Structure and regulation of MARK, a kinase involved in abnormal phosphorylation of Tau protein
© Timm et al. 2008
Published: 3 December 2008
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© Timm et al. 2008
Published: 3 December 2008
Protein kinases of the MARK family phosphorylate tau protein in its repeat domain and thereby regulate its affinity for microtubules and affect the aggregation of tau into Alzheimer paired helical filaments. We are searching for low molecular weight compounds to interfere with the activity of MARK and its pathways. Here we summarize structural features of MARK and cellular pathways of regulation.
Kinases of the MARK (microtubule-associated protein (MAP)-microtubule affinity regulating kinase)/Par-1 family play important roles in several contexts relevant for Alzheimer's disease (AD) research. The best-known property is that these kinases phosphorylate the tau protein in its repeat domain. The consequence is that tau looses its affinity for microtubules and detaches from them. The microtubules become destabilized, and the unbound tau is free to undergo abnormal aggregation. This process represents one of the hallmarks of AD. Furthermore, the sites on tau phosphorylated by MARK (KXGS motifs in the repeat domain) appear early in AD , MARK protein is elevated in neurofibrillary tangles in AD brain , and MARK phosphorylation sites on tau are elevated early in transgenic mouse models of tauopathy [3, 4].
Apart from neurodegeneration, MARK and its homologues belong to a set of conserved proteins that are essential for establishing cellular polarity, which is crucial for the development of an organism. Because of this property, these proteins were initially named Par (for 'partition-defective'), since mutations in the gene products lead to defects in the partitioning of the Caenorhabditis elegans zygote . The combination of Par genes has since been discovered and studied in many contexts, notably in polarity development in the fruit fly  and in the establishment of polarized epithelial cells . Besides tau, a number of other MARK target proteins have been identified. They include the MAPs related to tau (for example, MAP2, MAP4 and their isoforms) , other MAPs (for example, doublecortin), or proteins involved in phosphorylation signaling and 14-3-3 binding (for example, Cdc25, PTPH1, and KSR1) . In particular, the Par genes are involved in neuronal differentiation , and the activity of MARK2/Par-1 is necessary for the outgrowth of cell processes, neurites, and dendritic spines [11–13].
Our search for MARK was prompted by the observation of the phoshorylated KXGS motifs in tau protein and their strong effect on microtubule affinity. The kinase was identified and cloned on the basis of this property [14, 15]. The kinase subfamily contains four members, termed MARK1–4, encoded on chromosomes 1, 11, 14, and 19 in the human genome. MARK belongs to the AMPK (adenosine-monophosphate activated protein kinase) branch of the CAMK (calcium/calmodulin-dependent protein kinase) group of kinases . The kinase is relatively large (nearly 800 residues), contains several domains, and appears to be regulated by multiple pathways. Two of these were already anticipated when the kinase was originally isolated because two residues in the so-called 'activation loop' of the catalytic domain were phosphorylated. Subsequent work showed that phosphorylation of the first site (T208 in the MARK2 sequence) activated the kinase, whereas the second site was inhibitory (S212). Activation by phosphorylation at T208 can be achieved by the upstream kinase MARKK/TAO-1 , or alternatively by the kinase LKB1 . Inactivation by phosphorylation at S212 is achieved by the glycogene synthase kinase 3β (GSK3β) . Because of the relationship to tau phosphorylation in neurofibrillary pathology, we are interested in low molecular weight compounds by which the activity of MARK can be modulated. One example is that of hymenialdisine, which inhibits MARK and other kinases by binding in the catalytic pocket [12, 20]. Here we summarize some structural features of MARK and mechanisms of regulation that may serve as entry points for pharmacological intervention.
The carboxy-terminal KA1 domains are only predicted for a subset of the AMPK family kinases, MARK1–4, AMPKα1/2, BRSK1/2 (alias SADK-A/B) and MELK (Figure 1B). Among the domains of MARK, three have been solved structurally at high resolution: the catalytic domain together with the UBA domain (by X-ray diffraction) [22–24]; and the tail domain (by NMR spectroscopy) . If not stated otherwise, residue numbers refer to MARK2 [UniProt: Q7KZI7], which is the isoform that has been resolved at the highest resolution so far [PDB: 2Y8G].
The catalytic domain has a carboxy-terminal extension that starts with a highly charged four residue motif (ExxE, x = E or D, except for MARK4, EGEE) that resembles the common docking (CD) site in MAP kinases  and may represent an attachment of upstream or downstream regulatory cofactors. This motif is followed by an extended chain of amino acid residues that ends with the UBA domain, a small, globular domain that binds close to the amino terminus of the catalytic domain. The generic UBA domain consists of three short helices (α1–3) folded in a characteristic helical bundle. In all MARK crystals that have been solved so far, including constructs from MARK isoforms 1, 2, and 3, the UBA domain is unusually folded: helix α3 is inverted compared to the typical fold (Figure 2B).
The activity of MARK is additionally regulated by several mechanisms that all lead to reduced activity (Figure 4) . First, the activity is modulated by interaction with other proteins. Using a yeast two-hybrid screen, we identified the Ste20-kinase PAK5, which can bind to the catalytic domain of MARK, resulting in inhibition . Furthermore, MARK can also interact with the scaffold protein 14-3-3/Par-5 (one of the conserved polarity genes). Two different modes have been proposed for this interaction: 14-3-3 can bind in a phosphorylation-independent manner to a fragment containing the catalytic domain and the linker to the UBA domain of MARK/Par-1 . Alternatively, 14-3-3 can bind to the spacer domain after phosphorylation by atypical protein kinase C [32, 33]. These interactions not only regulate MARK spatially by altering its localization, but also inhibit the catalytic activity of the enzyme, probably by stabilizing the inhibitory interaction of the tail domain with the amino-terminal header or the catalytic domain (, and our unpublished data). The structural analysis of MARK  suggests further regulatory interactions with yet unknown proteins and the UBA domain – for example, poly-ubiquitin – leading to intracellular signaling. Finally, the CD site is known in MAP kinases for multiple interactions with upstream and downstream effectors ; this motif can be found in all MARK isoforms .
adenosine monophosphate-activated protein kinase
brain-selective kinase 1/2
glycogene synthase kinase 3β
MAP-microtubule affinity regulating kinase
maternal embryonic leucine zipper kinase
sucrose non-fermenting 1
This work was supported by the Max-Planck-Gesellschaft (MPG), the Deutsche Forschungsgemeinschaft and the Alzheimer Drug Discovery Foundation (ADDF, New York).
This article has been published as part of BMC Neuroscience Volume 9 Supplement 2: 2008 Proceedings of the 8th International Conference on Alzheimer's Disease Drug Discovery The full contents of the supplement are available online at http://www.biomedcentral.com/1471-2202/9?issue=S2.
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