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

Comparison of the phosphorylation of microtubule-associated protein tau by non-proline dependent protein kinases


Microtubule-associated protein tau from Alzheimer brain has been shown to be phosphorylated at several ser/thr-pro and ser/thr-X sites (Hasegawa, M. et al., J. Biol. Chem, 267, 17047–17054, 1992). Several proline-dependent protein kinases (PDPKs) (MAP kinase, cdc2 kinase, glycogen synthase kinase-3, tubulin-activated protein kinase, and 40 kDa neurofilament kinase) are implicated in the phosphorylation of the ser-thr-pro sites. The identity of the kinase(s) that phosphorylate that ser/thr-X sites are unknown. To identify the latter kinase(s) we have compared the phosphorylation of bovine tau by several brain protein kinases. Stoichiometric phosphorylation of tau was achieved by casein kinase-1, calmodulin-dependent protein kinase II, Gr kinase, protein kinase C and cyclic AMP-dependent protein kinase, but not with casein kinase-2 or phosphorylase kinase. Casein kinase-1 and calmodulin-dependent protein kinase II were the best tau kinases, with greater than 4 mol and 3 mol32P incorporated, respectively, into each mol of tau. With the sequential addition of these two kinases,32P incorporation approached 6 mol. Peptide mapping revealed that the different kinases largely phosphorylate different sites on tau. After phosphorylation by casein kinase-1, calmodulin-dependent protein kinase II, Gr kinase, cyclic AMP-dependent protein kinase and casein kinase-2, the mobility of tau isoforms as detected by SDS-PAGE was decreased. Protein kinase C phosphorylation did not produce such a mobility shift. Our results suggest that one or more of the kinases studied here may participate in the hyperphosphorylation of tau in Alzheimer disease. Such phosphorylation may serve to modulate the activaties of other tau kinases such as the PDPKs.

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



paired helical filaments


cyclic AMP-dependent protein kinase

CaM kinase II:

calcium/calmodulin-dependent protein kinase II


calcium-phospholipid-dependent protein kinase


casein kinase-1


casein kinase-2

Gr kinase:

calcium/calmodulin-dependent protein kinase from rat cerebellum


glycogen synthase kinase-3

MAP kinase:

mitogen-activated protein kinase


sodium dodecyl sulfate-polyacrylamide gel electrophoresis


  1. 1.

    Goedert M, Spillantini MG, Jakes R, Rutherford D, Crowther RA: Multiple isoforms of human microtubule-associated protein tau: Sequences and localization in neurofibrillary tangles of Alzhemer's disease. Neuron 3: 519–526, 1989

  2. 2.

    Himmler A: Structure of the bovine tau gene: Alternatively spliced transcripts generate a protein family. Mol Cell Biol 9: 1389–1396, 1989

  3. 3.

    Brion JP, Hanger DP, Bruce MT, Couck AM, Flament-Durant J, Anderton BT: Tau in Alzheimer's neurofibrillary tangles. Biochem J 273: 127–133, 1991

  4. 4.

    Greenberg SG, Davies P: A preparation of Alzheimer's paired helical filaments that displays distinct τ proteins by polyacrylamide gel electrophoresis. Proc Natl Acad Sci USA 87: 5827–5831, 1990

  5. 5.

    Grundke-Iqbal I, Iqbal K, Quinlan M, Tung Y-C, Zaidi MS, Wski HM: Microtubule-associated protein tau: a component of Alzheimer's paired helical filaments. J Biol Chem 261: 6084–6089, 1986

  6. 6.

    Grundke-Iqbal, I, Iqbal K, Tung Y-C, Quinlan M, Wisniewski HM, Binder LI: Abnormal phosphorylation of the microtubule-associated protein tau in Alzheimer cytoskeletal pathology. Proc Natl Acad Sci USA 83 4913–4917, 1986

  7. 7.

    Iqbal K, Grundke-Iqbal I, Smith AJ, George L, Tung Y-C, Zaidi T: Identification and localization of a tau peptide to paired helical filaments of Alzheimer disease. Proc Natl Acad Sci USA 86: 5646–5650, 1989

  8. 8.

    Lee VMY, Balin BJ, Otvos L, Trojanowski JQ: A68: a major subunit of paired helical filaments and derivatized forms of normal tau. Science 251: 675–678, 1991

  9. 9.

    Ksiezak-Reding H, Liu W-K, Yen S-H: Phosphate analysis and dephosphorylation of modified tau associated with paired helical filaments. Brain Res 597: 209–219, 1992

  10. 10.

    Gustke N, Steiner B, Mandelkow E-M, Biernat J, Meyer HE, Goedert M, Mandelkow E: The Alzheimer-like phosphorylation of tau protein reduces microtubule binding and involves ser-pro and thr-pro motifs. FEBS Lett 307: 199–205, 1992

  11. 11.

    Hasegawa M, Morishima-Kawashima M, Takio K, Sukuki M, Titani K, Ihara Y: Protein sequence and mass spectrometric analyses of tau in the Alzheimer's disease brain. J Biol Chem 267: 17047–17054, 1992

  12. 12.

    Drewes G, Lichtenberg-Kraag B, Coring F, Mandelkow E-M, Biernat J, Goris J, Doree M, Mandelkow E: Mitogen-activated protein (MAP) kinase transforms tau protein into an Alzheimer-like state. EMBO J 11: 2131–2138, 1992

  13. 13.

    Vulliet R, Halloran SM, Braun RK, Smith AJ, Lee G: Proline-directed phosphosphorylation of human tau protein. J Biol Chem 267: 22570–22574, 1992

  14. 14.

    Roder HM, Ingram VM: Two novel kinases phosphorylate tau and the KSP site of heavy neurofilament subunits in high stoichiometries. J Neurosci 11: 3323–3343, 1991

  15. 15.

    Mandelkow E-M, Drewes G, Biernat J, Gustke N, Van Lint J, Vandenheede JR, Mandelkow E: Glycogen synthase kinase-3 and the Alzheimer-like state of microtubule-associated protein tau. FEBS Lett 314:315–321, 1992

  16. 16.

    Ishiguro K, Takamatsu M, Tomizawa K, Omori A, Takahashi M, Arioka M, Uchida T, Imahori K: Tau protein kinase I converts normal tau protein into A68-like component of paired helical filaments. J Biol Chem 267: 10897–10901, 1992

  17. 17.

    Litersky JM, Johnson GVW: Phosphorylation by cAMP-dependentent protein kinase inhibits the degradation of tau by calpain. J Biol Chem 267: 1563–1568, 1992

  18. 18.

    Baudier J, Cole RD: Phosphorylation of tau proteins to a state like that in Alzheimer's brain is catalyzed by calcium/calmodulin-dependent protein kinase and modulated by phospholipids. J Biol Chem 262: 17577–17583, 1991

  19. 19.

    Baudier J, Lee S-H, Cole RD: Separation of the different microtubule-associated tau protein species from bovine brain and their mode II phosphorylation by Ca2+/phospholipid-dependent protein kinase. J Biol Chem 262: 17584–17590, 1987

  20. 20.

    Steiner B, Mandelkow E-M, Biernat J, Gustke N, Meyer HE, Schmidt B, Mieskes G, Soling HD, Drechsel D, Kirschner MW, Goedert M, Mandelkow E: Phosphorylation of microtubule-associated protein tau: identification of the site for Ca2+/calmodulin dependent kinase and relationship with tau phosphorylation in Alzheimer tangles. EMBO J 9: 3539–3544, 1990

  21. 21.

    Singh TJ: Polyamines stimulate the activity of glycogen synthase (casein) kinase-1 from bovine kidney and different rat tissues. Arch Biochem Biophys 267: 167–175, 1988

  22. 22.

    Cohen P: The subunit structure of rabbit skeletal muscle phosphorylase kinase and the molecular basis of its activation reactions. Eur J Biochem 34: 1–14, 1973

  23. 23.

    Ohmstede C-A, Jensen KJ, Sahyoun NE: Ca2+/calmodulin-dependent protein kinase enriched in cerebellar granule cells. J Biol Chem 264: 5866–5875, 1989

  24. 24.

    Huang K-P, Chang K-FJ, Singh TJ, Nakabayashi H, Huang FL: Autophosphorylation of rat brain Ca2+-activated and phospholipid-dependent protein kinase. J Biol Chem 261: 12134–12140, 1986

  25. 25.

    Binder LI, Frankfurter A, Rebhun LI: The distribution of tau in, the mammalian central nervous system. J Cell Biol 101: 1371–1378, 1985

  26. 26.

    Laemmli UK: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227: 680–685, 1970

  27. 27.

    Cleveland DW, Fischer SG, Kirschner MW, Laemmli UK: Peptide mapping by limited proteolysis in sodium dodecyl sulfate and analysis by gel electrophoresis. J Biol Chem 252: 1102–1106, 1977

  28. 28.

    Biernat J, Mandelkow E-M, Schroter C, Lichtenberg-Kraag B, Steiner B, Berling B, Meyer H, Mercken M, Vandermeeren A, Goedert M, Mandelkow E: The switch of tau protein to an Alzheimer-like state includes the phosphorylation of two serinesproline motifs upstream of the microtubule binding region. EMBO J 11: 1593–1597, 1992

  29. 29.

    Lichtenberg-Kraag B, Mandelkow E-M, Biernat J, Steiner B, Schroter C, Gustke N, Meyer HE, Mandelkow E: Phosphorylation dependent epitopes of neurofilament antibodies on tau protein and relationship with Alzheimer tau. Proc Natl Acad Sci USA 89 5384–5388, 1992

  30. 30.

    Arioka M, Tsukamoto M, Ishiguro K, Kato R, Sato K, Imahori K, Uchida T: Tau protein kinase II is involved in the regulation of the normal phosphorylation state of tau protein. J Neurochem 60: 461–468, 1993

  31. 31.

    Pierre M, Nunez JL: Multisite phosphorylation of τ proteins from rat brain. Biochem Biophys Res Commun 115: 212–219, 1983

  32. 33.

    Scott CW, Spreen RC, Herman JL, Chow FP, Davison MD, Young J, Caputo CB: Phosphorylation of recombinant tau by cAMP-dependent protein kinase. J Biol Chem 268: 1166–1173, 1993

  33. 33.

    Correas I, Diaz-Nido J, Avila J: Microtubule-associated protein tau is phosphorylated by protein kinase C on its tubulin binding domain. J Biol Chem 267: 15721–15728, 1992

  34. 34.

    Vincent IJ, Davies P: A protein kinase associated with paired helical filaments in Alzheimer disease. Proc Natl Acad Sci USA 89: 2878–2882 1992

  35. 35.

    Singh TJ: Inhibition of glycogen synthase (casein) kinase-1 by heparin. Arch Biochem Biophys 260: 661–666, 1988

  36. 36.

    Singh TJ, Huang K-P: Glycogen synthase (casein) kinase-1: tissue distribution and subcellular localization. FEBS Lett 190: 84–88, 1985

  37. 37.

    Roder HM, Eden PA, Ingram VM: Brain protein kinase PK40erk converts tau into a PHF-like form as found in Alzheimer disease. Biochem Biophys Res Commun 193: 639–647, 1993

  38. 38.

    Ishiguro K, Shiratsuchi A, Sato S, Omori A, Arioka M, Kobayashi S, Uchida T, Imahori K: Glycogen synthase kinase 3β is identical to tau protein kinase 1, generating several epitopes of paired helical flaments. FEBS Lett 325: 167–172, 1993

Download references

Author information

Correspondence to Khalid Iqbal.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Singh, J., Grundke-Iqbal, I., McDonald, B. et al. Comparison of the phosphorylation of microtubule-associated protein tau by non-proline dependent protein kinases. Mol Cell Biochem 131, 181–189 (1994).

Download citation

Key words

  • tau phosphorylation
  • microtubules
  • protein kinases
  • Alzheimer disease
  • Tau-1 antibody