[Show abstract][Hide abstract] ABSTRACT: Microtubule-dependent transport of vesicles and organelles appears saltatory because particles switch between periods of rest, random Brownian motion, and active transport. The transport can be regulated through motor proteins, cargo adaptors, or microtubule tracks. We report here a mechanism whereby microtubule associated proteins (MAPs) represent obstacles to motors which can be regulated by microtubule affinity regulating kinase (MARK)/Par-1, a family of kinases that is known for its involvement in establishing cell polarity and in phosphorylating tau protein during Alzheimer neurodegeneration. Expression of MARK causes the phosphorylation of MAPs at their KXGS motifs, thereby detaching MAPs from the microtubules and thus facilitating the transport of particles. This occurs without impairing the intrinsic activity of motors because the velocity during active movement remains unchanged. In primary retinal ganglion cells, transfection with tau leads to the inhibition of axonal transport of mitochondria, APP vesicles, and other cell components which leads to starvation of axons and vulnerability against stress. This transport inhibition can be rescued by phosphorylating tau with MARK.
Preview · Article · Nov 2004 · The Journal of Cell Biology
[Show abstract][Hide abstract] ABSTRACT: The MARK protein kinases were originally identified by their ability to phosphorylate a serine motif in the microtubule-binding
domain of tau that is critical for microtubule binding. Here, we report the cloning and expression of a novel human paralog,
MARK4, which shares 75% overall homology with MARK1-3 and is predominantly expressed in brain. Homology is most pronounced
in the catalytic domain (90%), and MARK4 readily phosphorylates tau and the related microtubule-associated protein 2 (MAP2)
and MAP4. In contrast to the three paralogs that all exhibit uniform cytoplasmic localization, MARK4 colocalizes with the
centrosome and with microtubules in cultured cells. Overexpression of MARK4 causes thinning out of the microtubule network,
concomitant with a reorganization of microtubules into bundles. In line with these findings, we show that a tandem affinity-purified
MARK4 protein complex contains α-, β-, and γ-tubulin. In differentiated neuroblastoma cells, MARK4 is localized prominently
at the tips of neurite-like processes. We suggest that although the four MARK/PAR-1 kinases might play multiple cellular roles
in concert with different targets, MARK4 is likely to be directly involved in microtubule organization in neuronal cells and
may contribute to the pathological phosphorylation of tau in Alzheimer's disease.
Preview · Article · Mar 2004 · Journal of Biological Chemistry
[Show abstract][Hide abstract] ABSTRACT: The interaction of the anti-cancer drug podophyllotoxin with a high-molecular-weight assembly of tubulin has been employed to produce three-dimensional crystals from avian erythrocyte tubulin as well as from pig brain tubulin. Avian erythrocyte tubulin crystals belong to the space group C2 with unit cell dimensions a = 740 A, b = 330 A, c = 460 A, beta = 128 degrees. The basis of these crystals is ring oligomers with a molecular mass of approximately 6 x 10(6) Da. So far, the crystals diffract to 8-A resolution and a first complete data set to 12-A resolution has been collected under cryogenic conditions. The crystals grew from conventionally purified tubulin consisting of multiple isoforms and different posttranslational modifications. Thus, the use of highly homogeneous tubulin preparations should improve the diffraction quality of these crystals.
No preview · Article · Jan 2000 · Journal of Structural Biology
[Show abstract][Hide abstract] ABSTRACT: We have performed a real time analysis of fluorescence-tagged vesicle and mitochondria movement in living CHO cells transfected with microtubule-associated protein tau or its microtubule-binding domain. tau does not alter the speed of moving vesicles, but it affects the frequencies of attachment and detachment to the microtubule tracks. Thus, tau decreases the run lengths both for plus-end and minus-end directed motion to an equal extent. Reversals from minus-end to plus-end directed movement of single vesicles are strongly reduced by tau, but reversals in the opposite direction (plus to minus) are not. Analogous effects are observed with the transport of mitochondria and even with that of vimentin intermediate filaments. The net effect is a directional bias in the minus-end direction of microtubules which leads to the retraction of mitochondria or vimentin IFs towards the cell center. The data suggest that tau can control intracellular trafficking by affecting the attachment and detachment cycle of the motors, in particular by reducing the attachment of kinesin to microtubules, whereas the movement itself is unaffected.
Preview · Article · Aug 1999 · Journal of Cell Science
[Show abstract][Hide abstract] ABSTRACT: The neuronal microtubule-associated protein tau plays an important role in establishing cell polarity by stabilizing axonal microtubules that serve as tracks for motor-protein-driven transport processes. To investigate the role of tau in intracellular transport, we studied the effects of tau expression in stably transfected CHO cells and differentiated neuroblastoma N2a cells. Tau causes a change in cell shape, retards cell growth, and dramatically alters the distribution of various organelles, known to be transported via microtubule-dependent motor proteins. Mitochondria fail to be transported to peripheral cell compartments and cluster in the vicinity of the microtubule-organizing center. The endoplasmic reticulum becomes less dense and no longer extends to the cell periphery. In differentiated N2a cells, the overexpression of tau leads to the disappearance of mitochondria from the neurites. These effects are caused by tau's binding to microtubules and slowing down intracellular transport by preferential impairment of plus-end-directed transport mediated by kinesin-like motor proteins. Since in Alzheimer's disease tau protein is elevated and mislocalized, these observations point to a possible cause for the gradual degeneration of neurons.
Full-text · Article · Dec 1998 · The Journal of Cell Biology
[Show abstract][Hide abstract] ABSTRACT: In Alzheimer's disease the neuronal microtubule-associated protein tau becomes highly phosphorylated, loses its binding properties, and aggregates into paired helical filaments. There is increasing evidence that the events leading to this hyperphosphorylation are related to mitotic mechanisms. Hence, we have analyzed the physiological phosphorylation of endogenous tau protein in metabolically labeled human neuroblastoma cells and in Chinese hamster ovary cells stably transfected with tau. In nonsynchronized cultures the phosphorylation pattern was remarkably similar in both cell lines, suggesting a similar balance of kinases and phosphatases with respect to tau. Using phosphopeptide mapping and sequencing we identified 17 phosphorylation sites comprising 80-90% of the total phosphate incorporated. Most of these are in SP or TP motifs, except S214 and S262. Since phosphorylation of microtubule-associated proteins increases during mitosis, concomitant with increased microtubule dynamics, we analyzed cells mitotically arrested with nocodazole. This revealed that S214 is a prominent phosphorylation site in metaphase, but not in interphase. Phosphorylation of this residue strongly decreases the tau-microtubule interaction in vitro, suppresses microtubule assembly, and may be a key factor in the observed detachment of tau from microtubules during mitosis. Since S214 is also phosphorylated in Alzheimer's disease tau, our results support the view that reactivation of the cell cycle machinery is involved in tau hyperphosphorylation.
Full-text · Article · Jul 1998 · Molecular Biology of the Cell
[Show abstract][Hide abstract] ABSTRACT: The phosphorylation of microtubule-associated proteins (MAPs) is thought to be a key factor in the regulation of microtubule stability. We have shown recently that a novel protein kinase, termed p110 microtubule-affinity regulating kinase ("MARK"), phosphorylates microtubule-associated protein tau at the KXGS motifs in the region of internal repeats and causes the detachment of tau from microtubules (Drewes, G., Trinczek, B., Illenberger, S., Biernat, J., Schmitt-Ulms, G., Meyer, H.E., Mandelkow, E.-M., and Mandelkow, E. (1995) J. Biol. Chem. 270, 7679-7688). Here we show that p110mark phosphorylates analogous KXGS sites in the microtubule binding domains of the neuronal MAP2 and the ubiquitous MAP4. Phosphorylation in vitro leads to the dissociation of MAP2 and MAP4 from microtubules and to a pronounced increase in dynamic instability. Thus, the phosphorylation of the repeated motifs in the microtubule binding domains of MAPs by p110mark might provide a mechanism for the regulation of microtubule dynamics in cells.
No preview · Article · Jun 1996 · Journal of Biological Chemistry
[Show abstract][Hide abstract] ABSTRACT: This paper summarizes recent structural and functional studies on tau protein, its interactions with microtubules, its self-assembly into paired helical filaments (PHF)-like fibers, and its modification by phosphorylation. The structure of tau in solution resembles that of a random coil. Both tau and Alzheimer PHFs have very little secondary structure, making it improbable that the assembly of tau into PHFs is based on interacting beta sheets. Tau's binding to microtubules can be described by a "jaws" effect. The domain containing the repeats binds very weakly, while the flanking regions (jaws) bind strongly, even without the repeats. However, only the combination of flanking regions and repeats makes binding productive in terms of microtubule nucleation and assembly. Although the majority of Alzheimer-like phosphorylation sites are outside the repeats they have only a weak influence on binding, whereas the phosphorylation at Ser262 inside the repeats inhibits binding and makes microtubules dynamically unstable. This site can be phosphorylated by kinases present in brain tissue, and it is uniquely phosphorylated in Alzheimer brain.
No preview · Article · Feb 1996 · Annals of the New York Academy of Sciences
[Show abstract][Hide abstract] ABSTRACT: The dynamic instability of microtubules is thought to be regulated by MAPs and phosphorylation. Here we describe the effect of the neuronal microtubule-associated protein tau by observing the dynamics of single microtubules by video microscopy. We used recombinant tau isoforms and tau mutants, and we phosphorylated tau by the neuronal kinases MARK (affecting the KXGS motifs within tau's repeat domain) and cdk5 (phosphorylating Ser-Pro motifs in the regions flanking the repeats). The variants of tau can be broadly classified into three categories, depending on their potency to affect microtubule dynamics. "Strong" tau variants have four repeats and both flanking regions. "Medium" variants have one to three repeats and both flanking regions. "Weak" variants lack one or both of the flanking regions, or have no repeats; with such constructs, microtubule dynamics is not significantly different from that of pure tubulin. N- or C-terminal tails of tau have no influence on dynamic instability. The two ends of microtubules (plus and minus) showed different activities but analogous behavior. These results are consistent with the "jaws" model of tau where the flanking regions are considered as targeting domains whereas the addition of repeats makes them catalytically active in terms of microtubule stabilization. The dominant changes in the parameters of dynamic instability induced by tau are those in the dissociation rate and in the catastrophe rate (up to 30-fold). Other rates change only moderately or not at all (association rate increased up to twofold, rates of rescue or rapid shrinkage decreased up to approximately twofold). The order of repeats has little influence on microtubule dynamics (i.e., repeats can be re-arranged or interchanged), arguing in favor of the "distributed weak binding" model proposed by Butner and Kirschner (1991); however, we confirmed the presence of a "hotspot" of binding potential involving Lys274 and Lys281 observed by Goode and Feinstein, 1994. Phosphorylation of Ser-Pro motifs by cdk5 (mainly Ser 202, 235, and 404) in the flanking regions had a moderate effect on microtubule dynamics while phosphorylation at the "Alzheimer"-site Ser262 MARK eliminated tau's interactions with microtubules. In both cases the predominant effects of phosphorylation are on the rates of tubulin dissociation and catastrophe whereas the effects on the rates of association or rescue are comparatively small.
Full-text · Article · Jan 1996 · Molecular Biology of the Cell
[Show abstract][Hide abstract] ABSTRACT: We consider the interactions of tau protein with microtubules from two points of view, phosphorylation and domain structure. Tau can be phosphorylated at many sites and by several kinases, notably by proline-directed kinases (MAPK, GSK-3, cdk5) which generate Alzheimer-like antibody epitopes. Other kinases phosphorylate Ser 262, a site that has a particularly pronounced influence on the affinity of tau for microtubules. All of these sites can be cleared by phosphatases PP-2a and calcineurin. The site Ser262 lies within the repeat domain of tau. However, when probing the domains of tau for their effects on microtubule binding, nucleation, assembly, or bundling, the repeat domain has only a weak influence. Whereas the repeat domain of tau binds to microtubules with low affinity, repeat-less tau binds strongly yet unproductively in terms of microtubule assembly. Productive binding of tau to microtubules depends on the combination of (some) repeats with the flanking regions, as if the flanking regions acted as "jaws" for the proper positioning of tau on the microtubule surface.
Full-text · Article · May 1995 · Neurobiology of Aging
[Show abstract][Hide abstract] ABSTRACT: 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.
No preview · Article · Apr 1995 · Journal of Biological Chemistry
[Show abstract][Hide abstract] ABSTRACT: 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 microtubule-associated proteins/microtubule arrays and their
pathological degeneration in Alzheimer's disease.
No preview · Article · Mar 1995 · Journal of Biological Chemistry
[Show abstract][Hide abstract] ABSTRACT: The role of the neuronal microtubule-associated protein tau has been studied by generating a series of tau constructs differing in one or several of its subdomains: length and composition of the repeat domains, extensions of the repeats in the N- or C-terminal direction, constructs without repeats, assembly vs projection domain, and number of N-terminal inserts. The interaction of the mutant tau proteins with microtubules was judged by several independent methods. (i) Direct binding assays between tau and taxol-stabilized microtubules yield dissociation constants and stoichiometries. (ii) Light scattering and X-ray scattering of assembling microtubule solutions reflect the capacity of tau to promote microtubule nucleation, elongation, and bundling in bulk solution. (iii) Dark field microscopy of assembling microtubules allows one to assess the efficiency of nucleation and bundling separately. The repeat region alone, the N-terminal domains alone, or the C-terminal tail alone binds only weakly to microtubules. However, binding is strongly enhanced by combinations such as the repeat region plus one or both of the flanking regions which could be viewed as "jaws" for tau on the microtubule surface (the proline-rich domain P upstream of the repeats and the "fifth" repeat R' downstream). Such combinations make tau's binding productive in terms of microtubule assembly and stabilization, while the combination of the flanking regions without repeats binds only unproductively. Efficient nucleation parallels strong binding in most cases, i.e., when a construct binds tightly to microtubules, it also nucleates them efficiently and vice versa. In addition, the proline-rich domain P in combination with the repeats R or the flanking domain R' causes pronounced bundling. This effect disappears when the N-terminal domains (acidic or basic) are added on, suggesting that the tau isoforms are not "bundling proteins" in the proper sense. In spite of the wide range of binding strength and nucleation efficiency, the stoichiometries of binding are rather reproducible (around 0.5 tau/tubulin dimer); this is in remarkable contrast to the effect of certain types of phosphorylation which can strongly reduce the stoichiometry.
[Show abstract][Hide abstract] ABSTRACT: Microtubules can adjust their length by the mechanism of dynamic instability, that is by switching between phases of growth and shrinkage. Thus far this phenomenon has been studied with microtubules that contain several components, that is, a mixture of tubulin isoforms, with or without a mixture of microtubule-associated proteins (MAPs), which can act as regulators of dynamic instability. Here we concentrate on the influence of the tubulin component. We have studied MAP-free microtubules from the marginal band of avian erythrocytes and compared them with mammalian brain microtubules. The erythrocyte system was selected because it represents a naturally stable aggregate of microtubules; second, the tubulin is largely homogeneous, in contrast to brain tubulin. Qualitatively, erythrocyte microtubules show similar features as brain microtubules, but they were found to be much less dynamic. The critical concentration of elongation, and the rates of association and dissociation of tubulin are all lower than with brain microtubules. Catastrophes are rare, rescues frequent, and shrinkage slow. This means that dynamic instability can be controlled by the tubulin isotype, independently of MAPs. Moreover, the extent of dynamic behavior is highly dependent on buffer conditions. In particular, dynamic instability is strongly enhanced in phosphate buffer, both for erythrocyte marginal band and brain microtubules. The lower stability in phosphate buffer argues against the hypothesis that a cap of tubulin.GDP.Pi subunits stabilizes microtubules. The difference in dynamics between tubulin isotypes and between the two ends of microtubules is preserved in the different buffer systems.
Preview · Article · Apr 1993 · Molecular Biology of the Cell