Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P10636 (tau protein)
 5,110 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A photoaffinity label for the identification of tubulin-binding proteins was synthesized from phosphocellulose-purified bovine brain tubulin and (N-hydroxysuccinimidyl)-4-azidosalicylic acid. The azidotubulin derivative retained the ability to undergo temperature-dependent microtubule assembly and disassembly. When incubated with purified tau protein, the azidotubulin and tau formed cross-linked complexes upon photoactivation. When 125I-labeled azidotubulin was used to photoaffinity label tubulin-binding proteins within the kinetochore of isolated mammalian chromosomes, a 130-kDa band was identified on autoradiographs of SDS-polyacrylamide gels of the 125I-labeled azidotubulin/chromosome preparations. The 130-kDa complex was isolated by antitubulin affinity chromatography and analyzed by immunoblotting using both antitubulin and kinetochore-specific sera obtained from human patients with the autoimmune disease scleroderma CREST. The immunoblots demonstrated that the 130-kDa band that was observed on autoradiographs was a complex of a subunit of the tubulin dimer and an 80-kDa CREST-specific kinetochore protein. The binding of azidotubulin to the 80-kDa kinetochore protein was significantly decreased when chromosomes were treated with a mixture of 9 parts underivatized tubulin to 1 part azidotubulin prior to photolysis. The formation of the 130-kDa azidotubulin/kinetochore protein complex was not inhibited by pretreating the chromosomes with CREST serum prior to incubation with azidotubulin. Azidotubulin should be a useful probe for the identification and characterization of tubulin-binding proteins.
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PMID:Synthesis of azidotubulin: a photoaffinity label for tubulin-binding proteins. 260 99

Bovine brain tau protein (tau) consists of four closely related phosphoproteins named tau 1, tau 2, tau 3 and tau 4, that range in size from 55 to 68 kDa (as determined by gel electrophoresis). Here we report an improved large-scale purification method for tau protein and the separation of the four individual tau protein species. The separation of the individual tau protein was accomplished by two chromatographic techniques: hydroxyapatite chromatography allowed the separation of two pairs of tau protein (tau 1 and tau 3) and (tau 2 and tau 4); fast protein liquid chromatography on a Mono Q column at basic pH achieved the resolution of the individual tau protein species in each pair derived from hydroxyapatite columns. Chromatography on the Mono Q column revealed that tau protein possesses previously unrecognized, highly reactive sulfhydryl groups that may oxidize to form intermolecular disulfide bridges. The isolation of individual species of tau in substantial quantities permitted an improved amino acid analysis that demonstrated the occurrence of cysteine and tryptophan in the protein. The availability of individual tau protein species greatly simplified the analysis for mode II phosphorylation of tau, which was found to be catalyzed by the calcium/phospholipid-dependent protein kinase C. The mode II phosphorylation of tau by protein kinase C was not associated with a mobility shift for tau protein in SDS-polyacrylamide gel electrophoresis, in contrast to mode I phosphorylation of tau by the Ca2+/calmodulin-dependent kinase, which produces a substantial shift in mobility.
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PMID:Separation of the different microtubule-associated tau protein species from bovine brain and their mode II phosphorylation by Ca2+/phospholipid-dependent protein kinase C. 312 2

Antibodies were raised to paired helical filament (PHF) enriched fractions obtained from brains of individuals with Alzheimer disease by extraction with ionic detergent followed by sucrose gradient centrifugation. Electron microscopic examination showed that the fractions were enriched in Alzheimer PHF but contained also lipofuscin, amyloid, granular material and membranous elements. Analysis of these fractions with SDS-PAGE stained with Coomassie blue showed only a faint band at approximately 60 kDa while most of the material was excluded from the stacking gel. BALB/c mice were injected weekly with 100 or 200 micrograms of these fractions or corresponding fractions from age-matched control brains. The 3 mice injected with Alzheimer brain, but not the 5 mice injected with control brain fractions, produced antibodies that reacted with central and peripheral nervous system axons, Alzheimer neurofibrillary tangles in intact tissue as well as with isolated, SDS-treated paired helical filaments. In gel strips antibodies from all 3 mice injected with Alzheimer brain fractions reacted with the 200-kDa and 168-kDa but not the 68-kDa neurofilament subunits. The 3 antisera reacted also with some forms of the microtubule-associated protein tau. Adsorptions with the insoluble fraction from Alzheimer but not from control brains blocked staining of axons and NFT by all 3 antisera. Adsorption with highly purified neurofilament proteins or with a preparation containing the 200-kDa and 168-kDa neurofilament subunits blocked axon and NFT immunostaining only in one antiserum. Adsorptions with microtubule protein, heat-stable microtubule-associated protein, or a preparation of tau did not completely block immunostaining by any of the 3 antisera. These results demonstrate that fractions enriched with Alzheimer paired helical filaments contain insoluble neurofilament, tau and other yet unidentified antigens.
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PMID:Antibodies to the neuronal cytoskeleton are elicited by Alzheimer paired helical filament fractions. 367 58

Alz-50, a monoclonal antibody originally prepared using Alzheimer brain homogenates, reacts with PHF-tau and normal tau on immunoblots, and stains specific neuronal populations in sections from Alzheimer's disease brain. Although the Alz-50 epitope has been mapped to amino acids 2-10 present in all human tau isoforms, minimal Alz-50 immunoreactivity is present in tissue from control brain, suggesting Alz-50 binding may be dependent on tau conformational differences. The absence of conclusive results concerning Alz-50 binding presents the possibility of Alz-50 immunoreactivity with proteins other than tau. The present study demonstrates Alz-50 cross-reactivity with denatured bovine serum albumin (BSA) and human serum albumin (HSA). Using LA-N-5 neuroblastoma cells, BSA from serum-containing media was present in cell homogenates and was found to be Alz-50-reactive on immunoblots. In fact, Alz-50 (0.1 microgram/ml) recognized as little as 78 ng of BSA and 312 ng of HSA. Since Alz-50 does not recognize native BSA, blocking of immunoblots with 3% BSA did not alter Alz-50 reactivity with tau from LA-N-5 cells. On SDS-polyacrylamide gels, HSA (approximately 69 kDa) migrates very closely to the pattern of A68 (PHF-tau) from Alzheimer brain homogenates. Hence, the presence of BSA or other albumins in cell or brain homogenates may be an important concern when using the Alz-50 antibody.
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PMID:Monoclonal antibody Alz-50 reacts with bovine and human serum albumin. 753 57

During purification of tau protein kinase I and II from the bovine brain extract, a new tau protein kinase was detected and purified with phosphocellulose, gel filtration, S-Sepharose and AF-Heparin column chromatography. The molecular mass of the enzyme was determined to be 32 kDa by gel filtration and activity staining on SDS-PAGE. The enzyme is a Ser/Thr protein kinase phosphorylating tau, beta-tubulin, MAP2 and alpha-casein. Employing many synthetic peptides, the recognition site of this enzyme appears to be -SR-. The enzyme requires no second messenger and is inhibited with high concentration of heparin, but not by inhibitors of CKI. These results indicate that this enzyme, tau-tubulin kinase is novel and distinct from TPKI, II and CKI, II.
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PMID:A novel tau-tubulin kinase from bovine brain. 755 43

Aberrant phosphorylation of the microtubule-associated protein tau is one of the pathological features of neuronal degeneration in Alzheimer's disease. The phosphorylation of Ser-262 within the microtubule binding region of tau is of particular interest because so far it is observed only in Alzheimer's disease (Hasegawa, M., Morishima-Kawashima, M., Takio, K., Suzuki, M., Titani, K., and Ihara, Y. (1992) J. Biol. Chem. 26, 17047-17054) and because phosphorylation of this site alone dramatically reduces the affinity for microtubules in vitro (Biernat, J., Gustke, N., Drewes, G., Mandelkow, E.-M., and Mandelkow, E. (1993) Neuron 11, 153-163). Here we describe the purification and characterization of a protein-serine kinase from brain tissue with an apparent molecular mass of 110 kDa on SDS gels. This kinase specifically phosphorylates tau on its KIGS or KCGS motifs in the repeat domain, whereas no significant phosphorylation outside this region was detected. Phosphorylation occurs mainly on Ser-262 located in the first repeat. This largely abolishes tau's binding to microtubules and makes them dynamically unstable, in contrast to other protein kinases that phosphorylate tau at or near the repeat domain. The data suggest a role for this novel kinase in cellular events involving rearrangement of the microtuble-associated proteins/microtubule arrays and their pathological degeneration in Alzheimer's disease.
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PMID:Microtubule-associated protein/microtubule affinity-regulating kinase (p110mark). A novel protein kinase that regulates tau-microtubule interactions and dynamic instability by phosphorylation at the Alzheimer-specific site serine 262. 770 16

The formation of neurofibrillary tangles (NFTs) and paired-helical filaments (PHFs) in Alzheimer's disease (AD) reflects a major disorganization of the cytoskeleton. The role of the neuronal membrane skeleton in the development of these abnormalities has not previously been investigated. In this study, we used 9 antibodies raised against the erythrocyte membrane skeleton protein 4.1 (P4.1) for immunocytochemical and immunoblot analyses to investigate whether or not the brain homologues of this protein were constituents of NFTs or PHFs. Our results show that 7 of the 9 monospecific antibodies against the human and pig erythrocyte P4.1 stained NFTs in the prefrontal cortex and hippocampus of AD brains. The P4.1 antibodies used here did not cross-react with tau protein isolated from AD brain, and preabsorption of these antibodies with tau protein did not cause loss of NFT staining. In age-matched control brains, these P4.1 antibodies stained neuronal cell bodies or nuclei. Six of the antibodies also stained isolated NFTs but the SDS-insoluble NFTs were immunostained only by two of the P4.1 antibodies. By using inositol hexaphosphate affinity chromatography and immunoblot analysis, we identified a 68-kDa protein as the most likely brain analogue of P4.1. When SDS-extracted proteins from the isolated NFTs were immunoblotted, a 50-kDa band was immunostained. The 68-kDa and 50-kDa proteins were not stained by tau protein and neurofilament subunit NF-H antibodies, that strongly stained NFTs. We conclude that brain protein 4.1 isoform(s) are constituents of NFTs in AD.
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PMID:Evidence for the association of protein 4.1 immunoreactive forms with neurofibrillary tangles in Alzheimer's disease brains. 780 27

Microtubule protein extracted from dogfish erythrocyte cytoskeletons by disassembly of marginal bands at low temperature formed linear microtubule (MT) bundles upon reassembly at 22 degrees C. The bundles, which were readily visible by video-enhanced phase contrast or DIC microscopy, increased in length and thickness with time. At steady state after 1 hour, most bundles were 6-11 microns in length and 2-5 MTs in thickness. No inter-MT cross-bridges were visible by negative staining. The bundles exhibited mechanical stability in flow as well as flexibility, in this respect resembling native marginal bands. As analyzed by SDS-PAGE and immunoblotting, our standard extraction conditions yielded MT protein preparations and bundles containing tau protein but not high molecular weight MAPs such as MAP-2 or syncolin. In addition, late fractions of MT protein obtained by gel filtration were devoid of high molecular weight proteins but still produced MT bundles. The marginal band tau was salt-extractable and heat-stable, bound antibodies to mammalian brain tau, and formed aggregates upon desalting. Antibodies to tau blocked MT assembly, but both assembly and bundling occurred in the presence of antibodies to actin or syncolin. The MTs were "unbundled" by subtilisin or by high salt (0.5-1 M KCl or NaCl), consistent with tau involvement in bundling. High salt extracts retained bundling activity, and salt-induced unbundling was reversible with desalting. However, reversibility was observed only after salt-induced MT disassembly had occurred. Reconstitution experiments showed that addition of marginal band tau to preassembled MTs did not produce bundles, whereas tau presence during MT reassembly did yield bundles. Thus, in this system, tau appears to play a role in both MT assembly and bundling, serving in the latter function as a coassembly factor.
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PMID:Assembly and bundling of marginal band microtubule protein: role of tau. 782 Aug 58

Microtubule-associated protein tau from Alzheimer brain has been shown to be phosphorylated at several ser/thr-pro and ser/thr-X sites (Hasegawa, M. et al., J. Biol. Chem. 267, 17047-17054, 1992). Several proline-dependent protein kinases (PDPKs) (MAP kinase, cdc2 kinase, glycogen synthase kinase-3, tubulin-activated protein kinase, and 40 kDa neurofilament kinase) are implicated in the phosphorylation of the ser-thr-pro sites. The identity of the kinase(s) that phosphorylate the ser/thr-X sites are unknown. To identify the latter kinase(s) we have compared the phosphorylation of bovine tau by several brain protein kinases. Stoichiometric phosphorylation of tau was achieved by casein kinase-1, calmodulin-dependent protein kinase II, Gr kinase, protein kinase C and cyclic AMP-dependent protein kinase, but not with casein kinase-2 or phosphorylase kinase. Casein kinase-1 and calmodulin-dependent protein kinase II were the best tau kinases, with greater than 4 mol and 3 mol 32P incorporated, respectively, into each mol of tau. With the sequential addition of these two kinases, 32P incorporation approached 6 mol. Peptide mapping revealed that the different kinases largely phosphorylate different sites on tau. After phosphorylation by casein kinase-1, calmodulin-dependent protein kinase II, Gr kinase, cyclic AMP-dependent protein kinase and casein kinase-2, the mobility of tau isoforms as detected by SDS-PAGE was decreased. Protein kinase C phosphorylation did not produce such a mobility shift. Our results suggest that one or more of the kinases studied here may participate in the hyperphosphorylation of tau in Alzheimer disease.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Comparison of the phosphorylation of microtubule-associated protein tau by non-proline dependent protein kinases. 803 84

Phosphorylation of tau protein has been suggested as a major mechanism regulating its functions. In assembled brain microtubules, tau is phosphorylated, but additional phosphorylation can be induced in vitro. Supply of excess ATP alone was sufficient to reduce migration of tau on SDS gels, diminish Tau-1 immunostaining, and induce the expression of epitopes recognized by the PHF-1 antibody, suggesting that Alzheimer-type phosphorylation may have occurred. Okadaic acid had no further effect. However, treatment with tyrosine kinase inhibitors modifies the phosphorylation profiles of tau proteins. Most evidently, migration of the largest tau isoform was further retarded on SDS gels and PHF-1 immunostaining was enhanced. The profound effect of tyrosine kinase inhibitors on tau phosphorylation was also demonstrated in living cells following microinjection of cultured hippocampal neurons. Identification of proline-directed protein kinases and their regulatory factors associated with assembled microtubules indicated the presence of multiple phosphorylation pathways in microtubules. Our results are consistent with the hypothesis that sequential phosphorylation of tau proteins is at least partially mediated through tyrosine phosphorylation/dephosphorylation mechanisms.
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PMID:Abnormal phosphorylation of tau proteins associated with bovine brain microtubules: activation by excess ATP and tyrosine dephosphorylation. 804 76


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