In Caenorhabditis elegans, an insulin-like signaling pathway to phosphatidylinositol 3-kinase (PI 3-kinase) and AKT negatively regulates the activity of DAF-16, a Forkhead transcription factor. We show that in mammalian cells, C. elegans DAF-16 is a direct target of AKT and that AKT phosphorylation generates 14-3-3 binding sites and regulates the nuclear/cytoplasmic distribution of DAF-16 as previously shown for its mammalian homologs FKHR and FKHRL1. In vitro, interaction

Insulin negatively regulates expression of the insulin-like growth factor binding protein 1 (IGFBP-1) gene by means of an insulinresponsive element (IRE) that also contributes to glucocorticoid stimulation of this gene. We find that the Caenorhabditis elegans protein DAF-16 binds the IGFBP-1⅐IRE with specificity similar to that of the forkhead (FKH) factor(s) that act both to enhance glucocorticoid responsiveness and to mediate the negative effect of insulin at this site. In HepG2 cells, DAF-16 and its mammalian homologs, FKHR, FKHRL1, and AFX, activate transcription through the IGFBP-1⅐IRE; this effect is inhibited by the viral oncoprotein E1A, but not by mutants of E1A that fail to interact with the coactivator p300͞CREB-binding protein (CBP). We show that DAF-16 and FKHR can interact with both the KIX and E1A͞SRC interaction domains of p300͞CBP, as well as the steroid receptor coactivator (SRC). A C-terminal deletion mutant of DAF-16 that is nonfunctional in C. elegans fails to bind the KIX domain of CBP, fails to activate transcription through the IGFBP-1⅐IRE, and inhibits activation of the IGFBP-1 promoter by glucocorticoids. Thus, the interaction of DAF-16 homologs with the KIX domain of CBP is essential to basal and glucocorticoid-stimulated transactivation. Although AFX interacts with the KIX domain of CBP, it does not interact with SRC and does not respond to glucocorticoids or insulin. Thus, we conclude that DAF-16 and FKHR act as accessory factors to the glucocorticoid response, by recruiting the p300͞CBP͞SRC coactivator complex to an FKH factor site in the IGFBP-1 promoter, which allows the cell to integrate the effects of glucocorticoids and insulin on genes that carry this site.I nsulin signaling via the phosphatidylinositol 3-kinase (PI 3-kinase)͞protein kinase B (PKB) pathway has diverse effects on cellular metabolism and apoptosis (1, 2). A major role of insulin is to act in opposition to the catabolic effects of cAMP and glucocorticoids, agents that stimulate liver gluconeogenesis. The rate-limiting step in gluconeogenesis is catalyzed by the phosphoenolpyruvate carboxykinase (GTP) (PEPCK; EC 4.1.1.32) gene. The insulin-like growth factor binding protein 1 (IGFBP-1) gene indirectly promotes gluconeogenesis by binding insulin-like growth factor (IGF)-I and -II and inhibiting their insulin-like effects. Expression of the PEPCK and IGFBP-1 genes is controlled at the transcriptional level by a complex regulatory mechanism in which glucocorticoids activate and insulin inhibits gene expression (3-6).In the case of the PEPCK gene, the response to both glucocorticoids and insulin is mediated by the accessory factor II (AFII) site, located upstream of the glucocorticoid-response element (GRE); this site is also referred to as the PEPCK insulin-response sequence, IRS-1 (7). Similarly the response of the IGFBP-1 promoter to glucocorticoids and insulin is mediated by one site, the insulin-response element (IRE) site located upstream of its GRE (5, 8). Biochemical evidence first showed that the forkhead (FK...

THE upstream region of the human glyceraldehyde-3-phosphate dehydrogenase gene contains an insulin-response element (IRE-A) responsible for insulin-dependent transcription of the gene. The open reading frame of a rat complementary DNA encoding a protein (IRE-ABP) that binds to this sequence contains an HMG box motif that is 67% identical to the mouse candidate gene for the testis-determining factor SRY, and 98% identical to the mouse SRY-like gene, a4. Here we report that IRE-ABP and SRY bind to IRE-A DNA with comparable specificity in a DNase-I footprinting assay. Two females with sex reversal were found to have a single amino-acid substitution in the HMG box domain of SRY at position 3 and 7, respectively. SRY derivatives containing corresponding mutations do not make contact with IRE-A DNA. These results are direct evidence that mouse SRY-like proteins are sequence-specific DNA-binding proteins and identify two amino acids critical to this interaction. Moreover, IRE-A is a candidate SRY-response element.