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Integrative Features of the Yeast Phosphoproteome and Protein–Protein Interaction Map

DOI: 10.1371/journal.pcbi.1001064

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Following recent advances in high-throughput mass spectrometry (MS)–based proteomics, the numbers of identified phosphoproteins and their phosphosites have greatly increased in a wide variety of organisms. Although a critical role of phosphorylation is control of protein signaling, our understanding of the phosphoproteome remains limited. Here, we report unexpected, large-scale connections revealed between the phosphoproteome and protein interactome by integrative data-mining of yeast multi-omics data. First, new phosphoproteome data on yeast cells were obtained by MS-based proteomics and unified with publicly available yeast phosphoproteome data. This revealed that nearly 60% of ~6,000 yeast genes encode phosphoproteins. We mapped these unified phosphoproteome data on a yeast protein–protein interaction (PPI) network with other yeast multi-omics datasets containing information about proteome abundance, proteome disorders, literature-derived signaling reactomes, and in vitro substratomes of kinases. In the phospho-PPI, phosphoproteins had more interacting partners than nonphosphoproteins, implying that a large fraction of intracellular protein interaction patterns (including those of protein complex formation) is affected by reversible and alternative phosphorylation reactions. Although highly abundant or unstructured proteins have a high chance of both interacting with other proteins and being phosphorylated within cells, the difference between the number counts of interacting partners of phosphoproteins and nonphosphoproteins was significant independently of protein abundance and disorder level. Moreover, analysis of the phospho-PPI and yeast signaling reactome data suggested that co-phosphorylation of interacting proteins by single kinases is common within cells. These multi-omics analyses illuminate how wide-ranging intracellular phosphorylation events and the diversity of physical protein interactions are largely affected by each other.


[1]  Hunter T (2000) Signaling–2000 and beyond. Cell 100: 113–127.
[2]  Manning G, Whyte DB, Martinez R, Hunter T, Sudarsanam S (2002) The protein kinase complement of the human genome. Science 298: 1912–1934.
[3]  Pawson T, Nash P (2000) Protein-protein interactions define specificity in signal transduction. Genes Dev 14: 1027–1047.
[4]  Aebersold R, Mann M (2003) Mass spectrometry-based proteomics. Nature 422: 198–207.
[5]  Cravatt BF, Simon GM, Yates JR 3rd (2007) The biological impact of mass-spectrometry-based proteomics. Nature 450: 991–1000.
[6]  Huang PH, White FM (2008) Phosphoproteomics: unraveling the signaling web. Mol Cell 31: 777–781.
[7]  Witze ES, Old WM, Resing KA, Ahn NG (2007) Mapping protein post-translational modifications with mass spectrometry. Nat Methods 4: 798–806.
[8]  Beausoleil SA, Jedrychowski M, Schwartz D, Elias JE, Villen J, et al. (2004) Large-scale characterization of HeLa cell nuclear phosphoproteins. Proc Natl Acad Sci U S A 101: 12130–12135.
[9]  Molina H, Horn DM, Tang N, Mathivanan S, Pandey A (2007) Global proteomic profiling of phosphopeptides using electron transfer dissociation tandem mass spectrometry. Proc Natl Acad Sci U S A 104: 2199–2204.
[10]  Olsen JV, Blagoev B, Gnad F, Macek B, Kumar C, et al. (2006) Global, in vivo, and site-specific phosphorylation dynamics in signaling networks. Cell 127: 635–648.
[11]  Sugiyama N, Masuda T, Shinoda K, Nakamura A, Tomita M, et al. (2007) Phosphopeptide enrichment by aliphatic hydroxy acid-modified metal oxide chromatography for nano-LC-MS/MS in proteomics applications. Mol Cell Proteomics 6: 1103–1109.
[12]  Olsen JV, Vermeulen M, Santamaria A, Kumar C, Miller ML, et al. (2010) Quantitative phosphoproteomics reveals widespread full phosphorylation site occupancy during mitosis. Sci Signal 3: ra3.
[13]  Villen J, Beausoleil SA, Gerber SA, Gygi SP (2007) Large-scale phosphorylation analysis of mouse liver. Proc Natl Acad Sci U S A 104: 1488–1493.
[14]  Chi A, Huttenhower C, Geer LY, Coon JJ, Syka JE, et al. (2007) Analysis of phosphorylation sites on proteins from Saccharomyces cerevisiae by electron transfer dissociation (ETD) mass spectrometry. Proc Natl Acad Sci U S A 104: 2193–2198.
[15]  Ficarro SB, McCleland ML, Stukenberg PT, Burke DJ, Ross MM, et al. (2002) Phosphoproteome analysis by mass spectrometry and its application to Saccharomyces cerevisiae. Nat Biotechnol 20: 301–305.
[16]  Holt LJ, Tuch BB, Villen J, Johnson AD, Gygi SP, et al. (2009) Global analysis of Cdk1 substrate phosphorylation sites provides insights into evolution. Science 325: 1682–1686.
[17]  Gnad F, Ren S, Cox J, Olsen JV, Macek B, et al. (2007) PHOSIDA (phosphorylation site database): management, structural and evolutionary investigation, and prediction of phosphosites. Genome Biol 8: R250.
[18]  Diella F, Gould CM, Chica C, Via A, Gibson TJ (2008) Phospho.ELM: a database of phosphorylation sites—update 2008. Nucleic Acids Res 36: D240–244.
[19]  UniProt Consortium (2010) The Universal Protein Resource (UniProt) in 2010. Nucleic Acids Res 38: D142–148.
[20]  Giot L, Bader JS, Brouwer C, Chaudhuri A, Kuang B, et al. (2003) A protein interaction map of Drosophila melanogaster. Science 302: 1727–1736.
[21]  Han JD, Bertin N, Hao T, Goldberg DS, Berriz GF, et al. (2004) Evidence for dynamically organized modularity in the yeast protein-protein interaction network. Nature 430: 88–93.
[22]  Ho Y, Gruhler A, Heilbut A, Bader GD, Moore L, et al. (2002) Systematic identification of protein complexes in Saccharomyces cerevisiae by mass spectrometry. Nature 415: 180–183.
[23]  Ito T, Chiba T, Ozawa R, Yoshida M, Hattori M, et al. (2001) A comprehensive two-hybrid analysis to explore the yeast protein interactome. Proc Natl Acad Sci U S A 98: 4569–4574.
[24]  Uetz P, Giot L, Cagney G, Mansfield TA, Judson RS, et al. (2000) A comprehensive analysis of protein-protein interactions in Saccharomyces cerevisiae. Nature 403: 623–627.
[25]  Li S, Armstrong CM, Bertin N, Ge H, Milstein S, et al. (2004) A map of the interactome network of the metazoan C. elegans. Science 303: 540–543.
[26]  Rual JF, Venkatesan K, Hao T, Hirozane-Kishikawa T, Dricot A, et al. (2005) Towards a proteome-scale map of the human protein-protein interaction network. Nature 437: 1173–1178.
[27]  Tarassov K, Messier V, Landry CR, Radinovic S, Serna Molina MM, et al. (2008) An in vivo map of the yeast protein interactome. Science 320: 1465–1470.
[28]  Barabasi AL, Albert R (1999) Emergence of scaling in random networks. Science 286: 509–512.
[29]  Girvan M, Newman ME (2002) Community structure in social and biological networks. Proc Natl Acad Sci U S A 99: 7821–7826.
[30]  Goh KI, Oh E, Jeong H, Kahng B, Kim D (2002) Classification of scale-free networks. Proc Natl Acad Sci U S A 99: 12583–12588.
[31]  Watts DJ, Strogatz SH (1998) Collective dynamics of ‘small-world’ networks. Nature 393: 440–442.
[32]  Hong EL, Balakrishnan R, Dong Q, Christie KR, Park J, et al. (2008) Gene Ontology annotations at SGD: new data sources and annotation methods. Nucleic Acids Res 36: D577–581.
[33]  Salwinski L, Miller CS, Smith AJ, Pettit FK, Bowie JU, et al. (2004) The Database of Interacting Proteins: 2004 update. Nucleic Acids Res 32: D449–451.
[34]  Ivanic J, Yu X, Wallqvist A, Reifman J (2009) Influence of protein abundance on high-throughput protein-protein interaction detection. PLoS One 4: e5815.
[35]  von Mering C, Krause R, Snel B, Cornell M, Oliver SG, et al. (2002) Comparative assessment of large-scale data sets of protein-protein interactions. Nature 417: 399–403.
[36]  Yu H, Zhu X, Greenbaum D, Karro J, Gerstein M (2004) TopNet: a tool for comparing biological sub-networks, correlating protein properties with topological statistics. Nucleic Acids Res 32: 328–337.
[37]  Newman JR, Ghaemmaghami S, Ihmels J, Breslow DK, Noble M, et al. (2006) Single-cell proteomic analysis of S. cerevisiae reveals the architecture of biological noise. Nature 441: 840–846.
[38]  Dosztanyi Z, Chen J, Dunker AK, Simon I, Tompa P (2006) Disorder and sequence repeats in hub proteins and their implications for network evolution. J Proteome Res 5: 2985–2995.
[39]  Dunker AK, Cortese MS, Romero P, Iakoucheva LM, Uversky VN (2005) Flexible nets. The roles of intrinsic disorder in protein interaction networks. FEBS J 272: 5129–5148.
[40]  Haynes C, Oldfield CJ, Ji F, Klitgord N, Cusick ME, et al. (2006) Intrinsic disorder is a common feature of hub proteins from four eukaryotic interactomes. PLoS Comput Biol 2: e100.
[41]  Gsponer J, Futschik ME, Teichmann SA, Babu MM (2008) Tight regulation of unstructured proteins: from transcript synthesis to protein degradation. Science 322: 1365–1368.
[42]  Landry CR, Levy ED, Michnick SW (2009) Weak functional constraints on phosphoproteomes. Trends Genet 25: 193–197.
[43]  Tan CS, Bodenmiller B, Pasculescu A, Jovanovic M, Hengartner MO, et al. (2009) Comparative analysis reveals conserved protein phosphorylation networks implicated in multiple diseases. Sci Signal 2: ra39.
[44]  Taylor IW, Linding R, Warde-Farley D, Liu Y, Pesquita C, et al. (2009) Dynamic modularity in protein interaction networks predicts breast cancer outcome. Nat Biotechnol 27: 199–204.
[45]  Tan CS, J?rgensen C, Linding R (2010) Roles of “junk phosphorylation” in modulating biomolecular association of phosphorylated proteins? Cell Cycle 9: 1276–1280.
[46]  Walhout AJ, Vidal M (2001) Protein interaction maps for model organisms. Nat Rev Mol Cell Biol 2: 55–62.
[47]  Fiedler D, Braberg H, Mehta M, Chechik G, Cagney G, et al. (2009) Functional organization of the S. cerevisiae phosphorylation network. Cell 136: 952–963.
[48]  Linding R, Jensen LJ, Ostheimer GJ, van Vugt MA, J?rgensen C, et al. (2007) Systematic discovery of in vivo phosphorylation networks. Cell 129: 1415–1426.
[49]  Gong W, Zhou D, Ren Y, Wang Y, Zuo Z, et al. (2008) PepCyber:P~PEP: a database of human protein protein interactions mediated by phosphoprotein-binding domains. Nucleic Acids Res 36: D679–683.
[50]  Yachie N, Saito R, Sugahara J, Tomita M, Ishihama Y (2009) In silico analysis of phosphoproteome data suggests a rich-get-richer process of phosphosite accumulation over evolution. Mol Cell Proteomics 8: 1061–1071.
[51]  Maslov S, Sneppen K (2002) Specificity and stability in topology of protein networks. Science 296: 910–913.
[52]  Walhout AJ, Reboul J, Shtanko O, Bertin N, Vaglio P, et al. (2002) Integrating interactome, phenome, and transcriptome mapping data for the C. elegans germline. Curr Biol 12: 1952–1958.
[53]  Ptacek J, Devgan G, Michaud G, Zhu H, Zhu X, et al. (2005) Global analysis of protein phosphorylation in yeast. Nature 438: 679–684.
[54]  Imamura H, Yachie N, Saito R, Ishihama Y, Tomita M (2010) Towards the systematic discovery of signal transduction networks using phosphorylation dynamics data. BMC Bioinformatics 11: 232.
[55]  Murray DB, Beckmann M, Kitano H (2007) Regulation of yeast oscillatory dynamics. Proc Natl Acad Sci U S A 104: 2241–2246.
[56]  Masuda T, Tomita M, Ishihama Y (2008) Phase transfer surfactant-aided trypsin digestion for membrane proteome analysis. J Proteome Res 7: 731–740.
[57]  Rappsilber J, Ishihama Y, Mann M (2003) Stop and go extraction tips for matrix-assisted laser desorption/ionization, nanoelectrospray, and LC/MS sample pretreatment in proteomics. Anal Chem 75: 663–670.
[58]  Rappsilber J, Mann M, Ishihama Y (2007) Protocol for micro-purification, enrichment, pre-fractionation and storage of peptides for proteomics using StageTips. Nat Protoc 2: 1896–1906.
[59]  Kyono Y, Sugiyama N, Imami K, Tomita M, Ishihama Y (2008) Successive and selective release of phosphorylated peptides captured by hydroxy acid-modified metal oxide chromatography. J Proteome Res 7: 4585–4593.
[60]  Ishihama Y, Rappsilber J, Andersen JS, Mann M (2002) Microcolumns with self-assembled particle frits for proteomics. J Chromatogr A 979: 233–239.
[61]  Shimizu K, Muraoka Y, Hirose S, Tomii K, Noguchi T (2007) Predicting mostly disordered proteins by using structure-unknown protein data. BMC Bioinformatics 8: 78.


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