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Beta interferon and oncostatin M activate Raf-1 and mitogen-activated protein kinase through a JAK1-dependent pathway
L F Stancato1, M Sakatsume, M David
1Laboratory of Cellular and Molecular Biology, National Cancer Institute, Bethesda, Maryland 20892, USA.
Abstract:
Activation of early response genes by interferons (IFNs) and other cytokines requires tyrosine phosphorylation of a family of transcription factors termed signal transducers and activators of transcription (Stats). The Janus family of tyrosine kinases (Jak1, Jak2, Jak3, and Tyk2) is required for cytokine-induced tyrosine phosphorylation and dimerization of the Stat proteins. In order for IFNs to stimulate maximal expression of Stat1alpha-regulated genes, phosphorylation of a serine residue in the carboxy terminus by mitogen-activated protein kinase (MAPK) is also required. In HeLa cells, both IFN-beta and oncostatin M (OSM) stimulated MAPK and Raf-1 enzyme activity, in addition to Stat1 and Stat3 tyrosine phosphorylation. OSM stimulation of Raf-1 correlated with GTP loading of Ras, whereas IFN-beta activation of Raf-1 was Ras independent. IFN-beta- and OSM-induced Raf-1 activity could be coimmunoprecipitated with either Jak1 or Tyk2. Furthermore, HeLa cells lacking Jak1 displayed no activation of STAT1alpha, STAT3, and Raf-1 by IFN-beta or OSM and also demonstrated no increase in the relative level of GTP-bound p21ras in response to OSM. The requirement for Jak1 for IFN-beta- and OSM-induced activation of Raf-1 was also seen in Jak1-deficient U4A fibrosarcoma cells. Interestingly, basal MAPK, but not Raf-1, activity was constitutively enhanced in Jak1-deficient HeLa cells. Transient expression of Jak1 in both Jak-deficient HeLa cells and U4A cells reconstituted the ability of IFN-beta and OSM to activate Raf-1 and decreased the basal activity of MAPK, while expression of a kinase-inactive form of the protein showed no effect. Moreover, U4A cells selected for stable expression of Jak1, or COS cells transiently expressing Jak1 or Tyk2 but not Jak3, exhibited enhanced Raf-1 activity. Therefore, it appears that Jak1 is required for Raf-1 activation by both IFN-beta and OSM. These results provide evidence for a link between the Jaks and the Raf/MAPK signaling pathways.
Insights
Interferons (IFNs) and cytokines activate genes by phosphorylating signal transducers and activators of transcription (Stats) via Janus kinases (Jaks). Jak1 is crucial for activating Raf-1 kinase, linking Jaks to MAPK signaling pathways.
Area of Science:
- Cellular signaling pathways
- Molecular biology
- Immunology
Background:
- Cytokine signaling, including interferons (IFNs), relies on signal transducers and activators of transcription (Stats) phosphorylation.
- Janus kinases (Jaks) are essential for cytokine-induced Stat tyrosine phosphorylation and dimerization.
- Maximal expression of Stat1alpha-regulated genes by IFNs requires mitogen-activated protein kinase (MAPK) phosphorylation of Stats.
Purpose of the Study:
- To investigate the role of Janus kinases (Jaks) in the activation of Raf-1 and MAPK signaling pathways by interferons (IFNs) and oncostatin M (OSM).
- To elucidate the relationship between Jak kinases and the Ras/Raf/MAPK cascade in response to cytokine stimulation.
Main Methods:
- Enzyme activity assays for MAPK and Raf-1.
- Tyrosine phosphorylation analysis of Stat1 and Stat3.
- GTP loading assays for Ras.
- Co-immunoprecipitation studies to assess protein interactions.
- Experiments using Jak1-deficient cell lines (HeLa and U4A) and reconstitution studies.
Main Results:
- Both IFN-beta and OSM stimulated MAPK and Raf-1 activity, alongside Stat1 and Stat3 tyrosine phosphorylation in HeLa cells.
- Jak1 deficiency abrogated IFN-beta and OSM-induced activation of STAT1alpha, STAT3, and Raf-1, and impaired OSM-induced Ras activation.
- Jak1 is demonstrated to be essential for IFN-beta and OSM-induced Raf-1 activation, establishing a link between Jaks and the Raf/MAPK pathway.
Conclusions:
- Jak1 plays a critical role in mediating the activation of the Raf-1 kinase by both IFN-beta and OSM.
- These findings reveal a significant connection between the Jak signaling pathway and the Ras/Raf/MAPK signaling cascade.
- Jak1 is indispensable for cytokine-induced activation of Raf-1, highlighting its importance in cellular responses to IFNs and OSM.