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K-252a inhibits nerve growth factor-induced trk proto-oncogene tyrosine phosphorylation and kinase activity
M M Berg1, D W Sternberg, L F Parada
1Department of Cell Biology and Anatomy, Cornell University Medical College, New York, New York 10021.
Abstract:
The rat pheochromocytoma PC12 cell line differentiates into a sympathetic neuronal phenotype upon treatment with either nerve growth factor (NGF) or basic fibroblast growth factor. The alkaloid-like compound K-252a has been demonstrated to be a specific inhibitor of NGF-induced biological responses in PC12 cells (Koizumi, S., Contreras, M. L., Matsuda, Y., Hama, T., Lazarovici, P., and Guroff, G. (1988) J. Neurosci. Res. 8, 715-721). NGF interacts with the protein product of the proto-oncogene trk and rapidly stimulates the tyrosine phosphorylation of both p140prototrk and a number of cellular substrates. Here we show that these phosphorylation events are directly inhibited in PC12 cells by K252a in a dose-dependent manner, indicating that the site of action of this inhibitor is at the NGF receptor level. K-252a inhibits p140prototrk activity in vitro, demonstrating that K-252a has a direct effect on the p140prototrk tyrosine kinase. Though many of the biochemical responses to NGF in PC12 cells are mimicked by basic fibroblast growth factor and epidermal growth factor, K-252a has no effect on the action of these growth factors in PC12 cells, demonstrating that the initial biological events initiated by NGF are distinctive during neuronal differentiation.
Insights
The compound K-252a specifically inhibits nerve growth factor (NGF)-induced neuronal differentiation in PC12 cells by targeting the NGF receptor and its associated tyrosine kinase activity.
Area of Science:
- Neuroscience
- Cell Biology
- Biochemistry
Background:
- PC12 cells differentiate into sympathetic neurons with nerve growth factor (NGF) or basic fibroblast growth factor.
- K-252a is a known specific inhibitor of NGF-induced responses in PC12 cells.
- NGF signaling involves the proto-oncogene trk receptor and tyrosine phosphorylation of substrates.
Purpose of the Study:
- To investigate the precise mechanism of K-252a inhibition on NGF signaling.
- To determine if K-252a affects other growth factor signaling pathways in PC12 cells.
Main Methods:
- Treatment of PC12 cells with NGF, K-252a, basic fibroblast growth factor, and epidermal growth factor.
- Analysis of tyrosine phosphorylation events.
- In vitro kinase assays to assess p140prototrk activity.
Main Results:
- K-252a inhibited NGF-induced phosphorylation events in a dose-dependent manner.
- K-252a directly inhibited the tyrosine kinase activity of p140prototrk in vitro.
- K-252a did not affect PC12 cell responses to basic fibroblast growth factor or epidermal growth factor.
Conclusions:
- K-252a acts at the NGF receptor level, directly inhibiting p140prototrk tyrosine kinase.
- NGF-initiated biological events leading to neuronal differentiation are distinct from those of other growth factors.