Tau Phosphorylation by Cyclin-dependent Kinase 5/p39 during Brain Development Reduces Its Affinity for Microtubules
2003; Elsevier BV; Volume: 278; Issue: 12 Linguagem: Inglês
10.1074/jbc.m211964200
ISSN1083-351X
AutoresSatoru Takahashi, Taro Saito, Shin‐ichi Hisanaga, Harish C. Pant, Ashok B. Kulkarni,
Tópico(s)Alzheimer's disease research and treatments
ResumoThe microtubule-associated protein tau is a developmentally regulated neuronal phosphoprotein. The phosphorylation of tau reduces its ability to bind and stabilize axonal microtubules during axonal growth. Although tau is phosphorylated by cyclin-dependent kinase 5 (Cdk5) in vitro, its in vivo roles remain unclear. Here, we show that tau is phosphorylated by Cdk5/p39 during brain development, resulting in a reduction of its affinity for microtubules. The activity of Cdk5 is tightly regulated by association with its neuronal activators, p35 or p39. The p35 and p39 expression levels were investigated in the developing mouse brain; the p39 expression level was higher in embryonic hind brain and spinal cord and in postnatal cerebral cortex, whereas that of p35 was most prominent in cerebral cortex at earlier stages of development. The ability of Cdk5 to phosphorylate tau was higher when in association with p39 than in association with p35. Tau phosphorylation at Ser-202 and Thr-205 was decreased in Cdk5−/− mouse brain but not in p35−/− mouse brain, suggesting that Cdk5/p39 is responsible for the in vivophosphorylation of tau at these sites. Our data suggest that tau phosphorylation by Cdk5 may provide the neuronal microtubules with dynamic properties in a region-specific and developmentally regulated manner. The microtubule-associated protein tau is a developmentally regulated neuronal phosphoprotein. The phosphorylation of tau reduces its ability to bind and stabilize axonal microtubules during axonal growth. Although tau is phosphorylated by cyclin-dependent kinase 5 (Cdk5) in vitro, its in vivo roles remain unclear. Here, we show that tau is phosphorylated by Cdk5/p39 during brain development, resulting in a reduction of its affinity for microtubules. The activity of Cdk5 is tightly regulated by association with its neuronal activators, p35 or p39. The p35 and p39 expression levels were investigated in the developing mouse brain; the p39 expression level was higher in embryonic hind brain and spinal cord and in postnatal cerebral cortex, whereas that of p35 was most prominent in cerebral cortex at earlier stages of development. The ability of Cdk5 to phosphorylate tau was higher when in association with p39 than in association with p35. Tau phosphorylation at Ser-202 and Thr-205 was decreased in Cdk5−/− mouse brain but not in p35−/− mouse brain, suggesting that Cdk5/p39 is responsible for the in vivophosphorylation of tau at these sites. Our data suggest that tau phosphorylation by Cdk5 may provide the neuronal microtubules with dynamic properties in a region-specific and developmentally regulated manner. microtubule-associated protein Alzheimer's disease cyclin-dependent kinase 5 n days of embryonic development n days postpartum 1,4-piperazinediethanesulfonic acid Neuronal development involves morphogenetic changes of neurons associated with complex regulatory mechanisms, which coordinate at the levels of gene expression and post-translational modifications. Current evidence supports the view that neuronal microtubule-associated proteins (MAPs)1 determine the microtubule rearrangements underlying neuronal morphogenesis (1Avila J. Dominguez J. Diaz-Nido J. Int. J. Dev. Biol. 1994; 38: 13-25PubMed Google Scholar). This process can be achieved through the regulation of the expression of particular MAP isoforms at specific subcellular locations and at distinct developmental stages as well as through the modification of MAPs by phosphorylation and dephosphorylation (2Riederer B.M. Histochem. J. 1992; 24: 783-790Crossref PubMed Scopus (30) Google Scholar). Among the neuronal MAPs, tau protein has attracted a particular interest due to its polar distribution in the axon as compared with the somatodendritic compartment, as well as its developmentally regulated expression and phosphorylation (3Arioka M. Tsukamoto M. Ishiguro K. Kato R. Sato K. Imahori K. Uchida T. J. Neurochem. 1993; 60: 461-468Crossref PubMed Scopus (87) Google Scholar, 4Caceres A. Potrebic S. Kosik K.S. J. Neurosci. 1991; 11: 1515-1523Crossref PubMed Google Scholar). Tau is one of several MAPs that regulate the assembly and stabilization of the microtubule network (5Hirokawa N. Curr. Opin. Cell Biol. 1994; 6: 74-81Crossref PubMed Scopus (348) Google Scholar). Multiple isoforms of tau are generated from a single gene by alternative splicing, leading to the developmentally regulated expression of different isoforms (6Goedert M. Spillantini M.G. Jakes R. Rutherford D. Crowther R.A. Neuron. 1989; 3: 519-526Abstract Full Text PDF PubMed Scopus (1858) Google Scholar). Phosphorylation provides tau with further molecular diversity; the function of tau as a microtubule-binding protein is regulated by the phosphorylation of specific residues (7Mandelkow E.M. Mandelkow E. Trends Cell Biol. 1998; 8: 425-427Abstract Full Text Full Text PDF PubMed Scopus (431) Google Scholar). In general, an increase in tau phosphorylation correlates inversely with its ability to bind and stabilize microtubules. Thus, phosphorylation of tau contributes an additional mechanism to control the balance between microtubule dynamics and stabilization in developing axons (8Bramblett 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, 9Goedert M. Jakes R. Crowther R.A. Six J. Lubke U. Vandermeeren M. Cras P. Trojanowski J.Q. Lee V.M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5066-5070Crossref PubMed Scopus (412) Google Scholar). Several developmental studies have already shown that phosphorylated tau is present in neurons at a high level only during the period of intense neuritic outgrowth and that it becomes barely detectable during the period of neurite stabilization and synaptogenesis (10Brion J.P. Octave J.N. Couck A.M. Neuroscience. 1994; 63: 895-909Crossref PubMed Scopus (116) Google Scholar, 11Riederer B.M. Binder L.I. Brain Res. Bull. 1994; 33: 155-161Crossref PubMed Scopus (31) Google Scholar). Phosphorylated tau is essential during brain development to maintain a certain degree of microtubule instability, thus affecting the growth of neurites. Tau phosphorylation at specific residues not only occurs during normal brain development but also during pathological conditions such as Alzheimer's disease (AD). In normal brain, the equilibrium between tau phosphorylation and dephosphorylation modulates the stability of the axonal cytoskeleton and thereby the axonal morphology (12Matsuo E.S. Shin R.W. Billingsley M.L. Van deVoorde A. O'Connor M. Trojanowski J.Q. Lee V.M. Neuron. 1994; 13: 989-1002Abstract Full Text PDF PubMed Scopus (553) Google Scholar). However, the breakdown of this equilibrium under pathological conditions results in tau dysfunction, which is considered to be one of the critical events leading to neuronal degeneration (13Mandelkow E.M. Biernat J. Drewes G. Gustke N. Trinczek B. Mandelkow E. Neurobiol. Aging. 1995; 16: 355-362Crossref PubMed Scopus (247) Google Scholar). It has been shown that hyperphosphorylation of tau reduces its affinity for microtubules and can contribute to the self-association of tau and the formation of neurofibrillary tangles, one of the major histopathological hallmarks of AD (14Goedert M. Trends Neurosci. 1993; 16: 460-465Abstract Full Text PDF PubMed Scopus (548) Google Scholar). The phosphorylation is interpreted as abnormal in the sense that this kind of tau phosphorylation has never been observed in normal aged human brain (15Grundke-Iqbal I. Iqbal K. Tung Y.C. Quinlan M. Wisniewski H.M. Binder L.I. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 4913-4917Crossref PubMed Scopus (2877) Google Scholar). Surprisingly, the phosphorylation of tau in neurofibrillary tangles has been found to be very similar to a transient hyperphosphorylation of tau that occurs during early development of the brain (8Bramblett 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, 9Goedert M. Jakes R. Crowther R.A. Six J. Lubke U. Vandermeeren M. Cras P. Trojanowski J.Q. Lee V.M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5066-5070Crossref PubMed Scopus (412) Google Scholar, 16Kanemaru K. Takio K. Miura R. Titani K. Ihara Y. J. Neurochem. 1992; 58: 1667-1675Crossref PubMed Scopus (191) Google Scholar, 17Brion J.P. Smith C. Couck A.M. Gallo J.M. Anderton B.H. J. Neurochem. 1993; 61: 2071-2080Crossref PubMed Scopus (170) Google Scholar, 18Kenessey A. Yen S.H. Brain Res. 1993; 629: 40-46Crossref PubMed Scopus (132) Google Scholar). Therefore, the biochemical pathways that play a role during early brain development are likely to be reactivated in AD brains. Tau is phosphorylated in vitroby several protein kinases, including cyclic AMP-dependent protein kinase (19Scott C.W. Spreen R.C. Herman J.L. Chow F.P. Davison M.D. Young J. Caputo C.B. J. Biol. Chem. 1993; 268: 1166-1173Abstract Full Text PDF PubMed Google Scholar), calcium/calmodulin-dependent protein kinase II (20Yamamoto H. Fukunaga K. Tanaka E. Miyamoto E. J. Neurochem. 1983; 41: 1119-1125Crossref PubMed Scopus (144) Google Scholar), protein kinase C (21Correas I. Diaz-Nido J. Avila J. J. Biol. Chem. 1992; 267: 15721-15728Abstract Full Text PDF PubMed Google Scholar), casein kinase I (22Pierre M. Nunez J. Biochem. Biophys. Res. Commun. 1983; 115: 212-219Crossref PubMed Scopus (36) Google Scholar), casein kinase II (21Correas I. Diaz-Nido J. Avila J. J. Biol. Chem. 1992; 267: 15721-15728Abstract Full Text PDF PubMed Google Scholar), and proline-directed protein kinases such as MAP kinase (23Drewes G. Lichtenberg-Kraag B. Doring F. Mandelkow E.M. Biernat J. Goris J. Doree M. Mandelkow E. EMBO J. 1992; 11: 2131-2138Crossref PubMed Scopus (494) Google Scholar), glycogen synthase kinase-3β (24Mandelkow E.M. Drewes G. Biernat J. Gustke N. Van Lint J. Vandenheede J.R. Mandelkow E. FEBS Lett. 1992; 314: 315-321Crossref PubMed Scopus (483) Google Scholar). and cyclin-dependent kinase 5 (Cdk5) (3Arioka M. Tsukamoto M. Ishiguro K. Kato R. Sato K. Imahori K. Uchida T. J. Neurochem. 1993; 60: 461-468Crossref PubMed Scopus (87) Google Scholar, 25Kobayashi S. Ishiguro K. Omori A. Takamatsu M. Arioka M. Imahori K. Uchida T. FEBS Lett. 1993; 335: 171-175Crossref PubMed Scopus (219) Google Scholar, 26Paudel H.K. Lew J. Ali Z. Wang J.H. J. Biol. Chem. 1993; 268: 23512-23518Abstract Full Text PDF PubMed Google Scholar, 27Patrick G.N. Zukerberg L. Nikolic M. de la Monte S. Dikkes P. Tsai L.H. Nature. 1999; 402: 615-622Crossref PubMed Scopus (1320) Google Scholar). Of these kinases, Cdk5 has been found to phosphorylate tau at sites implicated in AD pathology (13Mandelkow E.M. Biernat J. Drewes G. Gustke N. Trinczek B. Mandelkow E. Neurobiol. Aging. 1995; 16: 355-362Crossref PubMed Scopus (247) Google Scholar, 28Evans D.B. Rank K.B. Bhattacharya K. Thomsen D.R. Gurney M.E. Sharma S.K. J. Biol. Chem. 2000; 275: 24977-24983Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). Cdk5 is an active enzyme in postmitotic neurons; the activation of Cdk5 requires its association with the neuronal activators, p35 or p39 (29Lew J. Huang Q.Q. Qi Z. Winkfein R.J. Aebersold R. Hunt T. Wang J.H. Nature. 1994; 371: 423-426Crossref PubMed Scopus (539) Google Scholar, 30Tsai L.H. Delalle I. Caviness Jr., V.S. Chae T. Harlow E. Nature. 1994; 371: 419-423Crossref PubMed Scopus (810) Google Scholar, 31Tang D. Yeung J. Lee K.Y. Matsushita M. Matsui H. Tomizawa K. Hatase O. Wang J.H. J. Biol. Chem. 1995; 270: 26897-26903Abstract Full Text Full Text PDF PubMed Scopus (297) Google Scholar). p35 was the first activator identified for Cdk5 (29Lew J. Huang Q.Q. Qi Z. Winkfein R.J. Aebersold R. Hunt T. Wang J.H. Nature. 1994; 371: 423-426Crossref PubMed Scopus (539) Google Scholar, 30Tsai L.H. Delalle I. Caviness Jr., V.S. Chae T. Harlow E. Nature. 1994; 371: 419-423Crossref PubMed Scopus (810) Google Scholar). Subsequently, an additional Cdk5 activator, p39, was identified based on its sequence homology to p35 (31Tang D. Yeung J. Lee K.Y. Matsushita M. Matsui H. Tomizawa K. Hatase O. Wang J.H. J. Biol. Chem. 1995; 270: 26897-26903Abstract Full Text Full Text PDF PubMed Scopus (297) Google Scholar). p35 and p39 share 57% amino acid identity (31Tang D. Yeung J. Lee K.Y. Matsushita M. Matsui H. Tomizawa K. Hatase O. Wang J.H. J. Biol. Chem. 1995; 270: 26897-26903Abstract Full Text Full Text PDF PubMed Scopus (297) Google Scholar). Cdk5 has been shown to play an important role in the development of the nervous system, including neuronal migration and neurite outgrowth (32Chae T. Kwon Y.T. Bronson R. Dikkes P. Li E. Tsai L.H. Neuron. 1997; 18: 29-42Abstract Full Text Full Text PDF PubMed Scopus (664) Google Scholar, 33Kwon Y.T. Tsai L.H. J. Comp. Neurol. 1998; 395: 510-522Crossref PubMed Scopus (151) Google Scholar, 34Nikolic M. Dudek H. Kwon Y.T. Ramos Y.F. Tsai L.H. Genes Dev. 1996; 10: 816-825Crossref PubMed Scopus (530) Google Scholar, 35Ohshima T. Ward J.M. Huh C.G. Longenecker G. Veeranna Pant H.C. Brady R.O. Martin L.J. Kulkarni A.B. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 11173-11178Crossref PubMed Scopus (808) Google Scholar). Insights into the critical function of Cdk5/p35 in brain development have been gained from gene targeting experiments (32Chae T. Kwon Y.T. Bronson R. Dikkes P. Li E. Tsai L.H. Neuron. 1997; 18: 29-42Abstract Full Text Full Text PDF PubMed Scopus (664) Google Scholar, 35Ohshima T. Ward J.M. Huh C.G. Longenecker G. Veeranna Pant H.C. Brady R.O. Martin L.J. Kulkarni A.B. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 11173-11178Crossref PubMed Scopus (808) Google Scholar). Cdk5−/− mice display extensive defects in all brain areas (35Ohshima T. Ward J.M. Huh C.G. Longenecker G. Veeranna Pant H.C. Brady R.O. Martin L.J. Kulkarni A.B. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 11173-11178Crossref PubMed Scopus (808) Google Scholar), whereas p35−/− mice display defects mostly confined to the forebrain (32Chae T. Kwon Y.T. Bronson R. Dikkes P. Li E. Tsai L.H. Neuron. 1997; 18: 29-42Abstract Full Text Full Text PDF PubMed Scopus (664) Google Scholar). This difference in the phenotypic severity suggests the functional importance of another Cdk5 activator, p39, in brain development. Although p39−/− mice have no obvious abnormalities in the brain, p35−/−p39−/− double knockout mice exhibit phenotypes identical to those of Cdk5−/− mice (36Ko J. Humbert S. Bronson R.T. Takahashi S. Kulkarni A.B. Li E. Tsai L.H. J. Neurosci. 2001; 21: 6758-6771Crossref PubMed Google Scholar). These findings suggest that p35 and p39 are major activators of Cdk5 in the brain and that their coexistence as Cdk5 activators may contribute to the in vivo activation of Cdk5 in a region-specific or developmentally regulated manner, as proposed by Wu et al.(37Wu D.C. Yu Y.P. Lee N.T.K. Yu A.C.H. Wang J.H.C. Han Y.F. Neurochem. Res. 2000; 25: 923-929Crossref PubMed Scopus (51) Google Scholar). Developmental regulation of tau phosphorylation is critical in maintaining the balance between microtubule plasticity and stability in developing axons. The phosphorylation pattern of tau in AD brains closely resembles that of tau in embryonic brains (8Bramblett 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, 9Goedert M. Jakes R. Crowther R.A. Six J. Lubke U. Vandermeeren M. Cras P. Trojanowski J.Q. Lee V.M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5066-5070Crossref PubMed Scopus (412) Google Scholar, 16Kanemaru K. Takio K. Miura R. Titani K. Ihara Y. J. Neurochem. 1992; 58: 1667-1675Crossref PubMed Scopus (191) Google Scholar, 17Brion J.P. Smith C. Couck A.M. Gallo J.M. Anderton B.H. J. Neurochem. 1993; 61: 2071-2080Crossref PubMed Scopus (170) Google Scholar, 18Kenessey A. Yen S.H. Brain Res. 1993; 629: 40-46Crossref PubMed Scopus (132) Google Scholar). Thus, the developing brain is a useful experimental system to study the mechanisms that control tau phosphorylation. Although Cdk5 has been found to phosphorylate tau in vitro, its in vivoroles remain to be examined. Here, we show that Cdk5 in association with p39 is involved in the in vivo phosphorylation of tau during brain development and that its phosphorylation reduces the ability of tau to bind to microtubules. In addition, Cdk5/p35 and Cdk5/p39 were found to exhibit different abilities for tau phosphorylation, with the higher activity in Cdk5/p39. Considering the temporal and spatial expression patterns of p35 and p39 reported here, the role of tau phosphorylation by Cdk5 is discussed with regard to the regulation of microtubule stability during brain development. Polyclonal antibodies to p35 (C-19) and Cdk5 (C-8) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). An affinity-purified rabbit polyclonal antibody against p39 was kindly provided by Dr. Tsai (Howard Hughes Medical Institute and the Department of Pathology, Harvard Medical School, Boston, MA). The phosphorylation-dependent tau antibody, AT-8, was obtained from Endogen (Woburn, MA). The phosphorylation-independent mouse monoclonal TAU-5 antibody was purchased from BIOSOURCE International, Inc. (Camarillo, CA). An antibody against α-tubulin was purchased from Sigma. Cell culture reagents were purchased from Invitrogen. The gene-targeting strategy and generation of Cdk5−/− and p35−/− mice have been reported previously (35Ohshima T. Ward J.M. Huh C.G. Longenecker G. Veeranna Pant H.C. Brady R.O. Martin L.J. Kulkarni A.B. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 11173-11178Crossref PubMed Scopus (808) Google Scholar, 38Ohshima T. Ogawa M. Veeranna Hirasawa M. Longenecker G. Ishiguro K. Pant H.C. Brady R.O. Kulkarni A.B. Mikoshiba K. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 2764-2769Crossref PubMed Scopus (113) Google Scholar). Mouse lines of Cdk5 and p35 mutants, as well as wild-type controls, were maintained in a C57BL/6 × 129/SvJ hybrid background. Conception was ascertained by the presence of a vaginal plug. The first 24 h following conception was considered day 0 of embryonic development (E0), and the first 24 h following birth was considered day 0 postpartum (P0). Mouse embryonic development takes ∼20 days. The genotypes of the mutants were determined by performing Southern blot and/or PCR analysis on genomic DNA isolated from tail biopsies as described earlier (35Ohshima T. Ward J.M. Huh C.G. Longenecker G. Veeranna Pant H.C. Brady R.O. Martin L.J. Kulkarni A.B. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 11173-11178Crossref PubMed Scopus (808) Google Scholar, 38Ohshima T. Ogawa M. Veeranna Hirasawa M. Longenecker G. Ishiguro K. Pant H.C. Brady R.O. Kulkarni A.B. Mikoshiba K. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 2764-2769Crossref PubMed Scopus (113) Google Scholar). Animal care and use practices conformed to the NIH Guide for Care and Use of Laboratory Animals. Embryonic and postnatal mouse brains were dissected on ice into various brain regions: cerebral cortex, cerebellum, brain stem, and spinal cord. For Northern blot analysis, total RNA was extracted from the various brain regions of wild-type mice with TRIzol reagent (Invitrogen), as recommended by the manufacturer. Total RNA concentration and purity were determined by UV absorbance at 260 and 280 nm. For Western blot analysis, each tissue from wild-type, p35−/−, and Cdk5−/− mice was washed in ice-cold PBS and homogenized in 10 volumes of lysis buffer at 4 °C. The lysis buffer consisted of 50 mm Tris-HCl (pH 7.5) containing 150 mm NaCl, 5 mm EDTA, 1% Triton X-100, 1 mmdithiothreitol, 1 mm phenylmethylsulfonyl fluoride, 1 μg/ml aprotinin, 1 μg/ml leupeptin, 20 mmβ-phosphoglycerate, 10 mm sodium fluoride, 1 mm sodium vanadate, and 1 μm okadaic acid. Following a 30-min incubation on ice, insoluble material was removed by centrifugation at 4 °C, and the protein concentration of the supernatant was determined as described (39Lowry O. Rosebrough N.J. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar). For Cdk5 kinase activity assay, the brain extracts were prepared with the same lysis buffer except with a NaCl concentration of 50 mm to allow Cdk5 association with p35 and p39. Each sample (20 μg of total RNA) was electrophoresed in a 1% agarose-formaldehyde gel and blotted onto a nylon membrane (Schleicher & Schuell). After UV cross-linking, the membrane was prehybridized for more than 3 h at 42 °C in the presence of 50% formamide, 10× Denhardt's solution, 5× SSPE, 0.1% SDS, 10% dextran sulfate, and 100 μg/ml boiled fragmented salmon sperm DNA. 32P-Labeled probes were added to the prehybridization buffer, and the membrane was incubated overnight at 42 °C. After hybridization, the membrane was washed two times in 2× SSC, 0.1% SDS at 42 °C for 10 min, and two times in 0.1× SSC, 0.1% SDS at 65 °C for 20 min. The washed membrane was exposed to x-ray film with double intensifying screens at −80 °C. After stripping the probe, the same membrane was used for hybridization with a new probe. The expression levels of Cdk5, p35, and p39 mRNAs were quantified by measuring the optical densities of specific bands using an image analysis system with NIH Image software, version 1.62. For detection of p35 mRNA, a 924-bp fragment of mouse p35 cDNA containing the entire coding region was used as a probe as previously described (40Ohshima T. Kozak C.A. Nagle J.W. Pant H.C. Brady R.O. Kulkarni A.B. Genomics. 1996; 35: 372-375Crossref PubMed Scopus (39) Google Scholar). A 275-bp fragment used as a p39 probe (nucleotides 891–1165 of the full-length mouse p39 cDNA) was generated by reverse transcription-PCR using the following primers: 5′-CAACGAGATCTCCTACCCGCTC-3′ and 5′-TCATAGTCCAGTGCTTGGCTCC-3′. This fragment excludes the region of homology between p35 and p39 cDNAs, thus minimizing the probability of cross-hybridization. A 560-bp fragment used as a Cdk5 probe (nucleotides 331–890 of the full-length mouse Cdk5 cDNA) was produced by reverse transcription-PCR using the following primers: 5′-AGCTGCAATGGTGACCTGGACC-3′ and 5′-TCCTCTGCTGAGATGCGCTGCA-3′. As an internal control, a 433-bp fragment used as a mouse glyceraldehyde-3-phosphate dehydrogenase probe was generated as described previously (41Wüllner U. Isenmann S. Gleichmann M. Klockgether T. Bahr M. Brain Res. Dev. Brain Res. 1998; 110: 1-6Crossref PubMed Scopus (15) Google Scholar). Equal amounts of protein for each experiment were separated by SDS-PAGE before being transferred onto a nitrocellulose membrane. The membranes were blocked in 1× PBS containing 5% skim milk and 0.05% Tween 20 and incubated with primary antibodies overnight at 4 °C. Incubation with peroxidase-conjugated anti-mouse or rabbit IgG (1:10,000) was performed at room temperature for 60 min. A signal was detected by enhanced chemiluminescence (Pierce), and relative optical densities of the bands were quantified as described above. For detection of p35, p39, and Cdk5 protein, membranes were incubated with anti-p35 antibody (1:1,000), anti-p39 antibody (1:1,000), and anti-Cdk5 antibody (1:1,000). The phosphorylation state of tau was examined using the AT-8 antibody (diluted to 5 μg/ml), which recognizes tau only when Ser-202 and Thr-205 (numbering based on longest human brain tau isoforms) are phosphorylated (42Biernat J. Mandelkow E.M. Schroter C. Lichtenberg-Kraag B. Steiner B. Berling B. Meyer H. Mercken M. Vandermeeren A. Goedert M. EMBO J. 1992; 11: 1593-1597Crossref PubMed Scopus (430) Google Scholar). To determine total tau levels, the phosphorylation-independent monoclonal antibody TAU-5 (1:5,000) was used (43LoPresti P. Szuchet S. Papasozomenos S.C. Zinkowski R.P. Binder L.I. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10369-10373Crossref PubMed Scopus (323) Google Scholar). The data obtained with the AT-8 antibody were normalized to total tau levels on the stripped and reprobed membranes. For reuse, the membranes were stripped for 30 min at 50 °C in 63 mm Tris-HCl (pH 6.8) containing 100 mm 2-mercaptoethanol and 2% SDS. For the detergent extraction assay, the amount of tau was determined with the TAU-5 antibody. Different regions of mouse brain were lysed in ice-cold lysis buffer as described above. The supernatants (brain extracts) were collected after centrifugation at 10,000 × g for 30 min at 4 °C and immunoprecipitated with either anti-Cdk5 (C-8), anti-p35 (C-19), or anti-p39 antibodies. The Cdk5 immunoprecipitate was prepared by incubation of 300 μl of the lysate (corresponding to 300 μg of protein) with anti-Cdk5 antibody (3 μg) overnight at 4 °C followed by further incubation with 25 μl of Protein A-agarose beads (50% slurry in lysis buffer; Santa Cruz Biotechnology) for 3 h at 4 °C. For the preparation of p35 or p39 immunoprecipitates, 500 μl of the lysate (corresponding to 1 mg of protein) was incubated with either anti-p35 antibody (3 μg) or anti-p39 antibody (3 μg) as described above. The immunoprecipitates were washed twice with the lysis buffer and twice with a kinase buffer consisting of 50 mm Tris-HCl (pH 7.4), 5 mm MgCl2, 1 mm EDTA, 1 mm EGTA, and 1 mmdithiothreitol and resuspended in 60 μl of the kinase buffer. Kinase activity assays were performed using either histone H1 or bacterially expressed human tau as a substrate as described previously (44Li B.S. Zhang L. Gu J. Amin N.D. Pant H.C. J. Neurosci. 2000; 20: 6055-6062Crossref PubMed Google Scholar). Tau was purified from the heat-stable supernatant of anEscherichia coli lysate by phosphocellulose column chromatography (45Kusakawa G. Saito T. Onuki R. Ishiguro K. Kishimoto T. Hisanaga S. J. Biol. Chem. 2000; 275: 17166-17172Abstract Full Text Full Text PDF PubMed Scopus (335) Google Scholar, 59Hashiguchi M. Saito T. Hisanaga S. Hashiguch T. J. Biol. Chem. 2002; 277: 44525-44530Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). Briefly, a total volume of 50 μl of kinase assay mixture was used, containing 50 mm Tris-HCl (pH 7.4), 1 mm EDTA, 1 mm EGTA, 1 mm dithiothreitol, 5 mm MgCl2, 0.5 mm microcystin LR, 10 μl of the immunoprecipitate, and either 10 μg of histone H1 or 5 μg of tau protein. The phosphorylation reaction was initiated by the addition of 0.1 mm [γ-32P]ATP and incubated at 30 °C for 60 min. The reaction was stopped by the addition of SDS-PAGE sample buffer and boiled immediately for 5 min. Samples were separated by SDS-PAGE on a 15% polyacrylamide gel, and autoradiography was used to detect the phosphorylation of histone H1 or tau. To quantify the Cdk5 levels in the p35 and p39 immunoprecipitates, 10 μl of each immunoprecipitate was immunodetected by Western blotting using an anti-Cdk5 antibody. Primary cultures of cerebellar neurons were prepared as described previously (4Caceres A. Potrebic S. Kosik K.S. J. Neurosci. 1991; 11: 1515-1523Crossref PubMed Google Scholar). Briefly, cerebella were dissected from E17 wild-type, p35−/−, and Cdk5−/− mouse brains; dissociated by trituration; counted; and plated at 1 × 105 cells/cm2 onto six-well culture plates previously coated with polyethylenimine (2 μg/ml in 0.1 mboric acid buffer, pH 7.4). After 1 h in 10% horse serum-containing medium, the cells were maintained with Dulbecco's modified Eagle's medium/F-12 with N2 supplement, 50 units/ml penicillin G, and 50 mg/ml streptomycin. Cultures were maintained in a 37 °C incubator with 5% CO2. The cells were used for experiments on the 7th day in culture. To investigate the effect of tau phosphorylation on its association with microtubules, a Triton X-100 extraction assay was utilized to separate the detergent-insoluble cytoskeletal component from the detergent-soluble cytosolic component. The fractionation of cell lysates was performed by a modification of a previously reported procedure (46Esmaeli-Azad B. McCarty J.H. Feinstein S.C. J. Cell Sci. 1994; 107: 869-879Crossref PubMed Google Scholar). Briefly, cultures were washed with prewarmed (37 °C) PBS followed by washing with prewarmed (37 °C) microtubule stabilization buffer (0.1 m PIPES, pH 6.8, 1 mmMgCl2, 2 mm EGTA, 30% glycerol, 1 mm dithiothreitol, 1 mm phenylmethylsulfonyl fluoride, 1 μg/ml aprotinin, 1 μg/ml leupeptin, 20 mmβ-phosphoglycerate, 10 mm sodium fluoride, 1 mm sodium vanadate, and 1 μm okadaic acid) and incubated at 37 °C for 10 min in the same buffer containing 0.1% Triton X-100. The lysates were centrifuged, and the detergent-soluble supernatants were collected. The supernatants were incubated in a boiling water bath for 5 min following the addition of 2× SDS sample buffer. The remaining cellular pellets were solubilized in 2× SDS sample buffer, sonicated, and incubated in a boiling water bath as above. The relative protein concentration of these samples was determined by densitometry of a Coomassie Blue-stained SDS-gel. Equal protein amounts were subsequently electrophoresed on 10% SDS-polyacrylamide gels, and immunodetection was performed with a TAU-5 antibody. Tau is predominantly localized in axons and has a specific function in the formation of the axonal cytoskeleton. A study that demonstrated the temporal and spatial expression patterns of tau mRNA in the rat brain suggested that its expression appeared to coincide with the region-specific onset of axogenesis (47Takemura R. Kanai Y. Hirokawa N. Neuroscience. 1991; 44: 393-407Crossref PubMed Scopus (26) Google Scholar). The onset of tau mRNA expression and its subsequent decrease in the brain stem occurred earlier than those in the cerebral cortex (47Takemura R. Kanai Y. Hirokawa N. Neuroscience. 1991; 44: 393-407Crossref PubMed Scopus (26) Google Scholar). Whereas studies have demonstrated the developmental regulation of tau phosphorylation in the brain, its spatial regulation remains less clear. To address this question, we examined the developmental changes in the expression and phosphorylation of tau in different brain regions, t
Referência(s)