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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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D685A and M686A Stat6 are resistant to the Stat6 protease activity. (A) Alanine substitutions of Stat6 (S683A, S684A, D685A, or M686A Stat6) were transfected to COS7 cells. Cell extracts were then incubated with or without Stat6−/− BMMC extract and blotted with anti-Stat6 (M200) antibody. A representative blot from four independent experiments is shown. (B) Relevant aa sequence of the putative cleavage site of Stat6 is shown in comparison with a reported cleavage site of Stat5a and Stat5b (17). (C) Similar to Fig. 5 B, COS7 cells were transfected with D685A, M686A, or WT Stat6 and the luciferase activity of TPU474 was measured in the presence or absence of IL-4. Data are means ± SD for four experiments.
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fig6: D685A and M686A Stat6 are resistant to the Stat6 protease activity. (A) Alanine substitutions of Stat6 (S683A, S684A, D685A, or M686A Stat6) were transfected to COS7 cells. Cell extracts were then incubated with or without Stat6−/− BMMC extract and blotted with anti-Stat6 (M200) antibody. A representative blot from four independent experiments is shown. (B) Relevant aa sequence of the putative cleavage site of Stat6 is shown in comparison with a reported cleavage site of Stat5a and Stat5b (17). (C) Similar to Fig. 5 B, COS7 cells were transfected with D685A, M686A, or WT Stat6 and the luciferase activity of TPU474 was measured in the presence or absence of IL-4. Data are means ± SD for four experiments.

Mentions: To address the role of Stat6 protease activity in mast cells, we tried to identify a mutant Stat6 that was resistant to the protease activity. Because Stat6 was cleaved between aa 673 and 695 and the mobility of 65-kD Stat6 was approximately in the middle of 695 and 673 Stat6 (Fig. 5 A), we prepared a series of point mutants of Stat6 by substituting each residues to alanine. The sensitivity of these mutants to the Stat6 protease activity was then examined by the coincubation assay. Interestingly, two of these mutants, D685A (aspartic acid at aa 685 to alanine) and M686A Stat6 (methionine at aa 686 to alanine), were resistant to the Stat6 protease activity (Fig. 6, A and B). In contrast, S683A and S684A Stat6 were as sensitive to the protease activity as WT Stat6 (Fig. 6 A).


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)

D685A and M686A Stat6 are resistant to the Stat6 protease activity. (A) Alanine substitutions of Stat6 (S683A, S684A, D685A, or M686A Stat6) were transfected to COS7 cells. Cell extracts were then incubated with or without Stat6−/− BMMC extract and blotted with anti-Stat6 (M200) antibody. A representative blot from four independent experiments is shown. (B) Relevant aa sequence of the putative cleavage site of Stat6 is shown in comparison with a reported cleavage site of Stat5a and Stat5b (17). (C) Similar to Fig. 5 B, COS7 cells were transfected with D685A, M686A, or WT Stat6 and the luciferase activity of TPU474 was measured in the presence or absence of IL-4. Data are means ± SD for four experiments.
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Related In: Results  -  Collection

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fig6: D685A and M686A Stat6 are resistant to the Stat6 protease activity. (A) Alanine substitutions of Stat6 (S683A, S684A, D685A, or M686A Stat6) were transfected to COS7 cells. Cell extracts were then incubated with or without Stat6−/− BMMC extract and blotted with anti-Stat6 (M200) antibody. A representative blot from four independent experiments is shown. (B) Relevant aa sequence of the putative cleavage site of Stat6 is shown in comparison with a reported cleavage site of Stat5a and Stat5b (17). (C) Similar to Fig. 5 B, COS7 cells were transfected with D685A, M686A, or WT Stat6 and the luciferase activity of TPU474 was measured in the presence or absence of IL-4. Data are means ± SD for four experiments.
Mentions: To address the role of Stat6 protease activity in mast cells, we tried to identify a mutant Stat6 that was resistant to the protease activity. Because Stat6 was cleaved between aa 673 and 695 and the mobility of 65-kD Stat6 was approximately in the middle of 695 and 673 Stat6 (Fig. 5 A), we prepared a series of point mutants of Stat6 by substituting each residues to alanine. The sensitivity of these mutants to the Stat6 protease activity was then examined by the coincubation assay. Interestingly, two of these mutants, D685A (aspartic acid at aa 685 to alanine) and M686A Stat6 (methionine at aa 686 to alanine), were resistant to the Stat6 protease activity (Fig. 6, A and B). In contrast, S683A and S684A Stat6 were as sensitive to the protease activity as WT Stat6 (Fig. 6 A).

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
Related in: MedlinePlus