WT splenocytes were incubated with cell extracts of BMMCs from Stat6−/− mice at 37°C for 20 min and analyzed by Western blotting with anti-Stat6 (M200) antibody (top) or anti-Stat6 (M20) antibody (bottom). As controls, cell extracts from WT BMMCs and Stat6−/− BMMCs were blotted with anti-Stat6 antibodies. Representative blots from four independent experiments are shown. (B) COS7 cells were transfected with Stat6 expression vector and their cell extracts were used as a source of Stat6 protein. Transfected Stat6 was incubated with cell extracts of thymocytes, splenocytes, or BMMCs from Stat6−/− mice at 37°C for 20 min and analyzed by Western blotting with anti-Stat6 (M200) antibody. A representative blot from four independent experiments is shown.
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Related In: Results  -  Collection

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fig1: A 65-kD isoform of Stat6 is produced by proteolytic processing. (A) Cell extracts from WT splenocytes were incubated with cell extracts of BMMCs from Stat6−/− mice at 37°C for 20 min and analyzed by Western blotting with anti-Stat6 (M200) antibody (top) or anti-Stat6 (M20) antibody (bottom). As controls, cell extracts from WT BMMCs and Stat6−/− BMMCs were blotted with anti-Stat6 antibodies. Representative blots from four independent experiments are shown. (B) COS7 cells were transfected with Stat6 expression vector and their cell extracts were used as a source of Stat6 protein. Transfected Stat6 was incubated with cell extracts of thymocytes, splenocytes, or BMMCs from Stat6−/− mice at 37°C for 20 min and analyzed by Western blotting with anti-Stat6 (M200) antibody. A representative blot from four independent experiments is shown.
Mentions: In previous reports, we and others have shown that a 65-kD isoform of Stat6 (65-kD Stat6) is expressed in BMMCs (13, 14). The 65-kD Stat6 in BMMCs is detected by anti-Stat6 (M200) antibody, which recognizes the middle portion of Stat6 (aa 280–480), but not by anti-Stat6 (M20) antibody, which recognizes the COOH terminus of Stat6 (13, 14). In addition, when BMMCs are stimulated with IL-4, the phosphorylated form of Stat6 is detected at 65 kD by anti-phospho Stat6 antibody, which recognizes the tyrosine residue at aa 641 (Y641) of Stat6 (13). These findings indicate that the 65-kD Stat6 lacks the COOH terminus but contains the Y641, which is essential for the homodimerization of Stat6 (3). To determine whether the 65-kD Stat6 is a product of protein processing, we first performed the coincubation assay in which the conventional 94-kD Stat6 from splenocytes was incubated with cell extracts of BMMCs and analyzed for the size of Stat6 protein by anti-Stat6 Western blotting. To eliminate the influence of endogenous Stat6 expression in BMMCs, we prepared whole cell extracts from BMMCs in Stat6−/− mice (Stat6−/− BMMCs) as a possible source of the protease(s). Interestingly, when conventional Stat6 (94-kD Stat6) was incubated with Stat6−/− BMMC extract, the 94-kD Stat6 was cleaved to 65 kD (Fig. 1 A, compare lanes 3 and 4). The cleaved Stat6 was detected by anti-Stat6 (M200) antibody (Fig. 1 A, top) but not by anti-Stat6 (M20) antibody (Fig. 1 A, bottom), suggesting that the cleaved Stat6 also lacks the COOH terminus. These results indicate that the 65-kD Stat6 is produced by the cleavage of the 94-kD Stat6 in BMMCs.

Bottom Line: 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.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.

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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