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With the amino acid sequences of all reported Akt kinase physiological substrates, the possible Akt kinase substrate specificity has been suggested. The serine/ threonine residue to be phosphorylated in these proteins is placed within stretches of amino acids with homology, and the arginine residues on the ؊5 and ؊3 positions and a hydrophobic amino acid on the ؉2 position are conserved relative to those of serine/threonine residues (XXRXRXXS/TXX . We observed the phosphorylation of hTERT peptide by the human melanoma cell lysate or the activated recombinant Akt kinase proteins in vitro. With the treatment of the growth factor deprivation or okadaic acid, we also observed the up-regulation of both hTERT peptide phosphorylation and the telomerase activity. We noticed that Wortmannin down-regulates hTERT peptide phosphorylation and telomerase activity together. In addition, we observed the enhancement of telomerase activity with the pretreatment of Akt kinase in vitro. Thus, these observations suggest that Akt kinase enhances human telomerase activity through phosphorylation of hTERT subunit as one of its substrate proteins.

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The protein serine-threonine kinase Akt mediates cell survival signaling initiated by various growth-promoting factors such as insulin. Here we report that SEK1 is a target of Akt in intact cells. Insulin inhibited the anisomycin-induced stimulation of both endogenous SEK1 and its substrate c-Jun N-terminal kinase (JNK), but not that of the upstream kinase MEKK1, in 293T cells. The inhibitory action of insulin on SEK1 or JNK1 activation was prevented by the phosphatidylinositol 3-kinase inhibitor LY294002. Expression of a constitutively active form of Akt also inhibited both SEK1 and JNK1 activation, but not that of MEKK1, in transfected 293T cells. Co-immunoprecipitation analysis revealed that endogenous Akt physically interacted with endogenous SEK1 in cells and that this interaction was promoted by insulin. In vitro and in vivo32 P labeling indicated that Akt phosphorylated SEK1 on serine 78. The SEK1 mutant SEK1(S78A) was resistant to Akt-induced inhibition. Finally, activated Akt inhibited SEK1-mediated apoptosis, and this effect of Akt was prevented by overexpression of SEK(S78A). Taken together, these results suggest that Akt suppresses stress-activated signaling by targeting SEK1.

We investigated whether the snake venom toxin (SVT) from Vipera lebetina turanica inhibits cell growth of human prostate cancer cells by inducing apoptosis and also studied possible signaling pathways involved in this cell death. SVT inhibited growth of PC-3 and DU145 cells, androgen-independent prostate cancer cells, but not LNCaP cells, a human androgen-dependent prostate cancer cell. Cells were arrested in the G 2 -M phase by SVT with a concomitant decrease in the expression of the G 2 -M phase regulatory protein cyclin B1 and were also arrested in the G 1 -S phase with decreasing expression of cyclin-dependent kinase 4, cyclin D1 and cyclin E. In addition to the growth-inhibitory effect, SVT increased the induction of apoptotic cell death. Untreated PC-3 cells show high DNA binding activity of nuclear factor KB (NF-KB), an antiapoptotic transcriptional factor, but this was inhibited by SVT and accompanied by a significant inhibition of p50 translocation into the nucleus, as well as phosphorylation of inhibitory KB. Consistent with the induction of apoptosis and inhibition of NF-KB, this toxin increased the expression of proapoptotic proteins such as p53, Bax, caspase-3, and caspase-9, but down-regulated antiapoptotic protein Bcl-2. However, SVT did not show an inhibitory effect on cell growth and caspase-3 activity in cells carrying mutant p50 and inhibitory KB kinase plasmids. Confocal microscopy analysis showed that SVT is taken up into the nucleus of the cells. These findings suggest that a nanogram concentration range of SVT from V. lebetina turanica could inhibit hormone-refractory human prostate cancer cell growth, and the effect may be related to NF-KB signal -mediated induction of apoptosis. [Mol Cancer Ther 2007;6(2):675 -83]

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Outer membrane vesicles (OMVs) derived from pathogenic Gram‐negative bacteria are an important vehicle for delivery of effector molecules to host cells, but the production of OMVs from Klebsiella pneumoniae, an opportunistic pathogen of both nosocomial and community‐acquired infections, and their role in bacterial pathogenesis have not yet been determined. In the present study, we examined the production of OMVs from K. pneumoniae and determined the induction of the innate immune response against K. pneumoniae OMVs. Klebsiella pneumoniae ATCC 13883 produced and secreted OMVs during in vitro culture. Proteomic analysis revealed that 159 different proteins were associated with K. pneumoniae OMVs. Klebsiella pneumoniae OMVs did not inhibit cell growth or induce cell death. However, these vesicles induced expression of proinflammatory cytokine genes such as interleukin (IL)‐1β and IL‐8 in epithelial cells. An intratracheal challenge of K. pneumoniae OMVs in neutropenic mice resulted in severe lung pathology similar to K. pneumoniae infection. In conclusion, K. pneumoniae produces OMVs like other pathogenic Gram‐negative bacteria and K. pneumoniae OMVs are a molecular complex that induces the innate immune response.

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