Biomedical subjects

Michael N Hall

Publications and source records attributed to Michael N Hall.

27 records · Page 2Linked to original sources

PubMed2004

Activation of the RAS/cyclic AMP pathway suppresses a TOR deficiency in yeast.

The TOR (target of rapamycin) and RAS/cyclic AMP (cAMP) signaling pathways are the two major pathways controlling cell growth in response to nutrients in yeast. In this study we examine the functional interaction between TOR and the RAS/cAMP pathway. First, activation of the RAS/cAMP signaling pathway confers pronounced resistance to rapamycin. Second, constitutive activation of the RAS/cAMP pathway prevents several rapamycin-induced responses, such as the nuclear translocation of the transcription factor MSN2 and induction of stress genes, the accumulation of glycogen, the induction of autophagy, the down-regulation of ribosome biogenesis (ribosomal protein gene transcription and RNA polymerase I and III activity), and the down-regulation of the glucose transporter HXT1. Third, many of these TOR-mediated responses are independent of the previously described TOR effectors TAP42 and the type 2A-related protein phosphatase SIT4. Conversely, TOR-controlled TAP42/SIT4-dependent events are not affected by the RAS/cAMP pathway. Finally, and importantly, TOR controls the subcellular localization of both the protein kinase A catalytic subunit TPK1 and the RAS/cAMP signaling-related kinase YAK1. Our findings suggest that TOR signals through the RAS/cAMP pathway, independently of TAP42/SIT4. Therefore, the RAS/cAMP pathway may be a novel TOR effector branch.

Antifungal Agents↗

PubMed2003

Quantitation of changes in protein phosphorylation: a simple method based on stable isotope labeling and mass spectrometry.

Reversible protein phosphorylation plays an important role in many cellular processes. However, a simple and reliable method to measure changes in the extent of phosphorylation is lacking. Here, we present a method to quantitate the changes in phosphorylation occurring in a protein in response to a stimulus. The method consists of three steps: (i) enzymatic digestion in H(2)16O or isotopically enriched H(2)18O to label individual pools of differentially phosphorylated proteins; (ii) affinity selection of phosphopeptides from the combined digests by immobilized metal-affinity chromatography; and (iii) dephosphorylation with alkaline phosphatase to allow for quantitation of changes of phosphorylation by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. We applied this strategy to the analysis of the yeast nitrogen permease reactivator protein kinase involved in the target of rapamycin signaling pathway. Alteration in the extent of phosphorylation at Ser-353 and Ser-357 could be easily assessed and quantitated both in wild-type yeast cells treated with rapamycin and in cells lacking the SIT4 phosphatase responsible for dephosphorylating nitrogen permease reactivator protein. The method described here is simple and allows quantitation of relative changes in the level of phosphorylation in signaling proteins, thus yielding information critical for understanding the regulation of complex protein phosphorylation cascades.

Electrophoresis, Polyacrylamide Gel↗

PubMed2003

Tor signalling in bugs, brain and brawn.

TOR--a highly conserved atypical protein kinase and the 'target of rapamycin', an immunosuppressant and anti-cancer drug--controls cell growth. TOR controls the growth of proliferating yeast, fly and mammalian cells in response to nutrients. Recent findings, however, indicate that TOR also controls the growth of non-proliferating cells, such as neurons and muscle cells. Furthermore, TOR, by associating with regulatory proteins and inhibiting phosphatases, controls the activity of multiphosphorylated effectors.

Animals↗

PubMed2002

Calmodulin controls organization of the actin cytoskeleton via regulation of phosphatidylinositol (4,5)-bisphosphate synthesis in Saccharomyces cerevisiae.

Phosphoinositides regulate a wide range of cellular processes, including proliferation, survival, cytoskeleton remodelling and membrane trafficking, yet the mechanisms controlling the kinases, phosphatases and lipases that modulate phosphoinositide levels are poorly understood. In the present study, we describe a mechanism controlling MSS4, the sole phosphatidylinositol (4)-phosphate 5-kinase in Saccharomyces cerevisiae. Mutations in MSS4 and CMD1, encoding the small Ca(2+)-binding protein calmodulin, confer similar phenotypes, including loss of viability and defects in endocytosis and in organization of the actin cytoskeleton. Overexpression of MSS4 suppresses the growth and actin defects of cmd1-226, a temperature-sensitive calmodulin mutant which is defective in the organization of the actin cytoskeleton. Finally, the cmd1-226 mutant exhibits reduced levels of phosphatidylinositol (4,5)-bisphosphate. These findings suggest that calmodulin positively controls MSS4 activity and thereby the actin cytoskeleton.

Actins↗

PubMed2002

The TOR-controlled transcription activators GLN3, RTG1, and RTG3 are regulated in response to intracellular levels of glutamine.

The essential, rapamycin-sensitive TOR kinases regulate a diverse set of cell growth-related readouts in response to nutrients. Thus, the yeast TOR proteins function as nutrient sensors, in particular as sensors of nitrogen and possibly carbon. However, the nutrient metabolite(s) that acts upstream of TOR is unknown. We investigated the role of glutamine, a preferred nitrogen source and a key intermediate in yeast nitrogen metabolism, as a possible regulator of TOR. We show that the glutamine synthetase inhibitor L-methionine sulfoximine (MSX) specifically provokes glutamine depletion in yeast cells. MSX-induced glutamine starvation caused nuclear localization and activation of the TOR-inhibited transcription factors GLN3, RTG1, and RTG3, all of which mediate glutamine synthesis. The MSX-induced nuclear localization of GLN3 required the TOR-controlled, type 2A-related phosphatase SIT4. Other TOR-controlled transcription factors, GAT1/NIL1, MSN2, MSN4, and an unknown factor involved in the expression of ribosomal protein genes, were not affected by glutamine starvation. These findings suggest that the TOR pathway senses glutamine. Furthermore, as glutamine starvation affects only a subset of TOR-controlled transcription factors, TOR appears to discriminate between different nutrient conditions to elicit a response appropriate to a given condition.

Basic Helix-Loop-Helix Leucine Zipper Transcriptio↗

PubMed2002

Two TOR complexes, only one of which is rapamycin sensitive, have distinct roles in cell growth control.

The target of rapamycin (TOR) proteins in Saccharomyces cerevisiae, TOR1 and TOR2, redundantly regulate growth in a rapamycin-sensitive manner. TOR2 additionally regulates polarization of the actin cytoskeleton in a rapamycin-insensitive manner. We describe two functionally distinct TOR complexes. TOR Complex 1 (TORC1) contains TOR1 or TOR2, KOG1 (YHR186c), and LST8. TORC2 contains TOR2, AVO1 (YOL078w), AVO2 (YMR068w), AVO3 (YER093c), and LST8. FKBP-rapamycin binds TORC1, and TORC1 disruption mimics rapamycin treatment, suggesting that TORC1 mediates the rapamycin-sensitive, TOR-shared pathway. FKBP-rapamycin fails to bind TORC2, and TORC2 disruption causes an actin defect, suggesting that TORC2 mediates the rapamycin-insensitive, TOR2-unique pathway. Thus, the distinct TOR complexes account for the diversity, specificity, and selective rapamycin inhibition of TOR signaling. TORC1 and possibly TORC2 are conserved from yeast to man.

Actins↗

PubMed2002

The RHO1-GAPs SAC7, BEM2 and BAG7 control distinct RHO1 functions in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, the small GTPase RHO1 plays an essential role in the control of cell wall synthesis and organization of the actin cytoskeleton. Several regulators for RHO1 are known, including the GTPase-activating proteins (GAPs) SAC7 and BEM2. Here we show that BAG7, identified as the closest homologue of SAC7, also acts as a GAP for RHO1 in vitro and in vivo. Furthermore, we find that BAG7, SAC7, and BEM2 are functionally different in vivo. Overexpression of BAG7 or SAC7,but not BEM2, suppresses the cold sensitivity of a sac7 mutation and the lethality of RHO1 hyperactivation in response to cell wall damage. In contrast, overexpression of BEM2 or SAC7, but not BAG7, downregulates the RHO1-controlled PKC1-MPK1 pathway, and disruption of BEM2 or SAC7, but not BAG7, results in increased MPK1 activation. We conclude that BEM2 and SAC7, but not BAG7, are involved in the control of the RHO1-mediated activation of MPK1, whereas BAG7 and SAC7, but not BEM2, are involved in the regulation of other RHO1 functions. This suggests that different RHO1GAPs control different RHO1 effector pathways, thus ensuring their individual regulation at the appropriate place and time.

Fungal Proteins↗

PubMed2002

Yeast protein kinases and the RHO1 exchange factor TUS1 are novel components of the cell integrity pathway in yeast.

The PKC1-associated mitogen-activated protein (MAP) kinase pathway of Saccharomyces cerevisiae regulates cell integrity by controlling the actin cytoskeleton and cell wall synthesis. Activation of PKC1 occurs via the GTPase RHO1 and the kinase pair PKH1 and PKH2. Here we report that YPK1 and YPK2, an essential pair of homologous kinases and proposed downstream effectors of PKH and sphingolipids, are also regulators of the PKC1-controlled MAP kinase cascade. ypk mutants display random distribution of the actin cytoskeleton and severely reduced activation of the MAP kinase MPK1. Upregulation of the RHO1 GTPase switch or the PKC1 effector MAP kinase pathway suppresses the growth and actin defects of ypk cells. ypk lethality is also suppressed by overexpression of an uncharacterized gene termed TUS1. TUS1 is a novel RHO1 exchange factor that contributes to cell wall integrity-mediated modulation of RHO1 activity. Thus, TUS1 and the YPKs add to the growing complexity of RHO1 and PKC1 regulation in the cell integrity signaling pathway. Furthermore, our findings suggest that the YPKs are a missing link between sphingolipid signaling and the cell integrity pathway.

Cell Wall↗

PubMed2002

Elucidating TOR signaling and rapamycin action: lessons from Saccharomyces cerevisiae.

TOR (target of rapamycin) is a phosphatidylinositol kinase-related protein kinase that controls cell growth in response to nutrients. Rapamycin is an immunosuppressive and anticancer drug that acts by inhibiting TOR. The modes of action of TOR and rapamycin are remarkably conserved from S. cerevisiae to humans. The current understanding of TOR and rapamycin is derived largely from studies with S. cerevisiae. In this review, we discuss the contributions made by S. cerevisiae to understanding rapamycin action and TOR function.

Animals↗