Familial FTDP-17 Missense Mutations Inhibit Microtubule Assembly-promoting Activity of Tau by Increasing Phosphorylation at Ser202 in Vitro
2009; Elsevier BV; Volume: 284; Issue: 20 Linguagem: Inglês
10.1074/jbc.m901095200
ISSN1083-351X
AutoresDong Cho Han, Hamid Y. Qureshi, Yifan Lu, Hemant K. Paudel,
Tópico(s)Prion Diseases and Protein Misfolding
ResumoIn Alzheimer disease (AD), frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17) and other tauopathies, tau accumulates and forms paired helical filaments (PHFs) in the brain. Tau isolated from PHFs is phosphorylated at a number of sites, migrates as ∼60-, 64-, and 68-kDa bands on SDS-gel, and does not promote microtubule assembly. Upon dephosphorylation, the PHF-tau migrates as ∼50–60-kDa bands on SDS-gels in a manner similar to tau that is isolated from normal brain and promotes microtubule assembly. The site(s) that inhibits microtubule assembly-promoting activity when phosphorylated in the diseased brain is not known. In this study, when tau was phosphorylated by Cdk5 in vitro, its mobility shifted from ∼60-kDa bands to ∼64- and 68-kDa bands in a time-dependent manner. This mobility shift correlated with phosphorylation at Ser202, and Ser202 phosphorylation inhibited tau microtubule-assembly promoting activity. When several tau point mutants were analyzed, G272V, P301L, V337M, and R406W mutations associated with FTDP-17, but not nonspecific mutations S214A and S262A, promoted Ser202 phosphorylation and mobility shift to a ∼68-kDa band. Furthermore, Ser202 phosphorylation inhibited the microtubule assembly-promoting activity of FTDP-17 mutants more than of WT. Our data indicate that FTDP-17 missense mutations, by promoting phosphorylation at Ser202, inhibit the microtubule assembly-promoting activity of tau in vitro, suggesting that Ser202 phosphorylation plays a major role in the development of NFT pathology in AD and related tauopathies. In Alzheimer disease (AD), frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17) and other tauopathies, tau accumulates and forms paired helical filaments (PHFs) in the brain. Tau isolated from PHFs is phosphorylated at a number of sites, migrates as ∼60-, 64-, and 68-kDa bands on SDS-gel, and does not promote microtubule assembly. Upon dephosphorylation, the PHF-tau migrates as ∼50–60-kDa bands on SDS-gels in a manner similar to tau that is isolated from normal brain and promotes microtubule assembly. The site(s) that inhibits microtubule assembly-promoting activity when phosphorylated in the diseased brain is not known. In this study, when tau was phosphorylated by Cdk5 in vitro, its mobility shifted from ∼60-kDa bands to ∼64- and 68-kDa bands in a time-dependent manner. This mobility shift correlated with phosphorylation at Ser202, and Ser202 phosphorylation inhibited tau microtubule-assembly promoting activity. When several tau point mutants were analyzed, G272V, P301L, V337M, and R406W mutations associated with FTDP-17, but not nonspecific mutations S214A and S262A, promoted Ser202 phosphorylation and mobility shift to a ∼68-kDa band. Furthermore, Ser202 phosphorylation inhibited the microtubule assembly-promoting activity of FTDP-17 mutants more than of WT. Our data indicate that FTDP-17 missense mutations, by promoting phosphorylation at Ser202, inhibit the microtubule assembly-promoting activity of tau in vitro, suggesting that Ser202 phosphorylation plays a major role in the development of NFT pathology in AD and related tauopathies. Neurofibrillary tangles (NFTs) 4The abbreviations used are: NFT, neurofibrillary tangle; AD, Alzheimer disease; Cdk5, cyclin-dependent protein kinase 5; AU, absorption unit; FTDP-17, frontotemporal dementia and Parkinsonism linked to chromosome 17; PHF, paired helical filament; PKA, cAMP-dependent protein kinase; Pipes, 1,4-piperazinediethanesulfonic acid; WT, wild type. and senile plaques are the two characteristic neuropathological lesions found in the brains of patients suffering from Alzheimer disease (AD). The major fibrous component of NFTs are paired helical filaments (PHFs) (for reviews see Refs. 1Iqbal K. Alonso Adel C. Chen S. Chohan M.O. El-Akkad E. Gong C.X. Khatoon S. Li B. Liu F. Rahman A. Tanimukai H. Grundke-Iqbal I. Biochim. Biophys. Acta. 2005; 1739: 198-210Crossref PubMed Scopus (739) Google Scholar, 2Lee V.M. Goedert M. Trojanowski J.Q. Annu. Rev. Neurosci. 2001; 24: 1121-1159Crossref PubMed Scopus (2144) Google Scholar, 3Avila J. Lucas J.J. Perez M. Hernandez F. Physiol. Rev. 2004; 84: 361-384Crossref PubMed Scopus (673) Google Scholar). Initially, PHFs were found to be composed of a protein component referred to as "A68" (4Lee V.M. Balin B.J. Otvos Jr., L. Trojanowski J.Q. Science. 1991; 251: 675-678Crossref PubMed Scopus (1251) Google Scholar). Biochemical analysis reveled that A68 is identical to the microtubule-associated protein, tau (4Lee V.M. Balin B.J. Otvos Jr., L. Trojanowski J.Q. Science. 1991; 251: 675-678Crossref PubMed Scopus (1251) Google Scholar, 5Greenberg S.G. Davies P. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 5827-5831Crossref PubMed Scopus (657) Google Scholar). Some characteristic features of tau isolated from PHFs (PHF-tau) are that it is abnormally hyperphosphorylated (phosphorylated on more sites than the normal brain tau), does not bind to microtubules, and does not promote microtubule assembly in vitro. Upon dephosphorylation, PHF-tau regains its ability to bind to and promote microtubule assembly (6Bramblett G.T. Goedert M. Jakes R. Merrick S.E. Trojanowski J.Q. Lee V.M. Neuron. 1993; 10: 1089-1099Abstract Full Text PDF PubMed Scopus (758) Google Scholar, 7Alonso A.C. Zaidi T. Grundke-Iqbal I. Iqbal K. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5562-5566Crossref PubMed Scopus (600) Google Scholar). Tau hyperphosphorylation is suggested to cause microtubule instability and PHF formation, leading to NFT pathology in the brain (1Iqbal K. Alonso Adel C. Chen S. Chohan M.O. El-Akkad E. Gong C.X. Khatoon S. Li B. Liu F. Rahman A. Tanimukai H. Grundke-Iqbal I. Biochim. Biophys. Acta. 2005; 1739: 198-210Crossref PubMed Scopus (739) Google Scholar, 2Lee V.M. Goedert M. Trojanowski J.Q. Annu. Rev. Neurosci. 2001; 24: 1121-1159Crossref PubMed Scopus (2144) Google Scholar, 3Avila J. Lucas J.J. Perez M. Hernandez F. Physiol. Rev. 2004; 84: 361-384Crossref PubMed Scopus (673) Google Scholar). PHF-tau is phosphorylated on at least 21 proline-directed and non-proline-directed sites (8Morishima-Kawashima M. Hasegawa M. Takio K. Suzuki M. Yoshida H. Titani K. Ihara Y. J. Biol. Chem. 1995; 270: 823-829Abstract Full Text Full Text PDF PubMed Scopus (529) Google Scholar, 9Hanger D.P. Betts J.C. Loviny T.L. Blackstock W.P. Anderton B.H. J. Neurochem. 1998; 71: 2465-2476Crossref PubMed Scopus (336) Google Scholar). The individual contribution of these sites in converting tau to PHFs is not entirely clear. However, some sites are only partially phosphorylated in PHFs (8Morishima-Kawashima M. Hasegawa M. Takio K. Suzuki M. Yoshida H. Titani K. Ihara Y. J. Biol. Chem. 1995; 270: 823-829Abstract Full Text Full Text PDF PubMed Scopus (529) Google Scholar), whereas phosphorylation on specific sites inhibits the microtubule assembly-promoting activity of tau (6Bramblett G.T. Goedert M. Jakes R. Merrick S.E. Trojanowski J.Q. Lee V.M. Neuron. 1993; 10: 1089-1099Abstract Full Text PDF PubMed Scopus (758) Google Scholar, 10Biernat J. Gustke N. Drewes G. Mandelkow E.M. Mandelkow E. Neuron. 1993; 11: 153-163Abstract Full Text PDF PubMed Scopus (648) Google Scholar). These observations suggest that phosphorylation on a few sites may be responsible and sufficient for causing tau dysfunction in AD. Tau purified from the human brain migrates as ∼50–60-kDa bands on SDS-gel due to the presence of six isoforms that are phosphorylated to different extents (2Lee V.M. Goedert M. Trojanowski J.Q. Annu. Rev. Neurosci. 2001; 24: 1121-1159Crossref PubMed Scopus (2144) Google Scholar). PHF-tau isolated from AD brain, on the other hand, displays ∼60-, 64-, and 68 kDa-bands on an SDS-gel (4Lee V.M. Balin B.J. Otvos Jr., L. Trojanowski J.Q. Science. 1991; 251: 675-678Crossref PubMed Scopus (1251) Google Scholar, 5Greenberg S.G. Davies P. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 5827-5831Crossref PubMed Scopus (657) Google Scholar, 11Crowther R.A. Goedert M. J. Struct. Biol. 2000; 130: 271-279Crossref PubMed Scopus (146) Google Scholar). Studies have shown that ∼64- and 68-kDa tau bands (the authors have described the ∼68-kDa tau band as an ∼69-kDa band in these studies) are present only in brain areas affected by NFT degeneration (12Flament S. Delacourte A. Delaere P. Duyckaerts C. Hauw J.J. Acta Neuropathol. 1990; 80: 212-215Crossref PubMed Scopus (43) Google Scholar, 13Flament S. Delacourte A. FEBS Lett. 1989; 247: 213-216Crossref PubMed Scopus (49) Google Scholar). Their amount is correlated with the NFT densities at the affected brain regions. Moreover, the increase in the amount of ∼64- and 68-kDa band tau in the brain correlated with a decline in the intellectual status of the patient. The ∼64- and 68-kDa tau bands were suggested to be the pathological marker of AD (12Flament S. Delacourte A. Delaere P. Duyckaerts C. Hauw J.J. Acta Neuropathol. 1990; 80: 212-215Crossref PubMed Scopus (43) Google Scholar, 13Flament S. Delacourte A. FEBS Lett. 1989; 247: 213-216Crossref PubMed Scopus (49) Google Scholar). Biochemical analyses determined that ∼64- and 68-kDa bands are hyperphosphorylated tau, which upon dephosphorylation, migrated as normal tau on SDS-gel (4Lee V.M. Balin B.J. Otvos Jr., L. Trojanowski J.Q. Science. 1991; 251: 675-678Crossref PubMed Scopus (1251) Google Scholar, 5Greenberg S.G. Davies P. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 5827-5831Crossref PubMed Scopus (657) Google Scholar, 11Crowther R.A. Goedert M. J. Struct. Biol. 2000; 130: 271-279Crossref PubMed Scopus (146) Google Scholar). Tau sites involved in the tau mobility shift to ∼64- and 68-kDa bands were suggested to have a role in AD pathology (12Flament S. Delacourte A. Delaere P. Duyckaerts C. Hauw J.J. Acta Neuropathol. 1990; 80: 212-215Crossref PubMed Scopus (43) Google Scholar, 13Flament S. Delacourte A. FEBS Lett. 1989; 247: 213-216Crossref PubMed Scopus (49) Google Scholar). It is not known whether phosphorylation at all 21 PHF-sites is required for the tau mobility shift in AD. However, in vitro the tau mobility shift on SDS-gel is sensitive to phosphorylation only on some sites (6Bramblett G.T. Goedert M. Jakes R. Merrick S.E. Trojanowski J.Q. Lee V.M. Neuron. 1993; 10: 1089-1099Abstract Full Text PDF PubMed Scopus (758) Google Scholar, 14Steiner B. Mandelkow E.M. Biernat J. Gustke N. Meyer H.E. Schmidt B. Mieskes G. Soling H.D. Drechsel D. Kirschner M.W. Goedert M. Mandelkow E. EMBO J. 1990; 9: 3539-3544Crossref PubMed Scopus (257) Google Scholar). It is therefore possible that in the AD brain, phosphorylation on some sites also causes a tau mobility shift. Identification of such sites will significantly enhance our knowledge of how NFT pathology develops in the brain. PHFs are also the major component of NFTs found in the brains of patients suffering from a group of neurodegenerative disorders collectively called tauopathies (2Lee V.M. Goedert M. Trojanowski J.Q. Annu. Rev. Neurosci. 2001; 24: 1121-1159Crossref PubMed Scopus (2144) Google Scholar, 11Crowther R.A. Goedert M. J. Struct. Biol. 2000; 130: 271-279Crossref PubMed Scopus (146) Google Scholar). These disorders include frontotemporal dementia and Parkinsonism linked to chromosome 17 (FTDP-17), corticobasal degeneration, progressive supranuclear palsy, and Pick disease. Each PHF-tau isolated from autopsied brains of patients suffering from various tauopathies is hyperphosphorylated, displays ∼60-, 64-, and 68-kDa bands on SDS-gel, and is incapable of binding to microtubules. Upon dephosphorylation, the above referenced PHF-tau migrates as a normal tau on SDS-gel, binds to microtubules, and promotes microtubule assembly (2Lee V.M. Goedert M. Trojanowski J.Q. Annu. Rev. Neurosci. 2001; 24: 1121-1159Crossref PubMed Scopus (2144) Google Scholar, 11Crowther R.A. Goedert M. J. Struct. Biol. 2000; 130: 271-279Crossref PubMed Scopus (146) Google Scholar). These observations suggest that the mechanisms of NFT pathology in various tauopathies may be similar and the phosphorylation-dependent mobility shift of tau on SDS-gel may be an indicator of the disease. The tau gene is mutated in familial FTDP-17, and these mutations accelerate NFT pathology in the brain (15Hutton M. Lendon C.L. Rizzu P. Baker M. Froelich S. Houlden H. Pickering-Brown S. Chakraverty S. 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In COS cells, although G272V, P301L, and V337M mutations do not show any significant affect, the R406W mutation caused a reduction in tau phosphorylation (21Sahara N. Tomiyama T. Mori H. J. Neurosci. Res. 2000; 60: 380-387Crossref PubMed Scopus (31) Google Scholar, 22Perez M. Lim F. Arrasate M. Avila J. J. Neurochem. 2000; 74: 2583-2589Crossref PubMed Scopus (60) Google Scholar). When expressed in SH-SY5Y cells subsequently differentiated into neurons, the R406W, P301L, and V337M mutations reduce tau phosphorylation (23Mack T.G. Dayanandan R. Van Slegtenhorst M. Whone A. Hutton M. Lovestone S. Anderton B.H. Neuroscience. 2001; 108: 701-712Crossref PubMed Scopus (25) Google Scholar). In contrast, in hippocampal neurons, R406W increases tau phosphorylation (24Krishnamurthy P.K. Johnson G.V. J. Biol. Chem. 2004; 279: 7893-7900Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). When phosphorylated by recombinant GSK3β in vitro, the P301L and V337M mutations do not have any effect, and the R406W mutation inhibits phosphorylation (25Connell J.W. Gibb G.M. Betts J.C. Blackstock W.P. Gallo J. Lovestone S. Hutton M. Anderton B.H. FEBS Lett. 2001; 493: 40-44Crossref PubMed Scopus (31) Google Scholar). However, when incubated with rat brain extract, all of the G272V, P301L, V337M, and R406W mutations stimulate tau phosphorylation (26Alonso Adel C. Mederlyova A. Novak M. Grundke-Iqbal I. Iqbal K. J. Biol. Chem. 2004; 279: 34873-34881Abstract Full Text Full Text PDF PubMed Scopus (249) Google Scholar). The mechanism by which FTDP-17 mutations promote tau phosphorylation leading to development of NFT pathology has remained unclear. Cyclin-dependent protein kinase 5 (Cdk5) is one of the major kinases that phosphorylates tau in the brain (27Paudel H.K. Lew J. Ali Z. Wang J.H. J. Biol. Chem. 1993; 268: 23512-23518Abstract Full Text PDF PubMed Google Scholar, 28Sobue K. Agarwal-Mawal A. Li W. Sun W. Miura Y. Paudel H.K. J. Biol. Chem. 2000; 275: 16673-16680Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). In this study, to determine how FTDP-17 missense mutations affect tau phosphorylation, we phosphorylated four FTDP-17 tau mutants (G272V, P301L, V337M, and R406W) by Cdk5. We have found that phosphorylation of tau by Cdk5 causes a tau mobility shift to ∼64- and 68 kDa-bands. Although the mobility shift to a ∼64-kDa band is achieved by phosphorylation at Ser396/404 or Ser202, the mobility shift to a 68-kDa band occurs only in response to phosphorylation at Ser202. We show that in vitro, FTDP-17 missense mutations, by promoting phosphorylation at Ser202, enhance the mobility shift to ∼64- and 68-kDa bands and inhibit the microtubule assembly-promoting activity of tau. Our data suggest that Ser202 phosphorylation is the major event leading to NFT pathology in AD and related tauopathies. cDNA Cloning—The longest isoform of human tau and FTDP-17 tau mutants G272V, P301L, V337M, and R406W, each in the pQE32 vector, were gifts from Dr. Peter Davies (Albert Einstein College of Medicine, Bronx, NY). Cloning of tau mutants S202A, T231A, and S396A in pcDNA3.1 vector is described previously (29Li T. Paudel H.K. Biochemistry. 2006; 45: 3125-3133Crossref PubMed Scopus (105) Google Scholar). Tau mutants S262A and S214A in pcDNA3.1 vector were gifts from Dr. Nicole Leclerc (University of Montreal). Each DNA fragment from the WT or mutant tau was amplified by PCR using pfu DNA polymerase (Stratagene), with a forward primer (5′-AAAAAACGCCATATGGCTGAGCCCCGC-3′) that contained an NdeI site and a reverse primer (5′-AAA AAA GGA TCC TCA CAA ACC CTG CTT GG-3′) that contained a BamHI site, and subcloned into bacterial expression vector pET9a (Promega). Various double mutants, each containing the indicated FTDP-17 and S202A mutations, were cloned by PCR using their respective FTDP-17 mutant in pET9a vector as the template and the QuikChange II site-specific mutagenesis kit (Stratagene) following the manufacturer's instruction manual. Primers used for PCR were 5′-CAG CGG CTA CAG CAG CCC CGG CGC CCC AGG CAC TCC CGG CAG CCG C-3′ and 5′-GCG GCT GCC GGG AGT GCC TGG GGC GCC GGG GCT GCT GTA GCC GCT G-3′. All cDNA clones and mutations were confirmed by DNA sequencing. Proteins and Enzymes—Tau(WT) and various tau mutants were purified from lysates of Escherichia coli overexpressing their respective tau species essentially as described previously (28Sobue K. Agarwal-Mawal A. Li W. Sun W. Miura Y. Paudel H.K. J. Biol. Chem. 2000; 275: 16673-16680Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). Briefly, tau expression was induced by adding isopropyl 1-thio-β-d-galactopyranoside (0.2 mm) to the overnight bacterial culture. The culture containing isopropyl 1-thio-β-d-galactopyranoside was allowed to grow for 3 h at 37 °C with shaking and then was centrifuged. The pellet was suspended in Pipes buffer (100 mm Pipes (pH 6.8), 1 mm EGTA, 1 mm MgSO4) containing 5 mg/ml benzamidine, 1 μg/ml leupeptine, 1 μg/ml pepstatin, 1 mm phenylmethylsulfonyl fluoride, and 20 μg/ml lysozyme. The bacterial suspension was lysed by sonication and then clarified by centrifugation (15,000 rpm, 15 min at 4 °C). The supernatant was placed in a boiling water bath for 20 min and subsequently centrifuged. The heat-stable proteins in the supernatant were loaded onto a Q-Sepharose Fast Flow column (∼1 ml; Amersham Biosciences) equilibrated previously in Pipes buffer. The flow-through containing tau was loaded onto an SP-Sepharose Fast Flow column (∼1 ml) equilibrated in Pipes buffer. The column was washed with ∼20 column volumes of the Pipes buffer and then eluted with Pipes buffer containing 0.2 m NaCl. Fractions containing tau were pooled, concentrated with Aquacade III (Calbiochem) by dialysis, dialyzed against Hepes buffer (25 mm Hepes (pH 7.2), 0.1 mm EDTA, 0.5 mm dithiothreitol, and 100 mm NaCl), and stored at –80 °C until use. Microtubules were purified from fresh bovine brain extract by three cycles of temperature-induced microtubule polymerization and depolymerization as described previously (28Sobue K. Agarwal-Mawal A. Li W. Sun W. Miura Y. Paudel H.K. J. Biol. Chem. 2000; 275: 16673-16680Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 30Sun W. Qureshi H.Y. Cafferty P.W. Sobue K. Agarwal-Mawal A. Neufield K.D. Paudel H.K. J. Biol. Chem. 2002; 277: 11933-11940Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). Tubulin was purified from purified microtubules through phosphocellulose chromatography (28Sobue K. Agarwal-Mawal A. Li W. Sun W. Miura Y. Paudel H.K. J. Biol. Chem. 2000; 275: 16673-16680Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 30Sun W. Qureshi H.Y. Cafferty P.W. Sobue K. Agarwal-Mawal A. Neufield K.D. Paudel H.K. J. Biol. Chem. 2002; 277: 11933-11940Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). Monoclonal tau 5 antibody against total tau and tau phosphorylation-dependent monoclonal antibodies AT8, PHF-1, MC6, and TG3 have been described previously (29Li T. Paudel H.K. Biochemistry. 2006; 45: 3125-3133Crossref PubMed Scopus (105) Google Scholar, 31Li T. Hawkes C. Qureshi H.Y. Kar S. Paudel H.K. Biochemistry. 2006; 45: 3134-3145Crossref PubMed Scopus (113) Google Scholar). Polyclonal antibodies pS202 and pT212 against tau phosphorylated at Ser202 and Thr212, respectively, were purchased from BIOSOURCE. Cdk5 was purified from the extract of fresh bovine brain (28Sobue K. Agarwal-Mawal A. Li W. Sun W. Miura Y. Paudel H.K. J. Biol. Chem. 2000; 275: 16673-16680Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). The active catalytic subunit of PKA was purchased from Sigma-Aldrich. Purification of protein phosphatase 1 (PP1) from E. coli extract overexpressing human PP1α has been described previously (32Agarwal-Mawal A. Paudel H.K. J. Biol. Chem. 2001; 276: 23712-23718Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, 33Li T. Chalifour L.E. Paudel H.K. J. Biol. Chem. 2007; 282: 6619-6628Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). Protein Concentrations—Tau(WT) concentration is based on its absorption at A280 nm as described previously (28Sobue K. Agarwal-Mawal A. Li W. Sun W. Miura Y. Paudel H.K. J. Biol. Chem. 2000; 275: 16673-16680Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). The concentrations of various FTDP-17 tau mutants were determined by Bio-Rad protein assay using tau(WT) as the standard. Concentrations of phosphorylated tau and tau mutants were also determined by Bio-Rad protein assay using tau(WT) as the standard. The concentration of Cdk5 is based on its activity (28Sobue K. Agarwal-Mawal A. Li W. Sun W. Miura Y. Paudel H.K. J. Biol. Chem. 2000; 275: 16673-16680Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). PKA concentration was determined by its dry weight. The concentrations of all other proteins were determined by Bio-Rad proteins assay using bovine serum albumin as the standard. Tau Phosphorylation—Tau(WT) and various tau mutants were phosphorylated by Cdk5 under identical conditions. Each phosphorylation mixture contained 25 mm Hepes (pH 7.2), 0.1 mm EDTA, 0.2 mm dithiothreitol, 0.1 m NaCl, 10 mm MgCl2, 0.5 mm [γ32P] ATP, 1.0 mg/ml tau, and 400 units/ml Cdk5. The reaction was initiated by adding an aliquot of Cdk5 to a vial containing the rest of the phosphorylation mixture at 30 °C. At the indicated time points, aliquots were withdrawn and analyzed for phosphorylation by filter paper assay (34Hashiguchi M. Sobue K. Paudel H.K. J. Biol. Chem. 2000; 275: 25247-25254Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar) or subjected to SDS-PAGE followed by Western blot analysis. Gel and blot bands were scanned, and the band intensity values were used to determine the relative amounts of various proteins. Phosphorylation of tau and tau mutants by PKA was also performed as described above, except Cdk5 was replaced by PKA at a concentration of 10 μg/ml each. Microtubule Assembly Assay—The microtubule assembly was monitored by measuring the increase of A350 by a spectrophotometer (35Gustke N. Trinczek B. Biernat J. Mandelkow E.M. Mandelkow E. Biochemistry. 1994; 33: 9511-9522Crossref PubMed Scopus (525) Google Scholar). The vial containing all of the components of the assay except tau was incubated at 37 °C for 1 min in a water bath. To the incubated vial, the indicated prewarmed tau species was added. After gentle mixing, the content of the vial was transferred immediately to a quartz cuvette placed in a spectrophotometer at 37 °C. The increase in the A350 of the transferred sample was recorded at 1-min intervals for 32 min. The final concentrations of various components in the assay were 0.75 mg/ml tubulin, 100 mm Pipes (pH 6.8), 1 mm EGTA, 1 mm dithiothreitol, 2 mm MgSO4, 1 mm GTP, 10 μm taxol, and 0.2 mg/ml tau (indicated species). The lag phase of polymerization is defined as the time at which the rise in the A350 is detected since initiation of the assay. The rate of polymerization is the A350 at the steady state divided by the minimum time required to achieve the steady state after the lag phase, and it is expressed as absorption units per min (AU/min). The amount of microtubule formed corresponds to the maximum A350 reached during the assay. Effect of Phosphorylation on Microtubule Assembly-promoting Activity of FTDP-17 Tau Mutants—In vitro, purified tubulin polymerizes in the presence of GTP/Mg2+ and assembles into microtubules. This process consists of an initial lag phase during which microtubules nucleate (35Gustke N. Trinczek B. Biernat J. Mandelkow E.M. Mandelkow E. Biochemistry. 1994; 33: 9511-9522Crossref PubMed Scopus (525) Google Scholar, 36Gelfand V.I. Bershadsky A.D. Annu. Rev. Cell Biol. 1991; 7: 93-116Crossref PubMed Scopus (177) Google Scholar). Following nucleation, microtubules polymerize and reach the steady state. In vitro microtubule assembly can be monitored spectrophotometrically by the light scattering technique, which measures the turbidity of the solution at 350 nm, which increases as the microtubules assemble (35Gustke N. Trinczek B. Biernat J. Mandelkow E.M. Mandelkow E. Biochemistry. 1994; 33: 9511-9522Crossref PubMed Scopus (525) Google Scholar, 37Hong M. Zhukareva V. Vogelsberg-Ragaglia V. Wszolek Z. Reed L. Miller B.I. Geschwind D.H. Bird T.D. McKeel D. Goate A. Morris J.C. Wilhelmsen K.C. Schellenberg G.D. Trojanowski J.Q. Lee V.M. Science. 1998; 282: 1914-1917Crossref PubMed Scopus (821) Google Scholar). When present, tau promotes microtubule assembly by influencing some or all of these parameters (35Gustke N. Trinczek B. Biernat J. Mandelkow E.M. Mandelkow E. Biochemistry. 1994; 33: 9511-9522Crossref PubMed Scopus (525) Google Scholar, 36Gelfand V.I. Bershadsky A.D. Annu. Rev. Cell Biol. 1991; 7: 93-116Crossref PubMed Scopus (177) Google Scholar, 37Hong M. Zhukareva V. Vogelsberg-Ragaglia V. Wszolek Z. Reed L. Miller B.I. Geschwind D.H. Bird T.D. McKeel D. Goate A. Morris J.C. Wilhelmsen K.C. Schellenberg G.D. Trojanowski J.Q. Lee V.M. Science. 1998; 282: 1914-1917Crossref PubMed Scopus (821) Google Scholar). To determine how phosphorylation affects the microtubule assembly promoting activity of various tau species, we phosphorylated WT and FTDP-17 mutants by Cdk5 under identical conditions. Each phosphorylated and nonphosphorylated tau species was included in the microtubule assembly mixture, and the assembly was monitored by light scattering assay. The concentration of tubulin was kept low so that no detectable turbidity was observed in the absence of tau. Microtubules in the presence of tau(WT) assembled with a lag time of 2 min at a rate of 0.0636 AU/min. When P301L was used, the lag phase was extended to 6 min and the polymerization rate was reduced to 0.0363 AU/min (Fig. 1 and supplemental Table S1). These data determined that in the presence of P301L, microtubule nucleation and polymerization occurred 3 and 1.75 times, respectively, slower than in the presence of tau(WT). This, in turn, indicates that the microtubule nucleation-promoting activity of P301L is 33.3% of that of the WT. Likewise, microtubule polymerization-promoting activity of P301L is 57% of that of the WT. In the presence of the WT, microtubule polymerization plateaued at A350 = 0.70, which represents the amount of microtubules formed (supplemental Table S1). This value is reduced to 0.40 in the presence of P301L. These data indicate that the microtubule formation-promoting activity of P301L is 57.1% of that of the WT. The microtubule nucleation, polymerization, and formation-promoting activities of V337M and R406W are also significantly less than that of the WT (Fig. 1 and supplemental Table S1). This observation is consistent with previous reports (37Hong M. Zhukareva V. Vogelsberg-Ragaglia V. Wszolek Z. Reed L. Miller B.I. Geschwind D.H. Bird T.D. McKeel D. Goate A. Morris J.C. Wilhelmsen K.C. Schellenberg G.D. Trojanowski J.Q. Lee V.M. Science. 1998; 282: 1914-1917Crossref PubMed Scopus (821) Google Scholar, 38Hasegawa M. Smith M.J. Goedert M. FEBS Lett. 1998; 437: 207-210Crossref PubMed Scopus (419) Google Scholar) and indicates that FTDP-17 mutations impair the microtubule assembly-promoting activity of tau. Phosphorylation reduced the microtubule nucleation, polymerization, and formation-promoting activities of tau(WT) to 33.3, 29.5, and 28.6%, respectively (Fig. 1 and supplemental Table S1). Compared with its nonphosphorylated counterpart, the microtubule nucleation, polymerization, and formation-promoting activities of phosphorylated P301L were 54.6, 30.5, and 50.0%, respectively, those of V337M were 36.4, 31.7, and 50.0%, respectively, and those of R406W were 58.5, 36.0, and 45.9%, respectively. T
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