Abstract
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/EBPbeta 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/EBPbeta, but not C/EBPalpha, and that N-terminal transactivation domains in C/EBPbeta are required. Studies with Gal4 fusion proteins reveal that insulin and PKB suppress transactivation by the major activation domain in C/EBPbeta (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/EBPbeta. Mammalian two-hybrid studies indicate that insulin and PKB disrupt interactions between this region of p300 and AD II and that Ser(1834) is critical for this effect. Signaling by PKB and phosphorylation of Ser(1834) 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.
Highlights
From the ‡University of Illinois at Chicago College of Medicine and Veterans Affairs Chicago Health Care System, Chicago, Illinois 60612, the ¶Research Genetics, Huntsville, Alabama 35801, and the **Eukaryotic Transcriptional Regulation Section, Regulation of Cell Growth Laboratory, NCI, Frederick Cancer Research and Development Center, Frederick, Maryland 20892-0822
Since a nucleoprotein complex containing CAAT/enhancer-binding proteins (C/EBPs) interacts with insulin response sequences (IRSs) in the insulin-like growth factor-binding protein-1 (IGFBP-1) and phosphoenolpyruvate carboxykinase (PEPCK) genes, we considered the possibility that C/EBP proteins might contribute to the ability of insulin to regulate promoter activity downstream from PI 3-kinase and protein kinase B (PKB)
Previous studies have shown that the ability of insulin to inhibit promoter activity via an IRS is mediated through the PI 3-kinase/PKB signaling pathway [12], and we asked whether signaling to C/EBP proteins might occur through this pathway
Summary
C/EBP, CAAT/enhancer-binding protein; CRE, cAMP response element; GST, glutathione S-transferase; IGFBP-1, insulin-like growth factor-binding protein-1; IRS, insulin response sequence; PEPCK, phosphoenolpyruvate carboxykinase; PKB, protein kinase B; PI 3-kinase, phosphatidylinositol 3Ј-kinase; CBP, CREB-binding protein; Myr, myristoylated; LZ, leucine zipper. Since p300/CBP proteins are important for transactivation by C/EBP, we considered the possibility that insulin signaling via PKB may modify interactions between this region of p300/ CBP and C/EBP
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