Abstract
Tau cDNAs from each of the six human isoforms were transfected into COS- 1 cells and, in every case, more than one peptide was observed. The diversity of expressed isoforms was due to different levels of tau phosphorylation. Tau phosphorylation results in a decrease of the protein electrophoretic mobility. The major contribution to this mobility shift is due to the phosphorylation at the at the C-terminus of the molecule, as inferred from the expression of tau fragments. Phosphorylation takes place in some of the sites modified in neural cells and in the basis of AD patients. Copolymerization studies indicate that the level of phosphorylation, as well as the localization of the modified residues, may affect the binding of the protein to microtubules. These results indicate that phosphorylation regulates tau function inside the cell.
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Cleveland CB, Sygowski LA, Scott CW, Sobel EI: Purification of tau, a microtubule-associated protein that induces assembly of microtubules from purified tubulin. J Mol Biol 116: 207–225, 1977
Himmler A: Structure of the bovine tau gene: alternatively spliced transcripts generate a protein family. Mol Cell Biol 9: 1389–1396, 1989
Mavilla C, Couchie D, Mattei MG, Nivez MP, Nunez J: High and low molecular weight tau proteins are differentially expressed from a single gene. J Neurochemistry 61: 1073–1081, 1993
Lindwall B, Cole RD: The purification of tau protein and the occurrence of two phosphorylation states of tau in brain. J Biol Chem 259: 12241–12245, 1984
Baudier J, Lee SH, 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 C. J Biol Chem 262: 17584–17590, 1987
García de Ancos J, Avila J: Differential distribution in white and gray matter of tau phosphoisoforms containing four tubulin binding motifs. Biochem J 296: 351–354, 1993
Butler MM, Cocham ML: Microheterogeneity of microtubule associated tau proteins is due to differences in phosphorylation. J Neurochem 47: 1517–1522, 1986
Drubin DG, Kirschner MW: Tau protein function in living cells. J Cell Biol 103: 2730–2746, 1986
Kanai Y, Takemura R, Oshima T, Mori H, Ihara Y, Yanagisawa M, Masaki T, Hirokawa N: Expression of multiple tau isoforms and microtubule bundle formation in fibroblasts transfected with a single tau cDNA. J Cell Biol 109: 1173–1184, 1989
Cáceres A, Kosik K: Inhibition of neurite polarity by tau antisense oligonucleotides in primary cerebellar neurons. Nature 343: 461–463, 1990
Weingarten MD, Lockwood AH, Hwo SY, Kirschner MW: A protein factor essential for microtubule assembly. Proc Natl Acad Sci USA 72: 1858–1862, 1975
Goedert M, Jakes R: Expression of separate isoforms of human tau protein: correlation with the tau pattern in brain and effects on tubulin polymerization. EMBO J 9: 4225–4230, 1990
García de Ancos J, Correas I, Avila J: Differences in microtubule binding and self-association abilities of bovine brain tau isoforms. J Biol Chem 268: 7976–7982, 1993
Hoshi M, Nishida E, Miyata Y, Sakai H, Miyoshi T, Ogawara H, Akiyama T: Protein kinase C phosphorylates tau and induces its functional alterations. FEBS Lett 217: 237–241, 1987
Yamamoto H, Fukunaga K, Goto S, Tanaka E, Miyamoto E: Ca2+, calmodulin-dependent regulation of microtubule formation via phosphorylation of MAP2, τ, and tubulin and comparison with the cAMP-dependent phosphorylation. J Neurochem 44: 759–768, 1985
Bancher C, Brunner C, Lassmann H, Budka H, Jellinger K, Wiche G, Seitelberger F, Grundke-Iqbal I, Iqbal K, Wisniewski HM: Accumulation of abnormally phosphorylated tau precedes the formation of neurofibrillary tangles in Alzheimer's disease. Brain Res 477: 90–99, 1989
Grundke-Iqbal I, Iqbal K, Tung YC, Quinlin M, Wisniewski HM, Binder LI: Abnormal phosphorylation of the microtubule-associated protein τ (tau) in Alzheimer cytoskeletal pathology. Proc Natl Acad Sci USA 83: 4913–4917, 1986
Flament S, Delacourte A, Hemon B, Defossez A: Characterization of two pathological tau protein variants in Alzheimer brain cortices. J Neurol Sci 92: 133–141, 1989
Lee VM-Y, 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
Takemura R, Okabe S, Umeyama T, Kanai Y, Cowan NJ, Hirokawa N: Increased microtubule stability and alpha tubulin acetylation in cells transfected with microtubule-associated proteins MAP1B, MAP2 or tau. J Cell Sci 103: 953–964, 1992
Lee G, Rook SL: Expression of tau protein in neuronal cells: structural requirements for microtubule binding and stabilization. J Cell Sci 102: 227–237, 1992
Lo MMS, Fieles AW, Norris TE, Dargis PG, Caputo CB, Scott CW, Lee VM-Y, Goedert M: Human tau isoforms confer distinct morphological and functional properties to stably transfected fibroblasts. Mol Brain Res 20: 209–220, 1993
Montejo de Garcini E, de la Luna S, Domínguez JE, Avila J: Overexpression of tau protein in COS-1 cells results in the stabilization of centrosome-independent microtubule and extension of cytoplasmic processes. Mol Cell Biochem 130: 187–196, 1994
Kanai Y, Chem J, Hirokawa N: Microtubule bundling by tau proteinsin vivo: analysis of functional domains. EMBO J 11L 3953–3961, 1992
Gallo JM, Hanger DP, Twist EC, Kosik KS, Anderton BH: Expression and phosphorylation of a three-repeat isoform of tau in transfected non-neuronal cells. Biochem J 286: 399–404, 1992
Sygowski LA, Fieles AW, Lo MMS, Scott CW, Caputo CB: Phosphorylation of tau protein in tau-transfected 3T3 cells. Mol Brain Res 20: 221–228, 1993
Correas I, Díaz-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
Kobayashi S, Ishiguro K, Amori A, Takamatsu M, Arioka M, Imahori K, Uchida T: A cdc2 related kinase PSSALRE/cdk5 is homologous with the 30 kD Subunit of tau protein kinase II, a proline-directed protein kinase associated with microtubule. FEBS Lett 335: 171–175, 1993
Ishiguro K, Shiratsuchi A, Sato S, Omoni A, Arioka M, Kobayashi S, Uchida T, Imahori K: Glycogen synthase kinase 3 beta is identical to tau protein kinase I generating several epitopes of paired helical filaments. FEBS Lett 325: 167–172, 1993
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
Drewes G, Lichtenberg-Kraag B, Doring F, Mandelkow EM, Biernat J, Goris J, Doree M, Mandelkow E: Mitogen activated protein (MAP) kinase transforms tau protein into an Alzheimer-like state. EMBO J 11: 2121–2138, 1992
Litersky JM, Johnson GV: Phosphorylation by cAMP-dependent protein kinase inhibits the degradation of tau by calpain. J Biol Chem 267: 1563–1568, 1992
Scott CW, Spreen RC, Herman JL, Chow FP, Davidson MD, Young J, Caputo CB: Phosphorylation of recombinant tau by cAMP-dependent protein kinase. J Biol Chem 268: 1166–1173, 1993
Biernat J, Mandelkow EM, Schroter C, Lichtenber-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 serine-proline motifs upstream of the microtubule binding region. EMBO J 11: 1593–1597, 1992
Goedert M, Jakes R, Crowther RA, Six J, Lubke V, Vandermeeren M, Cras P, Trojanowski JQ, Lee VMY: The abnormal phosphorylation of tau protein at Ser-202 in Alzheimer disease recapitulates phosphorylation during development. Proc Natl Acad Sci USA 90: 5066–5070, 1993
Kosik KS, Orecchio LD, Binder LI, Trojanowski J, Lee V, Lee G: Epitoses that span the tau molecule are shared with paired helical filaments. Neuron 1: 817–825, 1988
Ledesma MD, Correas I, Avila J, Díaz-Nido J: Implication of cdc2 and MAP2 kinases in the phosphorylation of tau in Alzheimer's disease. FEBS Letters 308: 218–224, 1992
Lichtenberg-Kraag B, Mandelkow EM, 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
Novak M, Jakes R, Edwards PC, Milstein C, Wischik CM: Difference between the tau protein of Alzheimer paired helical filament core and normal tau revealed by epitope analysis of monoclonal antibodies 423 and 7.51. Proc Natl Acad Sci USA 88: 5837–5841, 1991
Papasozomenos SC, Binder LI: Phosphorylation determines two distinct species of tau in the central nervous system. Cell Motil Cytoskel 8: 210–226, 1987
Montejo de Garcini, Serrano L, Avila J: Self assembly of microtubule associated protein into filaments resembling those found in Alzheimer's disease. Biochem Biophys Res Commun 141: 790–796, 1986
Shelanski ML, Gaskin F, Cantor CR: Microtubule assembly in the absence of added nucleotides. Proc Natl Acad Sci USA 70: 765–768, 1973
Vallee RB: A taxol dependent procedure for the isolation of microtubules and microtubule associated proteins. J Cell Biol 92: 435–442, 1982
González P, Correas I, Avila J: Solubilization and fractionation of paired helical filaments. Neuroscience 50: 491–499, 1992
Towbin H, Staehelin T, Gordon J: Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 76: 4350–4354, 1979
Gluzman Y: SV40-transformed simian cells support the replication of early SV40 mutants. Cell 23: 175–182, 1981
Black MM, Cocham JM, Kurdyla JT: Solubility properties of neuronal tubulin: evidence for labile and stable microtubules. Brain Res 295: 255–263, 1984
Butner KA, Kirschner MW: Tau protein binds to microtubules through a flexible array of distributed weak sites. J Cell Biol 115: 717–730, 1991
Lovestone S, Reynolds CH, Latimer D, Davis DR, Anderton BH, Gallo JM, Hanger D, Mulot S, Marquardt B, Stabel S, Woodgett JR, Miller CCJ: Alzheimer's Disease-like phosphorylation of the microtubule-passociated protein tau by glycogen synthase kinase-3 in transfected mammalian dells. Curr Biol 4: 1077–1086, 1994
Cohen P, Holmes CFB, Tsukitani Y: Okadaic acid: a new probe for the study of cellular regulation. TIBS 15: 98–102, 1990
Lee G, Neve RL, Kosik KS: The microtubule binding domain of tau protein. Neuron 2: 1615–1624, 1989
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
Nieto A, Correas I, López-Otín C, Avila J: Tau related protein present in paired helical filaments has a decreased tubulin binding capacity as compared with microtubule associated protein tau. Biochem Biophys Acta 1096: 197–204, 1991
Ennulat DJ, Liem RK, Hashim GA, Shelanski ML: Two separate 18-amino acid domains of tau promote the polymerization of tubulin. J Biol Chem 264: 5327–5330, 1989
Aizawa H, Kawasaki H, Murofushi H, Kotani S, Suzuki K, Sakai H: A common amino acid sequence in 190-KD microtubule-associated protein and tau for the promotion of microtubule assembly. J Biol Chem 264: 5885–5890, 1989
Maccioni RB, Vera JC, Domínguez J, Avila J: A discrete repeated sequence defines a tubulin binding domain on microtubule-associated protein tau. Arch Biochem Biophys 275: 568–579, 1989
Drechsel DN, Hyman AA, Cobb MH, Kirschner MW: Modulation of the dynamic instability of tubulin assembly by the microtubule associated protein tau. Mol Biol Cell 3: 1141–1154, 1992
Kabsch W, Vandekerckhove J: Structure and function of actin. Ann Rev Biophys Biomol Struct 21: 49–76, 1992
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Medina, M., Montejo de Garcini, E. & Avila, J. The role of tau phosphorylation in transfected COS-1 cells. Mol Cell Biochem 148, 79–88 (1995). https://doi.org/10.1007/BF00929506
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DOI: https://doi.org/10.1007/BF00929506