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Modulation of Kit/stem cell factor receptor-induced signaling by protein kinase C
P Blume-Jensen1, L Rönnstrand, I Gout
1Ludwig Institute for Cancer Research, Biomedical Center, Uppsala, Sweden.
Abstract:
The Kit/stem cell factor receptor (Kit/SCF-R) is a transmembrane tyrosine kinase receptor of importance for the normal development of hemopoietic cells, melanoblasts, and germ cells. We recently reported that protein kinase C (PKC) is involved in a negative feedback loop regulating the Kit/SCF-R by direct phosphorylation on serine residues in the receptor. Inhibition of PKC led to increased SCF-induced tyrosine kinase activity and mitogenicity, but PKC was necessary for SCF-induced motility. In this report we have further examined the modulatory role of PKC on SCF-induced signaling. The ligand-activated Kit/SCF-R associated weakly with GRB2 and induced only little tyrosine phosphorylation of phospholipase C-gamma in porcine aortic endothelial cells transfected with Kit/SCF-R. In contrast, the SCF-stimulated Kit/SCF-R associated efficiently with, and induced tyrosine phosphorylation of, the p85 alpha regulatory subunit of phosphatidyl inositide-3'-kinase (PI-3'-kinase). Both receptor association and tyrosine phosphorylation of p85 alpha were increased after inhibition of PKC, while its serine phosphorylation was decreased. Concomitantly, the specific activity of receptor-associated PI-3'-kinase activity was increased. Inhibition of PI-3'-kinase with wortmannin inhibited SCF-induced mitogenicity. SCF-induced phosphorylation of Raf-1 and activation of ERK2 still occurred after PKC inhibition but was not increased. In conclusion, SCF-induced PI-3'-kinase activation paralleled the increased SCF-induced mitogenicity after inhibition of PKC.
Insights
Protein kinase C (PKC) negatively regulates the Kit receptor. Inhibiting PKC enhances stem cell factor-induced (SCF) signaling, particularly phosphatidylinositol-3
Area of Science:
- Cell Signaling
- Molecular Biology
- Receptor Tyrosine Kinases
Background:
- The Kit/stem cell factor receptor (Kit/SCF-R) is a transmembrane tyrosine kinase crucial for hemopoietic cells, melanoblasts, and germ cells.
- Protein kinase C (PKC) acts in a negative feedback loop, phosphorylating Kit/SCF-R on serine residues.
- Previous studies showed PKC inhibition increases SCF-induced tyrosine kinase activity and mitogenicity but is essential for SCF-induced motility.
Purpose of the Study:
- To further investigate the modulatory role of PKC on SCF-induced signaling pathways.
- To elucidate the specific downstream effectors regulated by PKC in response to SCF stimulation.
Main Methods:
- Transfection of porcine aortic endothelial cells with Kit/SCF-R.
- Analysis of Kit/SCF-R association with GRB2 and phospholipase C-gamma.
- Assessment of Kit/SCF-R association with phosphatidylinositol-3'-kinase (PI-3'-kinase) p85 alpha subunit.
- Measurement of PI-3'-kinase activity and downstream signaling components (Raf-1, ERK2).
- Inhibition of PI-3'-kinase using wortmannin.
Main Results:
- Ligand-activated Kit/SCF-R showed weak association with GRB2 and minimal phospholipase C-gamma phosphorylation.
- Kit/SCF-R efficiently associated with and phosphorylated the PI-3'-kinase p85 alpha subunit upon SCF stimulation.
- PKC inhibition increased Kit/SCF-R association with and tyrosine phosphorylation of p85 alpha, decreasing its serine phosphorylation.
- Receptor-associated PI-3'-kinase activity increased following PKC inhibition.
- Inhibition of PI-3'-kinase with wortmannin abrogated SCF-induced mitogenicity.
- SCF-induced Raf-1 phosphorylation and ERK2 activation occurred after PKC inhibition but were not enhanced.
Conclusions:
- SCF-induced PI-3'-kinase activation is a key pathway mediating mitogenicity.
- PKC negatively regulates SCF-induced PI-3'-kinase activation.
- Enhanced PI-3'-kinase activation following PKC inhibition directly correlates with increased SCF-induced mitogenicity.