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Activation of Raf-1 by interferon gamma and oncostatin M requires expression of the Stat1 transcription factor
L F Stancato1, C R Yu, E F Petricoin
1Laboratory of Cellular and Molecular Biology, NCI, National Institutes of Health, Bethesda, Maryland 20892, USA.
Abstract:
A primary signaling cascade responsible for the expression of cytokine-stimulated immediate early genes involves the activation of the Jak/Stat pathway. In addition to being tyrosine-phosphorylated, several signal transducers and activators of transcription (Stats), including Stat1alpha, Stat3, and Stat4, are phosphorylated on a conserved serine residue, which is a consensus phosphorylation site for mitogen-activated protein kinases (MAPKs). Serine phosphorylation of Stat1alpha is required for maximal transcriptional activation of early response genes by interferon gamma (IFNgamma) as well as the antiviral and antigrowth actions of this cytokine. Incubation of cells with either IFNgamma or oncostatin M (OSM) activates Raf-1, a serine/threonine kinase responsible for the ultimate activation of p42 MAPK. To examine whether any of the signaling components that are required for activation of the Jak/Stat pathway are also necessary for activation of Raf-1 by IFNs and OSM, we examined activation of Raf-1 in cell lines that are deficient in either Stat1alpha or Stat2. Unexpectedly, incubation of Stat1-deficient, but not Stat2-deficient cells with IFNgamma or OSM for 5 min displayed no increase in Raf-1 activity. In peripheral blood lymphocytes Raf-1 was associated with Stat1, and this interaction was disrupted after incubation of cells with IFNgamma. Stat1-negative cells reconstituted with either Stat1alpha or Stat1alpha with a point mutation in the site where it is serine-phosphorylated displayed normal activation of Raf-1 by IFNgamma and OSM. However, activation of Raf-1 was not observed in lines that expressed Stat1alpha containing a mutation in its tyrosine phosphorylation site or in its SH2 domain. These results provide the first example of a novel role of Stat1alpha not as a transcription factor, but as a protein which may function to scaffold signaling components required for activation of the distinct Raf/MEK/MAPK signaling cascade.
Insights
Signal transducer and activator of transcription 1-alpha (Stat1alpha) acts as a scaffold protein, not a transcription factor, facilitating the activation of the Raf/MEK/MAPK signaling cascade in response to cytokines like interferon gamma.
Area of Science:
- Cellular signaling pathways
- Molecular biology
- Immunology
Background:
- The Jak/Stat pathway is crucial for cytokine-induced gene expression.
- Signal transducers and activators of transcription (Stats) are phosphorylated on serine residues by mitogen-activated protein kinases (MAPKs).
- Stat1alpha serine phosphorylation is essential for interferon gamma's (IFNγ) antiviral and antigrowth effects.
Purpose of the Study:
- To investigate the role of Stat1alpha in the activation of the Raf/MEK/MAPK signaling cascade.
- To determine if components of the Jak/Stat pathway are necessary for Raf-1 activation by IFNs and oncostatin M (OSM).
Main Methods:
- Examined Raf-1 activation in Stat1-deficient and Stat2-deficient cell lines.
- Analyzed Raf-1 association with Stat1 in peripheral blood lymphocytes.
- Utilized Stat1-negative cells reconstituted with wild-type or mutated Stat1alpha.
Main Results:
- Stat1-deficient cells showed no increase in Raf-1 activity upon IFNγ or OSM stimulation.
- Raf-1 was associated with Stat1, and this interaction was disrupted by IFNγ.
- Stat1alpha mutations in tyrosine phosphorylation or SH2 domains abolished Raf-1 activation, while serine phosphorylation site mutations did not.
Conclusions:
- Stat1alpha plays a novel scaffolding role in the activation of the Raf/MEK/MAPK signaling cascade.
- Stat1alpha's function extends beyond transcription regulation, influencing distinct signaling pathways.
- This finding reveals a new mechanism for cytokine-mediated cellular responses.