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G Drewes

Publications and source records attributed to G Drewes.

At least 19 recordsLinked to original sources
PubMed2006

Microtubule affinity-regulating kinase 2 functions downstream of the PAR-3/PAR-6/atypical PKC complex in regulating hippocampal neuronal polarity.

The PAR-3/PAR-6/atypical PKC (aPKC) complex is required for axon-dendrite specification of hippocampal neurons. However, the downstream effectors of this complex are not well defined. In this article, we report a role for microtubule affinity-regulating kinase (MARK)/PAR-1 in axon-dendrite specification. Knocking down MARK2 expression with small interfering RNAs induced formation of multiple axon-like neurites and promoted axon outgrowth. Ectopic expression of MARK2 caused phosphorylation of tau (S262) and led to loss of axons, and this phenotype was rescued by expression of PAR-3, PAR-6, and aPKC. In contrast, the polarity defects caused by an MARK2 mutant (T595A), which is not responsive to aPKC, were not rescued by the PAR-3/PAR-6/aPKC complex. Moreover, polarity was abrogated in neurons overexpressing a mutant of MARK2 with a deleted kinase domain but an intact aPKC-binding domain. Finally, suppression of MARK2 rescued the polarity defects induced by a dominant-negative aPKC mutant. These results suggest that MARK2 is involved in neuronal polarization and functions downstream of the PAR-3/PAR-6/aPKC complex. We propose that aPKC in complex with PAR-3/PAR-6 negatively regulates MARK(s), which in turn causes dephosphorylation of microtubule-associated proteins, such as tau, leading to the assembly of microtubules and elongation of axons.

Animals↗
PubMed2000

Microtubule-affinity regulating kinase (MARK) is tightly associated with neurofibrillary tangles in Alzheimer brain: a fluorescence resonance energy transfer study.

Paired helical filaments, the main structural components of the neurofibrillary tangles in Alzheimer disease, consist of phosphorylated tau protein. Because the levels and degree of phosphorylation are significantly higher in paired helical filament (PHF)-derived tau than in normal adult tau, and because phosphorylation of tau severely disrupts microtubule stability, it is postulated that tau phosphorylation is an important step in PHF formation. The kinases and/or phosphatases that act in vivo to help induce such a pathological state of tau, however, are not yet known. In this study we implicate the non-proline directed kinase MARK in PHF-tau phosphorylation, by virtue of its close intermolecular association with the phosphorylated Ser262 epitope on PHF-tau as assessed by fluorescence resonance energy transfer. Moreover, because this tight enzyme-substrate association is observed in neurofibrillary tangles in Alzheimer tissue, we suggest that PHF-tau phosphorylation may occur to some extent on assembled PHF filaments.

Alzheimer Disease↗
PubMed1999

Phosphorylation of MAP2c and MAP4 by MARK kinases leads to the destabilization of microtubules in cells.

Microtubules serve as transport tracks in molecular mechanisms governing cellular shape and polarity. Rapid transitions between stable and dynamic microtubules are regulated by several factors, including microtubule-associated proteins (MAPs). We have shown that MAP/microtubule affinity regulating kinases (MARK) can phosphorylate the microtubule-associated-proteins MAP4, MAP2c, and tau on their microtubule-binding domain in vitro. This leads to their detachment from microtubules (MT) and an increased dynamic instability of MT. Here we show that MARK protein kinases phosphorylate MAP2 and MAP4 on their microtubule-binding domain in transfected CHO cells. In CHO cells expressing MARK1 or MARK2 under control of an inducible promoter, MARK2 phosphorylates an endogenous MAP4-related protein. Prolonged expression of MARK2 results in microtubule-disruption, detachment of cells from the substratum, and cell death. Concomitant with microtubule disruption, we also observed a breakdown of the vimentin network, whereas actin fibers remained unaffected. Thus, MARK seems to play an important role in controlling cytoskeletal dynamics.

Actins↗
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↗
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↗
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

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↗
PubMed1993

Dephosphorylation of tau protein and Alzheimer paired helical filaments by calcineurin and phosphatase-2A.

We have shown previously that brain tissue contains protein kinases which can phosphorylate tau protein to a state reminiscent of the pathological state of Alzheimer paired helical filaments (PHFs); these include proline-directed kinases which phosphorylate SP or TP motifs (such as MAP kinase and GSK-3) [Drewes et al. (1992); Mandelkow et al. (1992)], as well as a novel kinase which phosphorylates S262 of tau protein and thereby strongly reduces the binding of tau to microtubules [Biernat et al. (1993)]. Here we report on the corresponding phosphatases in brain which normally keep the 'pathological' sites free of phosphate. The major phosphatases acting on tau are calcineurin and PP-2A, but not PP-1. Both are present and active in brain extracts, they can dephosphorylate recombinant tau after prior phosphorylation with either MAP kinase, GSK-3, or brain extract, and the course of dephosphorylation can be monitored with antibodies diagnostic of the pathological state of tau. Both phosphatases also act directly on PHF tau isolated from Alzheimer brains.

Alzheimer Disease↗
PubMed1993

Microtubule-associated protein tau, paired helical filaments, and phosphorylation.

This paper summarizes our recent studies on microtubule-associated protein tau and its pathological state resembling that of the paired helical filaments of Alzheimer's disease. The Alzheimer-like state of tau protein can be identified and analyzed in terms of certain phosphorylation sites and phosphorylation-dependent antibody epitopes. It can be induced by protein kinases which tend to phosphorylate serine or threonine residues followed by a proline; this includes mitogen-activated protein kinase (MAPK) and glycogen-synthase kinase 3 (GSK-3). Both of these are tightly associated with microtubules as well as with paired helical filaments. Structurally, tau appears as a rod-like molecule; it tends to self-associate into dimers whose monomers are antiparallel. Constructs of truncated tau made up of antiparallel dimers of the microtubule binding domain can be assembled into paired helical filaments in vitro.

Alzheimer Disease↗
PubMed1993

Probing the phalloidin binding site of actin.

Phallotoxins form tight complexes with filamentous actin and stabilize the polymer against shearing stress. In the present study a phalloidin derivative containing a thiol-capturing moiety was prepared and reacted with single thiol groups of monomeric muscle actin. Sites of attachment in the protein were Cys-374 next to the C-terminus and Cys-10, close to the N-terminus; the latter was recently shown to be uncovered during a slow but reversible conformational transition occurring in ADP-G-actin. Phalloidin bound to Cys-374 stabilizes filaments against shearing stress almost as effectively as free phalloidin, indicating that the phalloidin binding site cannot be far from the C-terminus of actin. Stabilization was also achieved when the phalloidin reagent was added to F-actin, however, the subsequent formation of a covalent linkage with Cys-374 was not observed, most likely due to a restricted mobility of the reactants. In contrast to the efficient stabilization of filaments by phalloidin linked to Cys-374 a destabilizing effect was observed when phalloidin was attached to Cys-10. It appears that phalloidin located close to the N-terminus is unable to bind to the normal binding site in its own filament. Pronounced gelification of this actin derivative suggests that the toxin is able to mediate crosslinking with neighbouring filaments. From these results we conclude that the phalloidin binding site of actin is distant from the N-terminus, but close to the C-terminus. Furthermore, the data provide evidence that binding of phalloidin reduces the mobility of the C-terminus.

Actins↗
PubMed1993

Cooperative effects on filament stability in actin modified at the C-terminus by substitution or truncation.

We have studied the contribution of the C-terminus of actin to filament stability by chemical modification and limited proteolysis. Formation of mixed disulfides of the penultimate C-terminal cysteine residue 374 with various low-molecular-mass thiols resulted in filament destabilization, as reflected by an increase in critical concentration and steady-state ATPase activity. These effects were fully reversed by the addition of phalloidin. Both the destabilization by glutathionylation and the reversal of it by phalloidin exhibited a high degree of cooperativity; half-maximal destabilization required the modification of four out of five actin subunits, and half-maximal restabilization by phalloidin was already reached when only one out of 20 actin subunits was complexed. C-terminal truncation by limited trypsinolysis of filamentous actin resulted in a similar destabilization of the polymer, as shown by a 2-3-fold increase in the steady-state ATPase activity. This effect was likewise cooperative and could be reversed by phalloidin. Since truncation of the C-terminus of actin has an effect on stability similar to that of chemical modification with bulky substituents, the possibility can be excluded that, in the latter case, destabilization was caused by steric hindrance. Rather, it seems that the highly conserved C-terminal part of actin plays an active role in establishing a tight contact between neighbouring subunits.

Actins↗
PubMed1993

Phosphorylation of Ser262 strongly reduces binding of tau to microtubules: distinction between PHF-like immunoreactivity and microtubule binding.

Tau protein, a component of Alzheimer paired helical filaments, can be phosphorylated by several kinases. Of particular interest is the phosphorylation at Ser/Thr-Pro motifs because the resulting state of tau is similar to that found in Alzheimer's disease, as judged by its immunoreactivity. This state can be mimicked by a brain extract kinase activity and by MAP kinase. We have now studied the effect of these modes of phosphorylation on the interaction between tau and microtubules. Although MAP kinase efficiently phosphorylates many Ser/Thr-Pro motifs of tau, its effect on microtubule binding is only moderate. By contrast, phosphorylation of a single residue, Ser262, has a major effect on binding. Ser262 is not phosphorylated by MAP kinase or other proline-directed kinases, but is phosphorylated by a 35/41 kd kinase in brain, whose purification is described.

Alzheimer Disease↗
PubMed1992

Glycogen synthase kinase-3 and the Alzheimer-like state of microtubule-associated protein tau.

The Alzheimer-like state of tau protein includes phosphorylation by a proline-directed Ser/Thr kinase present in normal or pathological human brain. Extending earlier results on MAP kinase, we show here that the proline-directed kinase, GSK3, can induce an Alzheimer-like immune response involving several distinct and phosphorylatable epitopes at Ser-Pro motifs, as well as a gel mobility shift, similar to MAP kinase. Both kinases behave like microtubule-associated proteins in that they co-purify through cycles of assembly and disassembly, and both kinases are directly associated with paired helical filaments.

Alzheimer Disease↗
PubMed1992

Interchain and intrachain crosslinking of actin thiols by a bifunctional thiol reagent.

From 1,9-nonylenedithiol and Ellman's Reagent the bifunctional asymmetric disulfide n-nonylene-1,9-bis-[5-dithio-(2-nitrobenzoic acid)] (NBDN) was prepared. By monovalent reaction with cysteine-374 the crosslinker could be introduced into monomeric actin, with release of one equivalent of yellow 2-nitro-5-thiobenzoate (NTB). From the monovalent actin derivative we prepared a crosslinked actin dimer (Cys-374-Cys-374') as well as a monomer with a crosslink between Cys-374 and Cys-10. Neither crosslinked actin species was able to polymerize the crosslinked monomer even in the presence of phalloidin. The crosslinked monomer polymerized on the addition of dithiothreitol, thus providing the first unpolymerizable actin species whose polymerizability can be restored under mild conditions. We suggest the use of NBDN as a thiol-specific crosslinker that reacts under spectrophotometric control and can be removed by the addition of thiols.

Actins↗
PubMed1992

Mitogen activated protein (MAP) kinase transforms tau protein into an Alzheimer-like state.

The microtubule-associated protein tau is a major component of the paired helical filaments (PHFs) observed in Alzheimer's disease brains. The pathological tau is distinguished from normal tau by its state of phosphorylation, higher apparent M(r) and reaction with certain antibodies. However, the protein kinase(s) have not been characterized so far. Here we describe a protein kinase from brain which specifically induces the Alzheimer-like state in tau protein. The 42 kDa protein belongs to the family of mitogen activated protein kinases (MAPKs) and is activated by tyrosine phosphorylation. It is capable of phosphorylating Ser-Pro and Thr-Pro motifs in tau protein (approximately 14-16 P1 per tau molecule). By contrast, other proline directed Ser/Thr kinases such as p34(cdc2) combined with cyclin A or B have only minor effects on tau phosphorylation. We propose that MAP kinase is abnormally active in Alzheimer brain tissue, or that the corresponding phosphatases are abnormally passive, due to a breakdown of the normal regulatory mechanisms.

Alzheimer Disease↗
PubMed1992

Alzheimer-like paired helical filaments and antiparallel dimers formed from microtubule-associated protein tau in vitro.

Recent evidence from several laboratories shows that the paired helical filaments of Alzheimer's disease brains consist mainly of the protein tau in an abnormally phosphorylated form, but the mode of assembly is not understood. Here we use EM to study several constructs derived from human brain tau and expressed in Escherichia coli. All constructs or tau isoforms are rodlike molecules with a high tendency to dimerize in an antiparallel fashion, as shown by antibody labeling and chemical crosslinking. The length of the rods is largely determined by the region of internal repeats that is also responsible for microtubule binding. One unit length of the repeat domain (three or four repeats) is around 22-25 nm, comparable to the cross-section of Alzheimer PHF cores. Constructs corresponding roughly to the repeat region of tau can form synthetic paired helical filaments resembling those from Alzheimer brain tissue. A similar self-assembly occurs with the chemically cross-linked dimers. In both cases there is no need for phosphorylation of the protein.

Alzheimer Disease↗