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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.

The dependence on thiol pK of the second-order rate constant (kS) for reaction of thiolate anions with MMTS was shown to follow the Brønsted equation log kS = log G + beta pK with log G = 1.44 and 3.54 and beta = 0.635 and 0.309 for aryl and alkyl thiols, respectively. The reactivity toward MMTS of the protonated thiol group was found to be negligible in comparison to that of the thiolate anion. For 2-mercaptoethanol the reactivity toward MMTS of the protonated form of the thiol group was shown to be at least 5 X 10(9) smaller than that of the thiolate anion. The pH dependence of the second-order rate constant for reaction of the thiolate group of Cys-25 at the active site of papain was determined and shown to be consistent with the previously determined low pK for Cys-25 and its electrostatic interaction with His-159. The small dependence of the reactivity of Cys-25 on thiol pK (beta approximately 0.09) suggested that the charge-charge interactions that act through space to perturb the pK of the nucleophile at the active site of papain and perhaps other enzymes may serve to increase the fraction of nucleophile present in the reactive basic form without introducing the decrease in nucleophilic reactivity seen in model systems where pK's are lowered primarily by charge-dipole interactions.

To determine the role of individual saccharide residues of a specific heparin pentasaccharide, denoted DEFGH, in the allosteric activation of the serpin, antithrombin, we studied the effect of deleting pentasaccharide residues on this activation. Binding, spectroscopic, and kinetic analyses demonstrated that deletion of reducing-end residues G and H or nonreducing-end residue D produced variable losses in pentasaccharide binding energy of ϳ15-75% but did not affect the oligosaccharide's ability to conformationally activate the serpin or to enhance the rate at which the serpin inhibited factor Xa. Rapid kinetic studies revealed that elimination of the reducing-end disaccharide marginally affected binding to the native low-heparin-affinity conformational state of antithrombin but greatly affected the conversion of the serpin to the activated high-heparinaffinity state, although the activated conformation was still favored. In contrast, removal of the nonreducingend residue D drastically affected the initial low-heparin-affinity interaction so as to favor an alternative activation pathway wherein the oligosaccharide shifted a preexisiting equilibrium between native and activated serpin conformations in favor of the activated state. These results demonstrate that the nonreducing-end residues of the pentasaccharide function both to recognize the native low-heparin-affinity conformation of antithrombin and to induce and stabilize the activated high-heparin-affinity conformation. Residues at the reducing-end, however, poorly recognize the native conformation and instead function primarily to bind and stabilize the activated antithrombin conformation. Together, these findings establish an important role of the heparin pentasaccharide sequence in preferential binding and stabilization of the activated conformational state of the serpin.

The abilities of three synthetic oligosaccharides to accelerate antithrombin inhibition of ten clotting or fibrinolytic proteinases were compared with those of unfractionated, fractionated high-affinity and low-molecular-weight heparins. The results show that the anticoagulant effects of the latter three heparins under conditions approximating physiologic are exerted almost exclusively by acceleration of the inactivation of thrombin, factor Xa and factor IXa to near diffusion-controlled rate constants of approximately 10(6) - 10(7) M(-1).s(-1). All other proteinases are inhibited with at least 20-fold lower rate constants. The anti-coagulant ability of the synthetic regular (fondaparinux) and high-affinity (idraparinux) pentasaccharides is due to a common mechanism, involving acceleration of only factor Xa inhibition to rate constants of approximately 10(6) M(-1).s(-1) . A synthetic hexadecasaccharide, containing both the pentasaccharide sequence and a proteinase binding site, exerts its anticoagulant effect by accelerating antithrombin inactivation of both thrombin and factor Xa to rate constants of approximately 10(6) - 10(7) M(-1).s(-1), although thrombin appears to be the more important target. In contrast, factor IXa inhibition is appreciably less stimulated. The conformational change of antithrombin induced both by the pentasaccharides and longer heparins contributes substantially, approximately 150-500-fold, to accelerating the inactivation of factors Xa, IXa and VIIa and moderately, approximately 50-fold, to that of factor XIIa and tissue plasminogen activator inhibition. The bridging effect due to binding of antithrombin and proteinase to the same, long heparin chain is dominating, approximately 1000-3000-fold, for thrombin inhibition and is appreciably smaller, although up to approximately 250-350-fold, for the inactivation of factors IXa and XIa. These results establish the proteinase targets of heparin derivatives currently used in or considered for thrombosis therapy and give new insights into the mechanism of heparin acceleration of antithrombin inhibition of proteinases.

Serpin family protein proteinase inhibitors regulate the activity of serine and cysteine proteinases by a novel conformational trapping mechanism that may itself be regulated by cofactors to provide a finely-tuned time and location-dependent control of proteinase activity. The serpin, antithrombin, together with its cofactors, heparin and heparan sulfate, perform a critical anticoagulant function by preventing the activation of blood clotting proteinases except when needed at the site of a vascular injury. Here, we review the detailed molecular understanding of this regulatory mechanism that has emerged from numerous X-ray crystal structures of antithrombin and its complexes with heparin and target proteinases together with mutagenesis and functional studies of heparin-antithrombinproteinase interactions in solution. Like other serpins, antithrombin achieves specificity for its target blood clotting proteinases by presenting recognition determinants in an exposed reactive center loop as well as in exosites outside the loop. Antithrombin reactivity is repressed in the absence of its activator because of unfavorable interactions that diminish the favorable RCL and exosite interactions with proteinases. Binding of a specific heparin or heparan sulfate pentasaccharide to antithrombin induces allosteric activating changes that mitigate the unfavorable interactions and promote template bridging of the serpin and proteinase. Antithrombin has thus evolved a sophisticated means of regulating the activity of blood clotting proteinases in a time and locationdependent manner that exploits the multiple conformational states of the serpin and their differential stabilization by glycosaminoglycan cofactors.