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Phosphorylation of c-Raf-1 by protein kinase A interferes with activation
K Schramm1, M Niehof, G Radziwill
1Max-Planck-Institut fuer Molekulare Genetik, Abt. Schuster, Berlin (Dahlem), FRG.
Abstract:
c-Raf-1 is a serine/threonine-specific protein kinase which is regulated by phosphorylation. A putative c-AMP dependent protein kinase PKA phosphorylation site with the consensus sequence RRXS, Ser43, and a predominant phosphorylation site of c-Raf-1, Ser259, can be phosphorylated by PKA in vitro as shown by comparison of phosphopeptide maps of recombinant wild-type c-Raf-1 and the corresponding mutants. In vivo stimulation of the PKA pathway by treatment of A431 cells with Forskolin results in increase of phosphorylation in Ser43. Forskolin reduces the upshift of c-Raf-1 induced by EGF-treatment. It inhibits the EGF-activation of the c-Raf-1 protein kinase activity tested in vitro with a peptide substrate.
Insights
Cyclic AMP-dependent protein kinase (PKA) directly phosphorylates c-Raf-1 at Ser43 and Ser259. This PKA activity inhibits epidermal growth factor (EGF)-induced c-Raf-1 activation and signaling.
Area of Science:
- Molecular Biology
- Cell Signaling
- Biochemistry
Background:
- c-Raf-1 is a key regulator in cellular signaling pathways.
- Protein phosphorylation is a critical mechanism for controlling protein activity.
- The role of cyclic AMP-dependent protein kinase (PKA) in c-Raf-1 regulation requires further elucidation.
Purpose of the Study:
- To investigate the direct phosphorylation of c-Raf-1 by PKA.
- To determine the effect of PKA-mediated phosphorylation on c-Raf-1 activity and signaling.
- To explore the interaction between PKA and the epidermal growth factor (EGF) signaling pathway.
Main Methods:
- In vitro kinase assays using recombinant wild-type and mutant c-Raf-1.
- Phosphopeptide mapping to identify phosphorylation sites.
- In vivo studies using A431 cells treated with Forskolin and EGF.
- In vitro kinase activity assays with peptide substrates.
Main Results:
- PKA directly phosphorylates c-Raf-1 at Ser43 and Ser259 in vitro.
- Forskolin treatment increases Ser43 phosphorylation in vivo.
- Forskolin treatment reduces EGF-induced c-Raf-1 upshift and inhibits its kinase activity.
Conclusions:
- PKA directly regulates c-Raf-1 activity through phosphorylation at specific sites.
- PKA acts as an inhibitor of EGF-stimulated c-Raf-1 activation.
- These findings reveal a novel cross-talk mechanism between PKA and Raf signaling pathways.