Dominant negative mutants implicate STAT5 in myeloid cell proliferation and neutrophil differentiation

STAT5 is a member of the signal transducers and activation of transcription (STAT) family of latent transcription factors activated in a variety of cy tokine signaling pathways. We introduced alanine substitution mutations in highly conserved regions of murine STAT5A and studied the mutants for dimer ization, DNA binding, transactivation, and dominant negative effects on ery thropoietin-induced STAT5-dependent transcriptional activation. The mutatio ns included two near the amino-terminus (W255KR-->AAA and R(290)QQ-->AAA), two in the DNA-binding domain (E437E-->AA and V466VV-->AAA), and a carboxy- terminal truncation of STAT5A (STAT5A/Delta 53C) analogous to a naturally o ccurring isoform of rat STAT5B. All of the STAT mutant proteins were tyrosi ne phosphorylated by JAK2 and heterodimerized with STAT5B except for the WK R mutant, suggesting an important role for this region in STAT5 for stabili zing dimerization. The WKR, EE, and VVV mutants had no detectable DNA-bindi ng activity, and the WKR and VVV mutants, but not EE, were defective in tra nscriptional induction. The VVV mutant had a moderate dominant negative eff ect on erythropoietin-induced STAT5 transcriptional activation, which was l ikely due to the formation of heterodimers that are defective in DNA bindin g. Interestingly, the WKR mutant had a potent dominant negative effect, com parable to the transactivation domain deletion mutant, Delta 53C. Stable ex pression of either the WKR or Delta 53C STAT5 mutants in the murine myeloid cytokine-dependent cell line 32D inhibited both interleukin-3-dependent pr oliferation and granulocyte colony-stimulating factor (G-CSF)-dependent dif ferentiation, without induction of apoptosis. Expression of these mutants i n primary murine bone marrow inhibited G-CSF-dependent granulocyte colony f ormation in vitro. These results demonstrate that mutations in distinct reg ions of STAT5 exert dominant negative effects on cytokine signaling, likely through different mechanisms, and suggest a role for STAT5 in proliferatio n and differentiation of myeloid cells. (C) 1999 by The American Society of Hematology.