2004/01/30 by Hideki Shimura, Daniel Schwartz, Steven P. Gygi +1 · 28 citations
Medicine · Biochemistry, Genetics and Molecular Biology · #Alzheimer's disease research and treatments #Ubiquitin and proteasome pathways #Parkinson's Disease Mechanisms and Treatments
paper · pdf · doi:10.1074/jbc.m305838200
The microtubule-binding protein tau has been implicated in the neurofibrillary pathology of Alzheimer's disease. Within affected cells, ubiquitinated and hyperphosphorylated tau assembles into massive filamentous polymers. Eventually these tangle-bearing neurons die. The formation of neurofibrillary tangles closely parallels the progression and anatomic distribution of neuronal loss in Alzheimer's disease, suggesting that these lesions play a role in the disease pathogenesis. Mutations in the human tau gene cause autosomal dominant neurodegenerative disorders. These and other neurodegenerative conditions are also characterized by extensive neurofibrillary pathology. The mechanisms underlying tau-mediated neurotoxicity remain unclear; however, phosphorylated tau is a strong candidate for a toxic molecule, particularly those isoforms phosphorylated by the kinases glycogen synthase kinase 3β and Cdk5. Here we show that Alzheimer tau binds to Hsc70, and its phosphorylation is a recognition requirement for the addition of ubiquitin (Ub) by the E3 Ub ligase CHIP (carboxyl terminus of the Hsc70-interacting protein) and the E2 conjugating enzyme UbcH5B. Other E3 Ub ligases including parkin and Cbl failed to ubiquitinate phosphorylated tau. CHIP could rescue phosphorylated tau-induced cell death, and therefore the CHIP-Hsc70 complex may provide a new therapeutic target for the tauopathies. The microtubule-binding protein tau has been implicated in the neurofibrillary pathology of Alzheimer's disease. Within affected cells, ubiquitinated and hyperphosphorylated tau assembles into massive filamentous polymers. Eventually these tangle-bearing neurons die. The formation of neurofibrillary tangles closely parallels the progression and anatomic distribution of neuronal loss in Alzheimer's disease, suggesting that these lesions play a role in the disease pathogenesis. Mutations in the human tau gene cause autosomal dominant neurodegenerative disorders. These and other neurodegenerative conditions are also characterized by extensive neurofibrillary pathology. The mechanisms underlying tau-mediated neurotoxicity remain unclear; however, phosphorylated tau is a strong candidate for a toxic molecule, particularly those isoforms phosphorylated by the kinases glycogen synthase kinase 3β and Cdk5. Here we show that Alzheimer tau binds to Hsc70, and its phosphorylation is a recognition requirement for the addition of ubiquitin (Ub) by the E3 Ub ligase CHIP (carboxyl terminus of the Hsc70-interacting protein) and the E2 conjugating enzyme UbcH5B. Other E3 Ub ligases including parkin and Cbl failed to ubiquitinate phosphorylated tau. CHIP could rescue phosphorylated tau-induced cell death, and therefore the CHIP-Hsc70 complex may provide a new therapeutic target for the tauopathies. Tau inclusions are a major feature of several neurodegenerative diseases including Alzheimer's disease (1Jellinger K.A. J. Neural Transm. Suppl. 2003; 65: 101-144Crossref PubMed Scopus (117) Google Scholar, 2Lansbury Jr., P.T. Kosik K.S. Chem. Biol. 2000; 7: R9-R12Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar). Normally, tau functions as a neuronal microtubule-binding protein, and its binding to microtubules is regulated both by alternative splicing within the binding region and by phosphorylation in and around the binding region (3Kosik K.S. Brain Microtubule-associated Proteins. Harwood Academic Publishers, Amsterdam1997: 43-52Google Scholar). The flanking region around the microtubule-binding repeats is phosphorylated by multiple different kinases. Up to 19 different sites can be phosphorylated on tau (4Johnson G.V. Jenkins S.M. J. Alzheimers Dis. 1999; 1: 307-328Crossref PubMed Scopus (41) Google Scholar, 5Buee L. Bussiere T. Buee-Scherrer V. Delacourte A. Hof P.R. Brain Res. Brain Res. Rev. 2000; 33: 95-130Crossref PubMed Scopus (1620) Google Scholar), thus creating a staggeringly high number of possible tau phosphoisoforms. Little is known about the function of these different phosphorylation states other than that they alter the binding kinetics of tau to microtubules. Not only are many different sites phosphorylated on tau, allowing for quite subtle regulation of microtubule binding, but the controls over the sites can be regulated through different signaling pathways as indicated by the many different kinases capable of phosphorylating tau. Among the kinases capable of phosphorylating tau in vitro are both proline-directed kinases and non-proline-directed kinases. They include glycogen synthase kinase 3β (GSK-3β), 1The abbreviations used are: GSKglycogen synthase kinasePHFpaired helical filamentE1ubiquitin-activating enzymeE2ubiquitin carrier proteinE3ubiquitin-protein isopeptide ligaseMES4-morpholineethanesulfonic acidGSTglutathione S-transferasePP2Aprotein phosphatase 2AEGFPenhanced green fluorescence proteinADAlzheimer's disease. extracellular signal regulated kinase, stress-activated protein kinase, cyclin-dependent kinase 5 (Cdk5), CDC2-cyclin A kinase, MARK kinase, Ca2+/calmodulin-dependent protein kinase, cyclic AMP-dependent protein kinase, protein kinase C, casein kinase I and II, double-stranded DNA-dependent protein kinase, microtubule-associated protein/microtubule affinity-regulating kinase, and tau-tubulin kinase (4Johnson G.V. Jenkins S.M. J. Alzheimers Dis. 1999; 1: 307-328Crossref PubMed Scopus (41) Google Scholar, 5Buee L. Bussiere T. Buee-Scherrer V. Delacourte A. Hof P.R. Brain Res. Brain Res. Rev. 2000; 33: 95-130Crossref PubMed Scopus (1620) Google Scholar). glycogen synthase kinase paired helical filament ubiquitin-activating enzyme ubiquitin carrier protein ubiquitin-protein isopeptide ligase 4-morpholineethanesulfonic acid glutathione S-transferase protein phosphatase 2A enhanced green fluorescence protein Alzheimer's disease. Because phosphorylation releases tau from microtubules and because tau in the paired helical filament (PHF) is highly phosphorylated, kinases have been viewed with interest for a possible role in pathogenesis. Fath et al. (6Fath T. Eidenmuller J. Brandt R. J. Neurosci. 2002; 22: 9733-9741Crossref PubMed Google Scholar) showed that replacement of certain amino acids at known sites of phosphorylation with a charged amino acid to create “pseudohyperphosphorylated” tau can mimic structural and functional aspects of hyperphosphorylated tau; in differentiated PC12 cells, PHF-tau exhibited reduced microtubule interaction and caused apoptotic cell death. In vivo evidence for an interaction with tau exists for Cdk-5 and GSK-3β. Overexpression of human p25 (an activator of Cdk5) in mice induced tau hyperphosphorylation and cytoskeletal disruptions reminiscent of AD, but no filamentous deposits (7Ahlijanian M.K. Barrezueta N.X. Williams R.D. Jakowski A. Kowsz K.P. McCarthy S. Coskran T. Carlo A. Seymour P.A. Burkhardt J.E. Nelson R.B. McNeish J.D. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 2910-2915Crossref PubMed Scopus (311) Google Scholar). Noble et al. (8Noble W. Olm V. Takata K. Casey E. Mary O. Meyerson J. Gaynor K. LaFrancois J. Wang L. Kondo T. Davies P. Burns M. Nixon Veeranna R. Dickson D. Matsuoka Y. Ahlijanian M. Lau L.F. Duff K. Neuron. 2003; 38: 555-565Abstract Full Text Full Text PDF PubMed Scopus (444) Google Scholar) crossed transgenic mice overexpressing the Cdk5 activator p25 with transgenic mice overexpressing mutant (P301L) human tau. Tau was hyperphosphorylated at several sites in the double transgenics, and a highly significant accumulation of aggregated tau occurred in the brainstem and cortex. Increased numbers of silver-stained neurofibrillary tangles accompanied these changes as well as an association of active GSK with insoluble tau (8Noble W. Olm V. Takata K. Casey E. Mary O. Meyerson J. Gaynor K. LaFrancois J. Wang L. Kondo T. Davies P. Burns M. Nixon Veeranna R. Dickson D. Matsuoka Y. Ahlijanian M. Lau L.F. Duff K. Neuron. 2003; 38: 555-565Abstract Full Text Full Text PDF PubMed Scopus (444) Google Scholar). Overexpression of GSK-3β under the control of a tetracycline-sensitive transactivator also induced tau hyperphosphorylation, somatodendritic mislocalization of tau, and neuronal apoptosis (9Lucas J.J. Hernandez F. Gomez-Ramos P. Moran M.A. Hen R. Avila J. EMBO J. 2001; 20: 27-39Crossref PubMed Scopus (822) Google Scholar). However, the complexity of the role of GSK-3β was underscored in transgenic mice that expressed a constitutively active mutant form of human GSK-3β and surprisingly did not show neurofibrillary pathology. In fact, when crossed with the htau40 transgenic mouse, they improved the axonal dilations and motoric problems observed in htau40 mice (10Spittaels K. Van den Haute C. Van Dorpe J. Geerts H. Mercken M. Bruynseels K. Lasrado R. Vandezande K. Laenen I. Boon T. Van Lint J. Vandenheede J. Moechars D. Loos R. Van Leuven F. J. Biol. Chem. 2000; 275: 41340-41349Abstract Full Text Full Text PDF PubMed Scopus (283) Google Scholar). Nevertheless, overall these studies implicate tau hyperphosphorylation in tau-related neurodegeneration. Ubiquitination is a cellular process by which short lived or damaged proteins are conjugated with multimers of Ub, marking them for degradation in the proteasome. Conjugation requires an enzymatic cascade system that includes E1 Ub-activating enzyme, E2 Ub-conjugating enzyme (Ubc), and E3 Ub ligase enzyme. Evidence in favor of the Ub-proteasome system involvement in the pathogenesis of neurodegeneration includes the following: 1) inclusion bodies found in a variety of neurodegenerative disorders contain Ub and ubiquitinated proteins; 2) mutations in two Ub pathway enzymes, parkin (11Kitada T. Asakawa S. Hattori N. Matsumine H. Yamamura Y. Minoshima S. Yokochi M. Mizuno Y. Shimizu N. Nature. 1998; 392: 605-608Crossref PubMed Scopus (4324) Google Scholar, 12Shimura H. Hattori N. Kubo S. Mizuno Y. Asakawa S. Minoshima S. Shimizu N. Iwai K. Chiba T. Tanaka K. Suzuki T. Nat. Genet. 2000; 25: 302-305Crossref PubMed Scopus (1741) Google Scholar, 13Imai Y. Soda M. Takahashi R. J. Biol. Chem. 2000; 275: 35661-35664Abstract Full Text Full Text PDF PubMed Scopus (663) Google Scholar, 14Zhang Y. Gao J. Chung K.K. Huang H. Dawson V.L. Dawson T.M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 13354-13359Crossref PubMed Scopus (849) Google Scholar) and ubiquitin carboxyl-terminal hydrolase L1 (15Maraganore D.M. De Andrade M. Lesnick T.G. Farrer M.J. Bower J.H. Hardy J.A. Rocca W.A. Movement Disorders. 2003; 18: 631-636Crossref PubMed Scopus (28) Google Scholar, 16Leroy E. Boyer R. Auburger G. Leube B. Ulm G. Mezey E. Harta G. Brownstein M.J. Jonnalagada S. Chernova T. Dehejia A. Lavedan C. Gasser T. Steinbach P.J. Wilkinson K.D. Polymeropoulos M.H. Nature. 1998; 395: 451-452Crossref PubMed Scopus (1400) Google Scholar), cause a form of Parkinson's disease (PD); 3) proteasome activity is decreased or impaired in PD and AD post-mortem brain (17McNaught K.S. Belizaire R. Isacson O. Jenner P. Olanow C.W. Exp. Neurol. 2003; 179: 38-46Crossref PubMed Scopus (516) Google Scholar); 4) a mutation in a multi-Ub gene is associated with AD (18De Vrij F.M. Sluijs J.A. Gregori L. Fischer D.F. Hermens W.T. Goldgaber D. Verhaagen J. Van Leeuwen F.W. Hol E.M. FASEB J. 2001; 15: 2680-2688Crossref PubMed Scopus (120) Google Scholar); and 5) the loss of function of the Ub ligase, Ube3a in SCA1 mice increases neurodegeneration (19Cummings C.J. Reinstein E. Sun Y. Antalffy B. Jiang Y. Ciechanover A. Orr H.T. Beaudet A.L. Zoghbi H.Y. Neuron. 1999; 24: 879-892Abstract Full Text Full Text PDF PubMed Scopus (447) Google Scholar). Cbl, a Ub ligase, selectively conjugates Ub onto tyrosine-phosphorylated target molecules such as platelet-derived growth factor receptor, epidermal growth factor receptor, Syk, and Fyn (20Rao N. Dodge I. Band H. J. Leukocyte Biol. 2002; 71: 753-763PubMed Google Scholar). We therefore hypothesized that hyperphosphorylated tau could be ubiquitinated by an unknown E3 Ub ligase(s) for proteasomal delivery and degradation. Here we show that a CHIP (carboxyl terminus of the Hsc70-interacting protein)-Hsc70 complex selectively ubiquitinates phosphorylated tau in with UbcH5B. CHIP phosphorylated tau-induced cell death. and in with and in a with the to the and from human brain the and at for at with protein A and protein to tau for at in the and the at for at The to as the The in and at for at The to as the in was to the and and as H. Hattori N. A. R. Mizuno Y. Kosik K.S. 2001; PubMed Scopus Google Scholar). We by and the that was from was as the the with with and with or was by conjugated to at for and with the to the with a system In Tau and GSK-3β was in of and kinase tau was with or as the tau was with phosphatase or protein phosphatase 2A for at In Ubiquitination of tau from that conjugated to protein and and proteins from the with and the to was by tau was in a of Ub-activating enzyme a Ub-conjugating enzyme or of of and and for at the from tau, we the with the in vitro the in vitro proteins with The by with In Ubiquitination of and phosphorylated by GSK-3β was in a of of of or of and of Hsc70, and and for at of Ub for from human brain and and in the of and at for at The over a phosphorylated and a by a in and and with proteins by and with and by ligase activity of the was by an in vitro phosphorylated tau or phosphorylated to was to human brain and at for with The proteins from the by of used J.H. Kosik K.S. J. Exp. Neurol. PubMed Scopus Google Scholar, K.S. L. G. Neuron. 1: Full Text PDF PubMed Scopus Google Scholar) tau which isoforms of tau; J. E.M. C. B. B. B. H. Mercken M. A. M. EMBO J. PubMed Scopus Google Scholar) a tau that phosphorylated and Davies P. J.D. J. Biol. Chem. Full Text PDF PubMed Google Scholar, Jr., L. PubMed Scopus Google Scholar), a tau that phosphorylated and Tau J. E.M. C. B. B. B. H. Mercken M. A. M. EMBO J. PubMed Scopus Google Scholar), a tau that and when a tau that isoforms of tau; and GSK-3β and and as H. Hattori N. Kubo S. Mizuno Y. Asakawa S. Minoshima S. Shimizu N. Iwai K. Chiba T. Tanaka K. Suzuki T. Nat. Genet. 2000; 25: 302-305Crossref PubMed Scopus (1741) Google Scholar). was as M. Kosik K.S. Biol. 2001; PubMed Scopus Google Scholar). mutant and from was a from GSK-3β was into GSK-3β was into a Hsc70, and phosphatase from AD Brain Tau in with tau from human brain is ubiquitinated in we on of from two and AD We used a tau that J.H. Kosik K.S. J. Exp. Neurol. PubMed Scopus Google Scholar, K.S. L. G. Neuron. 1: Full Text PDF PubMed Scopus Google Scholar) and tau Tau was and therefore the insoluble of not be observed by both and tau proteins however, tau was We tau from the tau and an in vitro which E2 and We molecules with and with the tau in an in vitro AD tau was and control tau was not a by the tau in A by the in tau was did not ubiquitinate tau in vitro tau in vitro was not indicated that the an E3 Ub ligase for tau. The high in these was ubiquitinated tau, and not aggregated tau, because the when did not a high with the tau The in vitro of AD tau, but not control tau, was on that E1 and E2 We in the in vitro and found that AD tau was and ubiquitinated with The other in the did not ubiquitinate AD tau. that the E2 conjugating enzyme for tau is UbcH5B. tau by in the in vitro observed only with tau that was from AD and not from control as not of Tau the for Tau to the of of tau are by the E3 Ub The are phosphorylation (4Johnson G.V. Jenkins S.M. J. Alzheimers Dis. 1999; 1: 307-328Crossref PubMed Scopus (41) Google Scholar, 5Buee L. Bussiere T. Buee-Scherrer V. Delacourte A. Hof P.R. Brain Res. Brain Res. Rev. 2000; 33: 95-130Crossref PubMed Scopus (1620) Google Scholar). showed that AD phosphorylation of tau is the signal for tau AD tau was from brain and with phosphatase or and in vitro with and with the tau tau can be in in the control and in the These indicated that phosphorylation of sites is an recognition signal for to the of tau phosphorylation in formation in AD or in the tauopathies. Alzheimer Tau to on the to the tau E3 Ub proteins in the by and those which observed in the AD tau by However, we could not the E3 Ub ligase in not We the on the that can recognition by the E3 Ub ligase and in vitro is in brain and is associated with microtubules J. T. L. Avila J. I. K. Brain Res. 2000; PubMed Scopus Google Scholar). brain as a of by acid induced an hyperphosphorylation and accumulation of tau E. V. G. Brandt R. C. J. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). of the sites that are by are phosphorylated by GSK-3β or Cdk5. These include sites and Y. J. 65: PubMed Scopus Google Scholar, E. V. G. Neuron. Full Text Full Text PDF PubMed Scopus Google Scholar, M. M. K. Suzuki M. H. K. Y. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, S. U. K. K. B. J. R. E.M. E. Biol. 1998; PubMed Scopus Google Scholar). We used of human tau in an in vitro phosphorylation with GSK-3β. phosphorylated, the tau protein on with Davies P. J.D. J. Biol. Chem. Full Text PDF PubMed Google Scholar, Jr., L. PubMed Scopus Google Scholar) and J. E.M. C. B. B. B. H. Mercken M. A. M. EMBO J. PubMed Scopus Google Scholar), that at sites and GSK-3β tau as an for in vitro and the from AD tau that GSK-3β can on tau that create recognition sites for an E3 Ub The not that GSK-3β has in fact, Cdk5 can also on tau that as an E3 Ub ligase recognition because tau phosphorylated by Cdk5 was also ubiquitinated in vitro not was phosphorylated by GSK-3β and to a human brain was over the The was and over a to the to was E3 Ub ligase activity for the phosphorylated tau The by to and of these was by The that the was the protein we the of the interaction phosphorylated tau and We conjugated phosphorylated and to and of from human We found a of in the with phosphorylated conjugated to the and with the suggesting that binds phosphorylated tau In Ubiquitination of the Tau of in the complex a degradation pathway that CHIP as the E3 ligase J. Y. D.M. J. C. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). the from the was with an CHIP and an of was observed not However, because we expressed can be from an and We into cells, and from over the phosphorylated and and by The was by with a and we These a by which phosphorylated tau is to a and the complex is by the E3 Ub ligase We ubiquitinates phosphorylated tau in vitro in the of and Ubiquitination of phosphorylated tau was as high by with in the of and CHIP Tau for in we CHIP ubiquitinates phosphorylated tau in We and GSK-3β or a GSK-3β with the mutation I. K. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar) into the with the proteasome or as a control for from in with or and with or and We observed high tau with and but not with in with or when and but not in the of or The phosphorylation of the high ubiquitinated tau was which phosphorylated and and which isoforms of tau in a with high ubiquitinated tau. which and was not that and of high tau phosphorylated, and enhanced accumulation of phosphorylated tau. These that CHIP ubiquitinates phosphorylated tau for degradation by the proteasome in We loss of function CHIP with an mutation in the or other ubiquitin such as parkin and Cbl, to ubiquitinate tau. and with mutant or in with or as a with a or an and with a with the tau high tau was observed in of cell from but not mutant and CHIP as the E3 ligase for phosphorylated tau. CHIP the also observed that of both and GSK-3β caused cell death. we and into to the of of tau on cell We from with a and in We found that the of both and GSK-3β the of cell with the of and mutant GSK-3β or GSK-3β indicated that cell on the of GSK-3β and phosphorylated tau, and phosphorylated tau cell expressed decreased the of cell caused by the of and GSK-3β that phosphorylated tau may cause cell and that CHIP can rescue of cell death. The by in and the insoluble in phosphorylated tau, with and but not was in that been by that expressed and reduced cell and phosphorylated tau in both the and the In phosphorylated tau was in the of the cells, which expressed and but not and a of cell death. These that accumulation of phosphorylated tau is insoluble ubiquitinated phosphorylated tau is not and CHIP ubiquitinates phosphorylated tau not only for degradation in the proteasome but also to tau into a insoluble for of cell death. of with in the accumulation of phosphorylated tau in the with a role for the of phosphorylated tau into an insoluble was the in the of with with and The accumulation of phosphorylated tau in the of these did not cell death. The the that insoluble ubiquitinated tau may not to cell death. in tau in the cells, was used to phosphorylated tau with or with the green observed in the of with and mutant GSK-3β an showed that to microtubules not In fluorescence of and with green in the and The also with and and and These that ubiquitinated phosphorylated tau is a of the and no found in with but not of phosphorylated showed that phosphorylated to the and These cellular with the in which insoluble ubiquitinated phosphorylated tau was found in cell that expressed and We showed that phosphorylated tau was ubiquitinated by CHIP to in with UbcH5B. We a in which phosphorylated tau induced cell when tau and GSK-3β CHIP the cell caused by phosphorylated tau. ubiquitinated phosphorylated tau and ubiquitinated phosphorylated in the cells, which expressed tau, and GSK-3β. In mutant CHIP the other ubiquitin parkin and Cbl, conjugated Ub onto hyperphosphorylated tau. studies have a protein, repeats to and its with thus the of a Ub such as the C. W. D.M. Nat. Biol. 2001; PubMed Scopus Google Scholar), P. N. Dodge A.L. N. H. Band V. Band H. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar), the receptor, and kinase J. S. C. J. Biol. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar), as target molecules of et al. that was ubiquitinated by CHIP and that selectively ubiquitinates by with S. Y. M. Chiba T. Tanaka K. EMBO 2001; PubMed Scopus Google Scholar). CHIP to play a role in to the proteasome. The structural the tau is however, certain tau phosphorylation to et al. F. K. F. A. P. H. Proc. Natl. Acad. Sci. U. S. A. 2003; PubMed Scopus Google Scholar) that and tau; however, they did not the phosphorylation of tau. the complex is by CHIP and selectively and ubiquitinated phosphorylated tau in vivo and in vitro in protein a variety of neurodegenerative such as Parkinson's disease, disease, and of these conditions with protein by protein which are However, of the has a for neuronal cell in neurodegenerative In fact, an protein into an may for from the loss of function of the Ub ligase, Ube3a in SCA1 which increases neurodegeneration the number of (19Cummings C.J. Reinstein E. Sun Y. Antalffy B. Jiang Y. Ciechanover A. Orr H.T. Beaudet A.L. Zoghbi H.Y. Neuron. 1999; 24: 879-892Abstract Full Text Full Text PDF PubMed Scopus (447) Google Scholar). SCA1 mice form in the and that are In these that of the can may from in the form of or Here we showed that with expressed and have insoluble and enhanced In with expressed and but CHIP no ubiquitinated and reduced These that conjugates Ub onto phosphorylated tau not only for degradation but also to form that may from the of phosphorylated tau. In the CHIP-Hsc70 complex conjugates Ub to hyperphosphorylated tau in with UbcH5B. may also cell by phosphorylated tau in favor of insoluble an for cell the CHIP-Hsc70 complex may new therapeutic to AD and other neurodegenerative disorders. We H. J. and M. for and We also M. for and for to human brain