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

H-K Liu,Hs Hundal,Cj Hastie,H Mclauchlan,D Finlay,C Sutherland,S Perrier,C Lipina

doi:10.1186/1471-2199-7-14

H-K Liu, Hs Hundal + Show 6 more

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https://doi.org/10.1186/1471-2199-7-14

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Journal: BMC molecular biology	Publication Date: Apr 6, 2006
Citations: 60	License type: cc-by

Affiliation: University of Dundee, Ninewells Hospital

Abstract

BackgroundGlycogen 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/EBPα) 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 CEBPα is a link between GSK3 and these gene promoters.ResultsC/EBPα represses the IGFBP1 thymine-rich insulin response element (TIRE), but mutation of T222 or T226 of C/EBPα to non-phosphorylatable alanines has no effect on C/EBPα 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/EBPα activity in 3T3 L1 preadipocytes was enhanced by mutation of T222/T226 and/or S230 to alanine residues. Finally, we demonstrate that C/EBPα is a very poor substrate for GSK3 in vitro and in cells.ConclusionThe work demonstrates an important role for this domain in the regulation of C/EBPα 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/EBPα activity is regulated by direct phosphorylation by GSK3.

Highlights

Glycogen Synthase Kinase-3 (GSK3) activity is repressed following insulin treatment of cells
T222/T226 of C/EBPD were identified as targets for GSK3 it has not previously been established whether S230 was a target for a 'priming' kinase, so we have investigated whether mutation of S230 influences C/EBPD regulation
C/EBPD regulates the TIRE independently of T222/226 phosphorylation We have previously shown that insulin, or pharmacological inhibition of GSK3, reduced the expression of endogenous Phosphoenolpyruvate Carboxykinase (PEPCK), Glucose-6 Phosphatase (G6Pase) and IGF binding protein-1 (IGFBP1) genes and the activity of the isolated thymine-rich insulin response element (TIRE) found in these gene promoters [10,11]

Summary

Introduction

Glycogen Synthase Kinase-3 (GSK3) activity is repressed following insulin treatment of cells. Glycogen synthase kinase-3 (GSK3), an insulin-inhibited protein kinase, has been linked to the control of many cellular processes [1]. Two highly related forms of GSK3 (GSK3D and GSK3E) are expressed from distinct genes [3,4]. They share greater than 95% identity in their kinase domains and appear to be ubiquitously expressed in mammals [3,4]. In resting cells GSK3 activity is high and inhibition of the kinase is achieved by at least two mechanisms, firstly through phosphorylation of an N-terminal serine residue (Ser-21 in GSK3D, Ser-9 in GSK3E) [5,6], and secondly through protein protein interaction [7]. Insulin promotes the phosphorylation of Ser-9/21 of GSK3 by activation of protein kinase B (PKB, known as c-AKT) [8], while canonical wnt signalling inhibits GSK3 independently of this N-terminal phosphorylation [9]

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