Structural characterization by nuclear magnetic resonance of the impact of phosphorylation in the proline‐rich region of the disordered Tau protein

Phosphorylation of the neuronal Tau protein is implicated in both the regulation of its physiological function of microtubule stabilization and its pathological propensity to aggregate into the fibers that characterize Alzheimer's diseased neurons. However, how specific phosphorylation events influence both aspects of Tau biology remains largely unknown. In this study, we address the structural impact of phosphorylation of the Tau protein by Nuclear Magnetic Resonance (NMR) spectroscopy on a functional fragment of Tau (Tau[Ser208–Ser324] = TauF4). TauF4 was phosphorylated by the proline‐directed CDK2/CycA3 kinase on Thr231 (generating the AT180 epitope), Ser235, and equally on Thr212 and Thr217 in the Proline‐rich region (Tau[Ser208‐Gln244] or PRR). These modifications strongly decrease the capacity of TauF4 to polymerize tubulin into microtubules. While all the NMR parameters are consistent with a globally disordered Tau protein fragment, local clusters of structuration can be defined. The most salient result of our NMR analysis is that phosphorylation in the PRR stabilizes a short α‐helix that runs from pSer235 till the very beginning of the microtubule‐binding region (Tau[Thr245‐Ser324] or MTBR of TauF4). Phosphorylation of Thr231/Ser235 creates a N‐cap with helix stabilizing role while phosphorylation of Thr212/Thr217 does not induce modification of the local transient secondary structure, showing that the stabilizing effect is sequence specific. Using paramagnetic relaxation experiments, we additionally show a transient interaction between the PRR and the MTBR, observed in both TauF4 and phospho‐TauF4. Proteins 2012. © 2011 Wiley Periodicals, Inc.     

The "jaws" of the tau-microtubule interaction

Tau is the major microtubule-associated protein in neuronal axons. It aggregates into "neurofibrillary tangles" during the course of Alzheimer disease. Binding to microtubules and microtubule assembly requires the "repeat domain" in the C-terminal half of Tau, as well as the two regions flanking the repeats. Here we report the NMR characterization of a 198-residue Tau fragment composed of the four tandem repeats and the flanking domains and containing the full microtubule binding and assembly activity of Tau. NMR secondary chemical shifts and dipolar couplings detect the highest propensity for beta-structure within the four-repeat region, whereas the flanking domains are largely random coil, with an increased rigidity in the proline-rich region. Chemical shift perturbation experiments identify two motifs in the upstream flanking domain, (225)KVAVVRT(231) and (243)LQTA(246), and one downstream of the repeats, (370)KIETHKTFREN(380), which strongly contribute to the binding to the acidic outside of microtubules, as well as to the binding of other polyanions such as heparin. This is consistent with the "jaws" model of Tau-microtubule interactions and highlights the importance of the regions flanking the repeats for both microtubule binding and pathological Tau aggregation.     

Taxol-stabilized microtubules promote the formation of filaments from unmodified full-length Tau in vitro

Tau is a neuronal protein that stabilizes the microtubule (MT) network, but it also forms filaments associated with Alzheimer''s disease. Understanding Tau–MT and Tau–Tau interactions would help to establish Tau function in health and disease. For many years, literature reports on Tau–MT binding behavior and affinity have remained surprisingly contradictory (e.g., 10-fold variation in Tau–MT affinity). Tau–Tau interactions have also been investigated, but whether MTs might affect Tau filament formation is unknown. We have addressed these issues through binding assays and microscopy. We assessed Tau–MT interactions via cosedimentation and found that the measured affinity of Tau varies greatly, depending on the experimental design and the protein concentrations used. To investigate this dependence, we used fluorescence microscopy to examine Tau–MT binding. Strikingly, we found that Taxol-stabilized MTs promote Tau filament formation without characterized Tau-filament inducers. We propose that these novel Tau filaments account for the incongruence in Tau–MT affinity measurements. Moreover, electron microscopy reveals that these filaments appear similar to the heparin-induced Alzheimer''s model. These observations suggest that the MT-induced Tau filaments provide a new model for Alzheimer''s studies and that MTs might play a role in the formation of Alzheimer''s-associated neurofibrillary tangles.     

Serum- and glucocorticoid-inducible kinase 1 (SGK1) increases neurite formation through microtubule depolymerization by SGK1 and by SGK1 phosphorylation of tau

Serum- and glucocorticoid-inducible kinase 1 (SGK1) is a member of the Ser/Thr protein kinase family that regulates a variety of cell functions. Recently, SGK1 was shown to increase dendritic growth but the mechanism underlying the increase is unknown. Here we demonstrated that SGK1 increased the neurite formation of cultured hippocampal neurons through microtubule (MT) depolymerization via two distinct mechanisms. First, SGK1 directly depolymerized MTs. In vitro MT depolymerization experiments revealed that SGK1, especially N-truncated SGK1, directly disassembled self-polymerized MTs and taxol-stabilized MTs in a dose-dependent and ATP-independent manner. The transfection of sgk1 to HeLa cells also inhibited MT assembly in vivo. Second, SGK1 indirectly depolymerized MTs through the phosphorylation of tau at Ser214. An in vitro kinase assay revealed that active SGK1 phosphorylated tau Ser214 specifically. In vivo transfection of sgk1 also phosphorylated tau Ser214 in HEK293T cells and hippocampal neurons. Further, sgk1 transfection significantly increased the number of primary neurites and shortened the length of the total process in cultured hippocampal neurons. These effects were antagonized by the cotransfection of the tauS214A mutant plasmid. Dexamethasone, a synthetic glucocorticoid, mimics the effect of sgk1 overexpression. Together, these results suggest that SGK1 enhances neurite formation through MT depolymerization by a direct action of SGK1 and by the SGK1 phosphorylation of tau.     

Parkinsonian neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and alpha-synuclein mutations promote Tau protein phosphorylation at Ser262 and destabilize microtubule cytoskeleton in vitro

In Parkinson disease (PD) brain, a progressive loss of dopaminergic neurons leads to dopamine depletion in the striatum and reduced motor function. Lewy bodies, the characteristic neuropathological lesions found in the brain of PD patients, are composed mainly of α-synuclein protein. Three point mutations in the α-synuclein gene are associated with familial PD. In addition, genome-wide association studies indicate that α-synuclein and Tau protein synergistically increase disease susceptibility in the human population. To determine the mechanism by which α-synuclein and Tau act together, we have used PD-causing neurotoxin MPTP and pathogenic α-synuclein mutants A30P, E46K, and A53T as models. We found that exposure of human neuroblastoma M17 cells to MPTP enhances the intracellular α-synuclein protein level, stimulates Tau protein phosphorylation at Ser(262), and induces apoptosis. In mouse brain, ablation of α-synuclein function significantly suppresses Tau phosphorylation at Ser(262). In vitro, α-synuclein binds to phosphorylated Ser(214) of Tau and stimulates PKA-catalyzed Tau phosphorylation at Ser(262). PD-associated α-synuclein mutations increase α-synuclein binding to Tau and stimulate Tau phosphorylation at Ser(262). In HEK-293 cells, α-synuclein and its all PD-associated mutants destabilize the microtubule cytoskeleton in a similar extent. In contrast, when co-expressed with Tau, these PD-associated mutants destabilize microtubules with significantly higher potency than WT. Our results demonstrate that α-synuclein is an in vivo regulator of Tau protein phosphorylation at Ser(262) and suggest that PD-associated risk factors such as environmental toxins and α-synuclein mutations promote Tau phosphorylation at Ser(262), causing microtubule instability, which leads to loss of dopaminergic neurons in PD brain.     

Identification of a neural cell specific variant of microtubule-associated protein 4

The microtubule-binding domain of MAP4, a ubiquitous microtubule-associated protein, contains a region rich in proline and basic residues (proline-rich region). We searched the bovine adrenal gland for MAP4 isoforms, and identified a novel variant lacking 72 consecutive amino acid residues within the proline-rich region, as compared with the full-length MAP4. The amino acid sequence of the missing region was highly conserved (about 85% identity/similarity) among the corresponding regions of bovine, human, mouse, and rat MAP4, which suggested the functional significance of this region. A comparison of the genomic sequence with the cDNA sequence revealed that the missing region is encoded by a single exon. A MAP4 variant cDNA homologous to the bovine form was also detected in rat cells, suggesting that the new variant can be generated by alternative splicing, not only in bovine but also in other mammalian species. The mRNA expression of the novel isoform was restricted to the brain and the adrenal medulla, suggesting that this isoform is specific to a certain cell type. Using a bacterially expressed fragment corresponding to the microtubule-binding domain of the novel isoform, we analyzed its in vitro characteristics. The fragment induced microtubule assembly and bound to preformed microtubules, but the activities were slightly lower than those of the conventional MAP4 fragment, which carries the full-length proline-rich region. The microtubules assembled in the presence of the fragment failed to be bundled. Instead, a constant spacing between neighboring microtubules was observed.     

First tau repeat domain binding to growing and taxol-stabilized microtubules,and serine 262 residue phosphorylation

Tau phosphorylation plays a crucial role in microtubule stabilization and in Alzheimer's disease. To characterize the molecular mechanisms of tau binding on microtubules, we synthesized the peptide R1 (QTAPVPMPDLKNVKSKIGSTENLKHQPGGGKVQI), reproducing the first tau microtubule binding motif. We thermodynamically characterized the molecular mechanism of tubulin assembly with R1 in vitro, and measured, for the first time, the binding parameters of R1 on both growing and taxol-stabilized microtubules. In addition, we obtained similar binding parameters with R1 phosphorylated on Ser262. These data suggest that the consequences of Ser262 phosphorylation on tau binding to microtubules and on tubulin assembly are due to large intramolecular rearrangements of the tau protein.     

Nonsaturable binding indicates clustering of tau on the microtubule surface in a paired helical filament-like conformation

Tau protein modulates microtubule dynamics and forms insoluble aggregates in Alzheimer's disease. Because there is a discrepancy between reported affinities of Tau to microtubules, we determined the interaction over a wide concentration range using a sensitive enzyme-linked immunosorbent assay. We found that the interaction is biphasic and not monophasic as assumed earlier. The first binding phase is typical for identical and noninteracting binding sites, with dissociation constants around 0.1 micrometer and stoichiometries around 0.2 Tau/tubulin dimer. Surprisingly, the second phase is nonsaturable and shows a nearly linear increase in bound Tau versus free Tau for free Tau concentrations higher than 2 micrometer. The slope is proportional to the microtubule concentration. From this we define an overloading parameter with values around 50 micrometer. The influence of Tau isoform, phosphorylation, and dimerization on both phases was investigated. Remarkably the overloading of Tau on microtubules leads to a thioflavin S fluorescence increase reminiscent of that seen with Tau aggregated into Alzheimer paired helical filaments. Because polyanions stimulate Tau aggregation and because the C-terminal domain of tubulin is polyanionic, we suggest that an early conformational change in Tau leading to paired helical filament aggregation occurs right on the microtubule surface.     

Tau protein binds to microtubules through a flexible array of distributed weak sites

Tau protein plays a role in the extension and maintenance of neuronal processes through a direct association with microtubules. To characterize the nature of this association, we have synthesized a collection of tau protein fragments and studied their binding properties. The relatively weak affinity of tau protein for microtubules (approximately 10(-7) M) is concentrated in a large region containing three or four 18 amino acid repeated binding elements. These are separated by apparently flexible but less conserved linker sequences of 13-14 amino acids that do not bind. Within the repeats, the binding energy for microtubules is delocalized and derives from a series of weak interactions contributed by small groups of amino acids. These unusual characteristics suggest tau protein can assume multiple conformations and can pivot and perhaps migrate on the surface of the microtubule. The flexible structure of the tau protein binding interaction may allow it to be easily displaced from the microtubule lattice and may have important consequences for its function.     

Tau-isoform dependent enhancement of taxol mobility through microtubules

Tau, a family of microtubule-associated proteins (MAPs), stabilizes microtubules (MTs) and regulates their dynamics. Tau isoforms regulate MT dynamic instability differently: 3-repeat tau is less effective than 4-repeat tau at suppressing the disassembly of MTs. Here, we report another tau-isoform-dependent phenomenon, revealed by fluorescence recovery after photobleaching measurements on a BODIPY-conjugated taxol bound to MTs. Saturating levels of recombinant full-length 3-repeat and 4-repeat tau both cause taxol mobility to be remarkably sensitive to taxol concentration. However, 3-repeat tau induces 2.5-fold faster recovery (∼450 s) at low taxol concentrations (∼100 nM) than 4-repeat tau (∼1000 s), indicating that 3-repeat tau decreases the probability of taxol rebinding to its site in the MT lumen. Finding no tau-induced change in the MT-binding affinity of taxol, we conclude that 3-repeat tau either competes for the taxol binding site with an affinity of ∼1 μM or alters the MT structure so as to facilitate the passage of taxol through pores in the MT wall.     

Structural impact of heparin binding to full-length Tau as studied by NMR spectroscopy

The neuronal Tau protein is involved in stabilizing microtubules but is also the major component of the paired helical filaments (PHFs), the intracellular aggregates that characterize Alzheimer's disease (AD) in neurons. In vitro, Tau can be induced to form AD-like aggregates by adding polyanions such as heparin. While previous studies have identified the microtubule binding repeats (MTBRs) as the major player in Tau aggregation, the fact that the full-length protein does not aggregate by itself indicates the presence of inhibitory factors. Charge and conformational changes are of uttermost importance near the second (R2) and third (R3) MTBR that are thought to be involved directly in the nucleation of the aggregation. Recently, the positively charged regions flanking the MTBR were proposed to inhibit PHF assembly, where hyperphosphorylation neutralizes these basic inhibitory domains, enabling Tau-Tau interactions. Here we present results of an NMR study on the interaction between intact full-length Tau and small heparin fragments of well-defined size, under conditions where no aggregation occurs. Our findings reveal (i) micromolar affinity of heparin to residues in R2 and R3, (ii) two zones of strong interaction within the positively charged inhibitory regions flanking the MTBR, and (iii) another interaction site upstream of the two inserts encoded by exons 2 and 3. Three-dimensional heteronuclear NMR experiments demonstrate that the interaction with heparin induces beta-strand structure in several regions of Tau that might act as nucleation sites for its aggregation but indicate as well alpha-helical structure in regions outside the core of PHF. In the PHF, the residues outside of the core maintain sufficient mobility for NMR detection and recover their unbound chemical shift values after an overnight incubation at 37 degrees C with heparin. Heparin thus becomes integrated into the rigid core region of the PHF, probably providing the charge compensation for the lysine-rich stretches that form upon the in-register, parallel stacking of the repeat regions.     

Tau Protein Diffuses along the Microtubule Lattice

Current models for the intracellular transport of Tau protein suggest motor protein-dependent co-transport with microtubule fragments and diffusion of Tau in the cytoplasm, whereas Tau is believed to be stationary while bound to microtubules and in equilibrium with free diffusion in the cytosol. Observations that members of the microtubule-dependent kinesin family show Brownian motion along microtubules led us to hypothesize that diffusion along microtubules could also be relevant in the case of Tau. We used single-molecule total internal reflection fluorescence microscopy to probe for diffusion of individual fluorescently labeled Tau molecules along microtubules. This allowed us to avoid the problem that microtubule-dependent diffusion could be masked by excess of labeled Tau in solution that might occur in in vivo overexpression experiments. We found that approximately half of the individually detected Tau molecules moved bidirectionally along microtubules over distances up to several micrometers. Diffusion parameters such as diffusion coefficient, interaction time, and scanned microtubule length did not change with Tau concentration. Tau binding and diffusion along the microtubule lattice, however, were sensitive to ionic strength and pH and drastically reduced upon enzymatic removal of the negatively charged C termini of tubulin. We propose one-dimensional Tau diffusion guided by the microtubule lattice as one possible additional mechanism for Tau distribution. By such one-dimensional microtubule lattice diffusion, Tau could be guided to both microtubule ends, i.e. the sites where Tau is needed during microtubule polymerization, independently of directed motor-dependent transport. This could be important in conditions where active transport along microtubules might be compromised.     

Identification of microtubule binding sites in the Ncd tail domain

Nonclaret disjunctional (Ncd) is a minus end-directed, C-terminal motor protein that is required for spindle assembly and maintenance during meiosis and early mitosis in Drosophila oocytes and early embryos. Ncd has an ATP-independent MT binding site in the N-terminal tail domain, and an ATP-dependent MT binding site in the C-terminal motor domain. The ability of Ncd to cross-link MTs through the action of these binding sites may be important for Ncd function in vivo. To identify the region(s) responsible for ATP-independent MT interactions of Ncd, 12 cDNAs coding various regions of Ncd tail domain were expressed in E. coli as C-terminal fusions to thioredoxin (Trx). Ncd tail fusion proteins (TrxNT) were purified by ion exchange (S-Sepharose) and/or Talon metal affinity chromatography. Purified TrxNT and NT proteins were analyzed in microtubule (MT) cosedimentation and bundling assays to identify which tail proteins were able to bind and bundle MTs. Based on the results of these experiments, all TrxNT and NT proteins that showed MT binding activity also bundled MTs, and there are two ATP-independent MT interaction sites in the tail region: one within amino acids 83-100 that exhibits conformation-independent, high-affinity MT binding activity; and another within amino acids 115-187 that exhibits conformation-dependent, lower affinity MT binding activity. It is possible that both of these MT interacting sites combine in the native protein to form a single MT binding site that allows the Ncd tail to bind cargo MTs in vivo.     

Microtubule-associated Protein-like Binding of the Kinesin-1 Tail to Microtubules

The kinesin-1 molecular motor contains an ATP-dependent microtubule-binding site in its N-terminal head domain and an ATP-independent microtubule-binding site in its C-terminal tail domain. Here we demonstrate that a kinesin-1 tail fragment associates with microtubules with submicromolar affinity. Binding is largely electrostatic in nature, and is facilitated by a region of basic amino acids in the tail and the acidic E-hook at the C terminus of tubulin. The tail binds to a site on tubulin that is independent of the head domain-binding site but overlaps with the binding site of the microtubule-associated protein Tau. Surprisingly, the kinesin tail domain stimulates microtubule assembly and stability in a manner similar to Tau. The biological function of this strong kinesin tail-microtubule interaction remains to be seen, but it is likely to play an important role in kinesin regulation due to the close proximity of the microtubule-binding region to the conserved regulatory and cargo-binding domains of the tail.     

Functional interactions between the proline-rich and repeat regions of tau enhance microtubule binding and assembly.

Tau is a neuronal microtubule-associated protein that promotes microtubule assembly, stability, and bundling in axons. Two distinct regions of tau are important for the tau-microtubule interaction, a relatively well-characterized "repeat region" in the carboxyl terminus (containing either three or four imperfect 18-amino acid repeats separated by 13- or 14-amino acid long inter-repeats) and a more centrally located, relatively poorly characterized proline-rich region. By using amino-terminal truncation analyses of tau, we have localized the microtubule binding activity of the proline-rich region to Lys215-Asn246 and identified a small sequence within this region, 215KKVAVVR221, that exerts a strong influence on microtubule binding and assembly in both three- and four-repeat tau isoforms. Site-directed mutagenesis experiments indicate that these capabilities are derived largely from Lys215/Lys216 and Arg221. In marked contrast to synthetic peptides corresponding to the repeat region, peptides corresponding to Lys215-Asn246 and Lys215-Thr222 alone possess little or no ability to promote microtubule assembly, and the peptide Lys215-Thr222 does not effectively suppress in vitro microtubule dynamics. However, combining the proline-rich region sequences (Lys215-Asn246) with their adjacent repeat region sequences within a single peptide (Lys215-Lys272) enhances microtubule assembly by 10-fold, suggesting intramolecular interactions between the proline-rich and repeat regions. Structural complexity in this region of tau also is suggested by sequential amino-terminal deletions through the proline-rich and repeat regions, which reveal an unusual pattern of loss and gain of function. Thus, these data lead to a model in which efficient microtubule binding and assembly activities by tau require intramolecular interactions between its repeat and proline-rich regions. This model, invoking structural complexity for the microtubule-bound conformation of tau, is fundamentally different from previous models of tau structure and function, which viewed tau as a simple linear array of independently acting tubulin-binding sites.     

Mammalian septins regulate microtubule stability through interaction with the microtubule-binding protein MAP4

Mammalian septins constitute a family of at least 12 GTP-binding proteins that can form hetero-oligomers and that are sometimes found in association with actin or microtubule filaments. However, their functions are not understood. Using RNA interference, we found that suppression of septin expression in HeLa cells caused a pronounced increase in microtubule stability. Mass spectroscopic analysis of proteins coprecipitating with Sept6 identified the microtubule-associated protein MAP4 as a septin binding partner. A small, proline-rich region in the C-terminal half of MAP4 bound directly to a Sept 2:6:7 heterotrimer, and to the Sept2 monomer. The trimer blocked the ability of this MAP4 fragment to bind and bundle microtubules in vitro. In intact cells, MAP4 was required for the stabilization of microtubules induced by septin depletion. Moreover, septin depletion increased the number of cells with abnormal nuclei, and this effect was blocked by gene silencing of MAP4. These data identify a novel molecular function for septins in mammalian cells: the modulation of microtubule dynamics through interaction with MAP4.     

Surface-decoration of microtubules by human tau

Tau is a neuronal, microtubule-associated protein that stabilizes microtubules and promotes neurite outgrowth. Tau is largely unfolded in solution and presumably forms mostly random coil. Because of its hydrophilic nature and flexible structure, tau complexed to microtubules is largely invisible by standard electron microscopy methods. We applied a combination of high-resolution metal-shadowing and cryo-electron microscopy to study the interactions between tau and microtubules. We used recombinant tau variants with different domain compositions, (1) full length tau, (2) the repeat domain that mediates microtubule binding (K19), and (3) two GFP-tau fusion proteins that contain a globular marker (GFP) attached to full-length tau at either end. All of these constructs bind exclusively to the outside of microtubules. Most of the tau-related mass appears randomly distributed, creating a "halo" of low-density mass spread across the microtubule surface. Only a small fraction of tau creates a periodic signal at an 8 nm interval, centered on alpha-tubulin subunits. Our data suggest that tau retains most of its disordered structure even when bound to the microtubule surface. Hence, it binds along, as well as across protofilaments. Nevertheless, even minute concentrations of tau have a strong stabilizing effect and effectively scavenge unpolymerized tubulin.     

Drosophila Nod protein binds preferentially to the plus ends of microtubules and promotes microtubule polymerization in vitro

Nod, a nonmotile kinesin-like protein, plays a critical role in segregating achiasmate chromosomes during female meiosis. In addition to localizing to oocyte chromosomes, we show that functional full-length Nod-GFP (Nod(FL)-GFP) localizes to the posterior pole of the oocyte at stages 9-10A, as does kinesin heavy chain (KHC), a plus end-directed motor. This posterior localization is abolished in grk mutants that no longer maintain the microtubule (MT) gradient in the oocyte. To test the hypothesis that Nod binds to the plus ends of MTs, we expressed and purified both full-length Nod (Nod(FL)) and a truncated form of Nod containing only the motor-like domain (Nod318) from Escherichia coli and assessed their interactions with MTs in vitro. Both Nod(FL) and Nod318 demonstrate preferential binding to the ends of the MTs, displaying a strong preference for binding to the plus ends. When Nod318-GFP:MT collision complexes were trapped by glutaraldehyde fixation, the preference for binding to plus ends versus minus ends was 17:1. Nod(FL) and Nod318 also promote MT polymerization in vitro in a time-dependent manner. The observation that Nod is preferentially localized to the plus ends of MTs and stimulates MT polymerization suggests a mechanism for its function.     

Adenomatous polyposis coli (APC) protein moves along microtubules and concentrates at their growing ends in epithelial cells

Adenomatous polyposis coli (APC) tumor suppressor protein has been shown to be localized near the distal ends of microtubules (MTs) at the edges of migrating cells. We expressed green fluorescent protein (GFP)-fusion proteins with full-length and deletion mutants of Xenopus APC in Xenopus epithelial cells, and observed their dynamic behavior in live cells. During cell spreading and wound healing, GFP-tagged full-length APC was concentrated as granules at the tip regions of cellular extensions. At higher magnification, APC appeared to move along MTs and concentrate as granules at the growing plus ends. When MTs began to shorten, the APC granules dropped off from the MT ends. Immunoelectron microscopy revealed that fuzzy structures surrounding MTs were the ultrastructural counterparts for these GFP signals. The COOH-terminal region of APC was targeted to the growing MT ends without forming granular aggregates, and abruptly disappeared when MTs began to shorten. The APC lacking the COOH-terminal region formed granular aggregates that moved along MTs toward their plus ends in an ATP-dependent manner. These findings indicated that APC is a unique MT-associated protein that moves along selected MTs and concentrates at their growing plus ends through their multiple functional domains.