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Schnurri-3 (KRC) interacts with c-Jun to regulate the IL-2 gene in T cells.

Oukka M, Wein MN, Glimcher LH - J. Exp. Med. (2004)

Bottom Line: In the immune system, AP-1 activity is highest in T cells, suggesting that a subset of T cell-specific coactivator proteins exist to selectively potentiate AP-1 function.Here, we describe that the expression of Schnurri-3, also known as kappa recognition component (KRC), is induced upon T cell receptor signaling in T cells and functions to regulate the expression of the interleukin 2 (IL-2) gene.KRC physically associates with the c-Jun transcription factor and serves as a coactivator to augment AP-1-dependent IL-2 gene transcription.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology and Infectious Diseases, Harvard School of Public Health, Boston, MA 02115, USA.

ABSTRACT
The activator protein 1 (AP-1) transcription factor is a key participant in the control of T cell proliferation, cytokine production, and effector function. In the immune system, AP-1 activity is highest in T cells, suggesting that a subset of T cell-specific coactivator proteins exist to selectively potentiate AP-1 function. Here, we describe that the expression of Schnurri-3, also known as kappa recognition component (KRC), is induced upon T cell receptor signaling in T cells and functions to regulate the expression of the interleukin 2 (IL-2) gene. Overexpression of KRC in transformed and primary T cells leads to increased IL-2 production, whereas dominant-negative KRC, or loss of KRC protein in KRC- mice, results in diminished IL-2 production. KRC physically associates with the c-Jun transcription factor and serves as a coactivator to augment AP-1-dependent IL-2 gene transcription.

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KRC physically interacts with c-Jun and acts as a transcriptional coactivator. (A) 293T cells were transfected with c-Jun and myc-KRCtr. 48 h later, lysates were immunoprecipitated with anti-Myc antibody. Immunoprecipitates were probed by Western blotting with anti–c-Jun antibody. (B) 293T cells were cotransfected with c-Jun and full length His-KRC (left). 48 h later, lysates were immunoprecipitated with anti-His antibody (DE8 Omniprobe), and precipitates were probed by Western blotting with anti–c-Jun antibody. In vitro–translated and S35-labeled c-Jun and His-KRCtr were mixed and immunoprecipitated with anti-His antibody (right). Recovered c-Jun protein was visualized by autoradiography. (C) Jurkat or EL4 T cells were stimulated with PMA plus ionomycin for 45 min. Lysates were immunoprecipitated with anti–c-Jun antibody, and immunoprecipitates were probed with specific anti-KRC rabbit antisera. (D) 293T cells were transfected with AP-1 luciferase along with c-Jun, c-Fos, and KRC (top). 24 h later, luciferase activity was determined as aforementioned. 293T cells were transfected with GAL4 luciferase along with GAL4, GAL4–c-Jun 1-224, or GAL4–c-Fos 208-313 (bottom). 24 h later, luciferase activity was determined as aforementioned.
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fig5: KRC physically interacts with c-Jun and acts as a transcriptional coactivator. (A) 293T cells were transfected with c-Jun and myc-KRCtr. 48 h later, lysates were immunoprecipitated with anti-Myc antibody. Immunoprecipitates were probed by Western blotting with anti–c-Jun antibody. (B) 293T cells were cotransfected with c-Jun and full length His-KRC (left). 48 h later, lysates were immunoprecipitated with anti-His antibody (DE8 Omniprobe), and precipitates were probed by Western blotting with anti–c-Jun antibody. In vitro–translated and S35-labeled c-Jun and His-KRCtr were mixed and immunoprecipitated with anti-His antibody (right). Recovered c-Jun protein was visualized by autoradiography. (C) Jurkat or EL4 T cells were stimulated with PMA plus ionomycin for 45 min. Lysates were immunoprecipitated with anti–c-Jun antibody, and immunoprecipitates were probed with specific anti-KRC rabbit antisera. (D) 293T cells were transfected with AP-1 luciferase along with c-Jun, c-Fos, and KRC (top). 24 h later, luciferase activity was determined as aforementioned. 293T cells were transfected with GAL4 luciferase along with GAL4, GAL4–c-Jun 1-224, or GAL4–c-Fos 208-313 (bottom). 24 h later, luciferase activity was determined as aforementioned.

Mentions: We have demonstrated recently that KRC interacts with the adaptor protein TRAF2 to inhibit both NFκB and JNK/SAPK-mediated responses, including apoptosis and TNFα cytokine gene expression (14). We asked whether KRC might, therefore, physically associate with c-Jun. Expression vectors encoded c-Jun and a truncated myc-tagged version of KRC encoding amino acids 204–1,055 (KRC tr), which includes the third zinc finger domain, one of the three acidic domains and the putative NLS sequence that were overexpressed in the 293T kidney epithelial cell line. Coimmunoprecipitation using a monoclonal anti-myc antibody revealed that KRC physically associated with c-Jun (Fig. 5 A). Furthermore, it demonstrated that the region of KRC shown to associate with TRAF2 (amino acids 204–1,055) also interacted with c-Jun. Similar results were obtained in coimmunoprecipitation of overexpressed full-length KRC with c-Jun, although the absolute amounts of c-Jun obtained were less, presumably because the full-length KRC protein is poorly expressed due to its large size (Fig. 5 B). Further mapping of c-Jun to delineate its interaction site with KRC revealed that KRC interacts with c-Jun amino acids 1–224 fused to the DNA binding domain of GAL4, which includes the transactivation domain (unpublished data). Furthermore, this association is direct and does not require posttranslational modifications as shown by the interaction of in vitro translated KRC and c-Jun proteins (Fig. 5 B, right). Finally, it was important to demonstrate that this association occurred under physiologic conditions. Untransfected Jurkat or EL4 T cell lines were stimulated with PMA/ionomycin for 45 min, and AP-1 complexes were purified by immunoprecipitating c-Jun. Fig. 5 C shows that endogenous KRC is readily detected in these complexes obtained from stimulated cells.


Schnurri-3 (KRC) interacts with c-Jun to regulate the IL-2 gene in T cells.

Oukka M, Wein MN, Glimcher LH - J. Exp. Med. (2004)

KRC physically interacts with c-Jun and acts as a transcriptional coactivator. (A) 293T cells were transfected with c-Jun and myc-KRCtr. 48 h later, lysates were immunoprecipitated with anti-Myc antibody. Immunoprecipitates were probed by Western blotting with anti–c-Jun antibody. (B) 293T cells were cotransfected with c-Jun and full length His-KRC (left). 48 h later, lysates were immunoprecipitated with anti-His antibody (DE8 Omniprobe), and precipitates were probed by Western blotting with anti–c-Jun antibody. In vitro–translated and S35-labeled c-Jun and His-KRCtr were mixed and immunoprecipitated with anti-His antibody (right). Recovered c-Jun protein was visualized by autoradiography. (C) Jurkat or EL4 T cells were stimulated with PMA plus ionomycin for 45 min. Lysates were immunoprecipitated with anti–c-Jun antibody, and immunoprecipitates were probed with specific anti-KRC rabbit antisera. (D) 293T cells were transfected with AP-1 luciferase along with c-Jun, c-Fos, and KRC (top). 24 h later, luciferase activity was determined as aforementioned. 293T cells were transfected with GAL4 luciferase along with GAL4, GAL4–c-Jun 1-224, or GAL4–c-Fos 208-313 (bottom). 24 h later, luciferase activity was determined as aforementioned.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC1887724&req=5

fig5: KRC physically interacts with c-Jun and acts as a transcriptional coactivator. (A) 293T cells were transfected with c-Jun and myc-KRCtr. 48 h later, lysates were immunoprecipitated with anti-Myc antibody. Immunoprecipitates were probed by Western blotting with anti–c-Jun antibody. (B) 293T cells were cotransfected with c-Jun and full length His-KRC (left). 48 h later, lysates were immunoprecipitated with anti-His antibody (DE8 Omniprobe), and precipitates were probed by Western blotting with anti–c-Jun antibody. In vitro–translated and S35-labeled c-Jun and His-KRCtr were mixed and immunoprecipitated with anti-His antibody (right). Recovered c-Jun protein was visualized by autoradiography. (C) Jurkat or EL4 T cells were stimulated with PMA plus ionomycin for 45 min. Lysates were immunoprecipitated with anti–c-Jun antibody, and immunoprecipitates were probed with specific anti-KRC rabbit antisera. (D) 293T cells were transfected with AP-1 luciferase along with c-Jun, c-Fos, and KRC (top). 24 h later, luciferase activity was determined as aforementioned. 293T cells were transfected with GAL4 luciferase along with GAL4, GAL4–c-Jun 1-224, or GAL4–c-Fos 208-313 (bottom). 24 h later, luciferase activity was determined as aforementioned.
Mentions: We have demonstrated recently that KRC interacts with the adaptor protein TRAF2 to inhibit both NFκB and JNK/SAPK-mediated responses, including apoptosis and TNFα cytokine gene expression (14). We asked whether KRC might, therefore, physically associate with c-Jun. Expression vectors encoded c-Jun and a truncated myc-tagged version of KRC encoding amino acids 204–1,055 (KRC tr), which includes the third zinc finger domain, one of the three acidic domains and the putative NLS sequence that were overexpressed in the 293T kidney epithelial cell line. Coimmunoprecipitation using a monoclonal anti-myc antibody revealed that KRC physically associated with c-Jun (Fig. 5 A). Furthermore, it demonstrated that the region of KRC shown to associate with TRAF2 (amino acids 204–1,055) also interacted with c-Jun. Similar results were obtained in coimmunoprecipitation of overexpressed full-length KRC with c-Jun, although the absolute amounts of c-Jun obtained were less, presumably because the full-length KRC protein is poorly expressed due to its large size (Fig. 5 B). Further mapping of c-Jun to delineate its interaction site with KRC revealed that KRC interacts with c-Jun amino acids 1–224 fused to the DNA binding domain of GAL4, which includes the transactivation domain (unpublished data). Furthermore, this association is direct and does not require posttranslational modifications as shown by the interaction of in vitro translated KRC and c-Jun proteins (Fig. 5 B, right). Finally, it was important to demonstrate that this association occurred under physiologic conditions. Untransfected Jurkat or EL4 T cell lines were stimulated with PMA/ionomycin for 45 min, and AP-1 complexes were purified by immunoprecipitating c-Jun. Fig. 5 C shows that endogenous KRC is readily detected in these complexes obtained from stimulated cells.

Bottom Line: In the immune system, AP-1 activity is highest in T cells, suggesting that a subset of T cell-specific coactivator proteins exist to selectively potentiate AP-1 function.Here, we describe that the expression of Schnurri-3, also known as kappa recognition component (KRC), is induced upon T cell receptor signaling in T cells and functions to regulate the expression of the interleukin 2 (IL-2) gene.KRC physically associates with the c-Jun transcription factor and serves as a coactivator to augment AP-1-dependent IL-2 gene transcription.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology and Infectious Diseases, Harvard School of Public Health, Boston, MA 02115, USA.

ABSTRACT
The activator protein 1 (AP-1) transcription factor is a key participant in the control of T cell proliferation, cytokine production, and effector function. In the immune system, AP-1 activity is highest in T cells, suggesting that a subset of T cell-specific coactivator proteins exist to selectively potentiate AP-1 function. Here, we describe that the expression of Schnurri-3, also known as kappa recognition component (KRC), is induced upon T cell receptor signaling in T cells and functions to regulate the expression of the interleukin 2 (IL-2) gene. Overexpression of KRC in transformed and primary T cells leads to increased IL-2 production, whereas dominant-negative KRC, or loss of KRC protein in KRC- mice, results in diminished IL-2 production. KRC physically associates with the c-Jun transcription factor and serves as a coactivator to augment AP-1-dependent IL-2 gene transcription.

Show MeSH
Related in: MedlinePlus