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Updated: Aug 9, 2026

Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay
Published on: May 3, 2018
Functional characterisation of the regulation of CAAT enhancer binding protein alpha by GSK-3 phosphorylation of
1Division of Pathology and Neurosciences, Ninewells Medical School, University of Dundee, Dundee, DD1 9SY, UK. hk.liu@nricm.edu.tw
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/EBPalpha) 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 CEBPalpha is a link between GSK3 and these gene promoters.
Results:
C/EBPalpha represses the IGFBP1 thymine-rich insulin response element (TIRE), but mutation of T222 or T226 of C/EBPalpha to non-phosphorylatable alanines has no effect on C/EBPalpha 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/EBPalpha activity in 3T3 L1 preadipocytes was enhanced by mutation of T222/T226 and/or S230 to alanine residues. Finally, we demonstrate that C/EBPalpha 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/EBPalpha 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/EBPalpha activity is regulated by direct phosphorylation by GSK3.
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