Gerard Drewes

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.

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 Alzheimers 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.

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 phoshorylatable epitopes at Ser—Pro motifs, as well as 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.

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.

Dephosphorylation of tau protein and Alzheimer paired helical filaments by calcmeurin 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 imcrotubules [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.

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.

Interactions of MAP/microtubule affinity regulating kinases with the adaptor complex AP-2 of clathrin-coated vesicles

MARK [microtubule-associated proteins (MAPs)/microtubule affinity regulating kinase]/Par-1 (partition defective) phosphorylate MAPs tau, MAP2 and MAP4 at KXGS motifs and thereby regulate microtubule dynamics and transport processes in cells [Drewes et al., Cell1997;89:297-308]. We report here that MARK copurifies with clathrin-coated vesicles (CCVs) via interaction with the adaptor complex AP-2. The adaptin binding site on MARK includes the regulatory loop of its catalytic domain. Immunofluorescence demonstrates the colocalization of MARK with AP-2 and clathrin, as well as other MARK-interacting proteins such as PAK5. The results are consistent with an observed influence of MARK on the trafficking of CCVs. Cell Motil. Cytoskeleton 2009. (c) 2009 Wiley-Liss, Inc.

Cloning and sequencing of a cDNA encoding rat brain mitogen-activated protein (MAP) kinase activator*

The complete cDNA sequence for mitogen-activated protein kinase activator from rat brain was cloned. It encodes a protein kinase of 393 amino acids with a calculated M(r) of 43,465.

Tau Protein and Alzheimer Paired Helical Filament Assembly: Interacting Domains and Control Residues

Tau protein can interact with tubulin, the subunits of microtubules, and it can interact with itself in the paired helical filaments of Alzheimer’s disease. Both interactions are thought to be caused by post-translational modifications of the protein. We have analyzed the domain structure of tau, the effect of the domains on tau-tubulin or tau-tau interactions, and the residues that are critically involved. Two key residues appear to be particularly important in switching between different states, Ser-262 and Cys-322. Ser-262 can be phosphorylated by the kinase pllO(MARK), which abolishes tau’s binding to microtubules. Cys-322 can be oxidized to disulfide bridges, which leads to the formation of antiparallel tau dimers and paired helical filaments. The results argue that the tau-microtubule interactions and the tau-tau interactions lead ing to PHFs are controlled by different chemical modifications.

Phosphorylation of Tau and Its Relationship with Alzheimer Paired Helical Filaments

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; these include mitogen-activated protein kinase (MAPK), glycogen-synthase kinase-3 (GSK-3), or cyclin-dependent kinase-5 (cdk5). These kinases are tightly associated with microtubules as well as with paired helical filaments. In addition, the phosphorylation of serine 262 has a pronounced influence on the binding of tau to microtubules. All of the phosphorylation sites can be cleared by the phosphatases calcineurin and PP-2A, but not by PP-1. 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 the microtubule binding domain can be assembled into paired helical filaments in vitro.