Summary Akt-deficiency causes resistance to replicative senescence, oxidative stress- or oncogenic Ras-induced premature senescence, and to reactive oxygen species (ROS)-mediated apoptosis. Akt activation induces premature senescence and sensitizes cells to ROS-mediated apoptosis by increasing intracellular ROS through increased oxygen consumption and by inhibiting the expression of ROS-scavengers downstream of FoxO, particularly sestrin3 expression. This uncovers an Achilles’ heel of Akt, since in contrast to its ability to inhibit apoptosis induced by multiple apoptotic stimuli; Akt could not inhibit ROS-mediated apoptosis. Furthermore, treatment with rapamycin that led to further Akt activation and resistance to etoposide, hypersensitized cancer cells to ROS-mediated apoptosis. Given that rapamycin alone is mainly cytostatic, this constitutes a strategy for cancer therapy that selectively eradicates cancer cells via Akt activation.

Summary FoxO transcription factors and TORC1 are conserved downstream effectors of Akt. Here we unraveled regulatory circuits underlying interplay between Akt, FoxO, and mTOR. Activated FoxO1 inhibits mTORC1 by TSC2-dependent and TSC2-independent mechanisms. First, FoxO1 binds the promoter region of Sestrin3 (Sesn3) gene and directly elevates Sesn3 expression, which in turn inhibits mTORC1 activity in Tsc2-proficient cells. Second, FoxO1 elevates the expression of Rictor leading to increased mTORC2 activity that consequently activates Akt. In Tsc2-deficient cells, the elevation of Rictor by FoxO increases mTORC2 assembly and activity at the expense of mTORC1, thereby activating Akt, while inhibiting mTORC1. FoxO may act as rheostat that maintains homeostatic balance between Akt and mTOR complexes activities. Indeed, in response to physiological stresses, FoxO is required to maintain high Akt activity and low mTORC1 activity. Thus, under stress conditions, FoxO inhibits the anabolic activity of mTORC1-a major consumer of cellular energy, while activating Akt, which increases cellular energy metabolism, thereby maintaining cellular energy homeostasis.

Akt is perhaps the most frequently activated oncoprotein in human cancers. Overriding cell cycle checkpoint in combination with the inhibition of apoptosis are two principal requirements for predisposition to cancer. Here we show that the activation of Akt is sufficient to promote these two principal processes, by inhibiting Chk1 activation with concomitant inhibition of apoptosis. These activities of Akt cannot be recapitulated by the knockdown of Chk1 alone or by overexpression of Bcl2. Rather the combination of Chk1 knockdown and Bcl2 overexpression is required to recapitulate Akt activities. Akt was shown to directly phosphorylate Chk1. However, we found that Chk1 mutants in the Akt phosphorylation sites behave like wildtype Chk1 in mediating G2 arrest, suggesting that the phosphorylation of Chk1 by Akt is either dispensable for Chk1 activity or insufficient by itself to exert an effect on Chk1 activity. Here we report a new mechanism by which Akt affects G2 cell cycle arrest. We show that Akt inhibits BRCA1 function that induces G2 cell cycle arrest. Akt prevents the translocation of BRCA1 to DNA damage foci and, thereby, inhibiting the activation of Chk1 following DNA damage.The serine/threonine kinase Akt is perhaps the most frequently hyperactivated oncoprotein in human cancer (1, 2). However, the reasons behind this frequent hyperactivation are not fully explored. One of the most established consequences of Akt activation is inhibition of apoptosis. Inhibition of apoptosis is a critical step in the process of tumorigenesis and is a prerequisite for the genetic instability occurring during this process. Genetic instability that increases mutation rate, and that is associated with predisposition to cancer, is usually acquired through the abrogation of cell cycle checkpoints. However, the abrogation of cell cycle checkpoints by itself could elicit cell death as mechanism to eliminate cells with damaged DNA. Therefore, the inhibition of cell death is required in conjunction with deregulated cell cycle checkpoint to increase mutation rate during tumorigenesis. Certain genetic lesions could provide both cell cycle checkpoint abrogation and inhibition of apoptosis. The best example of such genetic lesion is the inactivation of p53 that overcomes cell cycle checkpoints with concomitant prevention of the elicited apoptosis, which could explain the frequent inactivation of p53 in cancer cells (3, 4). Our previous results suggest that, by analogy to p53 inactivation, the frequent activation of Akt in cancer cells could elicit both inhibition of apoptosis and abrogation of a G2 cell cycle checkpoint in a p53-independent manner (5). However, the mechanism by which Akt exerts its effect on the G2 cell cycle checkpoint is largely unknown.The p53-independent G2 cell cycle checkpoint following genotoxic stress is mediated by the cell cycle checkpoint protein Chk1. Upon DNA damage, the PI3 kinase-like kinases, ATM, ATR, and DNA-PK are activated and phosphorylate histone H2AX. The phosphorylated H2AX (␥H2AX) mediates the ...

A novel proteinase, PrtR, produced by the human vaginal isolate Lactobacillus rhamnosus strain BGT10 was identified and genetically characterized. The prtR gene and flanking regions were cloned and sequenced. The deduced amino acid sequence of PrtR shares characteristics that are common for other cell envelope proteinases (CEPs) characterized to date, but in contrast to the other cell surface subtilisin-like serine proteinases, it has a smaller and somewhat different B domain and lacks the helix domain, and the anchor domain has a rare sorting signal sequence. Furthermore, PrtR lacks the insert domain, which otherwise is situated inside the catalytic serine protease domain of all CEPs, and has a different cell wall spacer (W) domain similar to that of the cell surface antigen I and II polypeptides expressed by oral and vaginal streptococci. Moreover, the PrtR W domain exhibits significant sequence homology to the consensus sequence that has been shown to be the hallmark of human intestinal mucin protein. According to its ␣ S1 -and ␤-casein cleavage efficacy, PrtR is an efficient proteinase at pH 6.5 and is distributed throughout all L. rhamnosus strains tested. Proteinase extracts of the BGT10 strain obtained with Ca 2؉ -free buffer at pH 6.5 were proteolytically active. The prtR promoter-like sequence was determined, and the minimal promoter region was defined by use of prtR-gusA operon fusions. The prtR expression is Casitone dependent, emphasizing that nitrogen depletion elevates its transcription. This is in correlation with the catalytic activity of the PrtR proteinase.Lactic acid bacteria (LAB) have multiple amino acid auxotrophies, and in order to grow in protein-rich media they depend on the expression of a complex proteolytic system. Most of the genetic and biochemical studies of the LAB proteolytic system have focused on strains used in production of fermented milk products, and these data have been extensively reviewed (28,31,34). Essentially, a cell wall-bound extracellular proteinase (CEP) is responsible for the breakdown of casein, the major milk protein, into oligopeptides. Oligopeptides are then transported via oligopeptide transport systems into the bacterial cell, where the intracellular peptidases hydrolyze different oligopeptides to free amino acids.Three distinctly different genes encoding CEPs, referred to as prtP, prtB, and prtH (50), have been cloned and sequenced from dairy LAB. The prtP genes from a number of different Lactococcus lactis strains and from Lactobacillus paracasei NCDO151 have been cloned and sequenced (31). The different prtP genes encode proteinases with greater than 95% sequence identity. Distinct prt genes have been found in thermophilic lactobacilli. Genes encoding PrtB and PrtH from Lactobacillus delbrueckii subsp. bulgaricus NCDO1489 and Lactobacillus helveticus CNRZ32, respectively, have been sequenced and characterized (15,42). In order to produce the enzymatically active PrtP, L. lactis and L. paracasei require the presence of an upstream-located and divergently ...