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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