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FKHR is a member of the FOXO subfamily of Forkhead transcription factors, which are important targets for insulin and growth factor signaling. FKHR contains three predicted protein kinase B phosphorylation sites (Thr-24, Ser-256, and Ser-319) that are conserved in other FOXO proteins. We have reported that phosphorylation of Ser-256 is critical for the ability of insulin and insulin-like growth factors to suppress transactivation by FKHR (Guo, S., Rena, G., Cichy, S., He, X., Cohen, P., and Unterman, T. (1999) J. Biol. Chem. 274, 17184 -17192) and for its exclusion from the nucleus (Rena, G., Prescott, A. R., Guo, S., Cohen, P., and Unterman, T. G. Recent studies have revealed that FOXO Forkhead transcription factors are important targets for mediating effects of insulin and growth factors on gene expression downstream from phosphatidylinositol 3-kinase (PI3K) and protein kinase B (PKB; also known as Akt) (1-13). Early findings in this laboratory revealed that Forkhead transcription factors interact with insulin response sequences (IRSs) in the insulin-like growth factor-binding protein-1 (IGFBP-1) and phosphoenolpyruvate carboxykinase (PEPCK) genes (14, 15) and that signaling by PI3K and PKB mediate the ability of insulin to suppress basal IGFBP-1 promoter activity through an IRS (16). Subsequent studies in Caenorhabditis elegans provided genetic evidence that DAF-16, a member of the FOXO subfamily of Forkhead transcription factors, is a major target for signaling by the insulin/IGF receptor-PI3K-PKB pathway in the nematode (17, 18). DAF-16 and its mammalian homologues, including FKHR (FOXO1), FKHRL1 (FOXO3a), and AFX (FOXO4) interact directly with IRSs from the IGFBP-1 promoter in a sequence-specific fashion in in vitro assays and in cells (1,5,9,19,20). In liver-derived cells, FOXO proteins stimulate the activity of promoters for IGFBP-1 (1), glucose-6 phosphatase (21,22), and PEPCK (23,24). In other cell types, FOXO proteins also stimulate the expression of proteins that inhibit cell cycle progression, including p27 Kip (25), Rb2 (26), and GADD45 (27,28), and proteins that promote cells death, including Bim (29) and Fas ligand (5). Thus, the ability to suppress transactivation by FOXO Forkhead proteins is important for insulin to regulate hepatic production of IGFBP-1 and glucose and for effects of growth factors on cell proliferation and survival.Several critical features distinguish FOXO proteins from other Forkhead family members and make them uniquely suited as mediators of insulin and growth factor action. X-ray crystallographic studies with the DBD of HNF-3␥ indicated that the DNA binding motif of Forkhead proteins, named the Forkhead box, or FOX box, contains three ␣-helices, a wing-like

The beta-form of antithrombin, lacking a carbohydrate side chain on Asn-135, is known to bind heparin more tightly than the fully glycosylated alpha-form. The molecular basis for this difference in affinity was elucidated by rapid-kinetic studies of the binding of heparin and the antithrombin-binding heparin pentasaccharide to plasma and recombinant forms of alpha- and beta-antithrombin. The dissociation equilibrium constant for the first step of the two-step mechanism of binding of both heparin and pentasaccharide to alpha-antithrombin was only slightly higher than that for the binding to the beta-form. The oligosaccharide at Asn-135 thus at most moderately interferes with the initial, weak binding of heparin to alpha-antithrombin. In contrast, the rate constant for the conformational change induced by heparin and pentasaccharide in the second binding step was substantially lower for alpha-antithrombin than for beta-antithrombin. Moreover, the rate constant for the reversal of this conformational change was appreciably higher for the alpha-form than for the beta-form. The carbohydrate side chain at Asn-135 thus reduces the heparin affinity of alpha-antithrombin primarily by interfering with the heparin-induced conformational change. These and previous results suggest a model in which the Asn-135 oligosaccharide of alpha-antithrombin is oriented away from the heparin binding site and does not interfere with the first step of heparin binding. This initial binding induces conformational changes involving extension of helix D into the adjacent region containing Asn-135, which are transmitted to the reactive-bond loop. The resulting decreased conformational flexibility of the Asn-135 oligosaccharide and its close vicinity to the heparin binding site destabilize the activated relative to the native conformation. This effect results in a higher energy for inducing the activated conformation in alpha-antithrombin, leading to a decrease in heparin binding affinity.