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 tyros ine residues upon stimulation by cytokines. Seven members of the STAT family are known, including the closely related STAT5A and STAT5B, whi ch are activated by various cytokines. Except for prolactin-dependent beta-casein production in mammary gland cells, the biological conseque nces of STAT5 activation in various systems are not clear. We applied PCR-driven random mutagenesis and a retrovirus-mediated expression scr eening system to identify constitutively active forms of STAT5. By thi s strategy, we have identified a constitutively active STAT5 mutant wh ich has two amino acid substitutions; one is located upstream of the p utative DNA binding domain (H299R), and the other is located in the tr ansactivation domain (S711F). The mutant STAT5 was constitutively phos phorylated on tyrosine residues, localized in the nucleus, and was tra nscriptionally active. Expression of the mutant STAT5 partially dispen ses with interleukin 3 (IL-3) as a growth stimulant of IL-3-dependent cell lines. Further analyses of the mutant STAT5 have demonstrated tha t both of the mutations are required for nuclear localization, efficie nt transcriptional activation, and induction of IL-3-independent growt h of an IL-3-dependent cell line, Ba/F3, and have indicated that a mol ecular basis for the constitutive activation is the stability of the p hosphorylated form of the mutant STAT5.