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Raf-1-associated protein phosphatase 2A as a positive regulator of kinase activation
1Vienna Biocenter, Institute of Microbiology and Genetics, Dr. Bohr Gasse 9, A 1030 Vienna, Austria.
Abstract:
The Raf-1 kinase plays a key role in relaying proliferation signals elicited by mitogens or oncogenes. Raf-1 is regulated by complex and incompletely understood mechanisms including phosphorylation. A number of studies have indicated that phosphorylation of serines 259 and 621 can inhibit the Raf-1 kinase. We show that both serines are hypophosphorylated during early mitogenic stimulation and that hypophosphorylation correlates with peak Raf-1 activation. Concentrations of okadaic acid that selectively inhibit protein phosphatase 2A (PP2A) induce phosphorylation of these residues and prevent maximal activation of the Raf-1 kinase. This effect is mediated via phosphorylation of serine 259. The PP2A core heterodimer forms complexes with Raf-1 in vivo and in vitro. These data identify PP2A as a positive regulator of Raf-1 activation and are the first indication that PP2A may support the activation of an associated kinase.
Insights
Protein phosphatase 2A (PP2A) activates the Raf-1 kinase by dephosphorylating inhibitory sites. This study reveals PP2A as a positive regulator of Raf-1, crucial for cell proliferation signaling.
Area of Science:
- Cellular signaling pathways
- Kinase regulation
- Molecular biology
Background:
- Raf-1 kinase is central to transmitting signals for cell proliferation.
- Raf-1 activity is modulated by phosphorylation, with serines 259 and 621 previously suggested as inhibitory sites.
- The precise mechanisms regulating Raf-1 phosphorylation remain incompletely understood.
Purpose of the Study:
- To investigate the role of protein phosphatase 2A (PP2A) in regulating Raf-1 kinase activity.
- To determine the impact of PP2A inhibition on Raf-1 phosphorylation and activation.
- To elucidate the interaction between PP2A and Raf-1.
Main Methods:
- Analysis of Raf-1 phosphorylation status during mitogenic stimulation.
- Treatment with okadaic acid to selectively inhibit PP2A.
- In vivo and in vitro complex formation assays between PP2A and Raf-1.
Main Results:
- Raf-1 serines 259 and 621 are hypophosphorylated during early mitogenic stimulation, correlating with peak kinase activation.
- Inhibition of PP2A by okadaic acid leads to increased phosphorylation of these inhibitory serines, particularly serine 259, and impairs Raf-1 activation.
- The PP2A core heterodimer directly complexes with Raf-1.
Conclusions:
- Protein phosphatase 2A (PP2A) acts as a positive regulator of Raf-1 kinase activation.
- PP2A dephosphorylates inhibitory serine residues on Raf-1, facilitating its activation.
- This study provides the first evidence for PP2A supporting the activation of an associated kinase, specifically Raf-1.