Regulation of Fas-mediated immune homeostasis by an activation-induced protein, Cyclon.

Activation-induced cell death (AICD) plays an essential role in the contraction of activated T cells after eradication of pathogen. Fas (APO-1/CD95) is one of the key cell surface proteins that mediate AICD in CD4(+) and CD8(+) T cells. Despite its prime importance in cell death, regulation of Fas expression in T cells is poorly understood. Here we show that Cyclon, a newly identified cytokine-inducible protein, is induced in T cells on T-cell receptor ligation and important for immune homeostasis. Transgenic expression of Cyclon ameliorated autoimmune phenotype in mice lacking subunits of IL-2R. Transgenic expression of Cyclon markedly enhanced AICD through increased expression of Fas whose expression is essential for Cyclon action. Finally, we demonstrated that activated but not resting CD4(+) T cells with targeted deletion of a Cyclon allele show reduced AICD and expression of Fas, indicating a critical role of Cyclon in Fas expression in activated T cells. We think that our data provide insight into expression regulation of Fas in T cells.

Cyclic AMP/GMP-dependent modulation of Ca2+ channels sets the polarity of nerve growth-cone turning.

Signalling by intracellular second messengers such as cyclic nucleotides and Ca2+ is known to regulate attractive and repulsive guidance of axons by extracellular factors. However, the mechanism of interaction among these second messengers in determining the polarity of the guidance response is largely unknown. Here, we report that the ratio of cyclic AMP to cyclic GMP activities sets the polarity of netrin-1-induced axon guidance: high ratios favour attraction, whereas low ratios favour repulsion. Whole-cell recordings of Ca2+ currents at Xenopus spinal neuron growth cones indicate that cyclic nucleotide signalling directly modulates the activity of L-type Ca2+ channels (LCCs) in axonal growth cones. Furthermore, cGMP signalling activated by an arachidonate 12-lipoxygenase metabolite suppresses LCC activity triggered by netrin-1, and is required for growth-cone repulsion mediated by the DCC-UNC5 receptor complex. By linking cAMP and cGMP signalling and modulation of Ca2+ channel activity in growth cones, these findings delineate an early membrane-associated event responsible for signal transduction during bi-directional axon guidance.

Lack of nuclear translocation of cytoplasmic domains of IL-2/IL-15 receptor subunits.

Some sensors of extracellular signaling molecules such as Notch and sterol response element binding protein (SREBP) receive ligand-induced intra-membrane proteolysis followed by nuclear translocation of their cytoplasmic domains to regulate gene expression programs in the nucleus. It has not been extensively examined whether ligand-induced intra-membrane proteolysis of type I cytokine receptors and nuclear translocation of cytoplasmic domains occur. Here, by using a sensitive reporter system, we examined this possibility for the interleukin-2 (IL-2) receptor (IL-2R) beta-chain (IL-2R beta) and the IL-15 receptor (IL-15R) alpha-chain (IL-15R alpha). Flowcytometric analysis revealed that ligand stimulation does not induce nuclear translocation of their cytoplasmic domains. In addition, overexpression of the cytoplasmic domain of the common cytokine receptor gamma-chain (gamma c) in an IL-2R-reconstituted Ba/F3-derived cell line did not affect any biological responses including cell survival, disproving potential roles of the cleaved cytoplasmic domain of gamma c as a signal transducer. Collectively, these results indicated that potential nuclear function of cleaved type I cytokine receptor subunits is not plausible.

Redundant promoter elements mediate IL-3-induced expression of a novel cytokine-inducible gene, cyclon.

Cytokines control cell differentiation, proliferation, and function by regulating gene expression program. Physiological roles and induction mechanisms of cytokine-inducible genes are not fully understood. Here, we identified a novel immediate-early cytokine-responsive gene, cyclon (cytokine-induced protein with coiled-coil domain), which is induced in a hematopoietic cell line by interleukin 3 (IL-3). cyclon gene encodes a phosphorylated nuclear protein consisting of repetitive sequences in the amino-terminus and a coiled-coil domain in the carboxyl-terminus. A novel transient reporter assay revealed that mouse cyclon promoter contains redundant elements for IL-3-induced gene expression.

Nuclear translocation of 2-amino-3-ketobutyrate coenzyme A ligase by cold and osmotic stress.

Cells are continuously exposed to environmental stresses and respond to them to maintain cellular homeostasis. Failure to respond to these stresses may cause pathological states such as renal failure, complications of diabetes, and autoimmune diseases. Signal transduction induced by osmotic and cold stresses is not fully understood. In addition, mechanisms of these stress responses are yet to be elucidated. Activation of many signaling pathways induces translocation of proteins into the nucleus to transduce signals and regulate nuclear functions. By using inducible translocation trap (ITT), a reporter gene-based screening technique, nuclear translocation of 2-amino-3-ketobutyrate coenzyme A ligase (KBL) was detected in response to cold and osmotic stresses. Rapid nuclear translocation of KBL was confirmed by biochemical analysis and fluorescent microscopy. A large region of KBL was required for stress-induced nuclear translocation. The KBL reporter system will be a useful tool for the investigation of cold and osmotic stress responses.

Insertional chromatin immunoprecipitation: a method for isolating specific genomic regions.

We established a novel method, insertional chromatin immunoprecipitation (iChIP), for isolation of specific genomic regions. In iChIP, specific genomic domains are immunoprecipitated with antibody against a tag, which is fused to the DNA-binding domain of an exogenous DNA-binding protein, whose recognition sequence is inserted into the genomic domains of interest. The iChIP method will be a useful tool for dissecting chromatin structure of genomic region of interest.

Temporal regulation of Stat5 activity in determination of cell differentiation program.

Although Stat5 is activated by various cytokines, only erythropoietin (Epo) and a small number of cytokines induce Stat5-dependent erythroid differentiation. Here, by using a reporter gene system to monitor transcriptional activity of Stat5, we showed that Epo but not interleukin (IL)-3 supports sustained activation of Stat5, which induces globin gene expression. IL-3 or IL-2 stimulation inhibits Epo-induced globin gene expression. The acidic region of the IL-2 receptor beta-chain was essential for this inhibition. These results underscore the importance of temporal regulation of Stat activity for regulation of cytokine-specific cell differentiation.

Regulation of cell proliferation by interleukin-3-induced nuclear translocation of pyruvate kinase.

Extracellular signaling molecules bound to cell surface receptors can regulate nuclear function with consequences for cell proliferation, differentiation, and function. To regulate nuclear function, signals must be transduced across the nuclear envelope to propagate the signal from the cytoplasm to the nucleus. Therefore, many signaling responses induce the nuclear translocation of transcription factors, kinases, and others. By using inducible translocation trap, a reporter gene-based system to detect inducible nuclear translocation, we found that the M2 isoform of pyruvate kinase, a key enzyme in glycolysis, translocates into the nucleus by interleukin-3, but not by epidermal growth factor, stimulation. The C domain of the M2 isoform of pyruvate kinase was sufficient for interleukin-3-induced nuclear translocation. Interleukin-3-induced nuclear translocation of the M2 isoform of pyruvate kinase was dependent on the activation of Jak2. Overexpression of the M2 isoform of pyruvate kinase protein fused with a nuclear localization signal enhanced cell proliferation in the absence of interleukin-3, suggesting that the nuclear pyruvate kinase plays an important role in cell proliferation.

Regulation of Stat1 protein expression by phenylalanine 172 in the coiled-coil domain.

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.

Inducible translocation trap: a system for detecting inducible nuclear translocation.

Here we report a general system, inducible translocation trap (ITT), for identification of proteins that translocate into the nucleus following signal transduction from cell surface receptors. ITT consists of a retroviral cDNA expression library of fusion proteins consisting of a LexA DNA binding domain, the transactivation domain of a transcriptional activator, and proteins encoded by cDNA inserts. The retroviral library is then transduced into cell lines containing a reporter gene with LexA binding sites in its promoter. Cells expressing the reporter gene by extracellular stimuli are then selected by flow-cytometric sorting. By using ITT, we identified cDNA encoding Stat1 in a screen of proteins which translocate into the nucleus by IFNgamma, indicating that this system can be used for isolation of nuclear translocating proteins induced by extracellular stimuli. ITT may be a useful tool for dissecting dynamic translocation in various biological systems.