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CART promoter CRE site binds phosphorylated CREB
A Lakatos1, G Dominguez, M J Kuhar
1Yerkes Regional Primate Research Center of Emory University, Division of Neuroscience, 954 Gatewood Rd. NE, Atlanta, GA 30329, USA.
Brain Research. Molecular Brain Research
|July 16, 2002
Summary
Phosphorylated CREB (P-CREB) likely regulates cocaine- and amphetamine-regulated transcript (CART) gene expression in GH3 cells. Forskolin treatment increased P-CREB levels, indicating P-CREB
Area of Science:
- Neuroscience
- Molecular Biology
- Gene Regulation
Background:
- Cocaine- and amphetamine-regulated transcript (CART) mRNA expression is tightly regulated in the brain.
- Protein kinase A (PKA) signaling influences CART expression in GH3 cells.
- A cyclic AMP-responsive element (CRE) is present in the CART gene promoter.
Purpose of the Study:
- To investigate if CRE binding protein (CREB) binds to the CART CRE site.
- To determine if CREB phosphorylation occurs in GH3 cells during enhanced CART gene expression.
Main Methods:
- Electromobility shift assays (EMSA) to detect DNA-protein interactions.
- Western blotting to assess protein levels, specifically phosphorylated CREB (P-CREB).
- Supershift analysis using a P-CREB antibody.
Main Results:
- EMSA confirmed nuclear factors bind to the CART CRE site.
- Forskolin treatment, known to enhance CART mRNA, increased P-CREB levels in GH3 cells.
- Supershift analysis demonstrated P-CREB interaction within the CART CRE DNA-protein complex.
Conclusions:
- Phosphorylated CREB (P-CREB) is a probable regulator of CART gene expression in GH3 cells.
- These findings elucidate a key molecular mechanism in CART gene regulation.