Apoptosis signal-regulating kinase 1 (ASK1) is a pivotal component of a signaling pathway induced by many death stimuli, including tumor necrosis factor a, Fas, and the anticancer drugs cisplatin and paclitaxel. Here we report that ASK1 proapoptotic activity is antagonized by association with 14-3-3 proteins. We found that ASK1 specifically bound 14-3-3 proteins via a site involving Ser-967 of ASK1. Interestingly, overexpression of 14-3-3 in HeLa cells blocked ASK1-induced apoptosis whereas disruption of the ASK1/14-3-3 interaction dramatically accelerated ASK1-induced cell death. Targeting of ASK1 by a 14-3-3-mediated survival pathway may provide a novel mechanism for the suppression of apoptosis.

Nattokinase has been reported as an oral health product for the prevention of atherosclerosis.We developed a novel strategy to express a nattokinase from Bacillus subtilis in a live delivery vehicle, Lactococcus lactis. Promoter PnisZ and signal peptide SPUsp were used for inducible and secretory expression of nattokinase in L. lactis. Western blotting analysis demonstrated that nattokinase was successfully expressed, and about 94% of the enzyme was secreted to the culture. The recombinant nattokinase showed potent fibrinolytic activity, equivalent to 41.7 urokinase units per milliliter culture. Expression and delivery of such a fibrinolytic enzyme in the food-grade vehicle L. lactis would facilitate the widespread application of nattokinase in the control and prevention of thrombosis diseases.

Exoenzyme S (ExoS) is a mono-ADP-ribosyltransferase secreted by the opportunistic pathogen Pseudomonas aeruginosa. ExoS requires a eukaryotic factor, the 14-3-3 protein, for enzymatic activity. Here, two aspects of the activation of the ADP-ribosyltransferase activity of ExoS by 14-3-3 proteins are examined. Initial studies showed that several isoforms of 14-3-3, including β, ξ, η, σ, and ô, activated ExoS with similar efficiency. This implicates a conserved structure in 14-3-3 that contributes to the interaction between 14-3-3 and ExoS. One candidate structure is the conserved amphipathic groove that mediates the 14-3-3/Raf-1 interaction. The next series of experiments examined the role of individual amino acids of the amphipathic groove of 14-3-3ξ in ExoS activation and showed that ExoS activation required the basic residues lining the amphipathic groove of 14-3-3ξ　 without extensive involvement of the hydrophobic residues. Strikingly, mutations of Val-176 of 14-3-3ξ that disrupted its interaction with Raf-1 did not affect the binding and activation of ExoS by 14-3-3. Thus, ExoS selectively employs residues in the Raf-binding groove for its association with 14-3-3 proteins.

14-3-3 proteins are a family of conserved dimeric molecules that bind to a range of cellular proteins involved in signal transduction and oncogenesis. Our solution of the crystal structure of 14-3-3ζrevealed a conserved amphipathic groove that may allow the association of 14-3-3 with diverse ligands (Liu, D., Bienkowska, J., Petosa, C., Collier, R. J., Fu, H., and Liddington, R. (1995) Nature 376, 191–194). Here, the contributions of three positively charged residues (Lys-49, Arg-56, and Arg-60) that lie in this Raf-binding groove were investigated. Two of the charge-reversal mutations greatly (K49E) or partially (R56E) decreased the interaction of 14-3-3ζwith Raf-1 kinase, whereas R60E showed only subtle effects on the binding. Interestingly, these mutations exhibited similar effects on the functional interaction of 14-3-3ζwith another target protein, exoenzyme S (ExoS), an ADPribosyltransferase from Pseudomonas aeruginosa. The EC50 values of 14-3-3ζrequired for ExoS activation increased by ;110-, 5-, and 2-fold for the K49E, R56E, and R60E mutants, respectively. The drastic reduction of 14-3-3ζ/ligand affinity by the K49E mutation is due to a local electrostatic effect, rather than the result of a gross structural alteration, as evidenced by partial proteolysis and circular dichroism analysis. This work identifies the first point mutation (K49E) that dramatically disrupts 14-3-3ζ/ligand interactions. The parallel effects of this single point mutation on both Raf-1 binding and ExoS activation strongly suggest that diverse associated proteins share a common structural binding determinant on 14-3-3ζ.

Apoptosis signal-regulating kinase 1 (ASK1) is a serine/ threonine kinase that mediates cell stress signaling initiatedby diverse stimuli, such as H2O2 andTNF a. Owing to its critical role in promoting apoptosis, ASK1 activity is highly controlledin cells. Phosphorylation of ASK1 at Thr-845 has been correlatedwith its activation, while phosphorylation at Ser-967 negatively controls its death promoting activity. Here, we report the identification of a novel phosphorylation site at Ser-1034 in the Cterminal regulatory domain of ASK1. Mutating Ser-1034 to an unphosphorylatable Ala ledto increased catalytic activity of ASK1 andenhancedproapop totic function of ASK1. Thus, the proapoptotic function of ASK1 is suppressedin part by phosphorylation at its C-terminal regulatory domain, which may couple upstream survival kinases to the death regulatory machinery.