ORCID Profile
0000-0003-0729-9335
Current Organisation
Flinders University
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Biochemistry and Cell Biology | Signal Transduction | Cell Metabolism | Cell and Nuclear Division
Nutrition | Expanding Knowledge in the Biological Sciences |
Publisher: Springer Science and Business Media LLC
Date: 20-01-2020
DOI: 10.1038/S42255-019-0157-1
Abstract: Central to cellular metabolism and cell proliferation are highly conserved signalling pathways controlled by mammalian target of rapamycin (mTOR) and AMP-activated protein kinase (AMPK)
Publisher: The Company of Biologists
Date: 2013
DOI: 10.1242/JCS.122531
Abstract: In all eukaryotes tight control of mitogen-activated protein kinase (MAPK) activity plays an important role in modulating intracellular signalling in response to changing environments. The fission yeast MAPK Sty1 (also known as Spc1 or Phh1) is highly activated in response to a variety of external stresses. To avoid segregation of damaged organelles or chromosomes, strong Sty1 activation transiently blocks mitosis and cell ision until such stresses have been dealt with. MAPK phosphatases dephosphorylate Sty1 to reduce kinase activity. Therefore, tight control of MAPK phosphatases is central for stress adaptation and for cell ision to resume. In contrast to Pyp1, the fission yeast Pyp2 MAPK phosphatase is under environmental control. Pyp2 has a unique sequence (the linker region) between the catalytic domain and the amino-terminal MAPK binding site. Here we show that the Pyp2 linker region is a destabilisation domain. Furthermore, the linker region is highly phosphorylated to increase Pyp2 protein stability and this phosphorylation is Sty1 dependent. Our data suggests that Sty1 activation promotes Pyp2 phosphorylation to increase the stability of the phosphatase. This MAPK-dependent Pyp2 stabilisation allows cells to attenuate MAPK signalling and resume cell ision, once stresses have been dealt with.
Publisher: The Company of Biologists
Date: 15-10-2011
DOI: 10.1242/JCS.083683
Abstract: The coordination of cell ision and growth in response to changes in nutrient supply is mediated by TOR signalling. In fission yeast, increased nutrient provision transiently delays mitotic onset without affecting growth. The result is an increase in cell size at ision. We find that this block to cell ision relies upon TOR and MAPK signalling and that mitotic entry during recovery from this block is regulated by the Aurora kinase Ark1. We show that Ark1 phosphorylation of polo kinase Plo1 within the linker region between the kinase domain and polo boxes drives Plo1 onto the spindle poles where it promotes mitosis. Interestingly, the use of Ark1 to phosphorylate Plo1 and promote mitotic entry is dependent on the environment.
Publisher: Elsevier BV
Date: 02-2015
Publisher: Elsevier BV
Date: 02-2022
Publisher: The Company of Biologists
Date: 06-2009
DOI: 10.1242/JCS.049387
Abstract: TOR signalling coordinates growth and ision to control cell size. Inhibition of Schizosaccharomyces pombe Tor1, in response to a reduction in the quality of the nitrogen source (nutrient stress), promotes mitotic onset through activation of the mitogen-activated protein kinase (MAPK) Sty1 (also known as Spc1). Here we show that `nutrient starvation' (complete withdrawal of nitrogen or leucine) blocks mitotic commitment by altering Sty1 signalling and that different degrees of Sty1 activation determine these differences in mitotic commitment decisions. Mammals contain one TOR kinase, whereas yeasts contain two. In each case, they comprise two distinct complexes: TORC1 and TORC2. We find that nutrient-stress-induced control of mitotic onset, through Tor1, is regulated through changes in TORC1 signalling. In minimal medium, Tor1 interacts with the TORC1 component Mip1 (raptor), and overexpression of tor1+ generates growth defects reminiscent of TORC1 mutants. Strains lacking the TORC2-specific components Sin1 and Ste20 (rictor) still advance mitotic onset in response to nutrient stress. By contrast, Mip1 and the downstream effector Gad8 (a S6K kinase homologue), like Tor1, are essential for nutrient stress to advance mitotic onset. We conclude that S. pombe Tor1 and Tor2 can both act in TORC1. However, it is the inhibition of Tor1 as part of TORC1 that promotes mitosis following nutrient stress.
Publisher: Springer Science and Business Media LLC
Date: 05-2005
DOI: 10.1038/NATURE03590
Abstract: Stress-activated mitogen-activated protein kinase cascades instigate a range of changes to enable eukaryotic cells to cope with particular insults. In Schizosaccharomyces pombe these responses include the transcription of specific gene sets and inhibition of entry into mitosis. The S. pombe stress response pathway (SRP) also promotes commitment to mitosis in unperturbed cell cycles to allow cells to match their rate of ision with nutrient availability. The nature of this SRP function in cell cycle control is unknown. Entry into mitosis is controlled by mitosis-promoting factor (MPF Cdc2/cyclin B) activity. Inhibitory phosphorylation of Cdc2 by Wee1 kinase inactivates MPF until Cdc25 removes this phosphate to promote mitosis. The balance between Wee1 and Cdc25 activities is influenced by the recruitment of polo kinase (Plo1) to the spindle pole body (SPB). The SPB component Cut12 mediates this recruitment. Hyper-activating mutations in either cut12 or plo1 enable Cdc25-defective cells to enter mitosis. The hyperactive cut12.s11 mutation suppresses cdc25.22, as it promotes recruitment of active Plo1 to interphase SPBs. Here we show that the SRP promotes phosphorylation of Plo1 on Ser 402. In unperturbed cell cycles, SRP-mediated phosphorylation of Ser 402 promotes Plo1 recruitment to SPBs and thus commitment to mitosis. Ser 402 phosphorylation also ensures efficient reinitiation of cell tip growth and cell ision during recovery from particular stresses. Thus, phosphorylation of Plo1 Ser 402 not only enables SRP signalling to modulate the timing of mitotic commitment in response to nutrient status in unperturbed cycles, but also promotes the return to normal cell cycle control after stress.
Publisher: Cold Spring Harbor Laboratory
Date: 03-2016
Abstract: Here, we summarize the composition and uses of Schizosaccharomyces pombe media and discuss key issues for consideration in the generation of S. pombe cultures. We discuss the concept of “culture memory,” in which the growth state and stress experienced by a strain during storage, propagation, and starter culture preparation can alter experimental outcomes at later stages. We also describe the triggers that are widely used to manipulate signaling through the environment sensing pathways.
Publisher: Rockefeller University Press
Date: 06-1998
Abstract: Formins are involved in erse aspects of morphogenesis, and share two regions of homology: FH1 and FH2. We describe a new formin homology region, FH3. FH3 is an amino-terminal domain that differs from the Rho binding site identified in Bni1p and p140mDia. The Schizosaccharomyces pombe formin Fus1 is required for conjugation, and is localized to the projection tip in cells of mating pairs. We replaced genomic fus1+ with green fluorescent protein (GFP)- tagged versions that lacked either the FH1, FH2, or FH3 domain. Deletion of any FH domain essentially abolished mating. FH3, but neither FH1 nor FH2, was required for Fus1 localization. An FH3 domain–GFP fusion protein localized to the projection tips of mating pairs. Thus, the FH3 domain alone can direct protein localization. The FH3 domains of both Fus1 and the S. pombe cytokinesis formin Cdc12 were able to localize GFP to the spindle pole body in half of the late G2 cells in a vegetatively growing population. Expression of both FH3-GFP fusions also affected cytokinesis. Overexpression of the spindle pole body component Sad1 altered the distribution of both Sad1 and the FH3-GFP domain. Together these data suggest that proteins at multiple sites can interact with FH3 domains.
Publisher: Elsevier BV
Date: 05-2019
Publisher: Springer Science and Business Media LLC
Date: 02-12-2021
DOI: 10.1038/S41587-021-01099-9
Abstract: Protein phosphorylation dynamically integrates environmental and cellular information to control biological processes. Identifying functional phosphorylation amongst the thousands of phosphosites regulated by a perturbation at a global scale is a major challenge. Here we introduce 'personalized phosphoproteomics', a combination of experimental and computational analyses to link signaling with biological function by utilizing human phenotypic variance. We measure in idual subject phosphoproteome responses to interventions with corresponding phenotypes measured in parallel. Applying this approach to investigate how exercise potentiates insulin signaling in human skeletal muscle, we identify both known and previously unidentified phosphosites on proteins involved in glucose metabolism. This includes a cooperative relationship between mTOR and AMPK whereby the former directly phosphorylates the latter on S377, for which we find a role in metabolic regulation. These results establish personalized phosphoproteomics as a general approach for investigating the signal transduction underlying complex biology.
Publisher: EMBO
Date: 27-10-2017
Publisher: Public Library of Science (PLoS)
Date: 08-03-2017
Publisher: Springer Science and Business Media LLC
Date: 06-02-2019
DOI: 10.1038/S41598-018-37509-3
Abstract: Ammonia can be utilised as an alternative nitrogen source to glutamine to support cell proliferation. However, the underlying molecular mechanisms and whether all cells have this ability is not fully understood. We find that eleven cancer and non-cancerous cell lines have opposite abilities to tolerate and utilise ammonia to support proliferation in a glutamine-depleted environment. HEK293, Huh7, T47D and MCF7 cells can use ammonia, when starved of glutamine, to support proliferation to varying degrees. Glutamine depletion reduced mTORC1 activity, while additional ammonia supplementation diminished this mTORC1 inhibition. Depletion of glutamine promoted a rapid and transient activation of AMPK, whereas, additional ammonia supplementation blocked this starvation-induced AMPK activation. As expected, drug-induced AMPK activation reduced cell proliferation in glutamine-depleted cells supplemented with ammonia. Surprisingly, mTORC1 activity was largely unchanged despite the enhanced AMPK activity, suggesting that AMPK does not inhibit mTORC1 signalling under these conditions. Finally, glutamate dehydrogenase (GDH) inhibition, a key enzyme regulating ammonia assimilation, leads to AMPK activation, mTORC1 inhibition and reduced proliferation. Ammonia provides an alternative nitrogen source that aids certain cancer cells ability to thrive in nutrient-deprived environment. The ability of cells to utilise ammonia as a nitrogen source is intricately linked to AMPK, mTORC1 and GDH.
Publisher: Elsevier BV
Date: 08-1998
DOI: 10.1016/S0960-9822(98)70397-5
Abstract: During the G1 phase of the cell cycle, cells of the fission yeast Schizosaccharomyces pombe can be induced to mate by nitrogen starvation and the presence of mating pheromones. Polarised growth towards cells of the opposite mating type (P or M) leads to the formation of a projection tip and, upon contact, localised cell wall degradation results in conjugation and cell fusion [1]. Here, we have investigated the role of microtubules in this process. We describe a previously unidentified microtubule-organising centre (MTOC) that forms at projection tips upon cell-to-cell contact, before cells fuse. Treatment of mating cells with the microtubule-destabilising drug thiabendazole (TBZ) showed that microtubule integrity was required for mating at two distinct stages: during projection tip formation and cell fusion. Projection tip formation requires filamentous (F) actin function [2] and microtubules are required for the localisation of F actin to the projection tip. We also identify a role during mating for Mad2--a mitotic checkpoint protein that is required in all eukaryotes to maintain the mitotic state in response to microtubule depolymerisation [3]. S. pombe mad2 mutant cells were compromised in their ability to mate upon removal of TBZ, indicating that in fission yeast, in the absence of microtubules, Mad2 is also required to maintain mating competence.
Publisher: The Royal Society
Date: 05-2018
DOI: 10.1098/RSOB.180015
Abstract: Nutrient fluctuations in the cellular environment promote changes in cell metabolism and growth to adapt cell proliferation accordingly. The target of rapamycin (TOR) signalling network plays a key role in the coordination of growth and cell proliferation with the nutrient environment and, importantly, nutrient limitation reduces TOR complex 1 (TORC1) signalling. We have performed global quantitative fitness profiling of the collection of Schizosaccharomyces pombe strains from which non-essential genes have been deleted. We identified genes that regulate fitness when cells are grown in a nutrient-rich environment compared with minimal environments, with varying nitrogen sources including ammonium, glutamate and proline. In addition, we have performed the first global screen for genes that regulate fitness when both TORC1 and TORC2 signalling is reduced by Torin1. Analysis of genes whose deletions altered fitness when nutrients were limited, or when TOR signalling was compromised, identified a large number of genes that regulate transmembrane transport, transcription and chromatin organization/regulation and vesicle-mediated transport. The ability to tolerate reduced TOR signalling placed demands upon a large number of biological processes including autophagy, mRNA metabolic processing and nucleocytoplasmic transport. Importantly, novel biological processes and all processes known to be regulated by TOR were identified in our screens. In addition, deletion of 62 genes conserved in humans gave rise to strong sensitivity or resistance to Torin1, and 29 of these 62 genes have novel links to TOR signalling. The identification of chromatin and transcriptional regulation, nutritional uptake and transport pathways in this powerful genetic model now paves the way for a molecular understanding of how cells adapt to the chronic and acute fluctuations in nutrient supply that all eukaryotes experience at some stage, and which is a key feature of cancer cells within solid tumours.
Publisher: Public Library of Science (PLoS)
Date: 18-05-2016
Publisher: Springer Science and Business Media LLC
Date: 21-10-2007
DOI: 10.1038/NCB1646
Abstract: The coupling of growth to cell cycle progression allows eukaryotic cells to ide at particular sizes depending on nutrient availability. In fission yeast, this coupling involves the Spc1/Sty1 mitogen-activated protein kinase (MAPK) pathway working through Polo kinase recruitment to the spindle pole bodies (SPBs). Here we report that changes in nutrients influence TOR signalling, which modulates Spc1/Sty1 activity. Rapamycin-induced inhibition of TOR signalling advanced mitotic onset, mimicking the reduction in cell size at ision seen after shifts to poor nitrogen sources. Gcn2, an effector of TOR signalling and modulator of translation, regulates the Pyp2 phosphatase that in turn modulates Spc1/Sty1 activity. Rapamycin- or nutrient-induced stimulation of Spc1/Sty1 activity promotes Polo kinase SPB recruitment and Cdc2 activation to advance mitotic onset. This advanced mitotic onset is abolished in cells depleted of Gcn2, Pyp2, or Spc1/Sty1 or on blockage of Spc1/Sty1-dependent Polo SPB recruitment. Therefore, TOR signalling modulates mitotic onset through the stress MAPK pathway via the Pyp2 phosphatase.
Publisher: Elsevier BV
Date: 04-2003
DOI: 10.1016/S0960-9822(03)00205-7
Abstract: The spindle checkpoint inhibits anaphase until all chromosomes have established bipolar attachment. Two kinetochore states trigger this checkpoint. The absence of microtubules activates the attachment response, while the inability of attached microtubules to generate tension triggers the tension/orientation response. The single aurora kinase of budding yeast, Ipl1, is required for the tension/orientation, but not attachment, response. In contrast, we find that the single aurora kinase of fission yeast, Ark1, is required for the attachment response. Having established that the initiator codon assigned to ark1(+) was incorrect and that Ark1-associated kinase activity depended upon survivin function and phosphorylation, we found that the loss of Ark1 from kinetochores by either depletion or use of a survivin mutant overides the checkpoint response to microtubule depolymerization. Ark1/survivin function was not required for the association of Bub1 or Mad3 with the kinetochores. However, it was required for two aspects of Mad2 function that accompany checkpoint activation: full-scale association with kinetochores and formation of a complex with Mad3. Neither the phosphorylation of histone H3 that accompanies chromosome condensation nor condensin recruitment to mitotic chromatin were seen when Ark1 function was compromised. Cytokinesis was not affected by Ark1 depletion or expression of the "kinase dead" ark1.K118R mutant.
Publisher: Public Library of Science (PLoS)
Date: 21-05-2014
Publisher: The Company of Biologists
Date: 2014
DOI: 10.1242/JCS.146373
Abstract: The Target Of Rapamycin TOR kinase regulates cell growth and ision. Rapamycin only inhibits a subset of TOR activities. Here we show that in contrast to the mild impact of rapamycin on cell ision, blocking the catalytic site of TOR with the Torin1 inhibitor completely arrests growth without cell death in S.pombe. A mutation of the Tor2 TORC1 glycine residue (G2040D) that lies adjacent to the key Torin interacting tryptophan provides Torin1 resistance, confirming Torin1's specificity for TOR. Using this mutation we show that Torin1 advanced mitotic onset before inducing growth arrest. In contrast to TOR inhibition with Rapamycin, regulation by either Wee1 or Cdc25 was sufficient for this Torin1 induced advanced mitosis. Torin1 promoted a Polo and Cdr2 kinase controlled drop in Wee1 levels. Experiments in human cell lines re-capitulated these yeast observations mTOR was inhibited by Torin1, Wee1 levels declined and mitotic commitment was advanced in HeLa cells. Thus, the regulation of the mitotic inhibitor Wee1 by TOR signalling is a conserved mechanism that helps to couple cell cycle and growth controls.
Publisher: The Royal Society
Date: 04-2021
DOI: 10.1098/RSOB.200405
Abstract: Fluctuations in TOR, AMPK and MAP-kinase signalling maintain cellular homeostasis and coordinate growth and ision with environmental context. We have applied quantitative, SILAC mass spectrometry to map TOR and nutrient-controlled signalling in the fission yeast Schizosaccharomyces pombe . Phosphorylation levels at more than 1000 sites were altered following nitrogen stress or Torin1 inhibition of the TORC1 and TORC2 networks that comprise TOR signalling. One hundred and thirty of these sites were regulated by both perturbations, and the majority of these (119) new targets have not previously been linked to either nutritional or TOR control in either yeasts or humans. Elimination of AMPK inhibition of TORC1, by removal of AMPK α ( ssp2::ura4 + ), identified phosphosites where nitrogen stress-induced changes were independent of TOR control. Using a yeast strain with an ATP analogue-sensitized Cdc2 kinase, we excluded sites that were changed as an indirect consequence of mitotic control modulation by nitrogen stress or TOR signalling. Nutritional control of gene expression was reflected in multiple targets in RNA metabolism, while significant modulation of actin cytoskeletal components points to adaptations in morphogenesis and cell integrity networks. Reduced phosphorylation of the MAPKK Byr1, at a site whose human equivalent controls docking between MEK and ERK, prevented sexual differentiation when resources were sparse but not eliminated.
Publisher: Informa UK Limited
Date: 07-1995
Publisher: The Company of Biologists
Date: 17-07-2012
DOI: 10.1242/BIO.20122022
Abstract: TOR (Target Of Rapamycin) signalling coordinates cell growth and ision in response to changes in the nutritional environment of the cell. TOR kinases form two distinct complexes: TORC1 and TORC2. In mammals, the TORC1 controlled S6K1 kinase phosphorylates the ribosomal protein S6 thereby co-ordinating cell size and nutritional status. We show that the Schizosaccharomyces pombe AGC kinase Gad8 co-immunoprecipitates with the ribosomal protein S6 (Rps6) and regulates its phosphorylation status. It has previously been shown that Gad8 is phosphorylated by TORC2. Consistent with this, we find that TORC2 as well as TORC1 modulates Rps6 phosphorylation. Therefore, S6 phosphorylation in fission yeast actually represents a read-out of the combined activities of TORC1 and TORC2. In contrast, we find that the in vivo phosphorylation status of Maf1 (a repressor of RNA polymerase III) specifically correlates with TORC1 activity.
Publisher: Springer Science and Business Media LLC
Date: 21-09-2017
DOI: 10.1038/S41598-017-09722-Z
Abstract: The glucocorticoid receptor (GR) is essential for the stress response in mammals. We investigated potential non-transcriptional roles of GR in cellular stress response using fission yeast as a model.We surprisingly discovered marked heat stress resistance in yeast ectopically expressing human GR, which required expression of both the N-terminal transactivation domain, and the C-terminal ligand binding domain, but not the DNA-binding domain of the GR. This effect was not affected by GR ligand exposure, and occurred without significant GR nuclear accumulation. Mechanistically, the GR survival effect required Hsp104, and, indeed, GR expression increased Hsp104 expression. Proteomic analysis revealed GR binding to translasome components, including eIF3, a known partner for Sty1, a pattern of protein interaction which we confirmed using yeast two-hybrid studies.Taken together, we find evidence for a novel pathway conferring stress resistance in yeast that can be activated by the human GR, acting by protein-protein mechanisms in the cytoplasm. This suggests that in organisms where GR is natively expressed, GR likely contributes to stress responses through non-transcriptional mechanisms in addition to its well-established transcriptional responses.
Publisher: American Society for Cell Biology (ASCB)
Date: 08-1999
Abstract: Polo kinases execute multiple roles during cell ision. The fission yeast polo related kinase Plo1 is required to assemble the mitotic spindle, the prophase actin ring that predicts the site for cytokinesis and for septation after the completion of mitosis ( Ohkuraet al., 1995 Bahler et al., 1998 ). We show that Plo1 associates with the mitotic but not interphase spindle pole body (SPB). SPB association of Plo1 is the earliest fission yeast mitotic event recorded to date. SPB association is strong from mitotic commitment to early anaphase B, after which the Plo1 signal becomes very weak and finally disappears upon spindle breakdown. SPB association of Plo1 requires mitosis-promoting factor (MPF) activity, whereas its disassociation requires the activity of the anaphase-promoting complex. The stf1.1 mutation bypasses the usual requirement for the MPF activator Cdc25 ( Hudson et al., 1990 ). Significantly, Plo1 associates inappropriately with the interphase SPB of stf1.1 cells. These data are consistent with the emerging theme from many systems that polo kinases participate in the regulation of MPF to determine the timing of commitment to mitosis and may indicate that pole association is a key aspect of Plo1 function. Plo1 does not associate with the SPB when septation is inappropriately driven by deregulation of the Spg1 pathway and remains SPB associated if septation occurs in the presence of a spindle. Thus, neither Plo1 recruitment to nor its departure from the SPB are required for septation however, overexpression ofplo1 + activates the Spg1 pathway and causes transient Cdc7 recruitment to the SPB and multiple rounds of septation.
Publisher: Rockefeller University Press
Date: 18-11-2013
Abstract: TOR (target of rapamycin) signaling coordinates cell growth, metabolism, and cell ision through tight control of signaling via two complexes, TORC1 and TORC2. Here, we show that fission yeast TOR kinases and mTOR are phosphorylated on an evolutionarily conserved residue of their ATP-binding domain. The Gad8 kinase (AKT homologue) phosphorylates fission yeast Tor1 at this threonine (T1972) to reduce activity. A T1972A mutation that blocked phosphorylation increased Tor1 activity and stress resistance. Nitrogen starvation of fission yeast inhibited TOR signaling to arrest cell cycle progression in G1 phase and promoted sexual differentiation. Starvation and a Gad8/T1972-dependent decrease in Tor1 (TORC2) activity was essential for efficient cell cycle arrest and differentiation. Experiments in human cell lines recapitulated these yeast observations, as mTOR was phosphorylated on T2173 in an AKT-dependent manner. In addition, a T2173A mutation increased mTOR activity. Thus, TOR kinase activity can be reduced through AGC kinase–controlled phosphorylation to generate physiologically significant changes in TOR signaling.
Publisher: The Royal Society
Date: 04-2023
DOI: 10.1098/RSOB.230021
Abstract: Expression and activity of the AMP-activated protein kinase (AMPK) α 1 catalytic subunit of the heterotrimeric kinase significantly correlates with poor outcome for colorectal cancer patients. Hence there is considerable interest in uncovering signalling vulnerabilities arising from this oncogenic elevation of AMPK α 1 signalling. We have therefore attenuated mammalian target of rapamycin (mTOR) control of AMPK α 1 to generate a mutant colorectal cancer in which AMPK α 1 signalling is elevated because AMPK α 1 serine 347 cannot be phosphorylated by mTORC1. The elevated AMPK α 1 signalling in this HCT116 α 1.S347A cell line confers hypersensitivity to growth inhibition by metformin. Complementary chemical approaches confirmed this relationship in both HCT116 and the genetically distinct HT29 colorectal cells, as AMPK activators imposed vulnerability to growth inhibition by metformin in both lines. Growth inhibition by metformin was abolished when AMPK α 1 kinase was deleted. We conclude that elevated AMPK α 1 activity modifies the signalling architecture in such a way that metformin treatment compromises cell proliferation. Not only does this mutant HCT116 AMPKα1-S347A line offer an invaluable resource for future studies, but our findings suggest that a robust biomarker for chronic AMPK α 1 activation for patient stratification could herald a place for the well-tolerated drug metformin in colorectal cancer therapy.
Publisher: The Royal Society
Date: 03-2016
DOI: 10.1098/RSOB.150189
Abstract: Cell proliferation, metabolism, migration and survival are coordinated through the tight control of two target of rapamycin (TOR) kinase complexes: TORC1 and TORC2. Here, we show that a novel phosphorylation of fission yeast Gad8 (AGC kinase) on the evolutionarily conserved threonine 6 (Thr6) prevents the physical association between Gad8 and TORC2. Accordingly, this block to protein interactions by Gad8 Thr6 phosphorylation decreases TORC2-controlled activation of Gad8. Likewise, phosphorylation of Gad8 Thr6, possibly by PKC, prevents the association of Gad8 with TORC2 thereby increasing TORC2 activity, because it reduces Gad8-mediated feedback inhibition of TORC2. Consistently, the introduction of a Gad8 T6D mutant, that mimics phosphorylation, increased TORC2 activity. Increased PKC Pck2 expression prevented Gad8–TORC2 binding and so reduced the TORC2-mediated phosphorylation of Gad8 serine 546 that activates Gad8. Interestingly, independent of the Ser546 phosphorylation status, Gad8 Thr6 phosphorylation is important for remodelling the actin cytoskeleton and survival upon potassium ion and heat stresses. In contrast, Ser546 phosphorylation is required for the control of G1 arrest, mating, cell length at ision and vascular size. Finally, these findings reveal a novel mode of TORC2 activation that is essential for cell survival following stress.
Publisher: The Company of Biologists
Date: 2016
DOI: 10.1242/JCS.190124
Abstract: The timing of cell ision is controlled by the coupled regulation of growth and ision. The TOR signalling network synchronises these processes with the environmental setting. Here we describe a novel interaction of the fission yeast TOR Complex 2 (TORC2) with the Cytokinetic Actomyosin Ring (CAR), and a novel role for TORC2 in regulating the timing and fidelity of cytokinesis. Disruption of TORC2 or its localisation results in defects in CAR morphology and constriction. We provide evidence that a myosin II, Myp2, and myosin V, Myo51, play roles in recruiting TORC2 to the CAR. We show that Myp2 and TORC2 are co-dependent upon each other for their normal localisation to the cytokinetic machinery. We go on to show that TORC2 dependent phosphorylation of Acp1 (Actin Capping Protein, a known regulator of cytokinesis) controls CAR stability and the modulation of CAPZA/BAcp1/2 heterodimer formation and is essential for survival upon stress. Thus TORC2 localisation to the CAR and TORC2 dependent CAPZAAcp1 phosphorylation contributes to timely control and fidelity of cytokinesis and cell ision.
Publisher: Wiley
Date: 15-03-2001
Publisher: Elsevier BV
Date: 12-2012
DOI: 10.1016/J.CEB.2012.08.003
Abstract: Tight coupling between cell growth and cell cycle progression allows cells to adjust their size to the demands of proliferation in varying nutrient environments. Target of rapamycin (TOR) signalling pathways co-ordinate cell growth with cell cycle progression in response to altered nutritional availability. To increase cell size the active TOR Complex 1 (TORC1) promotes cell growth to delay mitosis and cell ision, whereas under limited nutrients TORC1 activity is decreased to reduce cell size. It remains unclear why cell size is subject to such tight control. Recent evidence suggests that in addition to modulating cell size, changes in nutrient availability also alter nuclear:cytoplasmic (N/C) ratios and may therefore compromise optimal cellular physiology. This could explain why cells increase their size when conditions are favourable, despite being competent to survive at a smaller size if necessary.
Publisher: The Company of Biologists
Date: 2019
DOI: 10.1242/JCS.223925
Abstract: AMP-activated kinase (AMPK) and Target Of Rapamycin (TOR) signalling coordinate cell growth, proliferation, metabolism, and cell survival with the nutrient environment of cells. The poor vasculature and nutritional stress experienced by cells in solid tumours raises the question: how do they assimilate sufficient nutrients to survive? Here, we show that human and fission yeast cells import ATP and AMP from their external environment to regulate AMPK and TOR signalling. Exposure of fission yeast and human cells to external AMP impeded cell growth, however, in yeast this restraining impact required AMPK. In contrast, external ATP rescued the growth defect of yeast mutants with reduced TORC1 signalling, furthermore, exogenous ATP transiently enhanced TORC1 signalling in both yeast and human cell lines. Addition of the PANX1 channel inhibitor probenecid blocked ATP import into human cell lines suggesting that this channel may be responsible for both ATP release and uptake in mammals. In light of these findings it is possible that the higher extracellular ATP concentration reported in solid tumours is both scavenged and recognized as an additional energy source beneficial for cells growth.
Publisher: Portland Press Ltd.
Date: 20-01-2009
DOI: 10.1042/BST0370273
Abstract: Cell growth and cell ision are coupled to control cell size and this co-ordination is often modulated by the availability of nutrients. In many eukaryotes, TOR (target of rapamycin) signalling is involved in coupling nutrient sensing to cell growth and ision controls. Nutrient stress inhibits TOR signalling to advance the timing of cell ision and thus leads to continued cell ision at reduced cell size. Most changes in the environment stimulate stress-activated MAPK (mitogen-activated protein kinase) signalling pathways. Several MAPKs also have a general role in the control of mitotic onset and cell ision. In the present paper, I discuss the interplay between two major signalling pathways, the TOR and the stress MAPK signalling pathways, in controlling mitotic commitment, with the main focus being on fission yeast (Schizosaccharomyces pombe).
Publisher: Life Science Alliance, LLC
Date: 04-02-2022
Abstract: Cells respond to changing nutrient environments by adjusting the abundance of surface nutrient transporters and receptors. This can be achieved by modulating ubiquitin-dependent endocytosis, which in part is regulated by the NEDD4 family of E3 ligases. Here we report novel regulation of Pub1, a fission yeast Schizosaccharomyces pombe member of the NEDD4-family of E3 ligases. We show that nitrogen stress inhibits Pub1 function, thereby increasing the abundance of the amino acid transporter Aat1 at the plasma membrane and enhancing sensitivity to the toxic arginine analogue canavanine. We show that TOR complex 2 (TORC2) signalling negatively regulates Pub1, thus TORC2 mutants under nutrient stress have decreased Aat1 at the plasma membrane and are resistant to canavanine. Inhibition of TORC2 signalling increases Pub1 phosphorylation, and this is dependent on Gsk3 activity. Addition of the Tor inhibitor Torin1 increases phosphorylation of Pub1 at serine 199 (S199) by 2.5-fold, and Pub1 protein levels in S199A phospho-ablated mutants are reduced. S199 is conserved in NEDD4 and is located immediately upstream of a WW domain required for protein interaction. Together, we describe how the major TORC2 nutrient-sensing signalling network regulates environmental control of Pub1 to modulate the abundance of nutrient transporters.
Start Date: 03-2018
End Date: 05-2021
Amount: $389,030.00
Funder: Australian Research Council
View Funded ActivityStart Date: 12-2022
End Date: 12-2025
Amount: $480,564.00
Funder: Australian Research Council
View Funded Activity