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

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-null 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.

Figure 1.

KRC expression increases in CD4 cells upon activation. Primary C57/B6 CD4⁺ T cells were stimulated with anti-CD3 (2.0 μg/ml)/anti-CD28 (1.0 μg/ml) antibodies for the indicated times. RNA was prepared and KRC expression was determined by RT-PCR, with β-actin as an internal control.

Figure 2.

KRC overexpression increases, whereas KRC loss decreases endogenous IL-2 production. (A) Jurkat T cells were stably transfected with vector (pEF) or KRC expression plasmids. Stable clones were stimulated for 18 h with 50 ng/ml PMA plus 2 μM ionomycin, and IL-2 production was measured by ELISA. (B) Primary CD4⁺ T cells were activated for 36 h and subsequently transduced with control (RV), KRC, or KRC dominant-negative (ZAS2) bicistronic GFP-expressing retroviruses. GFP-positive cells were sorted and stimulated for 24 h with anti-CD3 or anti-CD3/anti-CD28 antibodies and IL-2 production was measured by ELISA. (C) CD4 T cells from KRC^+/+ or ^−/− mice were stimulated with anti-CD3 (1.0 μg/ml)/CD28 (0.5 μg/ml) antibodies for 24 h, and IL-2 production was measured by ELISA. (D) CD4 T cells from KRC^+/+ or ^−/− mice were stimulated with anti-CD3/CD28 antibodies for 72 h in the presence of 200 U/ml of human IL-2. IFNγ production was measured by ELISA. Data shown are representative of four independent experiments with similar results.

Figure 3.

KRC overexpression increases the transcription of the IL-2 gene. (A) Stably transfected Jurkat T cell clones with vector (vec) or KRC (JURKAT-KRC) were stimulated with 50 ng/ml PMA plus 2 μM ionomycin for 6 h. IL-2 mRNA abundance was determined by RT-PCR with tubulin as an internal control. (B) Jurkat cells were transiently transfected with an IL-2–luciferase reporter (IL-2-LUC) along with Vector, KRC, or KRCtr (amino acids 204–1,055) and, in all cases, a CMV–β-Gal reporter as an internal control (Materials and Methods). 24 h later, cells were stimulated with PMA plus ionomycin for 6 h (top) or Raji cells loaded with SEE for 8 h (bottom). Luciferase activity was determined and normalized for β-galactosidase activity. (C) Jurkat cells were transiently transfected with NFAT/AP-1–, NFAT-, or AP-1–Luciferase reporters and treated as aforemtioned.

Figure 4.

KRC does not modulate MAPK activity. (A) Jurkat cells were transiently transfected with AP-1–luciferase reporter along with KRC and RasN17 DN vectors. 24 h later, cells were pretreated with 10 μM rottlerin and stimulated for 6 h with PMA plus ionomycin. Luciferase activity was measured as described in Materials and Methods. (B) Jurkat cells were transfected with a GAL4 luciferase reporter along with a GAL4 DNA binding domain, GAL4-ATF2, or GAL4-ELK1 with or without KRC. 24 h later, cells were stimulated with PMA plus ionomycin and analyzed for luciferase activity as described in Materials and Methods. (C) Jurkat cells were transiently transfected with FLAG-JNK2, and either Vector, KRC, or MKK7. 48 h later, cells were stimulated with PMA plus ionomycin for 6 h, and JNK activity was determined by immunoprecipitation/kinase assay. Equal amounts of FLAG-Jnk2 protein were immunoprecipitated, as judged by anti-FLAG Western blot (bottom).

Figure 5.

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.