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ERK2- and p90(Rsk2)-dependent pathways regulate the CCAAT/enhancer-binding protein-beta interaction with serum
M Hanlon1, T W Sturgill, L Sealy
1Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA.
The Journal of Biological Chemistry
|August 14, 2001
Summary
Ras signaling activates the CCAAT/enhancer-binding protein-beta (C/EBPbeta) and serum response factor (SRF) interaction. Mitogen-activated protein kinase (MAPK) pathways, including ERK2 and p90(Rsk2), are key Ras effectors regulating this interaction.
Area of Science:
- Molecular Biology
- Cell Signaling
- Gene Regulation
Background:
- The serum response element (SRE) in the c-fos promoter integrates mitogenic signals.
- Transcription factors like SRF, ternary complex factors, and C/EBPbeta regulate SRE activity.
- Ras activation is known to stimulate C/EBPbeta-mediated SRE transactivation and interaction with SRF, but downstream effectors were unclear.
Purpose of the Study:
- To identify the specific Ras effectors that regulate C/EBPbeta activity.
- To elucidate the signaling pathways linking Ras to C/EBPbeta and SRF interaction.
- To investigate the role of MAPK pathways in Ras-mediated SRE regulation.
Main Methods:
- Analysis of Ras effector constructs to dissect signaling pathways.
- Site-directed mutagenesis of a consensus MAPK site in C/EBPbeta.
- In vitro kinase assays using recombinant C/EBPbeta and MAPK family members.
- Use of dominant-negative constructs for ERK1 and ERK2 to assess pathway involvement.
Main Results:
- A consensus MAPK phosphorylation site on C/EBPbeta is essential for Ras-stimulated C/EBPbeta-SRF interaction and SRE transactivation.
- Activated Raf and phosphatidylinositol 3-kinase stimulate C/EBPbeta-SRF interaction.
- ERK2, but not ERK1, selectively mediates Ras-induced C/EBPbeta-SRF interaction, and ERK2 directly phosphorylates C/EBPbeta in vitro.
- p90(Rsk2) is required for the regulation of C/EBPbeta-SRF interaction.
Conclusions:
- Multiple Ras effectors, including Raf, PI3K, ERK2, and p90(Rsk2), converge to regulate C/EBPbeta and SRF association.
- Mitogen-activated protein kinase (MAPK) signaling, particularly via ERK2, plays a critical role in mediating Ras effects on C/EBPbeta.
- Phosphorylation of C/EBPbeta at a specific MAPK site is a key mechanism for Ras-dependent SRE regulation.
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