Tau Protein pp 179-213 | Cite as

The Study of Posttranslational Modifications of Tau Protein by Nuclear Magnetic Resonance Spectroscopy: Phosphorylation of Tau Protein by ERK2 Recombinant Kinase and Rat Brain Extract, and Acetylation by Recombinant Creb-Binding Protein

  • Haoling Qi
  • Clément Despres
  • Sudhakaran Prabakaran
  • François-Xavier Cantrelle
  • Béatrice Chambraud
  • Jeremy Gunawardena
  • Guy Lippens
  • Caroline Smet-Nocca
  • Isabelle Landrieu
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1523)

Abstract

Nuclear magnetic resonance (NMR) spectroscopy can be used as an analytical tool to investigate posttranslational modifications of protein. NMR is a valuable tool to map the interaction regions of protein partners. Here, we present protocols that have been developed in the course of our studies of the neuronal Tau protein. Tau is found aggregated in the neurons of Alzheimer’s disease patients. Development of the disease is accompanied by increased, abnormal phosphorylation and acetylation of Tau. We have used NMR to investigate how these posttranslational modifications of Tau affect the interactions with its partners. We present here detailed protocols of in vitro phosphorylation of Tau by recombinant kinase, ERK2, or kinase activity of rat brain extracts, and acetylation by recombinant Creb-binding protein (CBP) acetyltransferase. The analytical characterization of the modified Tau by NMR spectroscopy is additionally described.

Key words

Phosphorylation Acetylation ERK kinase Creb-binding protein Acetyltransferase NMR spectroscopy Recombinant proteins 

References

  1. 1.
    Martin L, Latypova X, Wilson CM, Magnaudeix A, Perrin ML, Yardin C, Terro F (2013) Tau protein kinases: involvement in Alzheimer’s disease. Ageing Res Rev 12:289–309. doi:10.1016/j.arr.2012.06.003, S1568-1637(12)00088-8 [pii]CrossRefPubMedGoogle Scholar
  2. 2.
    Hasegawa M, Morishima-Kawashima M, Takio K, Suzuki M, Titani K, Ihara Y (1992) Protein sequence and mass spectrometric analyses of tau in the Alzheimer’s disease brain. J Biol Chem 267:17047–17054PubMedGoogle Scholar
  3. 3.
    Morishima-Kawashima M, Hasegawa M, Takio K, Suzuki M, Yoshida H, Titani K, Ihara Y (1995) Proline-directed and non-proline-directed phosphorylation of PHF-tau. J Biol Chem 270:823–829CrossRefPubMedGoogle Scholar
  4. 4.
    Morris M, Knudsen GM, Maeda S, Trinidad JC, Ioanoviciu A, Burlingame AL, Mucke L (2015) Tau post-translational modifications in wild-type and human amyloid precursor protein transgenic mice. Nat Neurosci 18:1183–1189CrossRefPubMedGoogle Scholar
  5. 5.
    Cohen TJ, Guo JL, Hurtado DE, Kwong LK, Mills IP, Trojanowski JQ, Lee VM (2011) The acetylation of tau inhibits its function and promotes pathological tau aggregation. Nat Commun 2:252CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Min SW, Cho SH, Zhou Y, Schroeder S, Haroutunian V, Seeley WW, Huang EJ, Shen Y, Masliah E, Mukherjee C, Meyers D, Cole PA, Ott M, Gan L (2010) Acetylation of tau inhibits its degradation and contributes to tauopathy. Neuron 67:953–966. doi:10.1016/j.neuron.2010.08.044, S0896-6273(10)00687-2 [pii]CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Qi H, Cantrelle F-X, Benhelli-Mokrani H, Smet-Nocca C, Buée L, Lippens G, Bonnefoy E, Galas M-C, Landrieu I (2015) Nuclear magnetic resonance spectroscopy characterization of interaction of Tau with DNA and its regulation by phosphorylation. Biochemistry (Mosc) 54:1525–1533. doi:10.1021/bi5014613 CrossRefGoogle Scholar
  8. 8.
    Landrieu I, Lacosse L, Leroy A, Wieruszeski JM, Trivelli X, Sillen A, Sibille N, Schwalbe H, Saxena K, Langer T, Lippens G (2006) NMR analysis of a Tau phosphorylation pattern. J Am Chem Soc 128:3575–3583. doi:10.1021/ja054656+ CrossRefPubMedGoogle Scholar
  9. 9.
    Amniai L, Barbier P, Sillen A, Wieruszeski J-M, Peyrot V, Lippens G, Landrieu I (2009) Alzheimer disease specific phosphoepitopes of Tau interfere with assembly of tubulin but not binding to microtubules. FASEB J 23:1146–1152CrossRefPubMedGoogle Scholar
  10. 10.
    Smet-Nocca C, Wieruszeski JM, Melnyk O, Benecke A (2010) NMR-based detection of acetylation sites in peptides. J Pept Sci 16:414–423. doi:10.1002/psc.1257 PubMedGoogle Scholar
  11. 11.
    Theillet F-X, Smet-Nocca C, Liokatis S, Thongwichian R, Kosten J, Yoon M-K, Kriwacki RW, Landrieu I, Lippens G, Selenko P (2012) Cell signaling, post-translational protein modifications and NMR spectroscopy. J Biomol NMR 54:217–236. doi:10.1007/s10858-012-9674-x CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Kamah A, Huvent I, Cantrelle FX, Qi H, Lippens G, Landrieu I, Smet-Nocca C (2014) Nuclear magnetic resonance analysis of the acetylation pattern of the neuronal Tau protein. Biochemistry (Mosc) 53:3020–3032. doi:10.1021/bi500006v CrossRefGoogle Scholar
  13. 13.
    Biernat J, Mandelkow EM, Schroter C, Lichtenberg-Kraag B, Steiner B, Berling B, Meyer H, Mercken M, Vandermeeren A, Goedert M et al (1992) The switch of tau protein to an Alzheimer-like state includes the phosphorylation of two serine-proline motifs upstream of the microtubule binding region. EMBO J 11:1593–1597PubMedPubMedCentralGoogle Scholar
  14. 14.
    Goedert M, Jakes R, Crowther RA, Six J, Lubke U, Vandermeeren M, Cras P, Trojanowski JQ, Lee VM (1993) The abnormal phosphorylation of tau protein at Ser-202 in Alzheimer disease recapitulates phosphorylation during development. Proc Natl Acad Sci U S A 90:5066–5070CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Alonso AC, Zaidi T, Grundke-Iqbal I, Iqbal K (1994) Role of abnormally phosphorylated tau in the breakdown of microtubules in Alzheimer disease. Proc Natl Acad Sci U S A 91:5562–5566CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Boulton TG, Yancopoulos GD, Gregory JS, Slaughter C, Moomaw C, Hsu J, Cobb MH (1990) An insulin-stimulated protein kinase similar to yeast kinases involved in cell cycle control. Science 249:64–67CrossRefPubMedGoogle Scholar
  17. 17.
    Anderson NG, Maller JL, Tonks NK, Sturgill TW (1990) Requirement for integration of signals from two distinct phosphorylation pathways for activation of MAP kinase. Nature 343:651–653. doi:10.1038/343651a0 CrossRefPubMedGoogle Scholar
  18. 18.
    Seger R, Ahn NG, Boulton TG, Yancopoulos GD, Panayotatos N, Radziejewska E, Ericsson L, Bratlien RL, Cobb MH, Krebs EG (1991) Microtubule-associated protein 2 kinases, ERK1 and ERK2, undergo autophosphorylation on both tyrosine and threonine residues: implications for their mechanism of activation. Proc Natl Acad Sci U S A 88:6142–6146CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Mansour SJ, Matten WT, Hermann AS, Candia JM, Rong S, Fukasawa K, Vande Woude GF, Ahn NG (1994) Transformation of mammalian cells by constitutively active MAP kinase kinase. Science 265:966–970CrossRefPubMedGoogle Scholar
  20. 20.
    Prabakaran S, Everley RA, Landrieu I, Wieruszeski JM, Lippens G, Steen H, Gunawardena J (2011) Comparative analysis of Erk phosphorylation suggests a mixed strategy for measuring phospho-form distributions. Mol Syst Biol 7:482. doi:10.1038/msb.2011.15, msb201115 [pii]CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Tini M, Benecke A, Um SJ, Torchia J, Evans RM, Chambon P (2002) Association of CBP/p300 acetylase and thymine DNA glycosylase links DNA repair and transcription. Mol Cell 9:265–277, doi: S1097276502004537 [pii]CrossRefPubMedGoogle Scholar
  22. 22.
    Bienkiewicz EA, Lumb KJ (1999) Random-coil chemical shifts of phosphorylated amino acids. J Biomol NMR 15:203–206CrossRefPubMedGoogle Scholar
  23. 23.
    Wishart DS, Bigam CG, Holm A, Hodges RS, Sykes BD (1995) 1H, 13C and 15N random coil NMR chemical shifts of the common amino acids. I. Investigations of nearest-neighbor effects. J Biomol NMR 5:67–81CrossRefPubMedGoogle Scholar
  24. 24.
    Schleucher J, Schwendinger M, Sattler M, Schmidt P, Schedletzky O, Glaser SJ, Sorensen OW, Griesinger C (1994) A general enhancement scheme in heteronuclear multidimensional NMR employing pulsed field gradients. J Biomol NMR 4:301–306CrossRefPubMedGoogle Scholar
  25. 25.
    Weisemann R, Ruterjans H, Bermel W (1993) 3D triple-resonance NMR techniques for the sequential assignment of NH and 15N resonances in 15N- and 13C-labelled proteins. J Biomol NMR 3:113–120CrossRefPubMedGoogle Scholar
  26. 26.
    Lippens G, Wieruszeski JM, Leroy A, Smet C, Sillen A, Buee L, Landrieu I (2004) Proline-directed random-coil chemical shift values as a tool for the NMR assignment of the tau phosphorylation sites. Chembiochem 5:73–78. doi:10.1002/cbic.200300763 CrossRefPubMedGoogle Scholar
  27. 27.
    Smet C, Leroy A, Sillen A, Wieruszeski JM, Landrieu I, Lippens G (2004) Accepting its random coil nature allows a partial NMR assignment of the neuronal Tau protein. Chembiochem 5:1639–1646. doi:10.1002/cbic.200400145 CrossRefPubMedGoogle Scholar
  28. 28.
    Mukrasch MD, Bibow S, Korukottu J, Jeganathan S, Biernat J, Griesinger C, Mandelkow E, Zweckstetter M (2009) Structural polymorphism of 441-residue tau at single residue resolution. PLoS Biol 7, e34CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2017

Authors and Affiliations

  • Haoling Qi
    • 1
    • 2
  • Clément Despres
    • 1
    • 2
  • Sudhakaran Prabakaran
    • 3
  • François-Xavier Cantrelle
    • 1
    • 2
  • Béatrice Chambraud
    • 4
  • Jeremy Gunawardena
    • 3
  • Guy Lippens
    • 1
    • 2
  • Caroline Smet-Nocca
    • 1
    • 2
  • Isabelle Landrieu
    • 5
  1. 1.Université de Lille, Sciences et Technologies, Unité de Glycobiologie Structurale et Fonctionnelle (UMR CNRS 8576)LilleFrance
  2. 2.CNRS, UMR 8576LilleFrance
  3. 3.Department of Systems BiologyHarvard Medical SchoolBostonUSA
  4. 4.INSERM, Paris-Saclay University, UMR1195Le Kremlin BicêtreFrance
  5. 5.UMR8576 CNRS-Université de Lille, Sciences et Technologies, Résonance Magnétique Nucléaire (RMN)LilleFrance

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