Tau Phosphorylation at Serine 396 and Serine 404 by Human Recombinant Tau Protein Kinase II Inhibits Tau's Ability to Promote Microtubule Assembly

In Alzheimer's disease, hyperphosphorylated tau is an integral part of the neurofibrillary tangles that form within neuronal cell bodies and fails to promote microtubule assembly. Dysregulation of the brain-specific tau protein kinase II is reported to play an important role in the pathogenesis of Alzheimer's disease (Patrick, G. N., Zukerberg, L., Nikolic, M., De La Monte, S., Dikkes, P., and Tsai, L.-H. (1999) Nature 402, 615–622). We report here that in vitro phosphorylation of human tau by human recombinant tau protein kinase II severely inhibits the ability of tau to promote microtubule assembly as monitored by tubulin polymerization. The ultrastructure of tau-mediated polymerized tubulin was visualized by electron microscopy and compared with phosphorylated tau. Consistent with the observed slower kinetics of tubulin polymerization, phosphorylated tau is compromised in its ability to generate microtubules. Moreover, we show that phosphorylation of microtubule-associated tau results in tau's dissociation from the microtubules and tubulin depolymerization. Mutational studies with human tau indicate that phosphorylation by tau protein kinase II at serine 396 and serine 404 is primarily responsible for the functional loss of tau-mediated tubulin polymerization. These in vitroresults suggest a possible role for tau protein kinase II-mediated tau phosphorylation in initiating the destabilization of microtubules. In Alzheimer's disease, hyperphosphorylated tau is an integral part of the neurofibrillary tangles that form within neuronal cell bodies and fails to promote microtubule assembly. Dysregulation of the brain-specific tau protein kinase II is reported to play an important role in the pathogenesis of Alzheimer's disease (Patrick, G. N., Zukerberg, L., Nikolic, M., De La Monte, S., Dikkes, P., and Tsai, L.-H. (1999) Nature 402, 615–622). We report here that in vitro phosphorylation of human tau by human recombinant tau protein kinase II severely inhibits the ability of tau to promote microtubule assembly as monitored by tubulin polymerization. The ultrastructure of tau-mediated polymerized tubulin was visualized by electron microscopy and compared with phosphorylated tau. Consistent with the observed slower kinetics of tubulin polymerization, phosphorylated tau is compromised in its ability to generate microtubules. Moreover, we show that phosphorylation of microtubule-associated tau results in tau's dissociation from the microtubules and tubulin depolymerization. Mutational studies with human tau indicate that phosphorylation by tau protein kinase II at serine 396 and serine 404 is primarily responsible for the functional loss of tau-mediated tubulin polymerization. These in vitroresults suggest a possible role for tau protein kinase II-mediated tau phosphorylation in initiating the destabilization of microtubules. Alzheimer's disease microtubules tau protein kinase II tau protein kinase I paired helical filaments dithiothreitol cyclin-dependent kinase 5 immobilized metal affinity chromatography polyacrylamide gel electrophoresis wild type 1,4-piperazinediethanesulfonic acid Six isoforms of human tau are expressed in adult human brain (1Goedert M. Spillantini M.G. Jakes R. Rutherford D. Crowther R.A. Neuron. 1989; 3: 519-526Abstract Full Text PDF PubMed Scopus (1872) Google Scholar). These arise from alternate splicing of the mRNA transcribed from a single gene located on the long arm of chromosome 17 (1Goedert M. Spillantini M.G. Jakes R. Rutherford D. Crowther R.A. Neuron. 1989; 3: 519-526Abstract Full Text PDF PubMed Scopus (1872) Google Scholar, 2Lee G. Cowan N. Kirschner M. Science. 1998; 239: 285-288Crossref Scopus (528) Google Scholar, 3Goedert M. Wischik C.M. Crowther R.A. Walker J.E. Klug A. Proc. Natl. Acad. Sci. U. S. 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The main fibrous component of all neurofibrillary lesions is paired helical filament (PHF), which contains predominantly the abnormally phosphorylated tau (13Bancher C. Brunner C. Lassmann H. Budka H. Jellinger K. Wiche G. Seiteberger F. Grundke-Iqbal I. Iqbal K. Wisniewski H.M. Brain Res. 1989; 477: 90-99Crossref PubMed Scopus (704) Google Scholar, 14Bondareff W. Wischik C.M. Novak M. Amos W.B. Kluf A. Roth M. Am. J. Pathol. 1990; 137: 711-723PubMed Google Scholar, 15Goedert M. Trends Neurosci. 1993; 16: 460-465Abstract Full Text PDF PubMed Scopus (549) Google Scholar, 16Greenberg S.G. Davies P. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 5827-5831Crossref PubMed Scopus (660) Google Scholar, 17Ksiezak-Reding H. Yen S.-H. Neuron. 1991; 6: 717-728Abstract Full Text PDF PubMed Scopus (117) Google Scholar, 18Lee V.M.-Y. Balin B.J. Otvos Jr., L. Trojanowski J.Q. Science. 1991; 251: 675-678Crossref PubMed Scopus (1253) Google Scholar). It has been hypothesized that aberrant phosphorylation of tau leads to its aggregation into PHF, resulting in destabilization of microtubules and the death of neurons (1Goedert M. Spillantini M.G. Jakes R. Rutherford D. Crowther R.A. Neuron. 1989; 3: 519-526Abstract Full Text PDF PubMed Scopus (1872) Google Scholar, 19Kosik K.S. Curr. Opin. Cell Biol. 1990; 2: 101-104Crossref PubMed Scopus (44) Google Scholar). Notably, hyperphosphorylated tau has been detected in other tau-positive filamentous lesions, in a group of diseases collectively known as the tauopathies. These neurodegenerative diseases include progressive supranuclear palsy, corticobasal degeneration, Down's syndrome, frontotemporal dementia and Parkinsonism linked to chromosome 17, and Pick's disease (20Spillantini M.G. Goedert M. Trends Neurosci. 1998; 21: 428-433Abstract Full Text Full Text PDF PubMed Scopus (613) Google Scholar). Recent studies show that these various phenotypes might be the result of phosphorylation of specific tau isoforms in different nerve cells in distinct brain regions (21Mailliot C. Sergeant N. Bussiere T. Caillet-Boudin M.L. Delacourte A. Buee L. FEBS Lett. 1998; 433: 201-204Crossref PubMed Scopus (100) Google Scholar). Tau-associated with PHF from AD brain is hyperphosphorylated at several serine/threonine sites that are followed by a proline (22Hanger D.P. Betts J.C. Loviny T.L.F. Blackstock W.P. Anderton B.H. J. Neurochem. 1998; 71: 2465-2476Crossref PubMed Scopus (337) Google Scholar). AD tau protein in its hyperphosphorylated state fails to promote microtubule (MT) assembly in vitro (23Lu Q. Wood J.G. J. Neurosci. 1993; 13: 508-515Crossref PubMed Google Scholar) and this phosphorylation-dependent tau dysfunction is considered one of the critical events leading to neuronal degeneration (24Roloff E.L.V. Platt B. Cell. Mol. Life Sci. 1999; 55: 601-616Crossref PubMed Scopus (31) Google Scholar). It is not known which kinase initiates tau hyperphosphorylation and MT disassembly and which phosphorylated amino acids in tau contribute to tau's dysfunction. Understanding the molecular basis for tau dysfunction requires identification and availability of highly purified candidate tau protein kinase, careful mapping of phosphorylation sites, mutational studies, and functional characterization of the corresponding phosphorylated tau mutants. There are two proline-directed kinases, tau protein kinase II (TPK II) and glycogen synthase kinase 3β (TPK I), which have been found to be associated with microtubules (25Ishiguro K. Takamatsu M. Tomizawa K. Omori A. Takahashi M. Arioka M. Uchida T. Imahori K. J. Biol. 1992; Full Text PDF PubMed Google Scholar, T. T. S. J. PubMed Scopus Google Scholar) and are known to tau in a U. M. J. Cell Sci. 1996; PubMed Google Scholar, G. M. M. K. K. Imahori K. A. 1998; PubMed Scopus Google Scholar). One of the is that II initiates tau hyperphosphorylation and MT the of a highly purified and of human recombinant II has not been the of II-mediated phosphorylation sites in human tau and role in tau dysfunction we have been in of highly purified human recombinant II by in vitro expressed and of this critical has to II phosphorylation sites in human tau by a combination of T. R. D. B. S. K. and W. R. in We found that the II-mediated phosphorylation sites in tau are and on the in the longest form of human tau (1Goedert M. Spillantini M.G. Jakes R. Rutherford D. Crowther R.A. Neuron. 1989; 3: 519-526Abstract Full Text PDF PubMed Scopus (1872) Google Scholar). The of the to the II for of tau hyperphosphorylation and MT disassembly and to the molecular basis for tau dysfunction and its to tauopathies. We report here that phosphorylation of human tau by II inhibits tau's ability to promote microtubule assembly and that II-mediated phosphorylation of microtubule-associated tau results in tau's dissociation from the microtubules and tubulin depolymerization. on the from mapping human II-mediated phosphorylation sites in human the molecular basis for this tau dysfunction has been mutational studies on human tau. We show that II-mediated tau phosphorylation at and is primarily responsible for the loss of tau in MT assembly and dissociation of tau from microtubules. These results are with to II as a for of tau hyperphosphorylation and the for with and (20Spillantini M.G. Goedert M. Trends Neurosci. 1998; 21: 428-433Abstract Full Text Full Text PDF PubMed Scopus (613) Google Scholar, C. Sergeant N. Bussiere T. Caillet-Boudin M.L. Delacourte A. Buee L. FEBS Lett. 1998; 433: 201-204Crossref PubMed Scopus (100) Google Scholar). and molecular from and Life The from The was from The from and cells from The was by The for human tau was from of the from the electrophoresis from glycogen synthase kinase 3β (TPK was from for the was from and the tubulin a from The full-length human gene was into the the of was three and a single was to for in vitro of human recombinant was purified by from The of human was and expressed as a an in and of human recombinant II the the was in D. A. T. 1991; 14: Google Scholar) the and for with The was to and at for and the was a immobilized metal affinity chromatography in The was with followed by and was with purified was with and the The was with and the was with II by gel electrophoresis and in dithiothreitol of the II was immobilized followed by affinity chromatography on an type tau with an was and expressed in by the of the as from the was and to cells for with for in D. A. T. 1991; 14: Google Scholar). by and the cell at and for as N. K. J. R. C. C. D. S. D. A. J. Biol. Full Text Full Text PDF PubMed Google Scholar). The tau protein was with and by at on and the and in the tau tau and the from and of the and tau as for wild type tau. Tau phosphorylation was by 5 tau with II for at in a and the on Nature. PubMed Scopus Google Scholar) and with to tubulin the tau into The tubulin was as by from was on and to with and The was in by of tubulin in a of of tau at a of The was with the of the was at in a and was for was polymerized in the presence of tau the II or II was to the microtubules and the was for the the from the and and of tau was to and The was with a at a of followed by at a of and detected type human tau was expressed at and purified by as the immobilized metal The of human tau in this the two N-terminal repeats, the four microtubule binding It the of proline-directed phosphorylation sites as the longest form of human tau. this the of phosphorylation sites in human recombinant tau is on the longest form of tau (1Goedert M. Spillantini M.G. Jakes R. Rutherford D. Crowther R.A. Neuron. 1989; 3: 519-526Abstract Full Text PDF PubMed Scopus (1872) Google Scholar). The of human recombinant tau that was of of purified recombinant a of and its The recombinant was on an and with purified was affinity on immobilized and highly purified and II was for in vitro tau phosphorylation studies in the presence of The tau was to which phosphorylation of tau at proline-directed sites and the of phosphorylation at each is these results with the of of of tau. It is known that microtubules (MT) are in of tubulin (23Lu Q. Wood J.G. J. Neurosci. 1993; 13: 508-515Crossref PubMed Google Scholar, N. PubMed Scopus Google Scholar, A. T. 1998; PubMed Scopus Google Scholar, M. Goedert M. FEBS Lett. 1998; PubMed Scopus Google Scholar, H. L. D. A. J.C. Trojanowski J.Q. Science. 1998; PubMed Scopus Google Scholar). of MT from tubulin be followed by an in at in the presence of and tau (23Lu Q. Wood J.G. J. Neurosci. 1993; 13: 508-515Crossref PubMed Google Scholar, N. PubMed Scopus Google Scholar, M. Goedert M. FEBS Lett. 1998; PubMed Scopus Google Scholar, H. L. D. A. J.C. 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We the of tau on tubulin The in was with tau for whereas tau MT assembly at all the tau not shown in of to tau from tau by its ability to promote tubulin polymerization. to the of tau on microtubule we electron microscopy at tubulin polymerization. electron of the MT assembly at state for tau and tau. microtubules in assembly by tau that the recombinant human tau is In MT assembly was in of and in the presence of tau In the tubulin not microtubule assembly was The results to the of II on tau. that of II and to MT a in as a of These results indicate as tau phosphorylated on MT assembly as monitored by a in II or was or in from the was the and dissociation from by followed by Tau was with known to the N-terminal on human tau. shown in the a molecular the that of tau from MT is to phosphorylation of tau by In a form of tau of the four MT binding repeats of the II phosphorylation sites, was for MT the of II on these in vitro results show that II phosphorylation functional of which is critical for the microtubule II is the known of a proline-directed kinase that the ability of tau to promote MT assembly and MT disassembly in vitro In the tau the of each of the phosphorylated sites on tubulin be in an to the molecular basis for the loss of functional of tau we a of tau on identification of II phosphorylation sites in tau. of these and for or whereas the for the tau was and this tau was in the tubulin for we to as a to the of II phosphorylation on tubulin and to and for These tau phosphorylated with and results for and are shown in A. in molecular was observed the was phosphorylated with II 5 as shown in the of II phosphorylation was in single and a at We these results to that II phosphorylation of tau at is responsible for the observed molecular in other as the of the or on other II sites be from these tau to tubulin not and the results with wild type tau we the of tau and on tubulin polymerization. we the of these on and of These studies wild type tau II phosphorylation of the and tau tau's ability to promote tubulin as by at shown in I for these three tau the and the of severely and these results to the tau shown in These results suggest that phosphorylation at the other II sites and might play an important role in MT assembly. the of tubulin was with the of and with tau these three sites, on mutational studies, phosphorylation at a on in this the of the of the was to the other single tau not the suggest that II phosphorylation of tau at and to be responsible for of microtubule of II phosphorylation of tau on tubulin tau and on tubulin the of state type and of tau in the presence of tubulin at and MT assembly was by for the The is as the the of a the of state in a type and of tau in the presence of tubulin at and MT assembly was by for the The is as the the of a The studies to and tau mutants. on tubulin are in I for with single mutants. the and the of tubulin the to the wild type tau shown in to the phosphorylated of and single an in tau not the or the of tubulin in the corresponding and tau mutants. These results suggest that to a phosphorylation at and is responsible for tau's to promote tubulin polymerization. I that the of tubulin was with the form of the whereas the was to the or the phosphorylated tau mutants. we that in vitro II phosphorylation of tau at and is primarily responsible for the of tau to promote microtubule assembly. brain II J. K. J. J. Biol. 1992; Full Text PDF PubMed Google Scholar, D. J. Biol. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, J. J. Biol. 1993; Full Text PDF PubMed Google Scholar) and of in the presence of various recombinant of the protein from and human have been reported L.-H. I. T. Nature. PubMed Scopus Google Scholar, J. R. T. Nature. PubMed Scopus Google Scholar, D. M. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar, P. M. Jr., J. Biol. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, J. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar, J. T. J. Biol. 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Neuron. 1989; 3: 519-526Abstract Full Text PDF PubMed Scopus (1872) Google and these are sites for proline-directed These sites are and II of these sites in human tau. in mutational and functional studies 5 and are the to that of the sites phosphorylated by phosphorylated and are critical in microtubule assembly. these studies are the to show that human in to tau phosphorylation of tau and dissociation of tau from microtubules J. Biol. 1991; Full Text PDF PubMed Google Scholar) indicate that I to to and N-terminal to and the is on the of a role of II in I sites be In studies K. K. Takamatsu M. J. Uchida T. Imahori K. Neurosci. Lett. PubMed Scopus Google Scholar, I. G. M. J. Neurosci. PubMed Google Scholar, A. J. Novak M. Q. Grundke-Iqbal I. Iqbal K. 1998; PubMed Scopus Google was that phosphorylation of tau by kinase be in the of by I. has been shown that phosphorylation of tau by II is to generate an by I A. J. Novak M. Q. Grundke-Iqbal I. 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