Functional characterisation of the regulation of CAAT enhancer binding protein alpha by GSK-3 phosphorylation of Threonines 222/226.
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.


CONCLUSIONS
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.
Liu
H-K
HK
Division of Pathology and Neurosciences, Ninewells Medical School, University of Dundee, Dundee, DD1 9SY, UK. hk.liu@nricm.edu.tw
Perrier
S
S
Lipina
C
C
Finlay
D
D
McLauchlan
H
H
Hastie
C J
CJ
Hundal
H S
HS
Sutherland
C
C
D041721:3T3-L1 Cells; D000256:Adenoviridae; D017667:Adipocytes / Q000378:metabolism; D000818:Animals; D022763:CCAAT-Enhancer-Binding Protein-alpha / Q000096:biosynthesis* / Q000235:genetics*; D002460:Cell Line; D045744:Cell Line, Tumor; D005786:Gene Expression Regulation; D015723:Gene Library; D038362:Glycogen Synthase Kinase 3 / Q000378:metabolism*; D022781:Hepatocytes / Q000378:metabolism; D006801:Humans; D007328:Insulin / Q000378:metabolism; D050356:Lipid Metabolism; D051379:Mice; D008954:Models, Biological; D009154:Mutation; D010766:Phosphorylation; D017434:Protein Structure, Tertiary; D012313:RNA / Q000378:metabolism; D051381:Rats; D020218:Response Elements; D012260:Ribonucleases / Q000378:metabolism; D013379:Substrate Specificity; D013912:Threonine / Q000737:chemistry*; D013941:Thymine / Q000378:metabolism; D014162:Transfection
D022763:CCAAT-Enhancer-Binding Protein-alpha; D007328:Insulin; D013912:Threonine; D012313:RNA; D038362:Glycogen Synthase Kinase 3; D012260:Ribonucleases; D013941:Thymine