Phosphorylation and nuclear exclusion of the forkhead transcription factor FKHR after epidermal growth factor treatment in human breast cancer cells.

Akt, when activated by IGF/insulin, can phosphorylate forkhead transcription factors. We undertook this study to determine whether epidermal growth factor (EGF) treatment could produce a signaling cascade resulting in phosphorylation of the forkhead transcription factor FKHR in a breast cancer cell line, MDA-MB-231. After establishing ErbB1, cbl, PI3 kinase and Akt were activated in EGF treated MDA-MB-231, we determined by immunoblot with FKHR antiserum that the electrophoretic mobility of FKHR was retarded after EGF treatment. This mobility retardation was reversible by treatment with alkaline phosphatase, and immunoblot with phospho-Ser256 FKHR antibody further confirmed phosphorylation on an Akt consensus site after EGF treatment. EGF stimulated FKHR phosphorylation was blocked by the PI3 kinase inhibitor LY294002, and the ErbB1 inhibitor AG1478. FKHR immunoblotting after purification of nuclear and cytoplasmic proteins showed that EGF induced a simultaneous increase of FKHR in the cytoplasm and decrease in the nucleus. This finding was confirmed by immunofluorescence staining. Treatment of cells with pharmacological inhibitors of PI3 kinase or ErbB1 blocked this effect. Thus, these results demonstrate the phosphorylation and nuclear exclusion of FKHR after EGF treatment by a PI3 kinase dependent mechanism, and represent the first report of growth factor regulation of endogenous FKHR localization.

Coagulation protein function. IV. Effect of acetaldehyde upon factor X and factor Xa, the proteins at the gateway to the common coagulation pathway.

Acetaldehyde (AcH) (447 mM) exerts an inhibition on Factor Xa, as followed by a clotting assay, but does not inhibit the hydrolysis of the synthetic fluorogenic substrate, N-tBOC-Ile-Glu-Gly-Arg-7-amido-4-methylcoumarin. These data suggest that AcH, although not reacting at the catalytic site of Factor Xa nor at the binding site for the synthetic substrate, does interact with the functional groups on the enzyme that bind to its natural substrate, prothrombin. As a consequence of such interaction, the charge and conformation of Factor Xa is altered, thereby limiting effective activation of prothrombin. Additionally, alkylation of factor Xa may also affect its capacity to associate with Factor Va for the activation of prothrombin. AcH also reacts with Factor X, prolonging clotting times when the zymogen is activated with Russell's viper venom (RVV). It also reduces the rate of hydrolysis of the fluorogenic substrate after activation of the alkylated zymogen by RVV. These data lead to the considerations that AcH-modified Factor X is no longer as effectively activated by RVV due to an alteration of its charge/conformation. Additional possibilities include a likely alkylation of the Factor Xa moiety of Factor X by AcH such that the activation product has an altered charge/conformation compared to native Factor Xa, including possible alkylation of its binding site(s) for prothrombin. The reduced rate of hydrolysis of the synthetic fluorogenic substrate for Factor Xa by the alkylated, activated Factor X lends further support to the generation of a modified Factor Xa by RVV, which may have a lower binding or catalytic rate for the fluorogenic substrate. These results support the suggestion that chronic consumption of alcohol may prolong the reported coagulation times as a result of reaction of alcohol's primary metabolite, AcH, with clotting factors, thereby reducing their physiological potential.

Coagulation protein function. III. Effect of acetaldehyde upon the activation of prothrombin.

When prothrombin is mixed with acetaldehyde (AcH), the primary metabolite of ethanol, at room temperature for 30 min, dialyzed to remove excess AcH, and then activated with Echis carinatus venom (ECV), a prolonged clotting time is observed. Similarly treated prothrombin, however, readily hydrolyzes the synthetic substrate, benzoyl-DL-arginine-beta-naphthylamide (BANA). These results suggest that AcH does not react with the catalytic site of thrombin, which is protected in the prothrombin molecule. However, it does react with susceptible sites on the prothrombin surface which remain alkylated during extensive dialysis to remove excess AcH and subsequent activation of the molecule by ECV. These alkylated sites on the newly formed thrombin molecule may inhibit or prevent the effective/efficient binding of fibrinogen at its binding sites, causing prolonged clotting times. The binding sites for accommodating fibrinogen on the thrombin molecule are apparently quite different from those that bind BANA. These data further suggest that prolonged clotting times that are reported in chronic alcoholics may be due, in part, to ready interaction of acetaldehyde with circulating prothrombin. Fibrinogen, which has been exposed to AcH and subsequently dialyzed to remove excess AcH, also responds with a prolonged clotting time in the presence of added thrombin, signifying that sites on fibrinogen that are essential for its function/conformation are susceptible to AcH.

Coagulation protein function. II. Influence of thiols upon acetaldehyde effects.

It has been reported that prolonged prothrombin time may be a result of the interaction of acetaldehyde (AcH) with clotting proteins to form alkylated inactive products. The current investigation focuses on the influence of L-cysteine (CysH), DL-homocysteine (HC), D-penicillamine, N-acetyl-L-cysteine (NAC), L-serine and L-alanine at 0.01 M concentrations, lactalbumin hydrolysate (2 mg/ml), and 1.0 mM dithiothreitol (DTT) on clotting time as well as their interaction with AcH. The sulfhydryl amino acids, as well as DTT prolonged clotting upon preincubation with plasma. Cysteine and NAC, upon addition to plasma prior to the addition of AcH, exhibited a prolongation of clotting time compared to that of AcH alone. On sequential addition of serine, alanine, or lactalbumin hydrolysate to plasma followed by the addition of acetaldehyde, a prolongation of clotting time comparable to that of AcH alone was exhibited. When HC and penicillamine were added to plasma prior to the addition of AcH, a prolonged clotting time was observed, which was significantly less than that of AcH alone. Premixing of serine, alanine, and lactalbumin hydrolysate with AcH for 20 min prior to addition to the plasma reduced the effectiveness of AcH in prolonging clotting time as compared to successive additions of the amino acid and AcH. Since CysH and penicillamine have been reported to form cyclic adducts with AcH, it is suggested that a similar possibility exists for penicillamine and for HC. The reversible cyclic adduct formation reported for CysH may explain why cysteine did not lower the prolonged clotting time induced by AcH.(ABSTRACT TRUNCATED AT 250 WORDS)