Mammalian TOR: a homeostatic ATP sensor.

The bacterial macrolide rapamycin is an efficacious anticancer agent against solid tumors. In a hypoxic environment, the increase in mass of solid tumors is dependent on the recruitment of mitogens and nutrients. When nutrient concentrations change, particularly those of essential amino acids, the mammalian Target of Rapamycin (mTOR) functions in regulatory pathways that control ribosome biogenesis and cell growth. In bacteria, ribosome biogenesis is independently regulated by amino acids and adenosine triphosphate (ATP). Here we demonstrate that the mTOR pathway is influenced by the intracellular concentration of ATP, independent of the abundance of amino acids, and that mTOR itself is an ATP sensor.

Drosophila S6 kinase: a regulator of cell size.

Cell proliferation requires cell growth; that is, cells only divide after they reach a critical size. However, the mechanisms by which cells grow and maintain their appropriate size have remained elusive. Drosophila deficient in the S6 kinase gene (dS6K) exhibited an extreme delay in development and a severe reduction in body size. These flies had smaller cells rather than fewer cells. The effect was cell-autonomous, displayed throughout larval development, and distinct from that of ribosomal protein mutants (Minutes). Thus, the dS6K gene product regulates cell size in a cell-autonomous manner without impinging on cell number.

Proliferation, but not growth, blocked by conditional deletion of 40S ribosomal protein S6.

Because ribosome biogenesis plays an essential role in cell proliferation, control mechanisms may have evolved to recognize lesions in this critical anabolic process. To test this possibility, we conditionally deleted the gene encoding 40S ribosomal protein S6 in the liver of adult mice. Unexpectedly, livers from fasted animals deficient in S6 grew in response to nutrients even though biogenesis of 40S ribosomes was abolished. However, liver cells failed to proliferate or induce cyclin E expression after partial hepatectomy, despite formation of active cyclin D-CDK4 complexes. These results imply that abrogation of 40S ribosome biogenesis may induce a checkpoint control that prevents cell cycle progression.

Phosphorylation and activation of p70s6k by PDK1.

Activation of the protein p70s6k by mitogens leads to increased translation of a family of messenger RNAs that encode essential components of the protein synthetic apparatus. Activation of the kinase requires hierarchical phosphorylation at multiple sites, culminating in the phosphorylation of the threonine in position 229 (Thr229), in the catalytic domain. The homologous site in protein kinase B (PKB), Thr308, has been shown to be phosphorylated by the phosphoinositide-dependent protein kinase PDK1. A regulatory link between p70s6k and PKB was demonstrated, as PDK1 was found to selectively phosphorylate p70s6k at Thr229. More importantly, PDK1 activated p70s6k in vitro and in vivo, whereas the catalytically inactive PDK1 blocked insulin-induced activation of p70s6k.

A heat-sensitive Arabidopsis thaliana kinase substitutes for human p70s6k function in vivo.

In mammalian cells, mitogen-induced phosphorylation of ribosomal protein S6 by p70s6k has been implicated in the selective translational upregulation of 5'TOP mRNAs. We demonstrate here that the homologous Arabidopsis thaliana protein, AtS6k2, ectopically expressed in human 293 cells or isolated from plant cells, phosphorylates specifically mammalian and plant S6 at 25 degrees C but not at 37 degrees C. When Arabidopsis suspension culture cells are shifted from 25 to 37 degrees C, the kinase becomes rapidly inactivated, consistent with the observation that heat shock abrogates S6 phosphorylation in plants. Treatment with potato acid phosphatase reduced the specific activity of immunoprecipitated AtS6k2 threefold, an effect which was blocked in the presence of 4-nitrophenyl phosphate. In quiescent mammalian cells, AtS6k2 is activated by serum stimulation, a response which is abolished by the fungal metabolite wortmannin but is resistant to rapamycin. Treatment of mammalian cells with rapamycin abolishes in vivo S6 phosphorylation by p70s6k; however, ectopic expression of AtS6k2 rescues the rapamycin block. Collectively, the data demonstrate that AtS6k2 is the functional plant homolog of mammalian p70s6k and identify a new signalling pathway in plants.

The principal rapamycin-sensitive p70(s6k) phosphorylation sites, T-229 and T-389, are differentially regulated by rapamycin-insensitive kinase kinases.

Mitogen-induced activation of p70(s6k) is associated with the phosphorylation of specific sites which are negatively affected by the immunosuppressant rapamycin, the fungal metabolite wortmannin, and the methylxanthine SQ20006. Recent reports have focused on the role of the amino terminus of the p85(s6k) isoform in mediating kinase activity, with the observation that amino-terminal truncation mutants are activated in the presence of rapamycin while retaining their sensitivity to wortmannin. Here we show that the effects of previously described amino- and carboxy-terminal truncations on kinase activity are ultimately reflected in the phosphorylation state of the enzyme. Mutation of the principal rapamycin-targeted phosphorylation site, T-389, to an acidic residue generates a form of the kinase which is as resistant to wortmannin or SQ20006 as it is to rapamycin, consistent with the previous observation that T-389 was a common target of all three inhibitors. Truncation of the first 54 residues of the amino terminus blocks the serum-induced phosphorylation of three rapamycin-sensitive sites, T-229 in the activation loop and T-389 and S-404 in the linker region. This correlates with a severe reduction in the ability of the kinase to be activated by serum. However, loss of mitogen activation conferred by the removal of the amino terminus is reversed by additional truncation of the carboxy-terminal domain, with the resulting mutant demonstrating phosphorylation of the remaining two rapamycin-sensitive sites, T-229 and T-389. In this double-truncation mutant, phosphorylation of T-229 occurs in the basal state, whereas mitogen stimulation is required to induce acute upregulation of T-389 phosphorylation. The phosphorylation of both sites proceeds unimpaired in the presence of rapamycin, indicating that the kinases responsible for the phosphorylation of these sites are not inhibited by the macrolide. In contrast, activation of the double-truncation mutant is blocked in the presence of wortmannin or SQ20006, and these agents completely block the phosphorylation of T-389 while having only a marginal effect on T-229 phosphorylation. When the T-389 site is mutated to an acidic residue in the double-truncation background, the activation of the resulting mutant is insensitive to the wortmannin and SQ20006 block, but interestingly, the mutant is activated to a significantly greater level than a control in the presence of rapamycin. These data are consistent with the hypothesis that T-389 is the principal regulatory phosphorylation site, which, in combination with hyperphosphorylation of the autoinhibitory domain S/TP sites, is acutely regulated by external effectors, whereas T-229 phosphorylation is regulated primarily by internal mechanisms.

p70(S6K) controls selective mRNA translation during oocyte maturation and early embryogenesis in Xenopus laevis.

In mammalian cells, p70(S6K) plays a key role in translational control of cell proliferation in response to growth factors. Because of the reliance on translational control in early vertebrate development, we cloned a Xenopus homolog of p70(S6K) and investigated the activity profile of p70(S6K) during Xenopus oocyte maturation and early embryogenesis. p70(S6K) activity is high in resting oocytes and decreases to background levels upon stimulation of maturation with progesterone. During embryonic development, three peaks of activity were observed: immediately after fertilization, shortly before the midblastula transition, and during gastrulation. Rapamycin, an inhibitor of p70(S6K) activation, caused oocytes to undergo germinal vesicle breakdown earlier than control oocytes, and sensitivity to progesterone was increased. Injection of a rapamycin-insensitive, constitutively active mutant of p70(S6K) reversed the effects of rapamycin. However, increases in S6 phosphorylation were not significantly affected by rapamycin during maturation. mos mRNA, which does not contain a 5'-terminal oligopyrimidine tract (5'-TOP), was translated earlier, and a larger amount of Mos protein was produced in rapamycin-treated oocytes. In fertilized eggs rapamycin treatment increased the translation of the Cdc25A phosphatase, which lacks a 5'-TOP. Translation assays in vivo using both DNA and RNA reporter constructs with the 5'-TOP from elongation factor 2 showed decreased translational activity with rapamycin, whereas constructs without a 5'-TOP or with an internal ribosome entry site were translated more efficiently upon rapamycin treatment. These results suggest that changes in p70(S6K) activity during oocyte maturation and early embryogenesis selectively alter the translational capacity available for mRNAs lacking a 5'-TOP region.

New acceptor cell for transfected genomic DNA: oncogene transfer into a mouse mammary epithelial cell line.

A line of mouse mammary epithelial cells (NMuMG) has been characterized for its ability to be stably transfected with exogenous DNA. A transfection frequency of at least 1 cell per 1,000 was obtained with the pSV2neo plasmid. Several thousand G418-resistant NMuMG cell clones can easily be generated in cotransfection of genomic DNA and pSV2neo. The NMuMG cells were isolated from normal mammary glands and do not form malignant lesions when injected into nude mice. We have cotransfected NMuMG cells with pSV2neo and genomic DNA from the human EJ bladder carcinoma line, a cell line which contains an activated c-rasH oncogene. When a pool of 4,700 G418-resistant colonies was injected into nude mice, tumors were obtained. These tumors contain a transfected human rasH gene. Genomic DNA transfection into a line of mouse epithelial cells, in combination with the selection of stable transfectants and tumor induction in nude mice, can be used to screen human tumor DNA for the presence of activated oncogenes.

Dual requirement for a newly identified phosphorylation site in p70s6k.

The activation of p70s6k is associated with multiple phosphorylations at two sets of sites. The first set, S411, S418, T421, and S424, reside within the autoinhibitory domain, and each contains a hydrophobic residue at -2 and a proline at +1. The second set of sites, T229 (in the catalytic domain) and T389 and S404 (in the linker region), are rapamycin sensitive and flanked by bulky aromatic residues. Here we describe the identification and mutational analysis of three new phosphorylation sites, T367, S371, and T447, all of which have a recognition motif similar to that of the first set of sites. A mutation of T367 or T447 to either alanine or glutamic acid had no apparent effect on p70s6k activity, whereas similar mutations of S371 abolished kinase activity. Of these three sites and their surrounding motifs, only S371 is conserved in p70s6k homologs from Drosophila melanogaster, Arabidopsis thaliana, and Saccharomyces cerevisiae, as well as many members of the protein kinase C family. Serum stimulation increased S371 phosphorylation; unlike the situation for specific members of the protein kinase C family, where the homologous site is regulated by autophosphorylation, S371 phosphorylation is regulated by an external mechanism. Phosphopeptide analysis of S371 mutants further revealed that the loss of activity in these variants was paralleled by a block in serum-induced T389 phosphorylation, a phosphorylation site previously shown to be essential for kinase activity. Nevertheless, the substitution of an acidic residue at T389, which mimics phosphorylation at this site, did not rescue mutant p70s6k activity, indicating that S371 phosphorylation plays an independent role in regulating intrinsic kinase activity.

Radioimmunoassay for glycoprotein gp47 of murine mammary tumor virus in organs and serum of mice and search for related antigens in human sera.

Murine mammary tumor virus main glycoprotein (gp47), prepared by diethylaminoethyl cellulose and hydroxyapatite chromatography of detergent-mercaptoethanol-KCI-disrupted virion, was used as labeled antigen in a highly specific and reproducible radioimmunoassay. Seven other (glyco) proteins of the virus were antigenically distinct from gp47. Serum and organs of unifected C57BL mice did not contain gp47, but sera of infected Swiss and RIII mice did contain the antigen. Despite the high content in the mammary gland, the level of gp47 in other organs was identical in male and female mice. The titer of gp47 in serum was high in tumor-bearing females, but it varied with the mouse strain. Anti-gp47 immunoglobulins could not be detected. The investigation included 314 human sera (107 normal, 65 benign mastopathy, 89 breast vancer, and 53 digestive cancer). None contained an antigen related to gp47. One of 20 human mammary cyst fluids was positive.

Radioimmunoassay for protein p28 of murine mammary tumor virus in organs and serum of mice and search for related antigens in human sera and breast cancer extracts.

The main protein of the core of murine mammary tumor virus, with a molecular weight of 28,000 (p28), was solubilized by deoxycholate treatment of the virus and purified by Ultrogel ACA-54 filtration and hydroxyapatite chromatography. This protein was used as labeled antigen in a highly specific and reproducible radioimmunoassay. Organ extracts of uninfected C57BL mice did not contain p28, but organ extracts of infected RIII mice did contain the antigen. Despite the high content in the mammary gland, the level of p28 in the other organs was identical in male and female mice. Sera of uninfected mice and the majority of the sera of infected mice did not contain the antigen. The investigation included 338 human sera (50, normal; 157, breast cancer; 77, polycystic disease; 32, benign mastopathy; 12, fibroadenoma; 10, at risk of developing breast cancer). None contained an antigen related to p28. Eight of 24 extracts of human breast cancer gave results that appeared weakly positive, possibly as a result of proteolysis. Extracts of healthy breast tissue and the serum from the breast arterial and venous blood of corresponding patients were negative.

Identification, partial purification and biochemical characterization of gamma-glutamyltranspeptidase present as a membrane component in skimmed milk and milk fat-globule membranes, and in mammary-tumour virus from the milk of infected mice.

The enzyme gamma-glutamyltranspeptidase was reproducibly found to be associated with mouse milk particles; it is present in milk fat-globule membranes and mouse mammary-tumour virus of infected Swiss mice, also in particles from the milk of uninfected mice. The enzymatic activities observed range among the highest reported for mammalian tissues. The enzyme was partially purified from mouse mammary-tumour virus, and from milk fat-globule membranes. The molecule requires the presence of detergents to remain soluble, behaves as a high molecular weight component, properties characterizing integral membrane proteins. Kinetics, and the effect of competitors as well as of specific inhibitors show this enzyme to be identical to the well-known kidney gamma-glutamyltranspeptidase ((gamma-glutamyl)-peptide:amino-acid gamma-glutamyltransferase, EC 2.3.2.2). Other oncornaviruses budding from cultured cells originally expressing the enzyme in their plasma membrane also incorporate the enzyme in their structure.

[Various foods of intermediate humidity on the Chilean market]. / Algunos alimentos de humedad intermedia en el mercado chileno.

Water activity, moisture content, pH, total sugars and sodium chloride content of commercial foods with reduced water content found in Chilean supermarkets were determined. The sample included meat, dairy, fruits and confectionary products. Principles involved in the reduction of water activity are discussed for each case.

mTOR regulates DNA damage response through NF-κB-mediated FANCD2 pathway in hematopoietic cells.

Hematopoietic stem/progenitor cells (HSPCs) function to give rise to mature blood cells. Effective DNA damage response (DDR) and maintenance of genomic stability are crucial for normal functioning of HSPCs. Mammalian target of rapamycin (mTOR) integrates signals from nutrients and growth factors to control protein synthesis, cell growth, survival and metabolism, and has been shown to regulate DDR in yeast and human cancer cells through the p53/p21 signaling cascade. Here, we show that gene targeting of mTOR in HSPCs causes a defective DDR due to a variety of DNA damage agents, mimicking that caused by deficient FANCD2, a key component of the Fanconi anemia (FA) DDR machinery. Mechanistically, mTOR(-/-) HSPCs express drastically reduced FANCD2. Consistent with these genetic findings, inactivation of mTOR in human lymphoblast cells by pp242 or Torin 1, mTOR kinase inhibitors, suppresses FANCD2 expression and causes a defective DDR that can be rescued by reconstitution of exogenous FANCD2. Further mechanistic studies show that mTOR deficiency or inactivation increases phosphorylation and nuclear translocation of nuclear factor (NF)-κB, which results in an enhanced NF-κB binding to FANCD2 promoter to suppress FANCD2 expression. Thus, mTOR regulates DDR and genomic stability in hematopoietic cells through a noncanonical pathway involving NF-κB-mediated FANCD2 expression.

p70s6k/p85s6k: mechanism of activation and role in mitogenesis.

The p70s6k/p85s6k represent two isoforms of the same kinase which are derived by differential splicing from a common gene. The p85s6k isoform is identical to p70s6k except for a 23 amino acid extension at its N-terminus, which constitutively targets it to the nucleus. Both isoforms are activated by multisite phosphorylation in response to mitogens and reside on the same signaling pathway, a pathway which is distinct from that of p42mapk/p44mapk pathway. Inhibitory p70s6k/p85s6k antibodies or the immunosuppressant rapamycin selectively inhibit kinase activity and repress or abolish cell growth depending on the inhibitory agent employed and the cell type examined. Recent studies imply that these effects are exerted through inhibition of 40S ribosomal protein S6 phosphorylation, the kinase target, which in turn suppresses the translation of a family of transcripts essential for cell growth.

A single gene encodes two isoforms of the p70 S6 kinase: activation upon mitogenic stimulation.

Previously, two cDNA clones were isolated from a rat liver or hepatoma cDNA library for the mitogenic-activated p70 S6 kinase (p70s6k). Except for a single amino acid change and a 23-amino acid N-terminal extension in the latter clone, the open reading frames of the two clones are identical. A probe common to both clones also revealed four distinct transcripts. Here, by using specific probes, it was possible to show which transcript corresponds to which clone and that both clones are derived from the same gene. Furthermore, analysis of in vitro translation products using specific antibodies demonstrates that both clones encode the p70s6k but that the clone harboring the 23-amino acid extension also encodes an additional isoform of the kinase, referred to as p85s6k. It could be shown by using the specific antibody to the p85s6k that this isoform of the kinase is present in rat liver and is activated after mitogenic stimulation of quiescent Swiss 3T3 cells.

Distribution of mouse mammary tumour virus antigens in RIII mice as detected by immunofluorescence on tissue sections and by immunoassays in sera and organ extracts.

Organs of RIII mice at various physiological stages were tested for mouse mammary tumour virus (MMTV) antigen expression. Indirect immunofluorescence was used with three monospecific antisera to localize one envelope glycoprotein, gp47, and two core proteins, p28 and p8. These virus-specific antigens gave characteristic fluorescent patterns in the mammary tissues and were also detected in thymus and salivary gland sections of some mice. The amounts of antigens gp47 and p28 were measured by immunoassay in sera and organ extracts of corresponding samples of mice. Sera of mice of both sexes contained virus antigens from the suckling age onwards. Although ingested virus could be traced in suckling mice, weanlings were characterized by the absence of virus expression except in lymph nodes. This location points to the possible role of lymphoid tissue in producing the antigens of the blood and in disseminating the infection. In adult animals, virus antigens were present in salivary glands, digestive tract, lymph nodes, male genital organs and female mammary glands. Antigen expression, even found in the mammary glands of virgin mice, was strikingly increased by pregnancy, lactation and (or) ageing, the highest values being found in mammary tumours. The results for milk-borne MMTV infection in RIII mice are compared with those obtained previously in Swiss albino mice.