The TOR signal transduction cascade controls cellular differentiation in response to nutrients.

Rapamycin binds and inhibits the Tor protein kinases, which function in a nutrient-sensing signal transduction pathway that has been conserved from the yeast Saccharomyces cerevisiae to humans. In yeast cells, the Tor pathway has been implicated in regulating cellular responses to nutrients, including proliferation, translation, transcription, autophagy, and ribosome biogenesis. We report here that rapamycin inhibits pseudohyphal filamentous differentiation of S. cerevisiae in response to nitrogen limitation. Overexpression of Tap42, a protein phosphatase regulatory subunit, restored pseudohyphal growth in cells exposed to rapamycin. The tap42-11 mutation compromised pseudohyphal differentiation and rendered it resistant to rapamycin. Cells lacking the Tap42-regulated protein phosphatase Sit4 exhibited a pseudohyphal growth defect and were markedly hypersensitive to rapamycin. Mutations in other Tap42-regulated phosphatases had no effect on pseudohyphal differentiation. Our findings support a model in which pseudohyphal differentiation is controlled by a nutrient-sensing pathway involving the Tor protein kinases and the Tap42-Sit4 protein phosphatase. Activation of the MAP kinase or cAMP pathways, or mutation of the Sok2 repressor, restored filamentation in rapamycin treated cells, supporting models in which the Tor pathway acts in parallel with these known pathways. Filamentous differentiation of diverse fungi was also blocked by rapamycin, demonstrating that the Tor signaling cascade plays a conserved role in regulating filamentous differentiation in response to nutrients.

Identification and characterization of a highly conserved calcineurin binding protein, CBP1/calcipressin, in Cryptococcus neoformans.

Calcineurin is the conserved target of the immunosuppressants cyclosporin A and FK506. Using the yeast two-hybrid system, we identified a novel calcineurin binding protein, CBP1, from the pathogenic fungus Cryptococcus neoformans. We show that CBP1 binds to calcineurin in vitro and in vivo, and FKBP12-FK506 inhibits CBP1 binding to calcineurin. Cryptococcus neoformans cbp1 mutant strains exhibit modest defects in growth under stress conditions and virulence, similar to but less severe than the phenotypes of calcineurin mutants. Saccharomyces cerevisiae mutants lacking the CBP1 homolog RCN1 are, like calcineurin mutants, sensitive to lithium cation stress. CBP1 shares a central peptide sequence motif, SPPxSPP, with related proteins in S.CEREVISIAE:, Schizosaccharomyces pombe, Drosophila melanogaster, Caenorhabditis elegans and humans, and peptides containing this motif altered calcineurin activity in vitro. Interestingly, the human CBP1 homolog DSCR1 is encoded by the Down's syndrome candidate region interval on chromosome 21, is highly expressed in the heart and central nervous system, and may play a role in calcineurin functions in heart development, neurite extension and memory.

Characterization of alcohol-induced filamentous growth in Saccharomyces cerevisiae.

Diploid cells of the budding yeast Saccharomyces cerevisiae starved for nitrogen differentiate into a filamentous growth form. Poor carbon sources such as starches can also stimulate filamentation, whereas haploid cells undergo a similar invasive growth response in rich medium. Previous work has demonstrated a role for various alcohols, by-products of amino acid metabolism, in altering cellular morphology. We found that several alcohols, notably isoamyl alcohol and 1-butanol, stimulate filamentous growth in haploid cells in which this differentiation is normally repressed. Butanol also induces cell elongation and changes in budding pattern, leading to a pseudohyphal morphology, even in liquid medium. The filamentous colony morphology and cell elongation require elements of the pheromone-responsive MAPK cascade and TEC1, whereas components of the nutrient-sensing machinery, such as MEP2, GPA2, and GPR1, do not affect this phenomenon. A screen for 1-butanol-insensitive mutants identified additional proteins that regulate polarized growth (BUD8, BEM1, BEM4, and FIG1), mitochondrial function (MSM1, MRP21, and HMI1), and a transcriptional regulator (CHD1). Furthermore, we have also found that ethanol stimulates hyperfilamentation in diploid cells, again in a MAPK-dependent manner. Together, these results suggest that yeast may sense a combination of nutrient limitation and metabolic by-products to regulate differentiation.

TOR kinase homologs function in a signal transduction pathway that is conserved from yeast to mammals.

Rapamycin is a natural product with potent antifungal and immunosuppressive activities. Rapamycin binds to the FKBP12 prolyl isomerase, and the resulting protein-drug complex inhibits the TOR kinase homologs. Both the FKBP12 and the TOR proteins are highly conserved from yeast to man, and genetic and biochemical studies reveal that these proteins are the targets of rapamycin in vivo. Treatment of yeast or mammalian cells with rapamycin inhibits translational initiation of a subset of mRNAs and dramatically represses ribosomal mRNA and tRNA transcription. Furthermore, rapamycin exposure blocks cell cycle progression in the early G1 phase of the cell cycle, driving cells into a G0 state and, ultimately, triggering autophagy. Recent findings reveal that the upstream factors regulating the TOR signaling cascade are involved in detecting amino acids, nutrients, or growth factors. These findings indicate that the TOR proteins function in a signal transduction pathway that coordinates nutritional and mitogenic signals to control protein biosynthesis and degradation.

The TOR signaling cascade regulates gene expression in response to nutrients.

Rapamycin inhibits the TOR kinases, which regulate cell proliferation and mRNA translation and are conserved from yeast to man. The TOR kinases also regulate responses to nutrients, including sporulation, autophagy, mating, and ribosome biogenesis. We have analyzed gene expression in yeast cells exposed to rapamycin using arrays representing the whole yeast genome. TOR inhibition by rapamycin induces expression of nitrogen source utilization genes controlled by the Ure2 repressor and the transcriptional regulator Gln3, and globally represses ribosomal protein expression. gln3 mutations were found to confer rapamycin resistance, whereas ure2 mutations confer rapamycin hypersensitivity, even in cells expressing dominant rapamycin-resistant TOR mutants. We find that Ure2 is a phosphoprotein in vivo that is rapidly dephosphorylated in response to rapamycin or nitrogen limitation. In summary, our results reveal that the TOR cascade plays a prominent role in regulating transcription in response to nutrients in addition to its known roles in regulating translation, ribosome biogenesis, and amino acid permease stability.

Signal-transduction cascades as targets for therapeutic intervention by natural products.

Many bacteria and fungi produce natural products that are toxic to other microorganisms and have a variety of physiological effects in animals. Recent studies have revealed that, in several cases, the targets of these agents are components of conserved signal-transduction cascades. This article looks at the mechanisms of action of five natural products--the immunosuppressants cyclosporin A, FK506 and rapamycin, and the antiproliferative agents wortmannin and geldanamycin. These mechanisms reveal the importance of signal-transduction cascades as targets for therapeutic intervention and the enormous utility of studies of natural-product action in simple model genetic systems.

STT4 is an essential phosphatidylinositol 4-kinase that is a target of wortmannin in Saccharomyces cerevisiae.

Wortmannin is a natural product that inhibits signal transduction. One target of wortmannin in mammalian cells is the 110-kDa catalytic subunit of phosphatidylinositol 3-kinase (PI 3-kinase). We show that wortmannin is toxic to the yeast Saccharomyces cerevisiae and present genetic and biochemical evidence that a phosphatidylinositol 4-kinase (PI 4-kinase), STT4, is a target of wortmannin in yeast. In a strain background in which stt4 mutants are rescued by osmotic support with sorbitol, the toxic effects of wortmannin are similarly prevented by sorbitol. In contrast, in a different strain background, STT4 is essential under all conditions and wortmannin toxicity is not mitigated by sorbitol. Overexpression of STT4 confers wortmannin resistance, but overexpression of PIK1, a related PI 4-kinase, does not. In vitro, the PI 4-kinase activity of STT4, but not of PIK1, was potently inhibited by wortmannin. Overexpression of the phosphatidylinositol 4-phosphate 5-kinase homolog MSS4 conferred wortmannin resistance, as did deletion of phospholipase C-1. These observations support a model for a phosphatidylinositol metabolic cascade involving STT4, MSS4, and phospholipase C-1 and provide evidence that an essential product of this pathway is the lipid phosphatidylinositol 4,5-bisphosphate.

Oxidation at C-1 controls the cytotoxicity of 1,1-dichloro-2,2- bis(p-chlorophenyl)ethane by rabbit and human lung cells.

Isolated rabbit Clara cells and a transformed human bronchial epithelial cell line, BEAS-2B, were used to investigate the mechanism of cytotoxicity of 1,1-dichloro-2,2-bis(p-chlorophenyl)ethane (DDD), a persistent insecticide and stable metabolite of 1,1,1-trichloro-2,2- bis(p-chlorophenyl)ethane. Both BEAS-2B cells and rabbit Clara cells were highly susceptible to DDD toxicity and were partially protected by 1-aminobenzotriazole, a suicide substrate inhibitor of cytochrome P450 enzymes. DDD (0.05 mM) killed 47 +/- 1.8% of rabbit Clara cells and 42 +/- 7.9% of BEAS-2B cells after 3 hr and 84 +/- 3.0% of rabbit Clara cells and 80 +/- 14% of BEAS-2B cells after 6 hr. Consequently, DDD is the most potent Clara cell toxicant recognized to date. The cytotoxicity of DDD to these cells was decreased by deuterium substitution at the C-1 position. Rabbit Clara cells and pulmonary microsomes incubated with 14C-DDD produced the fully oxidized acetic acid metabolite 2,2'-bis(p- chlorophenyl)acetic acid (DDA), but DDA was not formed by Clara cells when DDD was coincubated with 1-aminobenzotriazole. These results support the hypothesis that the cytotoxicity of DDD to susceptible subpopulations of rabbit and human lung cells is, at least in part, caused by cytochrome P450-mediated oxidation of DDD at C-1. A required step for the production of the cytotoxic intermediate is proposed to be the formation of a highly reactive acyl halide intermediate that is readily hydrolyzed to a stable, nontoxic metabolite, DDA.