Phosphorylation of Raf by ceramide-activated protein kinase
1Laboratory of Signal Transduction, Memorial Sloan-Kettering Cancer Center, New York, New York 10021, USA.
Nature
|November 16, 1995
Summary
Ceramide-activated protein (CAP) kinase phosphorylates and activates Raf1, a key enzyme in cell signaling. This finding reveals CAP kinase as a crucial link in the tumor necrosis factor-alpha (TNF-alpha) pathway.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- The sphingomyelin pathway is crucial for mediating tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 beta signaling.
- Ceramide-activated protein (CAP) kinase is involved in this pathway but its specific role in Raf1 activation is unclear.
Purpose of the Study:
- To investigate the role of CAP kinase in the activation of Raf1 within the TNF-alpha signaling pathway.
- To identify the specific mechanism by which CAP kinase influences Raf1 activity.
Main Methods:
- In vitro kinase assays to determine CAP kinase substrate specificity.
- Analysis of protein-protein interactions between CAP kinase and Raf1.
- Phosphorylation site mapping of Raf1.
- Experiments in intact HL-60 cells stimulated with TNF and ceramide analogues.
Main Results:
- CAP kinase phosphorylates Raf1 at Thr 269, enhancing its activity towards MEK (MAP kinase/ERK kinase).
- CAP kinase forms complexes with Raf1 in HL-60 cells.
- TNF and ceramide analogues stimulate CAP kinase-mediated phosphorylation and activation of Raf1.
Conclusions:
- CAP kinase directly phosphorylates and activates Raf1, identifying it as a specific Raf kinase.
- CAP kinase acts as a critical link between the TNF receptor and Raf1 activation in the sphingomyelin pathway.
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