STAT (signal transducers and activators of transcription) proteins are transcription factors which are activated by phosphorylation on tyrosine residues upon stimulation by cytokines. Seven members of the STAT family are known, including the closely related STAT5A and STAT5B, which are activated by various cytokines. Except for prolactin-dependent ␤-casein production in mammary gland cells, the biological consequences of STAT5 activation in various systems are not clear. We applied PCR-driven random mutagenesis and a retrovirus-mediated expression screening system to identify constitutively active forms of STAT5. By this strategy, we have identified a constitutively active STAT5 mutant which has two amino acid substitutions; one is located upstream of the putative DNA binding domain (H299R), and the other is located in the transactivation domain (S711F). The mutant STAT5 was constitutively phosphorylated on tyrosine residues, localized in the nucleus, and was transcriptionally active. Expression of the mutant STAT5 partially dispenses with interleukin 3 (IL-3) as a growth stimulant of IL-3-dependent cell lines. Further analyses of the mutant STAT5 have demonstrated that both of the mutations are required for nuclear localization, efficient transcriptional activation, and induction of IL-3-independent growth of an IL-3-dependent cell line, Ba/F3, and have indicated that a molecular basis for the constitutive activation is the stability of the phosphorylated form of the mutant STAT5.Stimulation of cytokine receptors leads to activation of multiple signal transduction pathways, including the Ras-Raf-MEK-mitogen-activated protein kinase (MAPK) and the JAK-STAT pathways (14,28,34,42,44). The latter signaling pathway was originally found downstream of the interferon receptors and is now recognized as a common pathway downstream of most cytokine receptors. Upon stimulation with cytokines, receptor-associated JAKs are activated and phosphorylate STAT factors on tyrosine residues. The phosphorylated STAT molecules then form homo-or heterodimers through SH2-mediated interactions and translocate into nuclei to activate transcription of various target genes. Seven members of the STAT family (STAT1 through 4, -5A, -5B, and -6) are known; STAT5A and STAT5B are closely related. With the exception of STAT4 and STAT6, which were shown to be specifically activated by only one or two cytokines, interleukin 12 (IL-12) or both IL-4 and IL-13, respectively (13, 15), most of the other STATs are activated by multiple cytokines. In particular, both STAT5A and STAT5B are activated by numerous cytokines, including prolactin, IL-2, IL-3, IL-5, IL-7, granulocyte-macrophage colony-stimulating factor (GM-CSF), G-CSF, M-CSF, erythropoietin (Epo), thrombopoietin, and growth hormone (GH).Using the receptor for the human GM-CSF as a model system, members of our group previously showed that activation of the Ras-Raf-MEK-MAPK pathway inhibits apoptosis while the region of the GM-CSF receptor, which is responsible for activation of JAK2 and STAT5 an...

We previously established a high-efficiency, retrovirus-mediated expression cloning method. Using this system, we now have developed an expression cloning method (FL-REX; fluorescence localization-based retrovirus-mediated expression cloning) in which cDNAs can be isolated based on the subcellular localization of their protein products. Complementary DNAs generated from mRNA using random hexamers were fused to the cDNA of green fluorescent protein (GFP) in the pMX retrovirus vector. The resulting cDNA-GFP fusion library was transfected into retrovirus-packaging cells, and the derived retroviruses were used to infect NIH 3T3 cells. Infected cells then were screened to identify cDNAs of interest through the subcellular localization of the GFP-fusion products. Using FL-REX, we have identified 25 cDNAs, most of which showed reasonable subcellular localization as GFP-fusion proteins, indicating that FL-REX is useful for identification of proteins that show specific intracellular localization.

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We report a patient with congenital Chagas disease in Japan. This report reemphasizes the role of neglected and emerging tropical diseases in the era of globalization. It also indicates the need for increased vigilance for detecting Chagas disease in non–disease-endemic countries.