It has been shown previously that: CART (cocaine- and amphetamine-regulated transcript) mRNA is tightly regulated in brain; protein kinase A (PKA) is involved in CART expression in GH3 cells; and a cyclic AMP-responsive element (CRE) site is present in the proximal promoter region of the CART gene. Thus, the goal of this study was to test if CRE binding protein (CREB) can bind to the consensus CRE site and if phosphorylation of CREB occurs in GH3 cells under conditions of enhanced CART gene expression. Electromobility shift assays showed that a 27-bp oligonucleotide containing the CART CRE site was indeed bound by nuclear factors. Western blotting showed that incubation of GH3 cells with forskolin, which enhances CART mRNA expression, caused an increase in phosphorylated CREB (P-CREB) levels. Supershift analyses indicated that the CART CRE oligo/protein complex interacted with a P-CREB antibody. Taken together, these data indicate that P-CREB is a likely regulator of CART expression in GH3 cells.
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