Phosphoproteomic Analysis Reveals Interconnected System-Wide Responses to Perturbations of Kinases and Phosphatases in Yeast

Sci. Signal., 21 December 2010
Vol. 3, Issue 153, p. rs4
DOI: 10.1126/scisignal.2001182

Phosphoproteomic Analysis Reveals Interconnected System-Wide Responses to Perturbations of Kinases and Phosphatases in Yeast

  1. Bernd Bodenmiller1,2,*,,
  2. Stefanie Wanka2,3,*,
  3. Claudine Kraft4,
  4. Jörg Urban5,
  5. David Campbell6,
  6. Patrick G. Pedrioli4,,
  7. Bertran Gerrits7,§,
  8. Paola Picotti1,
  9. Henry Lam8,
  10. Olga Vitek9,
  11. Mi-Youn Brusniak6,
  12. Bernd Roschitzki7,
  13. Chao Zhang10,
  14. Kevan M. Shokat10,
  15. Ralph Schlapbach7,
  16. Alejandro Colman-Lerner11,
  17. Garry P. Nolan12,
  18. Alexey I. Nesvizhskii13,
  19. Matthias Peter4,
  20. Robbie Loewith5,
  21. Christian von Mering3, and
  22. Ruedi Aebersold1,6,14,||
  1. 1Institute of Molecular Systems Biology, ETH Zurich, 8093 Zurich, Switzerland.
  2. 2Zurich PhD Program in Molecular Life Sciences, 8057 Zurich, Switzerland.
  3. 3Institute of Molecular Life Sciences and Swiss Institute of Bioinformatics, University of Zurich, 8057 Zurich, Switzerland.
  4. 4Institute of Biochemistry, ETH Zurich, 8093 Zurich, Switzerland.
  5. 5Department of Molecular Biology, University of Geneva, Geneva 1211, Switzerland.
  6. 6Institute for Systems Biology, Seattle, WA 98103, USA.
  7. 7Functional Genomics Center Zurich, University Zurich and ETH Zurich, 8057 Zurich, Switzerland.
  8. 8Department of Chemical and Biomolecular Engineering, Hong Kong University of Science and Technology, Clear Water Bay, Hong Kong.
  9. 9Departments of Statistics and Computer Science, Purdue University, West Lafayette, IN 47107, USA.
  10. 10Howard Hughes Medical Institute and Department of Cellular and Molecular Pharmacology, University of California, San Francisco, CA 94158–2280, USA.
  11. 11Facultad de Ciencias Exactas y Naturales, University of Buenos Aires, C1428EHA Buenos Aires, Argentina.
  12. 12Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA 94305, USA.
  13. 13Department of Pathology, University of Michigan, Ann Arbor, MI 48109, USA.
  14. 14Faculty of Science, University of Zurich, 8057 Zurich, Switzerland.
  1. ||To whom correspondence should be addressed. E-mail: aebersold{at}imsb.biol.ethz.ch
  • * These authors contributed equally to this work.

  • Present address: Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA 94305, USA.

  • Present address: Scottish Institute for Cell Signalling, Sir James Black Centre, University of Dundee, Dundee, Scotland DD1 5EH, UK.

  • § Present address: Novartis Institute for Biomedical Research, Novartis International AG, CH-4002 Basel, Switzerland.

Abstract

The phosphorylation and dephosphorylation of proteins by kinases and phosphatases constitute an essential regulatory network in eukaryotic cells. This network supports the flow of information from sensors through signaling systems to effector molecules and ultimately drives the phenotype and function of cells, tissues, and organisms. Dysregulation of this process has severe consequences and is one of the main factors in the emergence and progression of diseases, including cancer. Thus, major efforts have been invested in developing specific inhibitors that modulate the activity of individual kinases or phosphatases; however, it has been difficult to assess how such pharmacological interventions would affect the cellular signaling network as a whole. Here, we used label-free, quantitative phosphoproteomics in a systematically perturbed model organism (Saccharomyces cerevisiae) to determine the relationships between 97 kinases, 27 phosphatases, and more than 1000 phosphoproteins. We identified 8814 regulated phosphorylation events, describing the first system-wide protein phosphorylation network in vivo. Our results show that, at steady state, inactivation of most kinases and phosphatases affected large parts of the phosphorylation-modulated signal transduction machinery—and not only the immediate downstream targets. The observed cellular growth phenotype was often well maintained despite the perturbations, arguing for considerable robustness in the system. Our results serve to constrain future models of cellular signaling and reinforce the idea that simple linear representations of signaling pathways might be insufficient for drug development and for describing organismal homeostasis.

Citation:

B. Bodenmiller, S. Wanka, C. Kraft, J. Urban, D. Campbell, P. G. Pedrioli, B. Gerrits, P. Picotti, H. Lam, O. Vitek, M.-Y. Brusniak, B. Roschitzki, C. Zhang, K. M. Shokat, R. Schlapbach, A. Colman-Lerner, G. P. Nolan, A. I. Nesvizhskii, M. Peter, R. Loewith, C. von Mering, and R. Aebersold, Phosphoproteomic Analysis Reveals Interconnected System-Wide Responses to Perturbations of Kinases and Phosphatases in Yeast. Sci. Signal. 3, rs4 (2010).

Rapid isolation and single-molecule analysis of ribonucleoproteins from cell lysate by SNAP-SiMPull
M. L. Rodgers, J. Paulson, and A. A. Hoskins
RNA 21, 1031-1041 (1 May 2015)

Dynamic phosphoproteomics reveals TORC1-dependent regulation of yeast nucleotide and amino acid biosynthesis
A. P. Oliveira, C. Ludwig, M. Zampieri, H. Weisser, R. Aebersold, and U. Sauer
Sci Signal 8, rs4-rs4 (28 April 2015)

Conditional regulation of Puf1p, Puf4p, and Puf5p activity alters YHB1 mRNA stability for a rapid response to toxic nitric oxide stress in yeast
J. Russo, and W. M. Olivas
Mol. Biol. Cell 26, 1015-1029 (15 March 2015)

2-Deoxyglucose Impairs Saccharomyces cerevisiae Growth by Stimulating Snf1-Regulated and {alpha}-Arrestin-Mediated Trafficking of Hexose Transporters 1 and 3
A. F. O'Donnell, R. R. McCartney, D. G. Chandrashekarappa, B. B. Zhang, J. Thorner, and M. C. Schmidt
Mol. Cell. Biol. 35, 939-955 (15 March 2015)

Differential Phosphorylation of a Regulatory Subunit of Protein Kinase CK2 by Target of Rapamycin Complex 1 Signaling and the Cdc-like Kinase Kns1
M. E. Sanchez-Casalongue, J. Lee, A. Diamond, S. Shuldiner, R. D. Moir, and I. M. Willis
J Biol Chem 290, 7221-7233 (13 March 2015)

Protein Kinase Ymr291w/Tda1 Is Essential for Glucose Signaling in Saccharomyces cerevisiae on the Level of Hexokinase Isoenzyme ScHxk2 Phosphorylation
S. Kaps, K. Kettner, R. Migotti, T. Kanashova, U. Krause, G. Rodel, G. Dittmar, and T. M. Kriegel
J Biol Chem 290, 6243-6255 (6 March 2015)

Ctk1 Function Is Necessary for Full Translation Initiation Activity in Saccharomyces cerevisiae
B. Coordes, K. M. Brunger, K. Burger, B. Soufi, J. Horenk, D. Eick, J. V. Olsen, and K. Strasser
Eukaryot Cell 14, 86-95 (1 January 2015)

Large-scale functional analysis of the roles of phosphorylation in yeast metabolic pathways
J. C. Schulz, M. Zampieri, S. Wanka, C. von Mering, and U. Sauer
Sci Signal 7, rs6-rs6 (25 November 2014)

Systematic lipidomic analysis of yeast protein kinase and phosphatase mutants reveals novel insights into regulation of lipid homeostasis
A. X. da Silveira dos Santos, I. Riezman, M.-A. Aguilera-Romero, F. David, M. Piccolis, R. Loewith, O. Schaad, and H. Riezman
Mol. Biol. Cell 25, 3234-3246 (15 October 2014)

Genetic Analysis of Resistance and Sensitivity to 2-Deoxyglucose in Saccharomyces cerevisiae
R. R. McCartney, D. G. Chandrashekarappa, B. B. Zhang, and M. C. Schmidt
Genetics 198, 635-646 (1 October 2014)

Structural Basis for Phosphorylation and Lysine Acetylation Cross-talk in a Kinase Motif Associated with Myocardial Ischemia and Cardioprotection
B. L. Parker, N. E. Shepherd, S. Trefely, N. J. Hoffman, M. Y. White, K. Engholm-Keller, B. D. Hambly, M. R. Larsen, D. E. James, S. J. Cordwell et al.
J Biol Chem 289, 25890-25906 (12 September 2014)

Single-step Enrichment by Ti4+-IMAC and Label-free Quantitation Enables In-depth Monitoring of Phosphorylation Dynamics with High Reproducibility and Temporal Resolution
E. L. de Graaf, P. Giansanti, A. F. M. Altelaar, and A. J. R. Heck
MCP 13, 2426-2434 (1 September 2014)

Calcineurin determines toxic versus beneficial responses to {alpha}-synuclein
G. Caraveo, P. K. Auluck, L. Whitesell, C. Y. Chung, V. Baru, E. V. Mosharov, X. Yan, M. Ben-Johny, M. Soste, P. Picotti et al.
Proc. Natl. Acad. Sci. USA 111, E3544-E3552 (26 August 2014)

Convergence of Ubiquitylation and Phosphorylation Signaling in Rapamycin-treated Yeast Cells
V. Iesmantavicius, B. T. Weinert, and C. Choudhary
MCP 13, 1979-1992 (1 August 2014)

Cross-talk between phosphorylation and lysine acetylation in a genome-reduced bacterium
V. van Noort, J. Seebacher, S. Bader, S. Mohammed, I. Vonkova, M. J. Betts, S. Kuhner, R. Kumar, T. Maier, M. O'Flaherty et al.
Mol Syst Biol 8, 571-571 (23 July 2014)

Regulation of yeast central metabolism by enzyme phosphorylation
A. P. Oliveira, C. Ludwig, P. Picotti, M. Kogadeeva, R. Aebersold, and U. Sauer
Mol Syst Biol 8, 623-623 (23 July 2014)

The quantitative proteome of a human cell line
M. Beck, A. Schmidt, J. Malmstroem, M. Claassen, A. Ori, A. Szymborska, F. Herzog, O. Rinner, J. Ellenberg, R. Aebersold et al.
Mol Syst Biol 7, 549-549 (23 July 2014)

Transcriptional regulation is insufficient to explain substrate-induced flux changes in Bacillus subtilis
V. Chubukov, M. Uhr, L. Le Chat, R. J. Kleijn, M. Jules, H. Link, S. Aymerich, J. Stelling, and U. Sauer
Mol Syst Biol 9, 709-709 (21 July 2014)

Phosphoproteomic analysis identifies proteins involved in transcription-coupled mRNA decay as targets of Snf1 signaling
K. A. Braun, S. Vaga, K. M. Dombek, F. Fang, S. Palmisano, R. Aebersold, and E. T. Young
Sci Signal 7, ra64-ra64 (8 July 2014)

Modulation of the Chromatin Phosphoproteome by the Haspin Protein Kinase
A. Maiolica, M. de Medina-Redondo, E. M. Schoof, A. Chaikuad, F. Villa, M. Gatti, S. Jeganathan, H. J. Lou, K. Novy, S. Hauri et al.
MCP 13, 1724-1740 (1 July 2014)

Nuclear Localization of Haa1, Which Is Linked to Its Phosphorylation Status, Mediates Lactic Acid Tolerance in Saccharomyces cerevisiae
M. Sugiyama, S.-P. Akase, R. Nakanishi, H. Horie, Y. Kaneko, and S. Harashima
Appl. Environ. Microbiol. 80, 3488-3495 (1 June 2014)

Cross-species Proteomics Reveals Specific Modulation of Signaling in Cancer and Stromal Cells by Phosphoinositide 3-kinase (PI3K) Inhibitors
V. Rajeeve, I. Vendrell, E. Wilkes, N. Torbett, and P. R. Cutillas
MCP 13, 1457-1470 (1 June 2014)

Deletion of conserved protein phosphatases reverses defects associated with mitochondrial DNA damage in Saccharomyces cerevisiae
G. Garipler, N. Mutlu, N. A. Lack, and C. D. Dunn
Proc. Natl. Acad. Sci. USA 111, 1473-1478 (28 January 2014)

Reconstitution of dynein transport to the microtubule plus end by kinesin
A. J. Roberts, B. S. Goodman, and S. L. Reck-Peterson
elife 3, e02641-e02641 (1 January 2014)

Status of Large-scale Analysis of Post-translational Modifications by Mass Spectrometry
J. V. Olsen, and M. Mann
MCP 12, 3444-3452 (1 December 2013)

Sucrose-induced Receptor Kinase SIRK1 Regulates a Plasma Membrane Aquaporin in Arabidopsis
X. N. Wu, C. Sanchez Rodriguez, H. Pertl-Obermeyer, G. Obermeyer, and W. X. Schulze
MCP 12, 2856-2873 (1 October 2013)

PhosphoChain: a novel algorithm to predict kinase and phosphatase networks from high-throughput expression data
W.-M. Chen, S. A. Danziger, J.-H. Chiang, and J. D. Aitchison
Bioinformatics 29, 2435-2444 (1 October 2013)

Identification of Direct Tyrosine Kinase Substrates Based on Protein Kinase Assay-Linked Phosphoproteomics
L. Xue, R. L. Geahlen, and W. A. Tao
MCP 12, 2969-2980 (1 October 2013)

In Scarcity and Abundance: Metabolic Signals Regulating Cell Growth
S. Saad, M. Peter, and R. Dechant
Physiology 28, 298-309 (1 September 2013)

Pyruvate decarboxylase activity is regulated by the Ser/Thr protein phosphatase Sit4p in the yeast Saccharomyces cerevisiae
L. J. de Assis, R. B. Zingali, C. A. Masuda, S. P. Rodrigues, and M. Montero-Lomeli
FEMS Yeast Res 13, 518-528 (1 September 2013)

Structure-function analysis of the 5' end of yeast U1 snRNA highlights genetic interactions with the Msl5*Mud2 branchpoint-binding complex and other spliceosome assembly factors
B. Schwer, J. Chang, and S. Shuman
Nucleic Acids Res 41, 7485-7500 (1 August 2013)

Structural and Functional Characterization of a Phosphatase Domain within Yeast General Transcription Factor IIIC
N. M. I. Taylor, S. Glatt, M. L. Hennrich, G. von Scheven, H. Grotsch, C. Fernandez-Tornero, V. Rybin, A.-C. Gavin, P. Kolb, C. W. Muller et al.
J Biol Chem 288, 15110-15120 (24 May 2013)

The PhosphoGRID Saccharomyces cerevisiae protein phosphorylation site database: version 2.0 update
I. Sadowski, B.-J. Breitkreutz, C. Stark, T.-C. Su, M. Dahabieh, S. Raithatha, W. Bernhard, R. Oughtred, K. Dolinski, K. Barreto et al.
Database 2013, bat026-bat026 (13 May 2013)

Mitochondrial association, protein phosphorylation, and degradation regulate the availability of the active Rab GTPase Ypt11 for mitochondrial inheritance
A. Lewandowska, J. Macfarlane, and J. M. Shaw
Mol. Biol. Cell 24, 1185-1195 (15 April 2013)

Response to Hyperosmotic Stress
H. Saito, and F. Posas
Genetics 192, 289-318 (1 October 2012)

Protein abundance is key to distinguish promiscuous from functional phosphorylation based on evolutionary information
E. D. Levy, S. W. Michnick, and C. R. Landry
Phil Trans R Soc B 367, 2594-2606 (19 September 2012)

An N-terminal acidic region of Sgs1 interacts with Rpa70 and recruits Rad53 kinase to stalled forks
A. M. Hegnauer, N. Hustedt, K. Shimada, B. L. Pike, M. Vogel, P. Amsler, S. M. Rubin, F. van Leeuwen, A. Guenole, H. van Attikum et al.
EMBO J. 31, 3768-3783 (12 September 2012)

Phosphoproteomic Analysis of Leukemia Cells under Basal and Drug-treated Conditions Identifies Markers of Kinase Pathway Activation and Mechanisms of Resistance
M. P. Alcolea, P. Casado, J.-C. Rodriguez-Prados, B. Vanhaesebroeck, and P. R. Cutillas
MCP 11, 453-466 (1 August 2012)

Impact of phosphoproteomics on studies of bacterial physiology
I. Mijakovic, and B. Macek
FEMS Microbiol Rev 36, 877-892 (1 July 2012)

Global Detection of Protein Kinase D-dependent Phosphorylation Events in Nocodazole-treated Human Cells
M. Franz-Wachtel, S. A. Eisler, K. Krug, S. Wahl, A. Carpy, A. Nordheim, K. Pfizenmaier, A. Hausser, and B. Macek
MCP 11, 160-170 (1 May 2012)

Computational Approaches for Analyzing Information Flow in Biological Networks
B. Kholodenko, M. B. Yaffe, and W. Kolch
Sci Signal 5, re1-re1 (17 April 2012)

Functional wiring of the yeast kinome revealed by global analysis of genetic network motifs
S. Sharifpoor, D. van Dyk, M. Costanzo, A. Baryshnikova, H. Friesen, A. C. Douglas, J.-Y. Youn, B. VanderSluis, C. L. Myers, B. Papp et al.
Genome Res 22, 791-801 (1 April 2012)

The importance of post-translational modifications in regulating Saccharomyces cerevisiae metabolism
A. P. Oliveira, and U. Sauer
FEMS Yeast Res 12, 104-117 (1 March 2012)

Phosphosite Mapping of P-type Plasma Membrane H+-ATPase in Homologous and Heterologous Environments
E. L. Rudashevskaya, J. Ye, O. N. Jensen, A. T. Fuglsang, and M. G. Palmgren
J Biol Chem 287, 4904-4913 (10 February 2012)

Dependence of Chs2 ER export on dephosphorylation by cytoplasmic Cdc14 ensures that septum formation follows mitosis
C. F. Chin, A. M. Bennett, W. K. Ma, M. C. Hall, and F. M. Yeong
Mol. Biol. Cell 23, 45-58 (1 January 2012)

Systematic Phosphorylation Analysis of Human Mitotic Protein Complexes
B. Hegemann, J. R. A. Hutchins, O. Hudecz, M. Novatchkova, J. Rameseder, M. M. Sykora, S. Liu, M. Mazanek, P. Lenart, J.-K. Heriche et al.
Sci Signal 4, rs12-rs12 (8 November 2011)

The Saccharomyces cerevisiae fermentation stress response protein Igd1p/Yfr017p regulates glycogen levels by inhibiting the glycogen debranching enzyme
C. J. Walkey, Z. Luo, C. H. Borchers, V. Measday, and H. J. J. van Vuuren
FEMS Yeast Res 11, 499-508 (1 September 2011)

Sequence, Structure, and Network Evolution of Protein Phosphorylation
C. S. H. Tan, Z. Luo, C. H. Borchers, V. Measday, and H. J. J. van Vuuren
Sci Signal 4, mr6-mr6 (19 July 2011)

ResponseNet: revealing signaling and regulatory networks linking genetic and transcriptomic screening data
A. Lan, I. Y. Smoly, G. Rapaport, S. Lindquist, E. Fraenkel, and E. Yeger-Lotem
Nucleic Acids Res 39, W424-W429 (1 July 2011)

Mitotic Substrates of the Kinase Aurora with Roles in Chromatin Regulation Identified Through Quantitative Phosphoproteomics of Fission Yeast
A. Koch, K. Krug, S. Pengelley, B. Macek, and S. Hauf
Sci Signal 4, rs6-rs6 (28 June 2011)

Network-Based Tools for the Identification of Novel Drug Targets
I. J. Farkas, T. Korcsmaros, I. A. Kovacs, A. Mihalik, R. Palotai, G. I. Simko, K. Z. Szalay, M. Szalay-Beko, T. Vellai, S. Wang et al.
Sci Signal 4, pt3-pt3 (17 May 2011)

Setting the Standards for Signal Transduction Research
J. Saez-Rodriguez, L. G. Alexopoulos, and G. Stolovitzky
Sci Signal 4, pe10-pe10 (15 February 2011)

Science Signaling Podcast: 21 December 2010
B. Bodenmiller, R. Aebersold, and A. M. VanHook
Sci Signal 3, pc23-pc23 (21 December 2010)

Science Signaling. ISSN 1937-9145 (online), 1945-0877 (print). Pre-2008: Science's STKE. ISSN 1525-8882