Biomedical subjects

E M Mandelkow

Publications and source records attributed to E M Mandelkow.

At least 37 records · Page 2Linked to original sources

PubMed1998

Sequential phosphorylation of Tau by glycogen synthase kinase-3beta and protein kinase A at Thr212 and Ser214 generates the Alzheimer-specific epitope of antibody AT100 and requires a paired-helical-filament-like conformation.

AT100 is a monoclonal antibody highly specific for phosphorylated Tau in Alzheimer paired helical filaments. Here we show that the epitope is generated by a complex sequence of sequential phosphorylation, first of Ser199, Ser202 and Thr205 (around the epitope of antibody AT8), next of Thr212 by glycogen synthase kinase (GSK)-3beta (a proline-directed kinase), then of Ser214 by protein kinase A (PKA). Conversely, if Ser214 is phosphorylated first it protects Thr212 and the Ser-Pro motifs around the AT8 site against phosphorylation, and the AT100 epitope is not formed. The generation of the AT100 epitope requires a conformation of tau induced by polyanions such as heparin, RNA or poly(Glu), conditions which also favor the formation of paired helical filaments. The Alzheimer-like phosphorylation can be induced by brain extracts. In the extract, the kinases responsible for generating the AT100 epitope are GSK-3beta and PKA, which can be inhibited by their specific inhibitors LiCl and RII, respectively. A cellular model displaying the reaction with AT100 is presented by Sf9 insect cells transfected with Tau. Knowledge of the events and kinases generating the AT100 epitope in cells might allow us to study the degeneration of the cytoskeleton in Alzheimer's disease.

Alzheimer Disease↗

PubMed1998

Interaction of monomeric and dimeric kinesin with microtubules.

The binding stoichiometry of kinesin to microtubules was determined using several biochemical and biophysical approaches (chemical crosslinking, binding assays, scanning transmission electron microscopy (STEM), image reconstruction, and X-ray scattering). The results show that each tubulin dimer associates with one kinesin head, irrespective of whether kinesin occurs in a monomeric or dimeric form in solution. Moreover, these heads appear to align along the protofilament axis generating a 16 nm periodicity of successive kinesin dimers. This is consistent with a "tightrope" model of movement where the first head of the dimer provides a guiding signal for the following one.

Animals↗

PubMed1998

Conformations of kinesin: solution vs. crystal structures and interactions with microtubules.

Recently, the molecular structures of monomeric and dimeric kinesin constructs in complex with ADP have been determined by X-ray crystallography (Kull et al. 1996; Kozielski et al. 1997 a; Sack et al. 1997). The "motor" or "head" domains have almost identical conformations in the known crystal structures, yet the kinesin dimer is asymmetric: the orientation of the two heads relative to the coiled-coil formed by their neck regions is different. We used small angle solution scattering of kinesin constructs and microtubules decorated with kinesin in order to find out whether these crystal structures are of relevance for kinesin's structure under natural conditions and for its interaction with microtubules. Our preliminary results indicate that the crystal structures of monomeric and dimeric kinesin are similar to their structures in solution, though in solution the center-of-mass distance between the motor domains of the dimer could be slightly greater. The crystal structure of dimeric kinesin can be interpreted as representing two equivalent conformations. Transitions between these or very similar conformational states may occur in solution. Binding of kinesin to microtubules has conformational effects on both, the kinesin and the microtubule. Solution scattering of kinesin decorated microtubules reveals a peak in intensity that is characteristic for the B-surface lattice and that can be used to monitor the axial repeat of the microtubules under various conditions. In decoration experiments, dimeric kinesin dissociates, at least partly, leading to a stoichiometry of 1:1 (one kinesin head per tubulin dimer; Thormählen et al. 1998a) in contrast to the stoichiometry of 2:1 reported for dimeric ncd. This discrepancy is possibly due to the effect of steric hindrance between kinesin dimers on adjacent binding sites.

Animals↗

PubMed1998

Mitotic phosphorylation of tau protein in neuronal cell lines resembles phosphorylation in Alzheimer's disease.

Tau protein, a neuronal microtubule-associated protein is phosphorylated on several sites when extracted from brain tissue and is a substrate for many protein kinases in vitro. In Alzheimer's disease it becomes hyperphosphorylated, notably at Ser-Pro or Thr-Pro motifs, and forms the paired helical filaments (PHFs). The increased phosphorylation can be detected by several antibodies raised against Alzheimer tau. We show here that a similar type of phosphorylation can be observed in cells of neuronal origin during mitosis. Murine neuroblastoma cells (N2a) were stably transfected with htau40, the largest of the six human tau isoforms in the brain. We used several antibodies reporting on the state of phosphorylation of tau (Tau-1, AT8, AT180, PHF-1, and T46) and the antibody MPM-2 that recognizes phosphorylated mitotic proteins. The results show that tau is in a state of low phosphorylation in interphase cells, whereas during mitosis it becomes highly phosphorylated. This behavior was also found for endogenous tau protein in human neuroblastoma cells (LAN-5). The similarity between tau phosphorylation in dividing neuronal cells and Alzheimer degenerating neurons may indicate that aging neurons exposed to inappropriate signals respond by an attempt to activate their machinery for regeneration.

Alzheimer Disease↗

PubMed1998

Tau in Alzheimer's disease.

Neurofibrillar protein aggregates containing tau are one of the major hallmarks of Alzheimer's disease (AD). In normal cells, tau stabilizes axonal microtubules, which are the tracks for intracellular traffic. In AD, tau becomes abnormally phosphorylated, aggregates into paired helical filaments and loses its ability to maintain the microtubule tracks. There is renewed interest in tau as a causative factor in neurodegenerative disease based on recently discovered mutations in the gene encoding tau. This article discusses how changes in tau protein could lead to retraction of neuronal processes and thus cell death and argues that tau pathology, rather than beta-amyloid, might be the most reliable indicative factor for AD.

Alzheimer Disease↗

PubMed1998

MAPs, MARKs and microtubule dynamics.

Microtubules (MTs) serve as tracks for cellular transport, and regulate cell shape and polarity. Rapid transitions between stable and dynamic forms of MTs are central to these processes. This dynamic instability is regulated by a number of cellular factors, including the structural MT-associated proteins (MAPs), which in turn are regulated by phosphorylation. MT-affinity-regulating kinases (MARKs) are novel mammalian serine/threonine kinases that phosphorylate the tubulin-binding domain of MAPs and thereby cause their detachment from MTs and increased MT dynamics. Molecular cloning of MARKs revealed a family of four closely related protein kinases that share homology with genes from the nematode Caenorhabditis elegans and fission yeast that are involved in the generation of cell shape and polarity. Hence, MARKs might play a role in the regulation of MT stability during morphogenesis.

Animals↗

PubMed1998

The endogenous and cell cycle-dependent phosphorylation of tau protein in living cells: implications for Alzheimer's disease.

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.

Alzheimer Disease↗

PubMed1997

The crystal structure of dimeric kinesin and implications for microtubule-dependent motility.

The dimeric form of the kinesin motor and neck domain from rat brain with bound ADP has been solved by X-ray crystallography. The two heads of the dimer are connected via a coiled-coil alpha-helical interaction of their necks. They are broadly similar to one another; differences are most apparent in the head-neck junction and in a moderate reorientation of the neck helices in order to adopt to the coiled-coil conformation. The heads show a rotational symmetry (approximately 120 degrees) about an axis close to that of the coiled-coil. This arrangement is unexpected since it is not compatible with the microtubule lattice. In this arrangement, the two heads of a kinesin dimer could not have equivalent interactions with microtubules.

Adenosine Diphosphate↗

PubMed1997

X-ray structure of motor and neck domains from rat brain kinesin.

We have determined the X-ray structure of rat kinesin head and neck domains. The folding of the core motor domain resembles that of human kinesin reported recently [Kull, F. J., et al. (1996) Nature 380, 550-554]. Novel features of the structure include the N-terminal region, folded as a beta-strand, and the C-terminal transition from the motor to the rod domain, folded as two beta-strands plus an alpha-helix. This helix is the beginning of kinesin's neck responsible for dimerization of the motor complex and for force transduction. Although the folding of the motor domain core is similar to that of a domain of myosin (an actin-dependent motor), the position and angle of kinesin's neck are very different from those of myosin's stalk, suggesting that the two motors have different mechanisms of force transduction. The N- and C-terminal ends of the core motor, thought to be responsible for the directionality of the motors [Case, R. B., et al. (1997) Cell 90, 959-966], take the form of beta-strands attached to the central beta-sheet of the structure.

Amino Acid Sequence↗

PubMed1997

Domains of neuronal microtubule-associated proteins and flexural rigidity of microtubules.

Microtubules are flexible polymers whose mechanical properties are an important factor in the determination of cell architecture and function. It has been proposed that the two most prominent neuronal microtubule-associated proteins (MAPs), tau and MAP2, whose microtubule binding regions are largely homologous, make an important contribution to the formation and maintenance of neuronal processes, putatively by increasing the rigidity of microtubules. Using optical tweezers to manipulate single microtubules, we have measured their flexural rigidity in the presence of various constructs of tau and MAP2c. The results show a three- or fourfold increase of microtubule rigidity in the presence of wild-type tau or MAP2c, respectively. Unexpectedly, even low concentrations of MAPs promote a substantial increase in microtubule rigidity. Thus at approximately 20% saturation with full-length tau, a microtubule exhibits >80% of the rigidity observed at near saturating concentrations. Several different constructs of tau or MAP2 were used to determine the relative contribution of certain subdomains in the microtubule-binding region. All constructs tested increase microtubule rigidity, albeit to different extents. Thus, the repeat domains alone increase microtubule rigidity only marginally, whereas the domains flanking the repeats make a significant contribution. Overall, there is an excellent correlation between the strength of binding of a MAP construct to microtubules (as represented by its dissociation constant Kd) and the increase in microtubule rigidity. These findings demonstrate that neuronal MAPs as well as constructs derived from them increase microtubule rigidity, and that the changes in rigidity observed with different constructs correlate well with other biochemical and physiological parameters.

Adsorption↗

PubMed1997

MARK, a novel family of protein kinases that phosphorylate microtubule-associated proteins and trigger microtubule disruption.

MARK phosphorylates the microtubule-associated proteins tau, MAP2, and MAP4 on their microtubule-binding domain, causing their dissociation from microtubules and increased microtubule dynamics. We describe the molecular cloning, distribution, activation mechanism, and overexpression of two MARK proteins from rat that arise from distinct genes. They encode Ser/Thr kinases of 88 and 81 kDa, respectively, and show similarity to the yeast kin1+ and C. elegans par-1 genes that are involved in the establishment of cell polarity. Expression of both isoforms is ubiquitous, and homologous genes are present in humans. Catalytic activity depends on phosphorylation of two residues in subdomain VIII. Overexpression of MARK in cells leads to hyperphosphorylation of MAPs on KXGS motifs and to disruption of the microtubule array, resulting in morphological changes and cell death.

Amino Acid Sequence↗

PubMed1997

The 'jaws' model of tau-microtubule interaction examined in CHO cells.

Tau is a neuronal microtubule-associated protein which promotes microtubule assembly. The C-terminal half of the protein contains three or four tandem repeats that are often considered to be the microtubule binding domain. This view is in conflict with in vitro data showing that the repeat domain binds only weakly to microtubules while the domains flanking the repeats bind strongly, even in the absence of the repeats. This has lead us to propose a 'jaws' model of tau whereby the regions flanking the repeats are considered as targetting domains, responsible for positioning tau on the microtubule surface, and the repeats which act as catalytic domains for microtubule assembly. To examine whether this model is appropriate in vivo we generated recombinant tau isoforms and microinjected them into CHO cells. Immunofluorescence microscopy of microtubules and tau shows that binding to microtubules, stabilization of microtubules and formation of bundles is not achieved by tau constructs comprising individual domains, but requires the combination of the flanking regions and the repeat domain. The results show that the jaws model describes the interactions between tau and microtubules in living cells. Since the targetting and catalytic domains are affected differently by phosphorylation the model provides a basis for studying the regulation of the interaction between microtubules and tau or other microtubule-associated proteins.

Animals↗

PubMed1996

RNA stimulates aggregation of microtubule-associated protein tau into Alzheimer-like paired helical filaments.

The microtubule-associated protein tau is the main component of the paired helical filaments (PHFs) of Alzheimer's disease, the most common senile dementia. To understand the origin of tau's abnormal assembly we have studied the influence of other cytosolic components. Here we report that PHF assembly is strongly enhanced by RNA. The RNA-induced assembly of PHFs is dependent on the formation of intermolecular disulfide bridges involving Cys322 in the third repeat of tau, and it includes the dimerization of tau as an early intermediate. Three-repeat constructs polymerize most efficiently, two repeat constructs are the minimum number required for assembly, and even all six full-length isoforms of tau can be induced to form PHFs by RNA.

Alzheimer Disease↗

PubMed1996

Phosphorylation of microtubule-associated proteins MAP2 and MAP4 by the protein kinase p110mark. Phosphorylation sites and regulation of microtubule dynamics.

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.

Animals↗

PubMed1996

Structure, microtubule interactions, and phosphorylation of tau protein.

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.

Microtubules↗

PubMed1995

Oxidation of cysteine-322 in the repeat domain of microtubule-associated protein tau controls the in vitro assembly of paired helical filaments.

One of the hallmarks of Alzheimer disease is the pathological aggregation of tau protein into paired helical filaments (PHFs) and neurofibrillary tangles. Here we describe the in vitro assembly of recombinant tau protein and constructs derived from it into PHFs. Though whole tau assembled poorly, constructs containing three internal repeats (corresponding to the fetal tau isoform) formed PHFs reproducibly. This ability depended on intermolecular disulfide bridges formed by the single Cys-322. Blocking the SH group, mutating Cys for Ala, or keeping tau in a reducing environment all inhibited assembly. With constructs derived from four-repeat tau (having the additional repeat no. 2 and a second Cys-291), PHF assembly was blocked because Cys-291 and Cys-322 interact within the molecule. PHF assembly was enabled again by mutating Cys-291 for Ala. The synthetic PHFs bound the dye thioflavin S used in Alzheimer disease diagnostics. The data imply that the redox potential in the neuron is crucial for PHF assembly, independently or in addition to pathological phosphorylation reactions.

Alzheimer Disease↗

PubMed1995

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.

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.

Alzheimer Disease↗

PubMed1995

Microtubules and microtubule-associated proteins.

Microtubule research is becoming increasingly diverse, reflecting the many isoforms and modifications of tubulin and the many proteins with which microtubules interact. Recent advances are particularly visible in four areas: microtubule motor proteins (their structures, stepping modes, and forces); microtubule nucleation (the roles of centrosomes and gamma-tubulin); tubulin folding (mediated by cytoplasmic chaperones); and the expanding list of microtubule-associated proteins, knowledge of their phosphorylation states, and information on their effects on microtubule dynamics.

Animals↗