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...

Expression cloning of cDNAs was first described a decade ago and was based on transient expression of cDNA libraries in COS cells. In contrast to transient transfection of plasmids, retroviral gene transfer delivers genes stably into a wide range of target cells. We utilize a simple packaging system for production of high-titer retrovirus stock from cDNA libraries to establish a cDNA expression cloning system. In two model experiments, murine interleukin (IL)-3-dependent Ba/F3 cells were infected with libraries of retrovirally expressed cDNA derived from human T-cell mRNA or human IL-3-dependent TF-1 cell line mRNA.These infected Ba/F3 cells were selected for the expression of CD2 by flow cytometry or for the a subunit of the human IL-3 receptor (hIL-3Ra) by factor-dependent growth. CD2 (frequency, 1 in 104) and hIL-3Ra (frequency, 1 in 1.5 x 105) cDNAs were readily detected in small-scale experiments, indicating this retroviral expression cloning system is efficient enough to clone low-abundance cDNAs by their expression or function.Expression cloning is a powerful tool with which to isolate a cDNA of interest when a phenotypic function of a protein is known but its amino acid sequence is not known. For instance, many cytokine cDNAs were cloned by assaying the growthpromoting activity of culture supematant of COS cells transiently transfected with subdivided cDNA libraries (1). The principle of the first cDNA expression cloning system (2) was to amplify expression vectors carrying the simian virus 40 (SV40) replication origin (ori) in mammalian cells stably expressing tumor antigen (T antigen) [i.e., a transformed African green monkey kidney cell line, COS (3)]. The presence of the SV40 large T antigen in COS cells allows replication of SV40 ori-containing plasmids, thus amplifying expression of the cDNA on the plasmid (2, 3). A notable alteration to this strategy was introduced by Seed and Aruffo (4), in which transfected COS cells were selected for the expression of a known antigen by panning transfected cells on antibody-coated plates. Genes encoding many surface markers for which antibodies were available have been cloned in this way (4,5). Alternatively, the cell sorter was also utilized to collect COS cells that express a cDNA of interest (6). The cDNAs for cytokine receptors have been also isolated from cDNA libraries by screening for cytokine binding (7-9). In addition, a transcription factor GATA-1 cDNA was isolated by COS-cell expression cloning using gel mobility shift assay as a screening procedure (10). Thus, expression cloning of genes expressing a desired protein or activity is limited only by the ingenuity of the experimental design.Despite many applications, most conventional expression cloning systems still suffer from the need for transient amplification of plasmids in particular cell lines expressing the SV40 (or polyoma) large T antigen. First of all, the function of the target gene has to be suited to transient detection. Moreover, target cells are restricted to cells that allow SV...

We previously identified a constitutively active form of STAT (signal transducer and activator of transcription) 5A by polymerase chain reaction-driven random mutagenesis followed by retrovirus-mediated expression screening, which had two point mutations in the DNA-binding and transcriptional activation domains, and was designated STAT5A1*6. STAT5A1*6 showed markedly elevated DNA binding and transactivation activities with stable tyrosine phosphorylation and nuclear accumulation, and conferred autonomous cell growth on interleukin 3-dependent Ba/F3 cells. We now report another constitutively active mutant, STAT5A-N642H which has a single point mutation (N642H) in its SH2 domain, identified using the same strategy as that used to identify STAT5A1*6. STAT5A-N642H showed identical properties to those of STAT5A1*6 both biochemically and biologically. Interestingly the mutation in STAT5A-N642H resulted in restoration of the conserved critical histidine which is involved in the binding of phosphotyrosine in the majority of SH2-containing proteins. Introduction of an additional mutation (Y694F) to STAT5A-N642H, which disrupted critical tyrosine 694 required for dimerization of STAT5, abolished all the activities manifested by the mutant STAT5A-N642H, which indicates that dimerization is required for the activity of STAT5A-N642H as was the case for the wild-type STAT5A. The present findings also show that different mutations rendered STAT5A constitutively active, through a common mechanism, which is similar to that of physiological activation.The STAT 1 protein is a transcription factor which is activated upon stimulation with various cytokines, and plays a central role in cytokine signaling (1-3). The STAT family consists of seven known members, including closely related STAT5A and STAT5B. Once ligands bind to their cognate receptors, Janus kinases (JAKs) and STATs are phosphorylated successively. The phosphorylated STAT protein forms homo-or heterodimer through intermolecular interaction between the SH2 domain and the phosphotyrosine of the STAT. The dimerized STAT then translocates into the nuclei and binds to promoter regions of target genes to activate transcription. Since phosphorylated STAT is rapidly dephosphorylated, transactivation of gene expression by STAT is generally transient (4). On the other hand, it was reported that human leukemias were frequently associated with the constitutive activation of STATs (5-8), albeit the role of activated STATs in leukemogenesis being unknown.Although gene targeting is a powerful strategy in analyzing biological roles of the gene product, redundancy of functional genes occasionally masks the phenotype of the null mutation of the gene. In the case of STAT5A and STAT5B-doubly disrupted mice, fetal anemia and apoptosis of erythroid progenitors occurred. However, no gross abnormalities were found in hematopoietic systems of adult mice (9 -12). Therefore, biological functions of STAT5 in hematopoietic cells have remained to be elucidated.Our group identified a constitutively a...

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Thrombopoietin and its receptor (MPL) are important regulators of megakaryopoiesis. We have identified an activating mutation of MPL using a combination of a retrovirus-mediated gene transfer and polymerase chain reaction-driven random mutagenesis. This point mutation causes a single amino acid substitution from Ser498 to Asn498 in the transmembrane region and abrogates factor-dependency of all interleukin-3-dependent cell lines tested. Murine interleukin-3- dependent Ba/F3 cells expressing the mutated but not the normal form of MPL were tumorigenic when transduced into syngeneic mice. Analysis of intracellular signaling pathways indicated that the mutant MPL protein constitutively activated two distinct signaling pathways, SHC-Raf-MAPK and JAK2-STAT3/STAT5.