Insulin suppresses transactivation by CAAT/Enhancer-binding proteins β (C/EBPβ). Signaling to p300/CREB-binding protein by protein kinase B disrupts interaction with the major activation domain of C/EBPβ

CAAT/enhancer-binding proteins (C/EBPs) play an important role in the regulation of gene expression in insulin-responsive tissues. We have found that a complex containing C/EBPβ interacts with an insulin response sequence in the insulin-like growth factor-binding protein-1 (IGFBP-1) gene and that a C/EBP-binding site can mediate effects of insulin on promoter activity. Here, we examined mechanisms mediating this effect of insulin. The ability of insulin to suppress promoter activity via a C/EBP-binding site is blocked by LY294002, a phosphatidylinositol 3-kinase inhibitor, but not by rapamycin, which blocks activation of p70^{S6 kinase}. Dominant negative phosphatidylinositol 3-kinase and protein kinase B (PKB) block the effect of insulin, while activated PKB suppresses promoter function via a C/EBP-binding site, mimicking the effect of insulin. Coexpression studies indicate that insulin and PKB suppress transactivation by C/EBPβ, but not C/EBPα, and that N-terminal transactivation domains in C/EBPβ are required. Studies with Gal4 fusion proteins reveal that insulin and PKB suppress transactivation by the major activation domain in C/EBPβ (AD II), located between amino acids 31 and 83. Studies with E1A protein indicate that interaction with p300/CBP is required for transactivation by AD II and the effect of insulin and PKB. Based on a consensus sequence, we identified a PKB phosphorylation site (Ser ¹⁸³⁴) within the region of p300/CBP known to bind C/EBPβ. Mammalian two-hybrid studies indicate that insulin and PKB disrupt interactions between this region of p300 and AD II and that Ser¹⁸³⁴ is critical for this effect. Signaling by PKB and phosphorylation of Ser¹⁸³⁴ may play an important role in modulating interactions between p300/CBP and transcription factors and mediate effects of insulin and related growth factors on gene expression.