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Dominant negative mutants implicate STAT5 in myeloid cell proliferation and neutrophil differentiation
R L Ilaria1, R G Hawley, R A Van Etten
1The Simmons Cancer Center, University of Texas Southwestern Medical School, Dallas, TX, USA.
Abstract:
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.
Insights
Mutations in signal transducers and activation of transcription 5 (STAT5) impact its dimerization, DNA binding, and transcriptional activity. STAT5 mutants interfere with cytokine signaling, affecting myeloid cell proliferation and differentiation.
Area of Science:
- Molecular Biology
- Cell Biology
- Signal Transduction
Background:
- Signal transducers and activation of transcription (STAT) proteins are key mediators of cytokine signaling.
- STAT5, a critical transcription factor, plays a role in various cellular processes.
- Understanding STAT5 function requires detailed analysis of its structural domains and their roles in signaling.
Purpose of the Study:
- To investigate the functional roles of specific regions within murine STAT5A.
- To characterize the effects of alanine substitution mutations on STAT5A dimerization, DNA binding, and transactivation.
- To determine the dominant negative effects of STAT5A mutants on cytokine-induced signaling pathways.
Main Methods:
- Site-directed mutagenesis was used to introduce alanine substitutions in conserved regions of murine STAT5A.
- Mutant STAT5A proteins were analyzed for tyrosine phosphorylation, dimerization with STAT5B, and DNA-binding activity.
- Transactivation assays and dominant-negative effect studies were performed using erythropoietin-induced signaling.
- Stable expression of STAT5A mutants in myeloid cell lines (32D) and primary bone marrow cells assessed effects on proliferation and differentiation.
Main Results:
- Mutations in the WKR region impaired dimerization, while WKR, EE, and VVV mutants lacked DNA-binding activity.
- WKR and VVV mutants showed defective transcriptional induction, with WKR exhibiting a potent dominant-negative effect.
- Stable expression of WKR or C-terminal truncated STAT5A inhibited IL-3-dependent proliferation and G-CSF-dependent differentiation in myeloid cells.
- STAT5 mutants also inhibited G-CSF-dependent granulocyte colony formation in primary bone marrow cells.
Conclusions:
- Distinct regions of STAT5A are crucial for its function in cytokine signaling.
- Mutations in STAT5A can exert dominant-negative effects through different mechanisms, impacting DNA binding and dimerization.
- STAT5 plays a significant role in the proliferation and differentiation of myeloid cells.