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Chemical Genetics of AGC-kinases Reveals Shared Targets of Ypk1, Protein Kinase A and Sch9*

  • Michael Plank
    Correspondence
    To whom correspondence may be addressed. Tel.: 41223796190;
    Footnotes
    Affiliations
    Department of Molecular Biology, University of Geneva, CH-1211, Geneva, Switzerland

    National Centre of Competence in Research - Chemical Biology, University of Geneva, CH-1211, Geneva, Switzerland
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  • Mariya Perepelkina
    Footnotes
    Affiliations
    Department of Molecular Biology, University of Geneva, CH-1211, Geneva, Switzerland
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  • Markus Müller
    Affiliations
    National Centre of Competence in Research - Chemical Biology, University of Geneva, CH-1211, Geneva, Switzerland

    Swiss Institute of Bioinformatics, CH-1015 Lausanne, Switzerland
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  • Stefania Vaga
    Footnotes
    Affiliations
    Department of Biology, Institute of Molecular Systems Biology, ETH Zürich, CH-8093 Zürich, Switzerland
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  • Xiaoming Zou
    Affiliations
    Department of Molecular Biology, University of Geneva, CH-1211, Geneva, Switzerland
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  • Clélia Bourgoint
    Affiliations
    Department of Molecular Biology, University of Geneva, CH-1211, Geneva, Switzerland
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  • Marina Berti
    Affiliations
    Department of Molecular Biology, University of Geneva, CH-1211, Geneva, Switzerland
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  • Jacques Saarbach
    Affiliations
    National Centre of Competence in Research - Chemical Biology, University of Geneva, CH-1211, Geneva, Switzerland

    Department of Organic Chemistry, University of Geneva, CH-1211, Geneva, Switzerland
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  • Steven Haesendonckx
    Affiliations
    Department of Molecular Biology, University of Geneva, CH-1211, Geneva, Switzerland
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  • Nicolas Winssinger
    Affiliations
    National Centre of Competence in Research - Chemical Biology, University of Geneva, CH-1211, Geneva, Switzerland

    Department of Organic Chemistry, University of Geneva, CH-1211, Geneva, Switzerland
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  • Ruedi Aebersold
    Affiliations
    Department of Biology, Institute of Molecular Systems Biology, ETH Zürich, CH-8093 Zürich, Switzerland

    Faculty of Science, University of Zurich, CH-8006, Zurich, Switzerland
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  • Robbie Loewith
    Correspondence
    To whom correspondence may be addressed. Tel.: 41223796190;
    Affiliations
    Department of Molecular Biology, University of Geneva, CH-1211, Geneva, Switzerland

    National Centre of Competence in Research - Chemical Biology, University of Geneva, CH-1211, Geneva, Switzerland
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  • Author Footnotes
    * The Loewith lab receives funds from the Canton of Geneva, the Swiss National Science Foundation (#51NF40-185898) and the European Research Council (#TORCH/TENDO) and is part of the National Center for Excellence in Research for Chemical Biology. We also acknowledge support from PhosphonetX and SignalX group grants of the Swiss National Science Foundation SystemsX program (#51RTP0_151037). The authors declare that they have no conflicts of interest with the contents of this article.
    This article contains supplemental Figures and Tables.
    ¶¶ These authors contributed equally to this work.
    ‖‖ Current address: Centre for Host-Microbiome Interactions, Dental Institute, King's College London, London, UK.
    ‡‡‡ Current address: Group of viral vectors screening, Advanced Cell Technology Division, BIOCAD, Svyazi str. 34-A, Strelna, 198515 Saint-Petersburg, Russian Federation.
Open AccessPublished:February 26, 2020DOI:https://doi.org/10.1074/mcp.RA120.001955
      Protein phosphorylation cascades play a central role in the regulation of cell growth and protein kinases PKA, Sch9 and Ypk1 take center stage in regulating this process in S. cerevisiae. To understand how these kinases co-ordinately regulate cellular functions we compared the phospho-proteome of exponentially growing cells without and with acute chemical inhibition of PKA, Sch9 and Ypk1. Sites hypo-phosphorylated upon PKA and Sch9 inhibition were preferentially located in RRxS/T-motifs suggesting that many are directly phosphorylated by these enzymes. Interestingly, when inhibiting Ypk1 we not only detected several hypo-phosphorylated sites in the previously reported RxRxxS/T-, but also in an RRxS/T-motif. Validation experiments revealed that neutral trehalase Nth1, a known PKA target, is additionally phosphorylated and activated downstream of Ypk1. Signaling through Ypk1 is therefore more closely related to PKA- and Sch9-signaling than previously appreciated and may perform functions previously only attributed to the latter kinases.

      Graphical Abstract

      Cell growth is dynamic and highly regulated by signaling pathways that are conserved across evolution. To accomplish this regulation, eukaryotes have developed intricate means to assess growth conditions and to rapidly communicate this information to the processes controlling the accumulation of mass, the modification of cellular volume and of membrane surface area. Although many signal transduction pathways are involved in this regulation, those employing AGC-family kinases (named after Protein Kinases A, G and C) are prominent.
      The target of rapamycin complexes 1 and 2 (TORC1 and TORC2) are central sensors of environmental conditions and regulators of cell growth (
      • Loewith R.
      • Hall M.N.
      Target of rapamycin (TOR) in nutrient signaling and growth control.
      ). Both complexes exert their functions by phosphorylating AGC-kinases as their main targets. In S. cerevisiae TORC1 primarily responds to changes in carbon and nitrogen availability and regulates ribosome biogenesis, cell cycle progression and stress responses via the AGC-kinase Sch9, like S6K downstream of mammalian TORC1 (
      • Loewith R.
      • Hall M.N.
      Target of rapamycin (TOR) in nutrient signaling and growth control.
      ,
      • Urban J.
      • Soulard A.
      • Huber A.
      • Lippman S.
      • Mukhopadhyay D.
      • Deloche O.
      • Wanke V.
      • Anrather D.
      • Ammerer G.
      • Riezman H.
      • Broach J.R.
      • Virgilio C.D.
      • Hall M.N.
      • Loewith R.
      Sch9 Is a Major Target of TORC1 in Saccharomyces cerevisiae.
      ).
      Another AGC-kinase, protein kinase A (PKA)
      The abbreviations used are:
      PKA
      Protein kinase A
      AGC-kinases
      Protein kinases of the family comprising PKA, PKG and PKC
      TORC1/TORC2
      TOR complex 1/2
      1NM-PP1
      C3–1′-naphthyl-methyl PP1
      FDR
      false-discovery rate
      GO
      gene ontology
      PSM
      peptide spectrum match.
      1The abbreviations used are:PKA
      Protein kinase A
      AGC-kinases
      Protein kinases of the family comprising PKA, PKG and PKC
      TORC1/TORC2
      TOR complex 1/2
      1NM-PP1
      C3–1′-naphthyl-methyl PP1
      FDR
      false-discovery rate
      GO
      gene ontology
      PSM
      peptide spectrum match.
      , performs many, if not most of its functions in parallel to Sch9 by regulating an overlapping set of functions and potentially by cross-talk (
      • Broach J.R.
      Nutritional control of growth and development in yeast.
      ,
      • Schmelzle T.
      • Beck T.
      • Martin D.E.
      • Hall M.N.
      Activation of the RAS/cyclic AMP pathway suppresses a TOR deficiency in yeast.
      ,
      • Zaman S.
      • Lippman S.I.
      • Schneper L.
      • Slonim N.
      • Broach J.R.
      Glucose regulates transcription in yeast through a network of signaling pathways.
      ). Indeed, Sch9 was originally identified by virtue of its ability to suppress growth phenotypes associated with loss of PKA activity (
      • Toda T.
      • Cameron S.
      • Sass P.
      • Wigler M.
      SCH9, a gene of Saccharomyces cerevisiae that encodes a protein distinct from, but functionally and structurally related to, cAMP-dependent protein kinase catalytic subunits.
      ). Reciprocally, hyper-activation of PKA signaling can suppress phenotypes linked to the loss of Sch9 activity (
      • Schmelzle T.
      • Beck T.
      • Martin D.E.
      • Hall M.N.
      Activation of the RAS/cyclic AMP pathway suppresses a TOR deficiency in yeast.
      ).
      Tpk1, Tpk2, and Tpk3 are the partially redundant paralogs of the catalytic subunit of PKA. When cells are starved of carbon, cAMP levels are low and, therefore, PKA is kept inactive by its regulatory subunit Bcy1. Glucose addition induces activation of the adenylate cyclase Cyr1/Cdc35 via the small GTPase Ras1/2 and the G protein-coupled receptor Gpr1. The subsequent increase in cAMP levels triggers the dissociation of Bcy1 from the Tpks allowing them to phosphorylate their substrates (
      • Broach J.R.
      RAS genes in Saccharomyces cerevisiae: signal transduction in search of a pathway.
      ,
      • Toda T.
      • Cameron S.
      • Sass P.
      • Zoller M.
      • Wigler M.
      Three different genes in S. cerevisiae encode the catalytic subunits of the cAMP-dependent protein kinase.
      ). Additionally, cAMP-independent activation of PKA has been reported (
      • Peeters T.
      • Louwet W.
      • Gelade R.
      • Nauwelaers D.
      • Thevelein J.M.
      • Versele M.
      Kelch-repeat proteins interacting with the G protein Gpa2 bypass adenylate cyclase for direct regulation of protein kinase A in yeast.
      ). In addition to cell growth, Tpk effectors influence many other processes including carbohydrate metabolism, cell cycle progression, sporulation, pseudohyphal development and longevity by controlling the activities of metabolic enzymes, transcription and autophagy factors (
      • Smith A.
      • Ward M.P.
      • Garrett S.
      Yeast PKA represses Msn2p/Msn4p-dependent gene expression to regulate growth, stress response and glycogen accumulation.
      ,
      • Shenhar G.
      • Kassir Y.
      A positive regulator of mitosis, Sok2, functions as a negative regulator of meiosis in Saccharomyces cerevisiae.
      ,
      • Longo V.D.
      The Ras and Sch9 pathways regulate stress resistance and longevity.
      ,
      • Van de Velde S.
      • Thevelein J.M.
      Cyclic AMP-protein kinase A and Snf1 signaling mechanisms underlie the superior potency of sucrose for induction of filamentation in Saccharomyces cerevisiae.
      ).
      Similarly to TORC1, TORC2 phosphorylates and activates AGC-kinases, including Ypk1 and its redundant paralog Ypk2, in their hydrophobic motif (
      • Kamada Y.
      • Fujioka Y.
      • Suzuki N.N.
      • Inagaki F.
      • Wullschleger S.
      • Loewith R.
      • Hall M.N.
      • Ohsumi Y.
      Tor2 directly phosphorylates the AGC kinase Ypk2 to regulate actin polarization.
      ,
      • Niles B.J.
      • Mogri H.
      • Hill A.
      • Vlahakis A.
      • Powers T.
      Plasma membrane recruitment and activation of the AGC kinase Ypk1 is mediated by target of rapamycin complex 2 (TORC2) and its effector proteins Slm1 and Slm2.
      ). Deletion of YPK2 produces no obvious phenotype suggesting that it only plays a minor role in cell growth control (
      • Chen P.
      • Lee K.S.
      • Levin D.E.
      A pair of putative protein kinase genes (YPK1 and YPK2) is required for cell growth in Saccharomyces cerevisiae.
      ). Recent work has demonstrated that TORC2 is regulated downstream of membrane tension (
      • Berchtold D.
      • Piccolis M.
      • Chiaruttini N.
      • Riezman I.
      • Riezman H.
      • Roux A.
      • Walther T.C.
      • Loewith R.
      Plasma membrane stress induces relocalization of Slm proteins and activation of TORC2 to promote sphingolipid synthesis.
      ), oxidative stress (
      • Niles B.J.
      • Powers T.
      TOR complex 2–Ypk1 signaling regulates actin polarization via reactive oxygen species.
      ) and carbon cues (
      • Hatano T.
      • Morigasaki S.
      • Tatebe H.
      • Ikeda K.
      • Shiozaki K.
      Fission yeast Ryh1 GTPase activates TOR Complex 2 in response to glucose.
      ). In turn, Ypk1, which is homologous to the mTORC2 substrate SGK in humans (
      • Eltschinger S.
      • Loewith R.
      TOR complexes and the maintenance of cellular homeostasis.
      ,
      • Casamayor A.
      • Torrance P.D.
      • Kobayashi T.
      • Thorner J.
      • Alessi D.R.
      Functional counterparts of mammalian protein kinases PDK1 and SGK in budding yeast.
      ), couples TORC2 signals to the regulation of membrane lipid biosynthesis and the regulation of cell surface area.
      Despite their central role in the regulation of fundamental cellular processes and intense efforts in understanding the functions controlled by these kinases, a systematic assessment of their targets has been lacking to date. A previous study aimed at systematically defining changes in the phospho-proteome associated with the absence of protein kinases, using kinase deletion strains (
      • Bodenmiller B.
      • Aebersold R.
      Quantitative analysis of protein phosphorylation on a system-wide scale by mass spectrometry-based proteomics.
      ). However, this approach is limited to the study of non-essential kinases and allows cells to adapt to the absence of a kinase.
      To overcome these limitations, we employed yeast strains expressing analog-sensitive (
      • Bishop A.C.
      • Buzko O.
      • Shokat K.M.
      Magic bullets for protein kinases.
      ) variants of PKA, Sch9 and Ypk1. Mutation of the gatekeeper residue of these kinases allows binding of the bulky ATP-analog C3–1′-naphthyl-methyl PP1 (1NM-PP1), thus preventing ATP-binding and rendering the enzymes inactive. Using this selective and acute way of kinase inhibition, we explored the phospho-protein targets downstream of each of these major AGC-kinases by means of quantitative mass spectrometry. In these phospho-proteomics data sets we identified both known and potentially new targets of each tested kinase. As expected, we found extensive substrate overlap between PKA and Sch9. Unexpected was our finding that several substrates were shared between PKA and/or Sch9 and Ypk1 and that many sites hypo-phosphorylated upon Ypk1 inhibition resided in an RRxS/T-motif, which has previously been associated with PKA and Sch9, rather than Ypk1.
      Among the numerous potentially new kinase-substrate relationships discovered in this study, we chose neutral trehalase Nth1 as a candidate for follow-up experiments. Nth1 has been employed as a model PKA-substrate in multiple previous studies (
      • van der Plaat J.B.
      Cyclic 3′,5′-adenosine monophosphate stimulates trehalose degradation in baker's yeast.
      ,
      • Schepers W.
      • Van Zeebroeck G.
      • Pinkse M.
      • Verhaert P.
      • Thevelein J.M.
      In vivo phosphorylation of Ser21 and Ser83 during nutrient-induced activation of the yeast protein kinase A (PKA) target trehalase.
      ). Trehalose is a disaccharide that functions as a stress-protectant and reserve carbohydrate under adverse conditions (
      • Panek A.
      Synthesis of trehalose by baker's yeast (Saccharomyces cerevisiae).
      ,
      • Singer M.A.
      • Lindquist S.
      Multiple effects of trehalose on protein folding in vitro and in vivo.
      ). Upon return to favorable conditions, trehalose is converted into two molecules of glucose by trehalases, including the neutral trehalase Nth1 (
      • Schepers W.
      • Van Zeebroeck G.
      • Pinkse M.
      • Verhaert P.
      • Thevelein J.M.
      In vivo phosphorylation of Ser21 and Ser83 during nutrient-induced activation of the yeast protein kinase A (PKA) target trehalase.
      ). The regulation of Nth1 by PKA has long been recognized as important for cell survival (
      • van der Plaat J.B.
      Cyclic 3′,5′-adenosine monophosphate stimulates trehalose degradation in baker's yeast.
      ,
      • Wera S.
      • De Schrijver E.
      • Geyskens I.
      • Nwaka S.
      • Thevelein J.M.
      Opposite roles of trehalase activity in heat-shock recovery and heat-shock survival in Saccharomyces cerevisiae.
      ). Here, we site-specifically validated that Ypk1-, as well as PKA-inhibition reduced its phosphorylation of an RRxS-site and showed that this is associated with reduced trehalase activity.
      These findings highlight the need for revisiting the Ypk1 consensus motif and prompt further investigation of the relationship of PKA and TORC1-Sch9 signaling on one hand and TORC2-Ypk1 on the other hand.

      DATA AVAILABILITY

      DDA data have been deposited to the ProteomeXchange Consortium via the PRIDEpartner repository with the data set identifier PXD015668 and 10.6019/PXD015668. Spectra were additionally deposited to MS-Viewer and are accessible using key 99yh6tladc.
      PRM data have been deposited at Panorama Public: https://panoramaweb.org/wMKBjd.url and are accessible at ProteomeXchange via identifier PXD015760.

      Acknowledgments

      We thank Claudio de Virgilio (University of Fribourg), Marko Kaksonen and Andreas Boland (both University of Geneva) for helpful advice and Johan Thevelein (KU Leuven) and Ji-Sook Hahn (Seoul National University) for reagents.

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