Identiﬁcation and Characterization of a Constitutively Active STAT5 Mutant That Promotes Cell Proliferation

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 b -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. Eco RI and Not I, and the excised 2.4-kbp STAT5A sequences harboring the PCR-generated mutations were ligated to the Eco RI and Not I sites of the pMX vector. The mixture of ligated plasmids was ampliﬁed in Escherichia coli DH5 a , and plasmid DNA was prepared with a Maxi prep kit (Qiagen) to be used as a library of mutated STAT5A sequences in the pMX vector. High-titer retroviruses containing the randomly mutated STAT5A sequences were produced from the library DNA with a transient retrovirus packaging cell line, BOSC23 (35), as described previously (19). For infection, Ba/F3 cells (2 3 10 6 ) were incubated with 5 ml of the retroviruses harboring the mutated STAT5A sequence library in the presence of 10 m g of Polybrene/ml and 10 ng of mIL-3/ml. After 8 h, 5 ml of fresh growth medium was added to the culture, and the incubation was extended for 16 h. Twenty-four hours after infection, cells were washed, refed with growth medium, and allowed to grow for one more day before being subjected to selection in the absence of mIL-3 in 96-well plates. Under these conditions, the efﬁciency of infection of Ba/F3 cells was 20 to 40% as assessed by parallel infections with the test construct pMX-hIL-3R a (31). Recovery and reintroduction of the integrated retroviruses. In order to re- cover retrovirally transduced STAT5A sequences, genomic DNA was isolated from each factor- independent (FI) clone, and the integrated STAT5 sequences were ampliﬁed from 10 ng of genomic DNAs by PCR with the retroviral vector primers described above. The PCR was run for 35 cycles as described above with Taq polymerase (Perkin-Elmer Cetus) under standard conditions with 100 m M deoxynucleoside triphosphates. The recovered PCR fragments were puriﬁed from the gel with QiaexII (Qiagen), digested with Eco RI and Not I, and inserted into the Eco RI and Not I sites of the pMX vector. The STAT5A