Stat1 plays an essential role in signal transduction and gene expression of various cytokines including interferons (IFNs). Although the mechanism of cytokine-induced activation of Stat1 and transcriptional regulation of Stat1 gene expression have been established, post-transcriptional regulation of Stat1 protein expression is not fully understood. Here, we report identification of a mutant of Stat1 that has decreased expression levels by using inducible translocation trap (ITT), a reporter gene-based detection system of nuclear translocation. The substitution of serine for phenylalanine 172 (F172S) in the coiled-coil domain causes marked decrease in Stat1 protein expression in various cell lines without decreasing its mRNA levels. Our results suggest that the decrease is caused by translational/post-translational mechanisms independent of proteasome machinery. These results suggest a novel potential mechanism of determination of specificity of Stat proteins and showed that the ITT system is a powerful technique to identify mutants of nuclear translocating signal transducers.

IntroductionActivation of acquired immunity induces rapid expansion of antigen-specific CD4 ϩ and CD8 ϩ T cells to eradicate the pathogens. After eradication of the pathogens, however, most activated T cells are removed. 1,2 The elimination of activated T cells is largely mediated by apoptotic cell death. 2 Activated T-cell autonomous death and activation-induced cell death (AICD) are the 2 mechanisms by which programmed death of activated T cells occurs. [3][4][5][6][7] Activated T-cell autonomous death is mediated by the intrinsic death pathway, [8][9][10] which is regulated by the B-cell lymphoma 2 (Bcl-2) family proteins. In contrast, the other mechanism of activated T-cell death, AICD, requires antigen restimulation of activated effector T cells and is triggered by the extrinsic death pathways regulated by signals of death receptors (DRs) of the tumor necrosis factor receptor (TNFR) family. 1,[11][12][13] Members of the DR subgroup include type I TNFR (TNFRI)/DR1, Fas/CD95/ Apo1/DR2, DR3, TNF-related apoptosis-inducing ligand receptor 1 (TRAIL-R1)/DR4, TRAIL-R2/DR5/Apo2, and DR6. These receptors contain extracellular cysteine-rich domains and an intracellular death domain, which is necessary for transducing death signals. [14][15][16] Binding of ligands to DRs triggers trimerization of the receptors, which is required for the recruitment of the adaptor protein Fas-associated death domain (FADD), 16 and subsequently procaspase 8, also known as FLICE, or procaspase 10. The ensemble constitutes what is known as the death-inducing signaling complex, leading to auto-processing of initiator procaspases into active caspases, which activate downstream effector caspases. 14,17,18 Other inhibitors of the pathway include the FLICE inhibitory protein (FLIP), which competes with procaspase 8 (FLICE) for binding to the DED of FADD. [19][20][21][22][23] Fas ligand (FasL) and TNF are the key death ligands that mediate AICD in mature T cells. 1,7,[24][25][26][27][28][29] Importance of the Fas/FasL system in regulation of immune homeostasis has been established by genetic studies showing that lpr mice, which have a loss-offunction mutation in the Fas gene, [30][31][32] and gld mice, which have a loss-of-function mutation in the Fasl gene, [33][34][35] develop severe lymphadenopathy caused by defects in Fas-mediated cell death. 36,37 In humans, defects in Fas and FasL cause similar abnormalities in the autoimmune lymphoproliferative syndrome type Ia and Ib, respectively. [38][39][40] Expression levels of Fas or FasL markedly impact T-cell death. Regulation of FasL expression in primary T cells has been extensively examined. [41][42][43] In contrast, surprisingly little is known about regulation of Fas expression in primary T cells. T-cell deathassociated gene 51 (TDAG51) was shown to be required for T-cell receptor (TCR)-mediated expression of Fas in a T-cell hybridoma. 44 However, it was later shown that TDAG51 is not essential for Fas regulation and apoptosis of primary mouse T cells in vivo. 45 NF-B was also implicated in ...

Binding of cytokines to their specific receptors induces activation of signal transduction pathways, many of which involve nuclear translocation of signaling proteins. In this review, an overview of cytokine-induced nuclear translocation of signaling proteins is provided. In addition, inducible translocation trap (ITT), a novel reporter-based system to detect nuclear translocation, and its application for identification of nuclear translocating proteins are elaborated. Finally, analysis of "nuclear translocatome", the entire set of proteins that translocate into or out of the nucleus in response to extracellular stimuli, by ITT is discussed.

Autoimmunity, which results from hyperreactivity and hypersensitivity of lymphocytes against self antigens, can be caused by various defects in lymphoid cells, including impaired activation induced cell death (AICD) in effector T and B cells. Elimination of activated self-reactive T and B cells may effectively cure autoimmune diseases. We recently identify a novel cytokine-inducible gene, cyclon, by DNA microarray analysis. Cyclon is induced in both CD4+ and CD8+ T cells by T cell receptor ligation. Our data show that transgenic expression of Cyclon has no significant effect on T cell development and functions in wild-type mice. However, transgenic expression of Cyclon normalizes the number of activated T cells and suppresses enlargement of peripheral lymphoid organs in some autoimmune mice such as mice lacking the α subunit or the common γ chain of Interleukin-2 receptor. in vitro assays also suggest that the transgenic expression of Cyclon decreases viability of activated T cells and enhances AICD. Collectively, these results indicate that Cyclon may play an important role in immunosuppression by enhancing AICD. This work is supported by NIH grant R01 AI059315 to H. F.