]>NBA5521S0197-4580(00)00248-710.1016/S0197-4580(00)00248-7Elsevier Science Inc.Fig. 1Purification and identification of the TG-3 antigen. (A) Purification and Western blotting of the TG-3 reactive 105 kDa protein. HEp-2 cells were synchronized with nocodazole, detergent extracted, and fractionated by sequential DEAE and Heparin chromatography. TG-3 immunoreactivity eluted in a single fraction from both columns with 0.4 M and 0.5 M NaCl respectively. (B) Western blot analysis (lanes 1–5) and immunoprecipitations with anti-nucleolin MS3 antibody (lanes 6, 7). Lysates of synchronized (lanes 4–7) and asynchronous (lanes 1–3) HEp-2 cells were analyzed with MS3 (lanes 1, 4) or TG-3 (lanes 2, 3, 5). Lane 3, cells collected by the mitotic shake technique (see methods).Fig. 2Identification of the TG-3 antigen as mitotic nucleolin by chemical cleavage. The TG-3 antigen was partially purified from synchronized cells as in Fig. 1, separated by SDS-PAGE, and subjected to partial chemical digestion with N-chlorosuccinimide (A) and hydroxylamine (B). The cleavage products of each digest were separated by SDS-PAGE and transferred onto two membranes. Each membrane was probed with the TG-3 antibody (lanes 1,2), stripped, tested by ECL, and reprobed with the MS3 antibody (lanes 3,4). Controls contained no cleaving reagent (lanes 1, 3).Fig. 3Phosphorylation of nucleolin by cdc2 confers TG-3 immunoreactivity. Nucleolin partially purified from asynchronous cells was phosphorylated by Cdc2/cyclinB in the presence of 32P labeled ATP. The phosphorylated nucleolin was further immunoprecipitated with anti-nucleolin monoclonal antibody MS3 and Western-blotted with either MS3 (panel B) or TG-3 (panel C) antibodies. (A) Autoradiograph of samples separated by SDS-PAGE (Lanes 1 and 2). Partially purified preparations were incubated with [32P]ATP in the presence (lane 1) and the absence (lane 2) of the cdc2/cyclinB complex. The 105 kDa TG-3 immunoreactive band appears only in the sample where Cdc2 was present (lane 1, panel C). (Lanes 3–6) Immunoprecipitations of nucleolin with MS3 from samples prepared as in lanes 1 and 2. 32P is incorporated into nucleolin both in the presence (lane 3, panel A) and absence (lane 4, panel A) of Cdc2. The 105 kDa, TG-3 immunoreactive protein is immunoprecipitated only in the sample where cdc2 was present (lane 3, panel C). No staining was detected when no nucleolin (lane 5) or no primary antibody (lane 6) was used in the experiment. (+) and (-) above lanes indicate presence or absence of the reagent.Fig. 4Distribution of TG-3 immunoreactive nucleolin throughout mitosis. Laser confocal micrograph of asynchronous HEp-2 cells dual labeled with neurofibrillary tangle monoclonal antibody, which recognizes nucleolin phosphorylated by Cdc2: TG-3 in red, monoclonal antibody to β-tubulin in green (A, D, G), and monoclonal antibodies to histone proteins in green (J). Colocalization is shown in yellow (C, F, I, L). Note the appearance of TG-3 immunoreactivity (B). Only the cells in mitosis are immunoreactive with TG-3 (B, H, K) and the immunoreactivity is localized to the nucleus and to the segregating centrosomes (A-arrow, B, C). During late prometaphase, TG-3 immunoreactivity becomes dispersed throughout the mitotic cytosol (E, F). The centrosomes retain TG-3 immunoreactivity (F). In metaphase, the aligned chromosomes are immunoreactive with TG-3 (H) and chromosomal TG-3 staining colocalizes with histone staining (K). The centrosomes lose their TG-3 immunoreactivity. In anaphase TG-3 immunoreactivity begins to decrease (H-cell 3). A marked difference in intensity of TG-3 immunoreactivity is observed between metaphase (K-cell 1) and telophase (K-cell 2).Fig. 5Distribution of TG-3 immunoreactive and non-reactive nucleolin in the early stages of mitosis. Laser confocal micrographs of asynchronous HEp-2 cells dual labeled with monoclonal antibody to nucleolin: MS3 (green) and TG-3 (red). A nuclear distribution of nucleolin is observed in interphase cells. In the early stages of mitosis, nucleoli are intensely immunoreactive with both TG-3 and MS3. Note that the overlap in the mitotic cells is partial (A, B), suggesting that not all nucleolin is phosphorylated by Cdc2 in these cells. DAPI insert indicates cell in prometaphase (D-arrow)Fig. 6Distribution of nucleolin in control and AD brains. Light and immunofluorescent micrographs of control (B-E) and AD (G-J) hippocampal sections immunostained with monoclonal antibody MS3. Control (A) and AD (F) brain sections immunostained with TG-3 were used as a reference for illustrative value. CA3-CA4 (B, C): Nucleolin is present in large neuronal nuclei and neuronal cytoplasm, and in small glial nuclei (B, bar = 0.05 mm; C, bar = 0.1 mm). Nucleolin is primarily nuclear in some neurons, primarily cytoplasmic in others and was detected in both compartments in still other neurons (B). In CA1 (D, F), note the intense immunoreactivity in the apical dendrites (D, bar = 0.05 mm). Note the cytoplasmic distribution of nucleolin and presence of nucleolin in early NFT in the control brain (E, bar = 0.05 mm). A decrease in nucleolin immunoreactivity in all regions of the AD hippocampus was consistently observed (G-J). In CA3-CA4 note infrequently observed immunoreactive plaque-like structures (G, arrows, bar = 0.2 mm), and the granular appearance of nucleolin staining (H, bar = 0.2 mm). CA1 (I, J) - note the abundance of nucleolin positive NFT. Type 1, 2, and 3 NFT were observed (bar = 0.05 mm).Fig. 7TG-3 reactive nucleolin in the AD brain. (A) Laser confocal micrograph of AD affected hippocampus dual labeled with antibody TG-3 (red) and anti-nucleolin mAb MS3 (green) (bar = 2.5 □). Green nuclei indicate TG-3 nonreactive nucleolin in neurons. Yellow NFT indicate overlap. TG-3 recognizes microtubule associated protein tau in neurites (red). (B, C) TG-3 recognition of phosphorylated Thr 231 on tau was blocked by preincubation with anti-tau monoclonal antibody AT-180 which recognizes phosphorylated Thr231 on tau, but not mitotic nucleolin. Blocked and control sections from the same region of the same brain were dual labeled with TG-3 and anti-nucleolin antiserum. Note the complete disappearance of TG-3 staining from compartments where no nucleolin was present (B). The amount of total TG-3 immunoreactivity and the ratio of TG-3/nucleolin overlap were quantitated and compared in blocked (+AT180) and control (-AT180) sections (C). Blocking tau from recognition by TG-3 decreased the amount of total TG-3 staining, but did not decrease the amount of TG-3/nucleolin overlap, suggesting that TG-3 recognizes nucleolin in tangles. The data are presented as the ratio of the amount of TG-3/nucleolin overlap to total TG-3.☆This paper is part of the special issue ‘Cell Cycle’ (Guest Editor Inez Vincent).Cdc2 phosphorylation of nucleolin demarcates mitotic stages and Alzheimer’s disease pathologyAlexDranovskyabInezVincentcLuisaGregoribAlexanderSchwarzmanbDavidColfleshdJanEnghildeWarrenStrittmatterePeterDaviescDmitryGoldgaberb*dgoldgaber@mail.psychiatry.sunysb.eduaMedical Scientist Training Program, State University of New York, Stony Brook, NY 11794, USAbDepartment of Psychiatry and Behavioral Science, State University of New York, Stony Brook, NY 11794, USAcDepartment of Pathology, Albert Einstein College of Medicine, Bronx, NY 10461, USAdUniversity Microscopy Imaging Center, State University of New York, Stony Brook, NY 11794, USAeDepartment of Neurology, Duke University, Durham, NC 27710, USA*Corresponding author. Tel.: +1-631-444-1369; fax: +1-631-444-7534AbstractNucleolin is a major multifunctional nuclear phosphoprotein that is phosphorylated by Cdc2 kinase in mitosis and that participates in a number of cellular processes. The monoclonal antibody TG-3 generated against neurofibrillary tangles (NFT) found in Alzheimer’s disease (AD) is highly specific for mitotic cells in culture. We here demonstrate that phosphorylation of nucleolin by Cdc2 kinase generates the TG-3 epitope. The unique pool of TG-3 immunoreactive nucleolin appears abruptly during the prophase. It is associated with chromosomes through the metaphase and it gradually disappears during separation of chromosomes and exit from mitosis. In the brain, nucleolin was localized not only to nuclei but also to neuronal cytoplasm, and it is a marker for early NFT. In patients with AD, Cdc2 phosphorylated nucleolin was present in NFT. These findings suggest that phosphorylation of nucleolin by Cdc2 kinase is a critical event and the point of convergence of two distinct pathways, mitosis and neurodegeneration.KeywordsNucleolinMitosisNeurodegenerationAlzheimer’s disease1IntroductionSeveral recent reports suggest that reactivation of certain cell cycle events in postmitotic neurons may underlie neurofibrillary degeneration in AD [6,25,28,29,43,44]. Evidence for involvement of cell cycle specific proteins in neurodegeneration stems from studies on the role of phosphorylation in the formation of neurofibrillary tangles (NFT), one of the neuropathological hallmarks of AD. NFT consist of paired helical filaments (PHF) and straight filaments. Phosphorylation by proline directed serine-threonine kinases results in PHF properties in tau (a structural component of PHF) [31]. Experiments in vitro have shown that many proline directed kinases phosphorylate recombinant tau [1,27,31,46]. However, the biologic relevance of such experiments is not clear due to the substrate promiscuity of these kinases [30] and the ability of tau to form PHF in the absence of phosphorylation in vitro [48].In vivo support for tau phosphorylation by mitotic proline directed kinases was provided when a kinase related to the major mitotic kinase Cdc2 was immunolocalized to NFT in the AD brain [26,50]. Moreover, Vincent and colleagues recently demonstrated that Cdc2 kinase was activated in AD neurons [43]. Furthermore, immunoreactivity of other cell cycle markers was detected in the brains of patients affected by AD [6,28–29]. Aberrant activation of mitotic mechanisms in postmitotic neurons may facilitate cellular events leading to NFT formation and neurodegeneration. Evidence was obtained for the involvement of mitotic mechanisms in NFT formation when monoclonal antibodies (TG/MC series) raised against immunopurified NFT showed specificity for both the AD brain in immunohistochemical studies, and for mitotic cells in culture [44]. The same study provided provocative evidence that monoclonal antibody MPM-2, which was raised against mitotic cells [10], was highly immunoreactive with AD, but not with control brains. Moreover, MPM-2 recognized NFT, the neuritic components of plaques, and morphologically healthy neurons in the AD brain, thus suggesting that it is a marker for the early stages of neurodegeneration. Similar to the TG/MC series, MPM-2 recognized mitotic phosphoepitopes [10,16,39]. The presence of mitotic phosphoepitopes in degenerating neurons suggests that mitotic and neurodegenerative pathways converge in AD. Therefore, antibodies that demonstrate high specificity for both AD pathology and mitotic events can serve as ideal tools for identifying common molecules within the two pathways.Of all the antibodies reported by Vincent and colleagues, TG-3 was the most specific for AD neurofibrillary pathology demonstrating no detectable immunoreactivity in control brains. Therefore, identifying and characterizing the TG-3 antigen or antigens are essential to our understanding of the convergence of neurofibrillary degeneration and mitosis. The first TG-3 immunoreactive protein in the brains of patients with AD was recently identified as an abnormally phosphorylated microtubule associated protein tau [20]. Other potentially biologically relevant TG-3 antigens in the brain remain unknown. However, in cultured cells synchronized in mitosis, TG-3 recognizes not tau but a 105 kDa protein [44]. We here report that the cellular TG-3 antigen is the major nucleolar phosphoprotein nucleolin after phosphorylation by Cdc2 kinase. We also report that in the human brain, nucleolin is present in neuronal cytoplasm and colocalizes with NFT in AD.2Materials and methods2.1AntibodiesMonoclonal antibody TG-3 (hybridoma medium) was described in a previous publication [44] and used at a 1:10 dilution for Western blotting. Anti-nucleolin monoclonal antibody MS3 (hybridoma medium) was kindly provided by Drs. H. Busch and L. Perlaky from Baylor College of Medicine, and used at a dilution of 1:10 for immunocytochemistry and at a 1:1000 dilution for Western blotting [42]. MS3 ascites fluid was used for immunoprecipitations at a 1:10 dilution. Monoclonal antibody D3 (hybridoma medium), kindly provided by Dr. J.-S. Deng from the University of Pittsburgh, was used at a 1:10 dilution for immunocytochemistry [11]. Anti-nucleolin monoclonal antibody CC98 (hybridoma medium), kindly provided by Dr. N.H. Yeh, National Yang Ming College, Taiwan, was used at a 1:5 dilution for immunocytochemistry [8]. Anti-nucleolin monoclonal antibody 4E2 (hybridoma medium) from Research Diagnostics (Flanders, NJ) was used at a 1:5 dilution for immunocytochemistry. Rabbit polyclonal antisera W15 generated against human nucleolin and nucleolin-maltose-binding protein fusion protein were generously provided by Dr. N. Maizels, Yale University [17]. Each polyclonal antiserum was used at a 1:1000 dilution. Non-immune rabbit serum was used as a control. Monoclonal antibody AT180 (Innogenetics, Belgium), which recognizes phospho-threonine 231 (Thr231) of tau [15], was used at a 1:10 dilution for the blocking experiments. Monoclonal antibodies against β-tubulin (Sigma, St. Louis, MO) and histone protein mAb052 (Chemicon, Temecula, CA) were used at a 1:200 final dilution. The specificity of each primary monoclonal and polyclonal antibody was characterized by the authors who generated the antibody and was described in the papers cited above. Secondary goat anti-mouse IgG1a and goat anti-mouse IgM antibodies conjugated to fluorescein and rhodamine (Jackson Immunochemicals, West Grove, PA) were used at a 1:200 final dilution for immunocytochemistry. Secondary antibodies conjugated to biotin along with streptavidin conjugated to CY3 and CY5 fluorochromes (Southern Biotech, Birmingham, AL) were all used at a 1:500 dilution for the immunohistochemistry of brain sections. Secondary antibodies conjugated to horseradish peroxidase (Amersham, Oakville, Ontario) were used at a 1:3000 final dilution for Western blotting.2.2Cell culture and immunocytochemistryHEp-2 human epithelial cells (ATCC, Rockville, MD) were cultured in DME containing 10% bovine calf serum, and penicillin-streptomycin (Gibco, Burlington, Ontario) was cultured at 37°C in 5% CO2. Cells were washed with PBS, harvested by trypsinization, counted and plated at 1.25 × 104 - 2.5 × 104 cells/cm2 on 8 well chambered coverslips (VWR, Piscataway, NJ). On the following day, cells were fixed in 2% paraformaldehyde/0.1% glutaraldehyde for 40 min, permeabilized with 0.5% Triton X-100 for 10 min and stored in PBS at 4°C for immunocytochemistry. Prior to immunocytochemistry, subconfluent monolayers were blocked with 5% bovine serum albumin (BSA) in PBS for 30 min and rinsed in 3 volumes of PBS 3 times followed by three 10 min washes in PBS. Immunostaining of monolayers was performed in the chambered coverslips. Primary antibodies were applied for 2 h at room temperature or overnight at 4°C in a high humidity incubation chamber. The monolayers were washed as described above and incubated with secondary antibodies in the presence of DAPI (Sigma, St. Louis, MO) for 30 min. The monolayers were washed as described above and stored in the ANTI-FADE reagent (Molecular Probes, Eugene, OR) at 4°C. Rodamine. Fluorescein was visualized by the Noran Confocal Odyssey system through a Nikon inverted Diaphot microscope, and images were captured using Image One software.2.3Protein purificationHEp-2 cells were grown in suspension according to the conditions described above. Five liters of logarithmically growing cells were synchronized in mitosis by treatment with 2 mg/ml of nocodazole (Sigma, St. Louis, MO) overnight. Nocodazole was diluted from a DMSO solution so that the final DMSO concentration was 0.05%. Cells were harvested by centrifugation at 250 × g for 2 min, rinsed with PBS, centrifuged, and cell pellets were stored at −80°C. All extraction and purification procedures were performed at 4°C. Cells were extracted in 5 volumes of buffer A for 15 min, gently vortexed, and centrifuged at 400 × g for 10 min. Buffer A contained 0.015 M Tris-Cl pH 7.4, 0.08 M KCl, 2 mM EDTA-KOH, 1% Nonidet P-40, 0.2 mM spermine, 0.5 mM spermidine, 1 mM phenylmethane-sulphonyl fluoride, 10 nM Microsystin-LR, 1 mM Na-o-vanadate (all from Sigma), and protease inhibitor cocktail (Boehringer Mannheim, Indianapolis, IN). The supernatant was chromatographed on DEAE-Sepharose (Pharmacia, Piscataway, NJ) preequilibrated with buffer A, washed, and eluted with a 0.1–0.5 M NaCl step gradient in buffer A using 0.1 M increments. TG-3 reactive fractions were identified by Western blotting using the ECL detection system (Amersham, Oakville Ontario). The TG-3 reactive fraction was diluted in 15 mM Tris, 2 mM EDTA, pH 7.4 (buffer B), chromatographed on HiTrap-Heparin mini column (Pharmacia, Piscataway, NJ) preequilibrated in buffer B, washed, and eluted with a 0.1–0.5 M NaCl step gradient using 0.1 M increments. TG-3 immunoreactivity was determined by Western blotting. The TG-3 reactive fraction was separated by SDS-PAGE and stained with Coomassie Brilliant Blue. The purified TG-3 reactive 105 kDa protein was subjected to N-terminal sequencing and the obtained sequence was compared by BLAST search against the Genbank database.2.4ImmunoprecipitationNocodazole-treated HEp-2 cells from 50 ml of culture media were collected by centrifugation and washed twice with PBS. The following procedures were performed at 4°C. Cells were lysed with 1 ml of RIPA buffer [18] and centrifuged at 8000 × g for 30 s. 30 μl of supernatant were mixed with 3 μl of MS3 ascites fluid. The reaction mixtures were incubated with rotation for 1 h, and 70 μl of anti-mouse IgG agarose (Sigma, St. Louis, MO) were added for an additional 30 min. The resin was washed three times with 1 ml of RIPA buffer, and the immunoprecipitated proteins were eluted by the addition of SDS-PAGE sample buffer containing 2-mercaptoethanol. The sample was divided into two equal parts and subjected to SDS-PAGE, transferred onto PVDF and probed using either MS3 antibodies or TG-3 antibodies.2.5“Mitotic shake”HEp-2 cells were grown in 175 mm2 flasks as described above. Mitotic cells were mechanically dislodged from the rest of the monolayer by dropping the flasks from three feet. The dislodged mitotic cells were harvested by centrifugation of the supernatant at 1000 × g for 5 min. The cell pellet was lysed and analyzed by Western blotting with TG-3 as described above.2.6Chemical cleavageTG-3 positive nucleolin was purified from nocodazole treated HEp-2 cells and subjected to SDS-PAGE. The TG-3 positive band was cut out of the gel, and the gel slice was subjected to chemical digestion with N-chlorosuccinimide, which specifically cleaves at tryptophane (positions 481 and 641 in nucleolin), or with hydroxylamine, which specifically cleaves between asparagine and glycine (positions 136, 137 and 519, 520 in nucleolin). N-chlorosuccinimide was used at 10 ml of 0.1 M in 2 M urea, and 50% acetic acid for 30 min at room temperature with rotation.Hydroxylamine was used at 2 M in 6 M guanidine-HCl at pH 9.0 for 12 h at room temperature with rotation. Gel slices were washed with 15 mM Tris, pH 6.8, 0.1% SDS, and 2-mercaptoethanol for 20 min. After chemical cleavage, the gel slice was placed on top of a second SS-gel and electrophoresed in a direction perpendicular with respect to the first gel. The separated cleavage products were transferred onto PVDF membrane and analyzed by Western blotting. The membranes were first immunostained with TG-3, then stripped with SDS and 2β- mercaptoethanol [18] and reprobed with MS3 to detect nucleolin. In the control experiments the cleaving reagent was omitted.2.7ImmunohistochemistryAll human subjects used in this study were evaluated both clinically and neuropathologically at the Albert Einstein College of Medicine as described elsewhere [44]. For neuropathological evaluation, tissue was immersion-fixed in 4% paraformaldehyde and independently evaluated with antibody Alz-50 and Thioflavin S. Brains from patients with clinical symptoms of AD showed marked neuropathological lesions. No clinical diagnosis of AD was noted in the control cases. Immunostaining was performed essentially as described elsewhere by free floating 50 μm vibratome sections in 24 well culture dishes [44]. For the blocking experiment the tissue was incubated overnight at 4°C with monoclonal antibody AT180 which recognized phosphorylated threonine 231 (Thr231) on tau and does not recognize nucleolin. The tissue was then washed as described elsewhere [44], incubated with unlabeled anti-mouse IgG1 overnight at 4°C, washed again, incubated with TG-3 and nucleolin rabbit antisera W15 overnight at 4°C, and washed. TG-3 was detected with TRITC conjugated anti-mouse IgM, while W15 was detected with biotinylated anti-rabbit antibodies followed by streptavidin conjugated CY5. All secondary incubations were performed as described above. Fluorochromes were visualized by the Bio-Rad Confocal system through a Nikon inverted Diaphot microscope.3Results3.1The TG-3 antigen in mitotic cells is nucleolinTG-3 consistently recognized a major 105 kDa band in extracts from HEp-2 cells that were synchronized in mitosis by treatment with nocodazole (Fig. 1  B, lane 5). This band was also observed in extracts from synchronized MSN neuroblastoma [44] and HeLa cells (data not shown). The appearance of this band was not an artifact of nocodazole treatment because an identical band was observed in extracts from mitotic HEp-2 cells that were enriched in mitotic cells by mechanical dislodging from the flask (Fig. 1B, lane 3). The TG-3 antigen was thus purified from nocodazole synchronized HEp-2 cells by sequential column chromatography and subjected to N-terminal sequencing (Fig. 1A). The sequence of the first 18 amino acids of the TG-3 antigen was identical to the N-terminal sequence of human nucleolin, a protein with a molecular mass of 105 kDa [37].To exclude the possibility that a minor protein of a similar size, copurifying with nucleolin, could account for the TG-3 immunoreactivity, we immunoprecipitated cell lysates with anti-nucleolin monoclonal antibody MS3 and analyzed the immunoprecipitate with TG-3. A band with the molecular weight of 105 kDa was immunoprecipitated by the MS3 antibody and detected by the TG-3 antibody (Fig. 1B, lane 7). These results support our initial finding that both MS3 and TG-3 antibodies recognize nucleolin in mitotic cells.As a further confirmation, the protein purified from nocodazole synchronized cells was subjected to chemical cleavage with either N-chlorosuccinimide or hydroxylamine (Fig. 2).  Human nucleolin has two recognition sites for each reagent, distributed in such a way that cleavage generates three fragments, two of which were predicted to be detectable with the antibodies TG-3 and MS3 (see methods). Major bands migrating at 92 kDa and 74 kDa were observed after cleavage with N-chlorosuccinimide (Fig. 2A) and hydroxylamine (Fig. 2B), respectively. It should be noted that although nucleolin contains two cleavage sites for each reagent, under our experimental conditions we detected the product of only one major cleavage event. After each cleavage reaction, both MS3 and TG-3 stained identical bands, suggesting that they recognized the same protein. Thus, the results from the chemical cleavage experiments, in conjunction with the sequencing and immunoprecipitation experiments, proved that the TG-3 antibody recognized nucleolin in mitotic cells.3.2TG-3 reactive nucleolin is phosphorylated by Cdc2Nucleolin is a major cellular phosphoprotein, which has nine putative phosphorylation sites for the major mitotic kinase Cdc2 and has been shown to be phosphorylated by Cdc2 both in vitro and in vivo [2]. Previous studies demonstrated that TG-3 recognizes phosphoepitopes [44,20].Since TG-3 recognizes mitotic and not interphase cells, the following experiments were performed in order to test if TG-3 immunoreactivity is the result of phosphorylation of nucleolin by Cdc2. Interphase nucleolin, which is not reactive with TG-3, was partially purified from asynchronous HEp-2 cells. Nucleolin enriched samples were phosphorylated with [32P]ATP in the presence or absence of Cdc2 kinase. After phosphorylation, nucleolin was immunoprecipitated with the MS3 antibody, separated by SDS-PAGE, and analyzed by autoradiography and immunoblotting with either MS3 or TG-3 antibodies (Fig. 3).  Autoradiography revealed 32P incorporation into numerous bands suggesting that the nucleolin enriched fraction contained many proteins that could be phosphorylated by Cdc2 kinase (Fig. 3A, lane 1). A significant incorporation of 32P into the protein immunoprecipitable with MS3 in the absence of Cdc2 suggested that nucleolin was also phosphorylated by copurified kinases (Fig. 3A, lane 4).While many bands showed Cdc2 dependent incorporation of 32P, only a single band migrating at 105 kDa was immunoreactive with TG-3 (Fig. 3C, lane 1). The appearance of the single TG-3 immunoreactive band at 105 kDa after phosphorylation with Cdc2 kinase demonstrated the antigenic specificity of the TG-3 phosphoepitope-specific antibody for the nucleolin protein in the relatively crude preparation. Furthermore, immunoprecipitation of nucleolin from the enriched fraction with non-phosphospecific MS3 (see Valdez et al., 1995 for epitope mapping) [42] revealed that nucleolin became immunoreactive with TG-3 only after phosphorylation by Cdc2 (Fig. 3C, lane 3). Phosphorylation reactions carried out in the absence of Cdc2 did not produce epitopes recognizable by TG-3 (Fig. 3C, lanes 2 and 4). Therefore, TG-3 is specific for phosphoepitopes produced by Cdc2 and not by copurifying endogenous kinases. Finally, phosphorylation was time and kinase concentration dependent, and TG-3 immunoreactivity was proportional to the amount of 32P incorporation (data not shown). Together, these data demonstrate the specificity of the TG-3 antibody for nucleolin phosphorylated by Cdc2.3.3TG-3 reactive nucleolin undergoes temporal and spatial changes during mitosis in cultureTG-3 immunoreactivity was examined in an asynchronous population of HEp-2 (Fig. 4  ) and HeLa cells (data not shown) by laser confocal microscopy. Dual labeling with monoclonal antibodies to either □-tubulin or to histone proteins was used to identify cells in different stages of mitosis. TG-3 reactive nucleolin (TG-3 nucleolin) appeared abruptly in cells entering mitosis. Interphase cells were not reactive with TG-3 (Fig. 4). TG-3 nucleolin was localized to the nuclei in cells in late prophase and early prometaphase at the beginning of centrosomal segregation (Figs. 4A–C). TG-3 nucleolin was also localized to the mitotic centrosomes starting from the earliest stages of polar migration (Figs. 4C and 4F). TG-3 nucleolin became dispersed throughout the mitotic cytosol as the prometaphase progressed (Figs. 4E and 4F). Optical sectioning revealed that the condensed chromosomes were immunoreactive with TG-3 as judged by the overlap with immunostaining that was detected by anti-histone antibody mAb053 (Fig. 4L).The chromosomal localization of TG-3 nucleolin became most apparent in metaphase when the majority of the immunoreactivity was localized to the aligned chromosomes (Figs. 4H and 4K). While chromosomal TG-3 immunoreactivity was most intense, some also localized to the mitotic cytosol. The intensity of cytosolic TG-3 immunoreactivity varied between the metaphase cells of the different cell lines but was always lower than immunoreactivity associated with chromosomes (data not shown). TG-3 immunoreactivity began to decrease at the earliest stages of chromosomal segregation, which was determined by histone specific mAb053 (Figs. 4J and 4L) and DAPI staining (data not shown). Whereas the intensity of TG-3 nucleolin immunoreactivity proceeded to decrease from metaphase to telophase, its spatial distribution did not change and the chromosomes persisted to be the predominant TG-3-immunoreactive structures (Figs. 4H, cell 3; Fig. 4K, cell 2). Cells in cytokinesis and interphase showed only background levels of immunostaining.3.4TG-3 reactive and non-reactive nucleolin are differentially distributed during mitosisAsynchronous HEp-2 cells were dual-labeled with phosphoepitope specific TG-3 and with MS3 which is specific for the region of nucleolin that is devoid of phosphorylation sites [42]. Whereas TG-3 was highly specific for nucleolin in cells in mitosis, MS3 was immunoreactive with nucleolin in all phases of the cell cycle (Fig. 5).  As expected, the most prominent MS3 immunoreactive structures in interphase cells were nuclei and nucleoli. MS3 immunoreactivity was more dispersed throughout the nucleus in the early stages of mitosis. In prophase, nuclear TG-3 immunoreactivity only partially colocalized with the MS3 immunoreactivity (Fig. 5A). In metaphase, MS3 immunoreactivity was distributed throughout the mitotic cell. MS3 and TG-3 immunoreactivity colocalized primarily around the chromosomes since chromosomal TG-3 immunoreactivity was most pronounced. MS3 immunoreactivity of cytosol persisted in anaphase, telophase, and cytokinesis, while TG-3 immunoreactivity gradually became undetectable (Fig. 5C). The partial overlap of MS3 and TG-3 immunoreactivities in all stages of mitosis suggests that TG3 reactive nucleolin represents a unique subpopulation of mitotic nucleolin.3.5Differential distribution of nucleolin in the normal human brain and in ADThe functions of nucleolin have been extensively studied in cultured cells and in vitro [14,41]. Nucleolin has been immunocytochemically localized primarily to the nucleolus and the nucleus. However, the distribution and the role of nucleolin in human brain cells remain unknown. Therefore, the distribution of nucleolin in the brain was examined in four individuals affected by AD and in eight age-matched controls. Identical immunolocalization was obtained with all examined anti-nucleolin monoclonal and polyclonal antibodies, strongly suggesting that the distribution of immunoreactivity reflected the true distribution of nucleolin and that it was not the result of cross reactivity with other proteins. The images on panels of Fig. 6 and 7   were obtained with anti-nucleolin monoclonal antibody MS3 and illustrate a typical distribution of nucleolin in the hippocampus. The specificity of TG-3 for AD brains has been previously described [44], and it is here presented for illustrative value (Fig. 6A and 6F).Cells corresponding to all major areas of the hippocampal formation, including the dentate gyrus, were positive for nucleolin. Nucleolin was detected in both large neuronal nuclei and smaller glial nuclei (Figs. 6C and 6D). The distribution of nucleolin in glia appeared primarily nuclear, while neuronal nucleolin was readily detected in both cytoplasm and nuclei. Moreover, some neurons displayed mostly cytoplasmic distribution of nucleolin, while in other neurons, nucleolin was primarily nuclear (Fig. 6B). In addition to staining the neuronal cell bodies, nucleolin was detected in the apical dendrites of the pyramidal cells (Fig. 6D). Neurons with predominantly nuclear, and neurons with predominantly cytoplasmic nucleolin were observed in CA3 and CA4, whereas more cytoplasmic and robust dendritic staining was observed in CA2 and CA1 (Figs. 6C and 6D). The variability in the distribution of nucleolin between cells is indicative of a dynamic function for the protein in neurons. Occasionally a small number of NFT were observed in the hippocampus from individuals who showed no cognitive impairments prior to their death. The appearance of these early pathological changes in the brain prior to development of clinical symptoms is believed to characterize AD progression. CA1 is the earliest region in the hippocampus to be affected by neurodegenerative changes [5]. Interestingly, an intense nucleolin immunoreactivity in densely packed material typical of NFT within some pyramidal cells in the CA 1 region was observed in four of our control cases (Fig. 6E, representative example).To further characterize the association of nucleolin with AD pathology, brains from 3 individuals affected by AD were immunostained with mAb MS3 (Figs. 6G,H,I, and J). The distribution of nucleolin in the AD brains paralleled the distribution of nucleolin in the control brains. However, the neurons in the AD brains exhibited a marked decrease in nucleolin immunoreactivity throughout the hippocampal formation (Fig. 6G and 6H). Nucleolin immunoreactivity, shown in red, labeled small granular structures in many of the neurons in the CA3 and CA4 regions (Fig. 6H). Nucleolin localized to NFT in the CA1 and CA2 regions of the hippocampal formation (Figs. 6I and 6J). Using the classification of NFT proposed by Wischik (1989) [49], we found that the NFT immunolabeled with MS3 had the appearance of type 1 (early intracellular), type 2 (late intracellular) and type 3 (extracellular) NFT. Type 1 NFT were observed as intensely fluorescent, densely packed material traversing the pyramidal neuron and surrounding the nucleus. Large swollen pyramidal neurons with intensely labeled NFT and a nucleus that was positioned near the plasma membrane resembled type 2 NFT. Finally, intense filament-like staining of cell remnants with no noticeable nucleus was consistent with type 3 NFT.3.6TG-3 reactive nucleolin is present in NFTA recent study demonstrated that in AD brains, TG-3 antibody recognized microtubule associated protein tau phosphorylated on Thr231 [20]. It has not been established, however, if TG-3 recognizes other proteins in AD brains. After we established that TG-3 is highly specific for mitotic nucleolin in cell cultures, it became important to determine if TG-3 immunreactive nucleolin was present in the brains of patients with AD and, if yes, to differentiate it from TG-3 immunoreactive tau.In order to identify a population of NFTs with TG-3 reactive nucleolin, dual-label confocal microscopy was first performed with TG-3 and MS3 antibodies on AD cases that showed abundant MS3 immunoreactive NFT (Fig. 7A). There were many TG-3 immunoreactive NFT and an intense neuritic staining by the TG-3 antibody. MS3 stained NFT and nuclei, but there was no staining of neurites. Thus, there were many NFT that were positive with both antibodies (Fig. 7A).We then took advantage of another monoclonal antibody, AT180, which is also specific for tau phosphorylated on Thr231 [15]. However, AT180 did not recognize nucleolin in mitotic cells in cell cultures by immunocytochemistry or immunoblotting (data not shown). In AD brains all cellular structures recognized by TG-3 were also recognized by AT180, indicating that the phosphorylated Thr231 on tau was accessible to both antibodies in the same cellular compartments (data not shown). Since AT180 recognizes phosphorylated tau, but not mitotic nucleolin, AD brain tissue sections were preincubated with AT180 to block phosphorylated Thr231 on tau from being recognized by TG-3. The tissue sections were subsequently dual labeled with TG-3 (to detect the TG-3 immunoreactive protein which was not tau) and anti-nucleolin polyclonal antisera (to detect how much of the residual TG-3 immunoreactivity overlapped nucleolin) (Figs. 7B and 7C). After preincubation with AT180, TG-3 immunoreactivity disappeared preferentially from areas where no nucleolin immunoreactivity was present. TG-3 immunoreactivity was completely blocked in dystrophic neurites and neuritic components of plaques (Fig. 7B). The laser confocal fluorescence was quantified, and the proportion of total TG-3 immunoreactivity represented by the overlap of TG-3 and nucleolin was computed (Fig. 7C). A six-fold increase in the overlap ratio was seen in the sample blocked by AT180 compared to the control sample, demonstrating that AT180 blocked TG-3 reactive tau, but not TG-3 reactivity colocalized with nucleolin. The overlapping of residual TG-3 with nucleolin indicated that TG-3 reactive nucleolin was present in the NFT.4Discussion4.1Nucleolin phosphorylated by Cdc2 is the TG-3 antigenNucleolin is a major multifunctional nuclear phosphoprotein [14,41] and was one of the first identified nuclear-cytoplasmic shuttling proteins [3]. It is thought to be intricately involved in ribosomal biogenesis and it is a marker for cellular proliferation [13,22]. Nucleolin is a physiological substrate for Cdc2 kinase [2,32], and its biologic functions are highly regulated by phosphorylation in a cell cycle specific manner [21,35]. Here we report the identification of the cellular antigen for the AD specific antibody TG-3 as a form of nucleolin that is phosphorylated by Cdc2 in mitosis.Nucleolin has 9 proline directed consensus sequences for Cdc2 recognition. Phosphorylation of nucleolin by Cdc2 and casein kinase II (CKII) has been reported in vitro and in vivo [2,32,24]. The incorporation of 32P in the absence of Cdc2 shown in the present study, complements the previous observation that CKII copurifies with and phosphorylates nucleolin [24]. However, our data indicate that in mitotic cells, TG-3 recognizes nucleolin phosphorylated by Cdc2 and not other kinases, and TG-3 does not recognize other phosphoproteins in cultured cells. These results firmly establish the TG-3 antibody as a novel and useful tool for studying nucleolin in mitosis.4.2Cdc2 phosphorylated nucleolin and mitosisThe abrupt appearance of TG-3 reactive nucleolin in early mitosis corresponds to activation of Cdc2, thus supporting the in vitro phosphorylation results discussed above. A recent report indicates that nucleolin is also phosphorylated by cell cycle kinases that are active prior to mitosis [33]. The specificity of TG-3 for cells in mitosis presents in situ evidence that TG-3 is specific for a phosphoepitope generated by Cdc2 and not for phosphoepitopes generated by other kinases. TG-3 immunoreactivity appears in the nucleus at the onset of mitosis. Here we report the translocation of nucleolin phosphorylated by Cdc2 to the cytoplasm during late prophase and early prometaphase. Schwab and Dreyer (1997) [35] recently demonstrated that Cdc2 phosphorylation localizes microinjected X.laevis nucleolin to the cytoplasm. The redistribution of Cdc2 phosphorylated nucleolin to the cytoplasm in late prophase provides functional in situ support for the above result and raises the possibility that phosphorylation may play a role in targeting nucleolin outside the nucleus at this stage of the cell cycle. Interestingly, the in situ detection of nucleolin in the cytoplasm has been elusive, suggesting that under normal conditions the amount of nucleolin in the cytoplasm of cultured cells is below the level detectable by immunocytochemical methods. However, in polio-virus infected cells [47] and in rat intestinal epithelial cells grown on a glass surface [51], nucleolin was found in the cytoplasm.The localization of Cdc2 phosphorylated nucleolin to the chromosomes is consistent with previously reported observations [12]. Moreover, colocalization of MS3 and TG-3 immunoreactivity in the perichromosomal region suggested that Cdc2 phosphorylation of nucleolin could be important for its chromosomal interaction. It has been hypothesized that interaction of Cdc2 phosphorylated nucleolin with histone H1 plays a role in chromatin condensation in mitosis [21].While most of the nucleolin is located in the mitotic cytosol, most of the TG-3 reactive nucleolin is localized to mitotic chromosomes. In every stage of mitosis, MS3 immunoreactivity only partially overlapped the TG-3 immunoreactivity, suggesting heterogeneity of mitotic nucleolin. This partial overlap also establishes that TG-3 reactive nucleolin represents a unique subpopulation of mitotic nucleolin. Functional studies with nucleolin mutated in its Cdc2 phosphorylation domains will help to elucidate the biologic role of phosphorylation in the interaction of nucleolin with chromosomes and in the cytoplasmic targeting of nucleolin.We observed a decrease in TG-3 immunoreactivity starting at anaphase, and it became undetectable in cytokinesis. The decrease in TG-3 immunoreactivity during mitotic exit may be due to dephosphorylation or degradation of nucleolin. Recent reports suggest that nucleolin is more susceptible to degradation in nonproliferating cells [our unpublished observations; 8]. Our observation that TG-3 immunoreactivity decreases in late mitosis while MS3 immunoreactivity remains unchanged suggests the possibility that TG-3 reactive nucleolin is first dephosphorylated and then degraded. Interestingly, the Saccharomyces pombe nucleolin homolog gar2 is essential for cytokinesis and phosphorylated by Cdc2 [23].Perhaps dephosphorylation and/or degradation of chromosome associated nucleolin has functional significance for chromosomal segregation or cell division. Activation of phosphatase Cdc14 triggers mitotic exit in budding yeast [36,45]. Similar events in mammalian cells may explain the disappearance of TG-3 phosphoepitope, which parallels the separation of chromosomes and mitotic exit. The recently demonstrated participation of nucleolin in special cases of recombination and replication may provide clues to the role of Cdc2 phosphorylated TG-3 immunoreactive nucleolin in mitosis [17,40,4,9]. Thus, our finding of the dynamic association of TG-3 reactive nucleolin with mitotic chromosomes may represent the long sought but elusive direct link between nucleolus and cell cycle regulation in higher organisms [7].4.3Nucleolin in the brainThis study represents the first characterization of the distribution of nucleolin in the human brain. The presented data were highly reproducible when several polyclonal antisera and monoclonal antibodies to nucleolin were used on sections of human brain tissues obtained from both archival biopsy and autopsy material.Three novel observations were made. First, nucleolin was detected in neuronal cytoplasm. Second, nucleolin was present in NFTs, including early tangles, and NFT-associated nucleolin was TG3-positive, suggesting that it was phosphorylated by Cdc2 kinase. Third, in AD, the overall level of nucleolin was dramatically decreased in the temporal cortex as well as in the CA1 and CA2 regions of the hippocampus. As reported by others in cultured cells [41,14,38] and as described here, nucleolin was found in nuclei and nucleoli of neurons and glia cells in the brain. Surprisingly, nucleolin was also present in the cytoplasm of granular neurons of the dentate gyrus and pyramidal neurons of the hippocampus and of the lower neocortical layers. Moreover, nucleolin was identified in the apical dendrites.The finding of nucleolin in the cytoplasm was surprising because it is generally believed that nucleolin, as the name indicates, is present in the nucleolus and that it can also be detected in the nucleus. Nucleolin has been found in the cytoplasm and plasma membrane by biochemical methods [41,14,38]. However, immunocytochemical detection visualized nucleolin normally in the nucleus and nucleolus of cultured cells suggesting that the quantity of nucleolin outside the nucleus is insufficient for its detection under normal conditions. Nucleolin can be detected in cytoplasm of cultured cells by immunocytochemical methods only under certain conditions [51,47]. Yu et al. [51] detected nucleolin in the cytoplasm of rat intestinal epithelial IEC-6 cells grown on glass slides. However, in cells grown on laminin, nucleolin was found in the usual location of the nucleus and nucleolus. Waggoner and Sarnow (1998) [47] reported a massive nucleolar-cytoplasmic relocalization of nucleolin in poliovirus-infected cells. Recently a nucleolin-related protein, probably nucleolin, was detected in the cytoplasm of bovine photoreceptor cells. These studies suggest that in response to certain as yet unidentified signals, nucleolin may relocalize to the cytoplasm. Thus, it is possible to speculate that in the brain, nucleolin is localized to the neuronal cytoplam in response to signals that are absent in cultured cells.The association of nucleolin with neuronal degeneration stems from the localization of nucleolin to the NFT. It is important to stress that brain tissue from some of the so called control age-matched cases, where no clinical symptoms of AD were apparent, had a small number of nucleolin positive, early (type 1) NFT in the CA1. Since CA1 is thought to be the earliest hippocampal region affected by NFT during the course of AD progression [5], nucleolin was inferred to be a morphological marker for early neurofibrillary changes.Dual labeling experiments with antibodies to nucleolin and TG-3 yielded partial colocalization. Recognition of hyperphosphorylated tau by TG-3 serves to explain the TG-3 immunoreactivity that does not overlap with nucleolin immunoreactivity. Blocking of tau from being recognized by TG-3 by preincubation with an excess of monoclonal antibody AT180 led to the disappearance of TG-3 immunoreactivity in dystrophic neurites and neuritic components of plaques, thus confirming tau presence in these structures. Persistence of residual TG-3 immunoreactivity in NFT suggests that in NFT, TG-3 primarily recognized proteins other than tau. Inefficient blocking of Thr231 on tau in NFT is not likely since AT180 is intensely immunoreactive with NFT. The colocalization of nucleolin immunoreactivity with the residual TG-3 immunoreactivity strongly suggests that Cdc2 phosphorylated nucleolin is one of the major TG-3 immunoreactive proteins in NFT.Detection of TG-3 immunoreactive nucleolin in NFT marks the first identification of a normal cellular substrate for mitotic kinases, which appears to undergo mitotic modification in AD. Several recent reports indicating that mitotic kinases are active in the AD brain contribute important information to our understanding of mitotic mechanisms that are reactivated in AD pathogenesis. However, in order to understand the mechanisms of the resulting neuronal degeneration, substrates for mitotic kinases in AD must be identified. It is important to reemphasize that nucleolin is a marker for early (pre-clinical) neurofibrillary changes and thus its mitotic modification may prove important in the early development of neuronal degeneration associated with AD.The possibility that nucleolin is involved in both chromosomal segregation during mitosis as well as AD may help to explain the observed co-occurrence of Down syndrome (DS) and AD within certain families [19,34]. The majority of DS cases are the result of non-dysjunction of maternal chromosome 21 in miosis, or a defect in chromosomal segregation. Therefore, the co-occurrence of DS and AD within specific families suggests a converging cellular mechanism between chromosomal segregation and neuronal degeneration. Specifically, proteins and pathways involved in chromosomal segregation and cell division in general may serve as signals for, or intermediates in, the degeneration of post-mitotic neurons in AD. The possible involvement of nucleolin in mitosis and AD makes it a candidate for such a protein.AcknowledgementsThis work was supported by NIA, Alzheimer’s Association, and Long Island Alzheimer Foundation (LIAF) to DG, AG/OD 12721 to IV and MSTP GM08444, AFAR, and LIAF fellowships to AD. The authors thank M. Cammer for assistance with confocal images; G. Jicha for assistance with a phosphorylation experiment; Drs. C. Dingwall, and S. Strickland for critical reading of the manuscript; Drs. E. Bromet, P. Fisher, W. Quitschke, S. Simon, and W. Van Nostrand for helpful comments on the manuscript.References[1]Baumann, K., E.M. Mandelkow, J. Biernat, H. Piwnica-Worms, and E. Mandelkow. Abnormal Alzheimer’-like phosphorylation of tau-protein by cyclin-dependent kinases cdk2 and cdk5. FEBS Lett. 1993;336:417–424.[2]Belenguer, P., M. Caizergues-Ferrer, J.C. Labbe, M. Doree, and F. Amalric. Mitosis-specific phosphorylation of nucleolin by p34Cdc2 protein kinase. Mol Cell Biol. 1990;10:3607–3618.[3]Borer, R.A., C.F. Lehner, H.M. Eppenberger, and E.A. Nigg. Major nucleolar proteins shuttle between nucleus and cytoplasm. Cell. 1989;56:379–390.[4]Borggrefe, T., M. Wabl, A.T. Akhmedov, and R. Jessberger. A B-cell-specific DNA recombination complex. J Biol Chem. 1998;273:17025–17035.[5]H.BraakE.BraakStaging of Alzheimer’s disease-related neurofibrillary changesNeurobiol Aging161995271278[6]Busser, J., D.S. Geldmacher, and K. Herrup. Ectopic cell cycle proteins predict the sites of neuronal cell death in Alzheimers disease brain. J Neurosc. 1998;18:2801–2807.[7]Carmo-Fonseca, M., L. Mendes-Soares, and I. Campos. To be or not to be in the nucleolus. Nature Cell Biol. 2:E107–E112, 2000.[8]Chen, C.M., S.Y. Chiang, and N.H. Yeh. Increased stability of nucleolin in proliferating cells by inhibition of its self-cleaving activity. J Biol Chem. 1991;266:7754–7758.[9]Daniely, Y, J.A. Borowiec. Formation of a complex between nucleolin and replication protein after cell stress prevents initiation of DNA replication. J. Cell Biol. 149:799–809, 2000.[10]Davis, F.M., T.Y. Tsao, S.K. Fowler, and P.N. Rao. Monoclonal antibodies to mitotic cells. Proc Nat Acad Sci. 1983;80:2926–2930.[11]Deng, J.S., B. Ballou, and J.K. Hofmeister. Internalization of anti-nucleolin antibody into viable HEp-2 cells. Mol Biol Rep. 1996;23:191–195.[12]Dundr, M., U.T. Meier, N. Lewis, D. Rekosh, M.L. Hammarskjold, and M.O.J. Olson. A class of nonribosomal nucleolar components is located in chromosome periphery and in nucleolus-derived foci during anaphase and telophase. Chromos. 1997;105:407–417.[13]Ghisolfi, L., G. Joseph, M. Erard, J.M. Escoubas, C. Mathieu, and F. Amalric. Nucleolin–pre-rRNA interactions and preribosome assembly. Mol Biol Rep. 1990;14:113–114.[14]Ginisty, H., H. Sicard, B. Roger, and P. Bouvet. Structure and functions of nucleolin. J Cell Sci. 1999;112:761–772.[15]Goedert, M., R. Jakes, R.A, Crowther, P. Cohen, E. Vanmechelen, M. Vandermeeren, and P. Cras. Epitope mapping of monoclonal antibodies to the paired helical filaments of Alzheimer’s disease: identification of phosphorylation sites in tau protein. Biochem J. 1994;301:871–877.[16]G.J.GorbskyCell cycle progression and chromosome segregation in mammalian cells cultured in the presence of the topoisomerase II inhibitors ICRF-187 [(+)-1,2-bis(3,5-dioxopiperazinyl-1-yl)propane; ADR-529] and ICRF-159 (Razoxane)Cancer Res54199410421048[17]Hanakahi, L.A., L.A. Dempsey, M.J. Li, and N. Maizels. Nucleolin is one component of the B cell-specific transcription factor and switch region binding protein, Lr1. Proc Natl Acad Sci. 1997;94:3605–3610.[18]Harlow, E, D. Lane. Antibodies (Cold Spring Harbor: Cold Spring Harbor Laboratory), 1988.[19]L.L.HestonA.R.MastriThe genetics of Alzheimer’s diseaseassociations with hematologic malignancy and Down’s syndromeArch Gen Psychiatry341977976981[20]Jicha, G.A., E. Lane, I. Vincent, L. Otvos, Jr., R. Hoffmann, and P. Davies. A conformation- and phosphorylation-dependent antibody recognizing the paired helical filaments of Alzheimer’s disease. J Neurochem. 1997;69:2087–2095.[21]Kharrat, A., J. Derancourt, M. Doree, F. Amalric, and M. Erard, M. Synergistic effect of histone H1 and nucleolin on chromatin condensation in mitosis: role of a phosphorylated heteromer. Biochem. 1991;30:10329–10336.[22]Lapeyre, B., H. Bourbon, and F. Amalric. Nucleolin, the major nucleolar protein of growing eukaryotic cells: an unusual protein structure revealed by the nucleotide sequence. Proc Natl Acad Sci USA. 1987;84:1472–1476.[23]Leger-Silvestre, I., M.P. Gulli, J. Noaillac-Depeyre, M. Faubladier, H. Sicard, M. Caizergues-Ferrer, and N. Gas. Ultrastructural changes in the Schizosaccharomyces pombe nucleolus following the disruption of the gar2+ gene, which encodes a nucleolar protein structurally related to nucleolin. Chromosoma. 1997;105:542–552.[24]Li, D., G. Dobrowolska, and E.G. Krebs. The physical association of casein kinase 2 with nucleolin. J Biol Chem. 1996;271:15662–15668.[25]Li, J.H., M. Xu, H. Zhou, J.Y. Ma, and H Potter. Alzheimer presenilins in the nuclear membrane, interphase kinetochores, and centrosomes suggest a role iIn chromosome segregation. Cell. 1997;90:917–927.[26]Liu, W.K., R.T. Williams, F.L. Hall, D.W. Dickson, and S.H. Yen. Detection of a cdc2-related kinase associated with Alzheimer paired helical filaments. Am J Path. 1995;146:228–238.[27]Mawal-Dewan, M., C.S. Parimal, M. Abdel-Ghani, D. Shalloway, and E. Racker. Phosphorylation of tau protein by purified p34 cdc28 and a related protein kinase. J Biol Chem. 1992;267:19705–19709.[28]McShea, A., P.L.R. Harris, K.R. Webster, A.F. Wahl, and M.A. Smith. Abnormal expression of the cell cycle regulators P16 and Cdk4 in Alzheimerı́s disease. Am J Path. 1997;150:1933–1939.[29]Nagy, Z., M.M. Esiri, and A.D. Smith. Expression of cell division markers in the hippocampus in Alzheimers disease and other neurodegenerative conditions. Acta Neuropath. 1997;93:294–300.[30]E.A.NiggCellular substrates of p34cdc2 and its companion cyclin-dependent kinasesTrends Cell Biol31993296301[31]Paudel, H., J. Lew, Z. Ali, and J.H. Wang. Brain proline-directed kinase phosphorylates tau on sites that are abnormally phosphorylated in tau associated with Alzheimer’s disease helical filaments. J Biol Chem. 1993;268:23512–23518.[32]Peter, M., J. Nakagawa, M. Doree, J.C. Labbe, and E.A. Nigg, E.A. Identification of major nucleolar proteins as candidate mitotic substrates of cdc2 kinase. Cell. 1990;60:791–801.[33]Sarcevic, B., R. Lilischkis, and R.L. Sutherland. Differential phosphorylation of T-47d human breast cancer cell substrates by D1-, D3-, E-, and a-type cyclin-Cdk complexes. J Biol Chem. 1997;272:33327–33337.[34]Schupf, N., D. Kapell, J.H. Lee, R. Ottman, and R. Mayeux. Increased risk of Alzheimer’s disease in mothers of adults with Down’s syndrome. Lancet. 1994;344:353–356.[35]M.S.SchwabC.DreyerProtein phosphorylation sites regulate the function of the bipartite NLS of nucleolinEur J Cell Biol731997287297[36]Shou, W., J.H. Seol, A. Shevchenko, C. Baskerville, D. Moazed, Z.W.S. Chen, J. Jang, A. Shevchenko, H. Charbonneau, and R.J. Deshaies. Exit from mitosis is triggered by Tem1-dependent release of the protein phosphatase Cdc14 from nucleolar RENT complex. Cell. 1999;97:233–244.[37]Srivastava, M., P.J. Fleming, H.B. Pollard, and A.L. Burns. Cloning and sequencing of the human nucleolin cDNA. FEBS Lett. 1989;250:99–105.[38]Srivastava, M, H.B. Pollard. Molecular dissection of nucleolin’s role in growth and cell proliferation: new insights. FASEB J. 1999;13:1911–1922.[39]Taagepera, S., M.S. Campbell, and G.J. Gorbsky. Cell-cycle-regulated localization of tyrosine and threonine phosphoepitopes at the kinetochores of mitotic chromosomes. Exp Cell Res. 1995;221:249–260.[40]Thyagarajan, B., R. Lundberg, M.C. Rafferty, and C. Campbell. Nucleolin promotes homologous DNA pairing in vitro. Somatic Cell Mol Genet. 1998;24:263–272.[41]Tuteja, R, N. Tuteja. Nucleolin: a multifunctional major nucleolar phosphoprotein. Crit Rev Biochem Mol Biol. 1999;33:407–436.[42]Valdez, B. C., D. Henning, R.K. Busch, M. Srivastava, and H. Busch. Immunodominant RNA recognition motifs of human nucleolin/C23. Mol Immunol. 1995;32:1207–1213.[43]Vincent, I., G. Jicha, M. Rosado, and D.W. Dickson. Aberrant expression of mitotic cdc2/cyclin B1 kinase in degenerating neurons of Alzheimers disease brain. J Neurosci. 1997;17:3588–3598.[44]Vincent, I., M. Rosado, and P. Davies. Mitotic mechanisms in Alzheimer’s disease? J Cell Biol. 1996;132:4134–4125.[45]Visinti, R., E.S. Hwang, and A. Amon. Cfi1 prevents premature exit from mitosis by anchoring Cdc14 phosphatase in the nucleolus. Nature. 1999;398:818–823.[46]Vulliet, R., S.M. Halloran, R.B., Braun, A.J. Smith, and G. Lee. Proline-directed phosphorylation of human tau protein. J Biol Chem. 1992;267:22570–22574.[47]Waggoner, S, P. Sarnow, P. Viral ribonucleoprotein complex formation and nucleolar cytoplasmic relocalization of nucleolin in poliovirus-infected cells. J Virol. 1998;72:6699–6709.[48]Wille, H., G. Drewes, J. Biernat, E.M. Mandelkow, and E. Mandelkow. Alzheimer-like paired helical filaments and antiparallel dimers formed from microtubule-associated protein tau in vitro. J Cell Biol. 1992;118:573–584.[49]C.M.WischikCell biology of the Alzheimer tangleCur Op Cell Biol11989115122[50]Yen, S.H., A. Kenessey, S.C. Lee, and D.W. Dickson. The distribution and biochemical properties of a Cdc2-related kinase, KKIALRE, in normal and Alzheimer brains. J Neurochem. 1995;65:2577–2584.[51]Yu, D.H., M.Z. Schwartz, and R. Petryshyn. Effect of laminin on the nuclear localization of nucleolin in rat intestinal epithelial Iec-6 cells. Biochem. Biophys. Res. Comm. 247:186–192;1998.