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

C/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.The 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.Glycogen synthase kinase-3 (GSK3), an insulin-inhibited protein kinase, has been linked to the control of many cellular processes [1]. It was originally identified as the protein kinase that phosphorylated and inactivated glycogen synthase in rabbit muscle [2]. Two highly related forms of GSK3 (GSK3α and GSK3β) 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 GSK3α, Ser-9 in GSK3β) [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, also known as c-AKT) [8], while canonical wnt signalling inhibits GSK3 independently of this N-terminal phosphorylation [9].In liver, inhibition of GSK3 reduces expres