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Carboxy-terminal truncated STAT5 is induced by interleukin-2 and GM-CSF in human neutrophils
P K Epling-Burnette1, Roy Garcia, Fanqi Bai
1Malignant Hematology Program, H. Lee Moffitt Cancer Center and Research Institute, 12902 Magnolia Drive, Tampa, FL 33612, USA. Burnetpk@moffitt.usf.edu
Abstract:
IL-2 and GM-CSF are potent activators of polymorphonuclear neutrophils (PMN) biologic activity. IL-2 and GM-CSF-mediated activation of STAT proteins was examined in nuclear extracts of human PMN. We found that both cytokines induced STAT5-like DNA-binding complexes that could not be supershifted using C-terminal-specific anti-STAT5 antibodies. Therefore, we performed oligoprecipitation experiments with a STAT5-biotinylated DNA probe (biotin-MGFe) and the precipitated proteins were identified by Western immunoblotting. We found that GM-CSF and IL-2 induced the DNA-binding activity of a C-terminal truncated isoform of STAT5. The truncated STAT5 form was present in the nucleus of PMN but the cytoplasmic extracts contained full-length STAT5, suggesting that PMN proteolytically process full-length STAT5 proteins. Proteolytic experiments demonstrated that PMN express a protease activity capable of producing C-terminal processed STAT5 proteins. In many settings, C-terminal truncation of the STAT5 protein leads to inhibition of STAT5 biological activity. Two known STAT5 regulated genes, encoding pim-1 and OSM proteins, failed to be induced by GM-CSF in PMN. These findings provide new insights to a mechanism by which PMN, a terminally differentiated cell, may regulate gene transcription by alternative proteolytic processing.
Insights
Interleukin-2 (IL-2) and granulocyte-macrophage colony-stimulating factor (GM-CSF) activate human polymorphonuclear neutrophils (PMN). These cytokines induce a truncated STAT5 protein in PMN nuclei, suggesting a novel gene regulation mechanism.
Area of Science:
- Immunology
- Molecular Biology
- Cell Biology
Background:
- Interleukin-2 (IL-2) and granulocyte-macrophage colony-stimulating factor (GM-CSF) are critical cytokines that activate polymorphonuclear neutrophils (PMN).
- Signal transducer and activator of transcription (STAT) proteins are key mediators of cytokine signaling.
- STAT5 activation is crucial for various cellular functions, but its regulation in PMN is not fully understood.
Purpose of the Study:
- To investigate the activation and function of STAT proteins in human PMN stimulated with IL-2 and GM-CSF.
- To identify the specific STAT proteins involved and elucidate their post-translational modifications.
- To explore the functional consequences of STAT protein processing on gene expression in PMN.
Main Methods:
- Nuclear extracts from human PMN treated with IL-2 or GM-CSF were analyzed for STAT protein DNA-binding activity.
- Oligoprecipitation assays using a STAT5-biotinylated DNA probe followed by Western immunoblotting were performed.
- Proteolytic processing experiments were conducted to identify protease activity in PMN.
- Expression of known STAT5-regulated genes (pim-1 and OSM) was assessed.
Main Results:
- Both IL-2 and GM-CSF induced STAT5-like DNA-binding complexes in human PMN nuclear extracts.
- These complexes contained a C-terminal truncated isoform of STAT5, not recognized by C-terminal specific antibodies.
- Full-length STAT5 was found in cytoplasmic extracts, while truncated STAT5 was in nuclear extracts, indicating proteolytic processing.
- PMN possess protease activity capable of generating C-terminal processed STAT5.
- GM-CSF failed to induce STAT5-regulated genes pim-1 and OSM in PMN, suggesting inhibited biological activity of truncated STAT5.
Conclusions:
- Human PMN process full-length STAT5 into a C-terminal truncated form in the nucleus upon stimulation with IL-2 and GM-CSF.
- This proteolytic processing may represent a mechanism for regulating STAT5-dependent gene transcription in terminally differentiated PMN.
- The truncated STAT5 isoform appears to have inhibited biological activity, as evidenced by the lack of induction of specific target genes.