Dominant negative mutants implicate STAT5 in myeloid cell proliferation and neutrophil differentiation.
- Author(s): Ilaria, R L, Jr
- Hawley, R G
- Van Etten, R A
- et al.
STAT5 is a member of the signal transducers and activation of transcription (STAT) family of latent transcription factors activated in a variety of cytokine signaling pathways. We introduced alanine substitution mutations in highly conserved regions of murine STAT5A and studied the mutants for dimerization, DNA binding, transactivation, and dominant negative effects on erythropoietin-induced STAT5-dependent transcriptional activation. The mutations included two near the amino-terminus (W255KR-->AAA and R290QQ-->AAA), two in the DNA-binding domain (E437E-->AA and V466VV-->AAA), and a carboxy-terminal truncation of STAT5A (STAT5A/triangle up53C) analogous to a naturally occurring isoform of rat STAT5B. All of the STAT mutant proteins were tyrosine phosphorylated by JAK2 and heterodimerized with STAT5B except for the WKR mutant, suggesting an important role for this region in STAT5 for stabilizing dimerization. The WKR, EE, and VVV mutants had no detectable DNA-binding activity, and the WKR and VVV mutants, but not EE, were defective in transcriptional induction. The VVV mutant had a moderate dominant negative effect on erythropoietin-induced STAT5 transcriptional activation, which was likely due to the formation of heterodimers that are defective in DNA binding. Interestingly, the WKR mutant had a potent dominant negative effect, comparable to the transactivation domain deletion mutant, triangle up53C. Stable expression of either the WKR or triangle up53C STAT5 mutants in the murine myeloid cytokine-dependent cell line 32D inhibited both interleukin-3-dependent proliferation and granulocyte colony-stimulating factor (G-CSF)-dependent differentiation, without induction of apoptosis. Expression of these mutants in primary murine bone marrow inhibited G-CSF-dependent granulocyte colony formation in vitro. These results demonstrate that mutations in distinct regions of STAT5 exert dominant negative effects on cytokine signaling, likely through different mechanisms, and suggest a role for STAT5 in proliferation and differentiation of myeloid cells.