Purification of tau,a microtubule-associated protein that induces assembly of microtubules from purified tubulin

Tau, a microtubule-associated protein which copurifies with tubulin through successive cycles of polymerization and depolymerization, has been isolated from tubulin by phosphocellulose chromatography and purified to near homogeneity. The purified protein is seen to migrate during electrophoresis on acrylamide gels as four closely spaced bands of apparent molecular weights between 55,000 and 62,000. Specific activity for induction of microtubule formation from purified tubulin has been assayed by quantitative electron microscopy and is seen to be enhanced three- to fourfold in the purified tau when compared with the unfractionated microtubule-associated proteins. Nearly 90% of available tubulin at 1 mg/ml is found to be polymerizable into microtubules with elevated levels of tau. Moreover, the critical concentration for polymerization of the reconstituted tau + tubulin system is seen to be a function of tau concentration and may be lowered to as little as 30 μg of tubulin per ml. Under depolymerizing conditions, 50% of the tubulin at only 1 mg/ml may be driven into ring structures. A separate purification procedure for isolation of tau directly from cell extracts has been developed and data from this purification suggest that tau is present in the extract in roughly the same proportion to tubulin as is found in microtubules purified by cycles of assembly and disassembly. Tau is sufficient for both nucleation and elongation of microtubules from purified tubulin and hence the reconstituted tau + tubulin system defines a complete microtubule assembly system under standard buffer conditions. In an accompanying paper (Cleveland et al., 1977) the physical and chemical properties of tau are discussed and a model by which tau may function in microtubule assembly is presented.     

Calmodulin binds to a tubulin binding site of the microtubule-associated protein tau

Previous studies have demonstrated that the microtubule - associated proteins MAP-2 and tau interact selectively with common binding domains on tubulin defined by the low-homology segments a (430–441) and (422–434). It has been also indicated that the synthetic peptide VRSKIGSTENLKHQPGGG corresponding to the first tau repetitive sequence represents a tubulin binding domain on tau. The present studies show that the calcium-binding protein calmodulin interacts with a tubulin binding site on tau defined by the second repetitive sequence VTSKCGSLGNIHHKPGGG. It was shown that both tubulin and calmodulin bind to tau peptide-Sepharose affinity column. Binding of calmodulin occurs in the presence of 1 mM Ca 2+ and it can be eluted from the column with 4 mM EGTA. These findings provide new insights into the regulation of microtubule assembly, since Ca 2+/calmodulin inhibition of tubulin polymerization into microtubules could be mediated by the direct binding of calmodulin to tau, thus preventing the interaction of this latter protein with tubulin.     

Bis-ANS as a specific inhibitor for microtubule-associated protein induced assembly of tubulin.

5,5'-Bis[8-(phenylamino)-1-naphthalenesulfonate] (bis-ANS), the fluorescent probe which binds to tubulin, inhibits its assembly into microtubules [Horowitz et al. (1984) J. Biol. Chem. 259, 14647-14650]. The results described in this paper demonstrate that bis-ANS is quite distinct from other well-known microtubule inhibitors in its specificity of action. The inhibitory potentials of bis-ANS and its three structural analogues ANS, Prodan [6-propionyl-2-(dimethylamino)naphthalene], and NSA (naphthalenesulfonic acid) have been compared. It is found that they can be arranged in the following order according to their polymerization inhibitory potentials: bis-ANS approximately equal to Prodan much greater than ANS greater than NSA. Interestingly, the naphthalene nucleus is sufficient to cause inhibition of polymerization. Detailed experiments were carried out to examine the mode of assembly inhibition by aminonaphthalenes at the molecular level, using bis-ANS as a representative. It was found that there was little or no effect of bis-ANS on the assembly of tubulin when polymerization was induced by assembly promoters like taxol, DMSO, or glutamate, or on the assembly of subtilisin-digested protein (tubulin S), for all of which half-maximal inhibition could not be achieved even at 120 microM bis-ANS. On the contrary, bis-ANS acts as an inhibitor in the case of MAP- (MAP2 and tau) and poly(L-lysine)-induced assembly of tubulin, with half-maximal inhibitory concentrations ranging from 1.5 to 7.6 microM. Our results place bis-ANS as a novel inhibitor, which seems to specifically inhibit C-termini-mediated assembly. Of all assembly inhibitors known so far, none exhibits such selection.(ABSTRACT TRUNCATED AT 250 WORDS)     

Kinetic analysis of tubulin assembly in the presence of the microtubule-associated protein TOGp

The microtubule-associated protein TOGp, which belongs to a widely distributed protein family from yeasts to humans, is highly expressed in human tumors and brain tissue. From purified components we have determined the effect of TOGp on thermally induced tubulin association in vitro in the presence of 1 mm GTP and 3.4 m glycerol. Physicochemical parameters describing the mechanism of tubulin polymerization were deduced from the kinetic curves by application of the classical theoretical models of tubulin assembly. We have calculated from the polymerization time curves a range of parameters characteristic of nucleation, elongation, or steady state phase. In addition, the tubulin subunits turnover at microtubule ends was deduced from tubulin GTPase activity. For comparison, parallel experiments were conducted with colchicine and taxol, two drugs active on microtubules and with tau, a structural microtubule-associated protein from brain tissue. TOGp, which decreases the nucleus size and the tenth time of the reaction (the time required to produce 10% of the final amount of polymer), shortens the nucleation phase of microtubule assembly. In addition, TOGp favors microtubule formation by increasing the apparent first order rate constant of elongation. Moreover, TOGp increases the total amount of polymer by decreasing the tubulin critical concentration and by inhibiting depolymerization during the steady state of the reaction.     

Phosphorylation of microtubule-associated protein tau by Ca2+/calmodulin-dependent protein kinase II in its tubulin binding sites

The paired helical filaments (PHF) found in Alzheimer's disease (AD) brain are composed mainly of the hyperphosphorylated form of microtubule-associated protein tau (PHF-tau). It is well known that tau is a good in vitro substrate for Ca(2+)/calmodulin-dependent protein kinase II (CaM kinase II). To establish the phosphorylation sites, the longest human tau (hTau40) was bacterially expressed and phosphorylated by CaM kinase II, followed by digestion with lysyl endoprotease. The digests were subjected to liquid chromatography/mass spectrometry. We found that 5 of 22 identified peptides were phosphorylated. From the tandem mass spectrometry, two phosphorylation sites (serines 262 and 356) were identified in the tubulin binding sites. When tau was phosphorylated by CaM kinase II, the binding of tau to taxol-stabilized microtubules was remarkably impaired. As both serines 262 and 356 are reportedly phosphorylated in PHF-tau, CaM kinase II may be involved in hyperphosphorylation of tau in AD brain.     

Contrasting roles of tau and microtubule-associated protein 2 in the vinblastine-induced aggregation of brain tubulin

Two different proteins, tau and microtubule-associated protein 2 (MAP 2), are able to stimulate tubulin polymerization into microtubules in vitro, but it is not certain if both proteins act by the same mechanism. We have examined the effects of tau and MAP 2 on the vinblastine-induced polymerization of tubulin into spiral filaments. In the presence of tau, vinblastine induced extensive aggregation of tubulin as shown by a large increase in turbidity. The increase in turbidity was accompanied by the formation of large numbers of spirals composed of a filament 40-60 A in diameter. The rate and extent of this aggregation into spirals were dependent on the concentrations of tubulin, tau, and vinblastine. Unlike normal microtubule assembly, this type of aggregation was not inhibited by colchicine or podophyllotoxin. In contrast, MAP 2, even at high concentrations, was less effective than tau at promoting the vinblastine-induced increase in turbidity of tubulin. In fact, MAP 2 strongly inhibited the effect of tau. These results indicate that tau and MAP 2 interact differently with the tubulin molecule in the presence of vinblastine and suggest that the two proteins may play different roles in regulating or promoting microtubule assembly. Vinblastine may thus be a useful probe in analyzing the modes of interactions of tau and MAP 2 with tubulin.     

A discrete repeated sequence defines a tubulin binding domain on microtubule-associated protein tau

The protein domain responsible for the interaction of tau with tubulin has been identified. Biophysical studies indicated that the synthetic peptide Val187-Gly204 (VRSKIG-STENLKHQPGGG) from the repetitive sequence on tau binds to two sites on the tubulin heterodimer and to one site on each of the microtubule-associated protein-interacting C-terminal tubulin peptides alpha(430-441) and beta(422-434). The binding data showed a relatively stronger interaction of Val187-Gly204 with beta(422-434) as compared to that with alpha(430-441). The interaction of this tau peptide with either alpha or beta tubulin peptides appears to be associated with conformational changes in both the tau and the tubulin peptides. The beta tubulin peptide also appears to induce a structural change of tau fragment Val218-Gly235. Interestingly, tau peptides Val187-Gly204 and Val218-Gly235 induced tubulin self-assembly in a cold-reversible fashion, and incorporated into the assembled polymers. The specificity of the interaction of the tau peptide was supported by the competition of tau protein for the interaction with the tubulin polymer. In addition, the tau peptide appears to contain the principal antigenic determinant(s) recognized by anti-idiotypic antibodies that react with the tubulin binding domains on microtubule-associated proteins. The present findings together with the demonstration of the presence of multiple sites for the binding of the alpha(430-441) and beta(422-434) tubulin fragments to tau, and the existence of repetitive sequences on tau, strongly support the hypothesis that the region of tau defined by the repetitive sequences is involved in its interaction with tubulin.     

Dephosphorylation of microtubule-associated protein tau by protein phosphatase 5

Protein phosphatase 5 (PP5) is a 58-kDa novel phosphoseryl/phosphothreonyl protein phosphatase. It is ubiquitously expressed in all mammalian tissues examined, with a high level in the brain, but little is known about its physiological substrates. We found that this phosphatase dephosphorylated recombinant tau phosphorylated with cAMP-dependent protein kinase and glycogen synthase kinase-3beta, as well as abnormally hyperphosphorylated tau isolated from brains of patients with Alzheimer's disease. The specific activity of PP5 toward tau was comparable to those reported with other protein substrates examined to date. The PP5 activity toward tau was stimulated by arachidonic acid by 30- to 45-fold. Immunostaining demonstrated that PP5 was primarily cytoplasmic in PC12 cells and in neurons of postmortem human brain tissue. A small pool of PP5 associated with microtubules. Expression of active PP5 in PC12 cells resulted in reduced phosphorylation of tau, suggesting that PP5 can also dephosphorylate tau in cells. These results suggest that PP5 plays a role in the dephosphorylation of tau and might be involved in the molecular pathogenesis of Alzheimer's disease.     

Phosphorylation of microtubule-associated protein tau by AMPK-related kinases

Microtubule-associated protein tau is abnormally hyperphosphorylated in the intracellular filamentous inclusions seen in neurodegenerative disorders with dementia, such as Alzheimer's disease and other tauopathies. Microtubule-associated protein/microtubule-affinity regulating kinases (MARKs) have previously been identified as kinases which phosphorylate KxGS motifs in the tandem repeats of tau. They are members of the 5'-AMP-activated protein kinase (AMPK)-related kinases in the Ca(2+)/calmodulin-dependent protein kinase group. In this study, we examined the ability of AMPK-related kinases, brain-specific kinases 1 and 2, maternal embryonic leucine-zipper kinase, MARK1, and salt-inducible kinase (SIK), to phosphorylate tau. We found that they phosphorylated S262 and S356 in KxGS motifs in the repeats of tau, thus resulting in immunoreactivity with antibody 12E8. MARK1 and SIK most effectively phosphorylated tau, and their down-regulation resulted in a reduction of 12E8-labelling. BX 795, an inhibitor of MARK1 and SIK, reduced 12E8-immunolabelling of tau in rat cortical neurons. These findings reveal a significant contribution of AMPK-related kinases to the phosphorylation of tau at S262/S356.     

The microtubule-associated protein tau is phosphorylated by Syk

Aberrant phosphorylation of tau protein on serine and threonine residues has been shown to be critical in neurodegenerative disorders called tauopathies. An increasing amount of data suggest that tyrosine phosphorylation of tau might play an equally important role in pathology, with at least three putative tyrosine kinases of tau identified to date. It was recently shown that the tyrosine kinase Syk could efficiently phosphorylate alpha-synuclein, the aggregated protein found in Parkinson's disease and other synucleinopathies. We report herein that Syk is also a tau kinase, phosphorylating tau in vitro and in CHO cells when both proteins are expressed exogenously. In CHO cells, we have also demonstrated by co-immunoprecipitation that Syk binds to tau. Finally, by site-directed mutagenesis substituting the tyrosine residues of tau with phenylalanine, we established that tyrosine 18 was the primary residue in tau phosphorylated by Syk. The identification of Syk as a common tyrosine kinase of both tau and alpha-synuclein may be of potential significance in neurodegenerative disorders and also in neuronal physiology. These results bring another clue to the intriguing overlaps between tauopathies and synucleinopathies and provide new insights into the role of Syk in neuronal physiology.     

Visualizing the microtubule-associated protein tau in the nucleus

Although tau is mainly known as an axonal microtubule-associated protein,many studies indicate that it is not restricted to this subcellular compartment.Assessing tau’s subcellular distribution,however,is not trivial as is evident from transgenic mouse studies.When human tau is over-expressed,it can be immunohistochemically localized to axons and the somatodendritic domain,modeling what is found in neurodegenerative diseases such as Alzheimer’s disease.Yet,in wild-type mice,despite its abundance,tau is difficult to visualize even in the axon.It is even more challenging to detect this protein in the nucleus,where tau has been proposed to protect DNA from damage.To establish a framework for future studies into tau’s nuclear functions,we compared several methods to visualize endogenous nuclear tau in cell lines and mouse brain.While depending on the fixation and permeabilization protocol,we were able to detect nuclear tau in SH-SY5Y human neuroblastoma cells,we failed to do so in N2a murine neuroblastoma cells.As a second method we used subcellular fractionation of mouse tissue and found that in the nucleus tau is mainly present in a hypophosphorylated form.When either full-length or truncated human tau was expressed,both accumulated in the cytoplasm,but were also found in the nuclear fraction.Because subcellular fractionation methods have their limitations,we finally isolated nuclei to probe for nuclear tau and found that the nuclei were free of cytoplasmic contamination.Together our analysis identifies several protocols for detecting tau in the nucleus where it is found in a less phosphorylated form.     

Phosphorylation of human microtubule-associated protein tau by protein kinases of the AGC subfamily

Intraneuronal inclusions made of hyperphosphorylated microtubule-associated protein tau are a defining neuropathological characteristic of Alzheimer's disease, and of several other neurodegenerative disorders. Many phosphorylation sites in tau are S/TP sites that flank the microtubule-binding repeats. Others are KXGS motifs in the repeats. One site upstream of the repeats lies in a consensus sequence for AGC kinases. This site (S214) is believed to play an important role in the events leading from normal, soluble to filamentous, insoluble tau. Here, we show that all AGC kinases tested phosphorylated S214. RSK1 and p70 S6 kinase also phosphorylated the neighbouring T212, a TP site that conforms weakly to the AGC kinase consensus sequence. MSK1 phosphorylated S214, as well as S262, a KXGS site in the first repeat, and S305 in the second repeat.     

Common antigenic determinants of the tubulin binding domains of the microtubule-associated proteins MAP-2 and tau.

The structural-functional aspects of the tubulin binding domain on the microtubule-associated protein MAP-2, and its relationship with the tubulin binding domain on tau, were studied using anti-idiotypic antibodies that react specifically with the epitope(s) on MAPs involved in their interaction with tubulin in addition to other tau and MAP-2 specific antibodies. Previous studies showed that MAP-2 and tau share common binding sites on tubulin defined by the peptide sequences alpha (430-441) and beta (422-434) of tubulin subunits. Furthermore, binding experiments revealed the existence of multiple sites for the interaction of the alpha- and beta-tubulin peptides with MAP-2 and tau. Most recent studies showed that the synthetic tau peptide Val187-Gly204 (VRSKIGSTENLKHQPGGG) from the repetitive sequence on tau defines a tubulin binding site on tau. Our present immunological studies using anti-idiotypic antibodies which interact with the synthetic tau peptide and antibodies against the Val187-Gly204 tau peptide indicate that MAP-2 and tau share common antigenic determinants at the level of their respective tubulin binding domains. These antigenic determinants appear to be present in the 35 kDa tubulin binding fragment of MAP-2 and in 18-20 kDa chymotryptic fragments containing the tubulin binding site(s) on MAP-2. These findings, along with structural information on these proteins, provide strong evidence in favor of the hypothesis that tubulin binding domains on MAP-2 and tau share similar structural features.     

Comparison of the phosphorylation of microtubule-associated protein tau by non-proline dependent protein kinases

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 that 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 mol³²P 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. Such phosphorylation may serve to modulate the activaties of other tau kinases such as the PDPKs.Abbreviations PHF paired helical filaments - A-kinase cyclic AMP-dependent protein kinase - CaM kinase II calcium/calmodulin-dependent protein kinase II - C-kinase calcium-phospholipid-dependent protein kinase - CK-1 casein kinase-1 - CK-2 casein kinase-2 - Gr kinase calcium/calmodulin-dependent protein kinase from rat cerebellum - GSK-3 glycogen synthase kinase-3 - MAP kinase mitogen-activated protein kinase - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

Interaction between microtubule-associated protein tau and spectrin

Tau factor, one of the microtubule-associated proteins (MAPs), is shown here to bind to spectrin. Evidence for an interaction between these two proteins is provided by spectrin affinity chromatography of brain MAPs, gel overlay of electrophoresed MAPs with ¹²⁵I-labelled spectrin, incorporation of tau factor in human erythrocyte ghosts, and demonstration that tau inhibits the F-actin cross-linking activity of tetrameric spectrin.The wide distribution of both tau and spectrin-like proteins in eukaryotic cells in in favor of the possible biological significance of this interaction. The results suggest that tau could be one of the proteins involved in the concerted regulation of microtubule and actin networks in the membrane vicinity.     

Tau-related protein present in paired helical filaments has a decreased tubulin binding capacity as compared with microtubule-associated protein tau

We have isolated, after exhaustive detergent treatments, a 33 kDa tau-related protein isolated from paired helical filaments from Alzheimer's disease patient brains. The N-terminal sequence of the 33 kDa protein begins at residue 71 of the sequence described for human fetal tau protein. This truncated form of tau is not the consequence of the translation of a tau RNA lacking a region at its 5' end, as measured by primer extension analyses, suggesting that the 33 kDa protein must be generated by proteolysis of previously synthesized tau. This tau-related protein has only one blocked cysteine residue and also has a decreased tubulin binding capacity as compared with that of tau protein.     

Estramustine-phosphate binds to a tubulin binding domain on microtubule-associated proteins MAP-2 and tau.

Estramustine-phosphate (EMP), a phosphorylated conjugate of estradiol and nor-nitrogen mustard binds to microtubule-associated proteins MAP-2 and tau. It was shown that this estramustine derivative inhibits the binding of the C-terminal tubulin peptide beta-(422-434) to both MAP-2 and tau. This tubulin segment constitutes a main binding domain for these microtubule-associated proteins. Interestingly, estramustine-phosphate interacted with the synthetic tau peptides V187-G204 and V218-G235, representing two major repeats within the conserved microtubule-binding domain on tau and also on MAP-2. This observation was corroborated by the inhibitory effects of estramustine-phosphate on the tau peptide-induced tubulin assembly into microtubules. On the other hand, the nonphosphorylated drug estramustine failed to block the MAP peptide-induced assembly, indicating that the negatively charged phosphate moiety of estramustine-phosphate is of importance for its inhibitory effect. These findings suggest that the molecular sites for the action of estramustine-phosphate are located within the microtubule binding domains on tau and MAP-2.     

Histone deacetylase 6 interacts with the microtubule-associated protein tau

Histone deacetylase 6 (HDAC6), a unique cytoplasmic deacetylase, likely plays a role in neurodegeneration by coordinating cell responses to abnormal protein aggregation. Here, we provide in vitro and in vivo evidence that HDAC6 interacts with tau, a microtubule-associated protein that forms neurofibrillary tangles in Alzheimer's disease. This interaction is mediated by the microtubule-binding domain on tau and the Ser/Glu tetradecapeptide domain on HDAC6. Treatment with tubacin, a selective inhibitor of tubulin deacetylation activity of HDAC6, did not disrupt HDAC6–tau interaction. Nonetheless tubacin treatment attenuated site-specific tau phosphorylation, as did shRNA-mediated knockdown of HDAC6. Proteasome inhibition potentiated HDAC6–tau interactions and facilitated the concentration and co-localization of HDAC6 and tau in a perinuclear aggresome-like compartment, independent of HDAC6 tubulin deacetylase activity. Furthermore, we observed that in Alzheimer's disease brains the protein level of HDAC6 was significantly increased. These findings establish HDAC6 as a tau-interacting protein and as a potential modulator of tau phosphorylation and accumulation.     

Erythrocyte microtubule assembly in vitro. Determination of the effects of erythrocyte tau, tubulin isoforms, and tubulin oligomers on erythrocyte tubulin assembly, and comparison with brain microtubule assembly

Two tubulin variants, isolated from chicken brain and erythrocytes and known to have different peptide maps and electrophoretic properties, are demonstrated to exhibit different assembly properties in vitro: 1) erythrocyte tubulin assembles with greater efficiency (lower critical concentration, greater elongation rate) but exhibits a lower nucleation rate than brain tubulin, and 2) erythrocyte tubulin readily forms oligomers whose presence significantly retards the rate of elongation, suggesting that tubulin oligomers may also be important for determining the rate of assembly and the length of microtubules in erythrocytes. Erythrocyte tubulin isolated by cycles of in vitro assembly-disassembly is also demonstrated to contain a 67-kDa tau factor that greatly enhances microtubule nucleation but has little effect on elongation rates or critical concentration. Immunofluorescence microscopy with tau antibody indicates that tau is specifically associated with marginal band microtubules, suggesting that it may be important for determining microtubule function in vivo.