Springer Nature is making SARS-CoV-2 and COVID-19 research free. View research | View latest news | Sign up for updates

Phosphorylation site mapping of soluble proteins: bioinformatical filtering reveals potential plastidic phosphoproteins in Arabidopsis thaliana

  • 320 Accesses

  • 31 Citations

Abstract

Protein phosphorylation is a major mode of regulation of metabolism, gene expression, and cell architecture. A combination of phosphopeptide enrichment strategies based on TiO2 and IMAC in addition to our MudPIT strategy revealed the detection of 181 phosphorylation sites which are located on 125 potentially plastidic proteins predicted by GoMiner, TargetP/Predotar in Arabidopsis thaliana. In our study phosphorylation on serine is favored over threonine and this in turn over phosphorylation on tyrosine residues, showing a percentage of 67.4% to 24.3% to 8.3% for pS:pT:pY. Four phosphorylated residues (S208, Y239, T246 and T330), identified by our approach have been fitted to the structure of the activated form of spinach RuBisCO, which are located in close proximity to the substrate binding site for ribulosebisphosphate. Potentially, these phosphorylation sites exert a direct influence on the catalytic activity of the enzyme. Such examples show nicely the value of the presented mass spectrometric dataset for further biochemical applications, since alternative mutation analysis often turns out to be unsuccessful, caused by mutations in essential proteins which result in lethal phenotypes.

This is a preview of subscription content, log in to check access.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Abbreviations

IMAC:

Immobilized metal ion affinity chromatography

MudPIT:

Multidimensional protein identification technology

RP:

Reversed phase

RuBisCo:

Ribulose-1,5-bisphosphate carboxylase

SCX:

Strong cation exchange

References

  1. Adams WW 3rd, Demmig-Adams B, Rosenstiel TN, Ebbert V (2001) Dependence of photosynthesis and energy dissipation activity upon growth form and light environment during the winter. Photosyn Res 67:51–62

  2. Allen JF (1992) How does protein phosphorylation regulate photosynthesis? Trends Biochem Sci 17:12–17

  3. Baginsky S, Tiller K, Link G (1997) Transcription factor phosphorylation by a protein kinase associated with chloroplast RNA polymerase from mustard (Sinapis alba). Plant Mol Biol 34:181–189

  4. Beausoleil SA, Jedrychowski M, Schwartz D, Elias JE, Villen J, Li J, Cohn MA, Cantley LC, Gygi SP (2004) Large-scale characterization of HeLa cell nuclear phosphoproteins. Proc Natl Acad Sci USA 101:12130–12135

  5. Bellafiore S, Barneche F, Peltier G, Rochaix JD (2005) State transitions and light adaptation require chloroplast thylakoid protein kinase STN7. Nature 433:892–895

  6. Bennett J (1977) Phosphorylation of chloroplast membrane polypeptides. Nature 269:344–346

  7. Benschop JJ, Mohammed S, O’Flaherty M, Heck AJ, Slijper M, Menke FL (2007) Quantitative phosphoproteomics of early elicitor signaling in Arabidopsis. Mol Cell Proteomics 6:1198–1214

  8. Bhalla P, Bennett J (1987) Chloroplast phosphoproteins: phosphorylation of a 12-kDa stromal protein by the redox-controlled kinase of thylakoid membranes. Arch Biochem Biophys 252:97–104

  9. Blom N, Gammeltoft S, Brunak S (1999) Sequence and structure-based prediction of eukaryotic protein phosphorylation sites. J Mol Biol 294:1351–1362

  10. Bonardi V, Pesaresi P, Becker T, Schleiff E, Wagner R, Pfannschmidt T, Jahns P, Leister D (2005) Photosystem II core phosphorylation and photosynthetic acclimation require two different protein kinases. Nature 437:1179–1182

  11. Chevalier D, Walker JC (2005) Functional genomics of protein kinases in plants. Brief Funct Genomic Proteomic 3:362–371

  12. Conrads TP, Hood BL, Petricoin EF 3rd, Liotta LA, Veenstra TD (2005) Cancer proteomics: many technologies, one goal. Expert Rev Proteomics 2:693–703

  13. Crooks GE, Hon G, Chandonia JM, Brenner SE (2004) WebLogo: a sequence logo generator. Genome Res 14:1188–1190

  14. Dannehl H, Alexandra Herbik A, Godde D (1995) Stress induced degradation of the photosynthetic apparatus is accompanied by changes in thylakoid protein turnover and phosphorylation. Physiol Plant 93:179–186

  15. Durr E, Yu J, Krasinska KM, Carver LA, Yates JR, Testa JE, Oh P, Schnitzer JE (2004) Direct proteomic mapping of the lung microvascular endothelial cell surface in vivo and in cell culture. Nat Biotechnol 22:985–992

  16. Ebbert V, Demmig-Adams B, Adams WW 3rd, Mueh KE, Staehelin LA (2001) Correlation between persistent forms of zeaxanthin-dependent energy dissipation and thylakoid protein phosphorylation. Photosyn Res 67:63–78

  17. Emanuelsson O, Nielsen H, Brunak S, von Heijne G (2000) Predicting subcellular localization of proteins based on their N-terminal amino acid sequence. J Mol Biol 300:1005–1016

  18. Foyer CH (1985) Stromal protein phosphorylation in spinach (Spinacia oleracea) chloroplasts. Biochem J 231:97–103

  19. Goshe MB, Conrads TP, Panisko EA, Angell NH, Veenstra TD, Smith RD (2001) Phosphoprotein isotope-coded affinity tag approach for isolating and quantitating phosphopeptides in proteome-wide analyzes. Anal Chem 73:2578–2586

  20. Guitton C, Mache R (1987) Phosphorylation in vitro of the large subunit of the ribulose-1, 5-bisphosphate carboxylase and of the glyceraldehyde-3-phosphate dehydrogenase. Eur J Biochem 166:249–254

  21. Heazlewood JL, Durek P, Hummel J, Selbig J, Weckwerth W, Walther D, Schulze WX (2008) PhosPhAt: a database of phosphorylation sites in Arabidopsis thaliana and a plant-specific phosphorylation site predictor. Nucleic Acids Res 36:D1015–D1021

  22. Hunter T (1995) Protein kinases and phosphatases: the yin and yang of protein phosphorylation and signaling. Cell 80:225–236

  23. Ihnatowicz A, Pesaresi P, Lohrig K, Wolters D, Muller B, Leister D (2008) Impaired photosystem I oxidation induces STN7-dependent phosphorylation of the light-harvesting complex I protein Lhca4 in Arabidopsis thaliana. Planta 227:717–722

  24. Initiative TAG (2000) Analysis of the genome sequence of the flowering plant Arabidopsis thaliana. Nature 408:796–815

  25. Jensen SS, Larsen MR (2007) Evaluation of the impact of some experimental procedures on different phosphopeptide enrichment techniques. Rapid Commun Mass Spectrom 21:3635–3645

  26. Kennelly PJ (2002) Protein kinases and protein phosphatases in prokaryotes: a genomic perspective. FEMS Microbiol Lett 206:1–8

  27. Kerk D, Conley TR, Rodriguez FA, Tran HT, Nimick M, Muench DG, Moorhead GB (2006) A chloroplast-localized dual-specificity protein phosphatase in Arabidopsis contains a phylogenetically dispersed and ancient carbohydrate-binding domain, which binds the polysaccharide starch. Plant J 46:400–413

  28. Kwon SJ, Choi EY, Seo JB, Park OK (2007) Isolation of the Arabidopsis phosphoproteome using a biotin-tagging approach. Mol Cells 24:268–275

  29. Larsen MR, Thingholm TE, Jensen ON, Roepstorff P, Jorgensen TJ (2005) Highly selective enrichment of phosphorylated peptides from peptide mixtures using titanium dioxide microcolumns. Mol Cell Proteomics 4:873–886

  30. Laurie S, Halford NG (2001) The role of protein kinases in the regulation of plant growth and development. Plant Growth Regul 34:253–265

  31. Leister D (2003) Chloroplast research in the genomic age. Trends Genet 19:47–56

  32. Lewandrowski U, Sickmann A, Cesaro L, Brunati AM, Toninello A, Salvi M (2008) Identification of new tyrosine phosphorylated proteins in rat brain mitochondria. FEBS Lett 582:1104–1110

  33. Link G (2003) Redox regulation of chloroplast transcription. Antioxid Redox Signal 5:79–87

  34. Liu H, Sadygov RG, Yates JR 3rd (2004) A model for random sampling and estimation of relative protein abundance in shotgun proteomics. Anal Chem 76:4193–4201

  35. Lohaus C, Nolte A, Bluggel M, Scheer C, Klose J, Gobom J, Schuler A, Wiebringhaus T, Meyer HE, Marcus K (2007) Multidimensional chromatography: a powerful tool for the analysis of membrane proteins in mouse brain. J Proteome Res 6:105–113

  36. Nuhse TS, Stensballe A, Jensen ON, Peck SC (2004) Phosphoproteomics of the Arabidopsis plasma membrane and a new phosphorylation site database. Plant Cell 16:2394–2405

  37. Oda Y, Nagasu T, Chait BT (2001) Enrichment analysis of phosphorylated proteins as a tool for probing the phosphoproteome. Nat Biotechnol 19:379–382

  38. Olsen JV, Blagoev B, Gnad F, Macek B, Kumar C, Mortensen P, Mann M (2006) Global, in vivo, and site-specific phosphorylation dynamics in signaling networks. Cell 127:635–648

  39. Peltier JB, Ytterberg AJ, Sun Q, van Wijk KJ (2004) New functions of the thylakoid membrane proteome of Arabidopsis thaliana revealed by a simple, fast, and versatile fractionation strategy. J Biol Chem 279:49367–49383

  40. Pesaresi P, Kleine T, Leister D (2009) Thylakoid protein phosphorylation and its impact on short- and long-term acclimation of photosynthesis. In: Buchner TB, Ewingen NH (eds) Theory and applications in energy, biotechnology and nanotechnology. Nova Science Publishers, Hauppauge (in press). ISBN: 978-1-60692-719-9

  41. Richly E, Leister D (2004) An improved prediction of chloroplast proteins reveals diversities and commonalities in the chloroplast proteomes of Arabidopsis and rice. Gene 329:11–16

  42. Rokka A, Aro EM, Herrmann RG, Andersson B, Vener AV (2000) Dephosphorylation of photosystem II reaction center proteins in plant photosynthetic membranes as an immediate response to abrupt elevation of temperature. Plant Physiol 123:1525–1536

  43. Romeis T, Ludwig AA, Martin R, Jones JD (2001) Calcium-dependent protein kinases play an essential role in a plant defence response. EMBO J 20:5556–5567

  44. Schaller F, Biesgen C, Mussig C, Altmann T, Weiler EW (2000) 12-Oxophytodienoate reductase 3 (OPR3) is the isoenzyme involved in jasmonate biosynthesis. Planta 210:979–984

  45. Schliebner I, Pribil M, Zühlke J, Dietzmann A, Leister D (2008) A survey of chloroplast protein kinases and phosphatases in Arabidopsis thaliana. Curr Genom 9:184–190

  46. Schubert M, Petersson UA, Haas BJ, Funk C, Schroder WP, Kieselbach T (2002) Proteome map of the chloroplast lumen of Arabidopsis thaliana. J Biol Chem 277:8354–8365

  47. Small I, Peeters N, Legeai F, Lurin C (2004) Predotar: a tool for rapidly screening proteomes for N-terminal targeting sequences. Proteomics 4:1581–1590

  48. Snyders S, Kohorn BD (1999) TAKs, thylakoid membrane protein kinases associated with energy transduction. J Biol Chem 274:9137–9140

  49. Soll J, Bennett J (1988) Localization of a 64-kDa phosphoprotein in the lumen between the outer and inner envelopes of pea chloroplasts. Eur J Biochem 175:301–307

  50. Sugiyama N, Nakagami H, Mochida K, Daudi A, Tomita M, Shirasu K, Ishihama Y (2008) Large-scale phosphorylation mapping reveals the extent of tyrosine phosphorylation in Arabidopsis. Mol Systems Biol 4:193

  51. Sun Q, Zybailov B, Majeran W, Friso G, Olinares PD, van Wijk KJ (2009) PPDB, the plant proteomics database at Cornell. Nucleic Acids Res 37:D969–D974

  52. Taylor TC, Andersson I (1997) The structure of the complex between rubisco and its natural substrate ribulose 1, 5-bisphosphate. J Mol Biol 265:432–444

  53. Taylor SW, Fahy E, Ghosh SS (2003) Global organellar proteomics. Trends Biotechnol 21:82–88

  54. Tetlow IJ, Wait R, Lu Z, Akkasaeng R, Bowsher CG, Esposito S, Kosar-Hashemi B, Morell MK, Emes MJ (2004) Protein phosphorylation in amyloplasts regulates starch branching enzyme activity and protein–protein interactions. Plant Cell 16:694–708

  55. Toroser D, Huber SC (1997) Protein phosphorylation as a mechanism for osmotic-stress activation of sucrose–phosphate synthase in spinach leaves. Plant Physiol 114:947–955

  56. Toroser D, McMichael R Jr, Krause KP, Kurreck J, Sonnewald U, Stitt M, Huber SC (1999) Site-directed mutagenesis of serine 158 demonstrates its role in spinach leaf sucrose–phosphate synthase modulation. Plant J 17:407–413

  57. van Bentem S, Anrather D, Dohnal I, Roitinger E, Csaszar E, Joore J, Buijnink J, Carreri A, Forzani C, Lorkovic ZJ, Barta A, Lecourieux D, Verhounig A, Jonak C, Hirt H (2008) Site-specific phosphorylation profiling of Arabidopsis proteins by mass spectrometry and peptide chip analysis. J Proteome Res 7:2458–2470

  58. Vener AV (2007) Environmentally modulated phosphorylation and dynamics of proteins in photosynthetic membranes. Biochim Biophys Acta 1767:449–457

  59. Vener AV, Ohad I, Andersson B (1998) Protein phosphorylation and redox sensing in chloroplast thylakoids. Curr Opin Plant Biol 1:217–223

  60. von Mering C, Jensen LJ, Kuhn M, Chaffron S, Doerks T, Kruger B, Snel B, Bork P (2007) STRING 7–recent developments in the integration and prediction of protein interactions. Nucleic Acids Res 35:D358–D362

  61. Waegemann K, Soll J (1996) Phosphorylation of the transit sequence of chloroplast precursor proteins. J Biol Chem 271:6545–6554

  62. Washburn MP, Wolters D, Yates JR 3rd (2001) Large-scale analysis of the yeast proteome by multidimensional protein identification technology. Nat Biotechnol 19:242–247

  63. Weiler EW (1997) Octadecanoid-mediated signal transduction in higher plants. Naturwissenschaften 84:340–349

  64. Zeeberg BR, Feng W, Wang G, Wang MD, Fojo AT, Sunshine M, Narasimhan S, Kane DW, Reinhold WC, Lababidi S, Bussey KJ, Riss J, Barrett JC, Weinstein JN (2003) GoMiner: a resource for biological interpretation of genomic and proteomic data. Genome Biol 4:R28

  65. Zhou H, Watts JD, Aebersold R (2001) A systematic approach to the analysis of protein phosphorylation. Nat Biotechnol 19:375–378

  66. Zybailov B, Rutschow H, Friso G, Rudella A, Emanuelsson O, Sun Q, van Wijk KJ (2008) Sorting signals, N-terminal modifications and abundance of the chloroplast proteome. PLoS ONE 3:e1994

Download references

Acknowledgments

We are grateful to the German Research Foundation (DFG) for generous financial support in The Arabidopsis Functional Genomics Network (AFGN); WO 1214/1-2, LE 1265/3-2.

Author information

Correspondence to Dirk Andreas Wolters.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary Table 1 (XLS 179 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Lohrig, K., Müller, B., Davydova, J. et al. Phosphorylation site mapping of soluble proteins: bioinformatical filtering reveals potential plastidic phosphoproteins in Arabidopsis thaliana . Planta 229, 1123–1134 (2009). https://doi.org/10.1007/s00425-009-0901-y

Download citation

Keywords

  • Arabidopsis
  • Chloroplast
  • Immobilized metal ion affinity chromatography (IMAC)
  • TiO2
  • Multidimensional protein identification technology (MudPIT)
  • Phosphorylation