Functional characterisation of the regulation of CAAT enhancer binding protein alpha by GSK-3 phosphorylation of Threonines

Background: Glycogen Synthase Kinase-3 (GSK3) activity is repressed following insulin treatment of cells. Pharmacological inhibition of GSK3 mimics the effect of insulin on Phosphoenolpyruvate Carboxykinase (PEPCK), Glucose-6 Phosphatase (G6Pase) and IGF binding protein-1 (IGFBP1) gene expression. CAAT/enhancer binding protein alpha (C/EBP ) regulates these gene promoters in liver and is phosphorylated on two residues (T222/T226) by GSK3, although the functional outcome of the phosphorylation has not been established. We aimed to establish whether CEBP is a link between GSK3 and these gene promoters. Results: C/EBP represses the IGFBP1 thymine-rich insulin response element (TIRE), but mutation of T222 or T226 of C/EBP to non-phosphorylatable alanines has no effect on C/EBP activity in liver cells (towards the TIRE or a consensus C/EBP binding sequence). Phosphorylation of T222/T226 is decreased by GSK3 inhibition, suggesting GSK3 does phosphorylate T222/226 in intact cells. However, phosphorylation was not altered by treatment of liver cells with insulin. Meanwhile C/EBP activity in 3T3 L1 preadipocytes was enhanced by mutation of T222/T226 and/ or S230 to alanine residues. Finally, we demonstrate that C/EBP is a very poor substrate for GSK3 in vitro and in cells. Conclusion: The work demonstrates an important role for this domain in the regulation of C/ EBP activity in adipocytes but not hepatocytes, however GSK3 phosphorylation of these residues does not mediate regulation of this C/EBP activity. In short, we find no evidence that C/EBP activity is regulated by direct phosphorylation by GSK3. Published: 06 April 2006 BMC Molecular Biology2006, 7:14 doi:10.1186/1471-2199-7-14 Received: 09 November 2005 Accepted: 06 April 2006 This article is available from: http://www.biomedcentral.com/1471-2199/7/14 © 2006Liu et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. BMC Molecular Biology 2006, 7:14 http://www.biomedcentral.com/1471-2199/7/14