Microtubule Affinity Regulating Kinases (MARK)
Microtubule affinity regulating kinase (MARK) was first identified as an activity in brain extract with the ability to phosphorylate the microtubule-associated protein (MAP) tau and inhibit its binding to microtubules (MTs) (Drewes et al. 1995). All four human MARKs phosphorylate tau and the structurally related proteins MAP 2 and MAP4 on their MT-binding domains, causing their dissociation from MTs in purified systems and in cells, which in turn leads to destabilization of MTs (Drewes et al. 1997, 1998). Since hyperphosphorylation and mislocalization of tau represent hallmarks of the neurofibrillar pathology of Alzheimer’s disease (AD), MARKs have attracted interest as potential therapeutic targets early on (Drewes 2004). Other clues for the function of MARKs in cytoskeletal regulation and cell polarity resulted from studies in epithelial cells (Bohm et al. 1997) and from MARK orthologues in fission yeast (Drewes and Nurse 2003) and the extensive data from the nematode Caenorhabditis elegans (Kemphues 2000).
Phylogeny, Structure, and Regulation
Structure: Several crystal structures have been reported including the catalytic and UBA domains of MARK1, MARK2, and MARK3 (Marx et al. 2010), and the KA domain (Moravcevic et al. 2010). The structures of the catalytic domain are all in the inactive state and indicate a propensity for dimer formation. The overall structure of the kinase domain resembles that of other eukaryotic protein kinases consisting of a smaller N-terminal lobe and a larger C-terminal lobe with the active site pocket with the nucleotide located in between (Marx et al. 2010). The larger lobe contains the activation segment which, depending on its phosphorylation state, inhibits or permits the proper access of the peptidic substrate to the MgATP in the active site (see below). The UBA domain is located at the C-terminal end of the large lobe and may be able to confer autoinhibition by directly binding to the N-terminal lobe. The catalytic and UBA domains are connected via an acidic stretch of 15 amino acids, which bears similarity to the common docking domain in MAPK family kinases where it functions to bind upstream activating kinases and inactivating phosphatases. The C-terminus of MARK kinases folds into a compact domain (“KA1 domain”) which is formed by five beta strands and two alpha helices and ends in an ELKL motif. This domain is less conserved in MARK4 which terminates with a DLEL sequence. The KA1 domain is a membrane association domain that binds acidic phospholipids like phosphatidylserine (Moravcevic et al. 2010).
Regulation: MARK kinases are subject to diverse regulatory mechanisms including posttranslational modifications and the association with inhibitory proteins. The posttranslational modifications include phosphorylation and ubiquitination (Al-Hakim et al. 2008; Brajenovic et al. 2004; Lizcano et al. 2004; Timm et al. 2003). MARK2 purified from tissue is phosphorylated on T208 and S212 in the activation loop (Drewes et al. 1997). Phosphorylation of T208 (and the homologous sites on other MARKs) is required for activation and can be induced by LKB1, which constitutes a mammalian orthologue of Drosophila melanogaster and C. elegans PAR-4, and by TAO1/MAP 3K16. Both upstream kinases increase MT dynamics through MARK activation (Kojima et al. 2007; Timm et al. 2003). Phosphorylation of S212 is mediated by GSK3β and leads to inactivation (Timm et al. 2008), although there is a conflicting report (Kosuga et al. 2005). Phosphorylation of S595 on MARK2 (and the homologous sites on other MARK isoforms) by aPKC (atypical PKC, a member of the Par-6 polarity complex) inactivates MARK by inducing its association with 14-3-3 proteins which in turn causes the delocalization from membranous compartments in polarized cells (Hurov and Piwnica-Worms 2007; Suzuki et al. 2004). MARK3 was shown to be inhibited by phosphorylation by Pim-1 (Bachmann et al. 2006) and MARK2 is inhibited by association with PAK5. It was proposed that the interplay between MARKs and the actin-modulating kinases PAK5 and TESK1 may provide a mechanism to route incoming signals to antagonistic MT network- or actin network-dependent cytoskeletal activity (Matenia et al. 2005). The proteomic analysis of MARK4 by tandem affinity purification revealed co-purification of several additional proteins including 14-3-3 proteins, the ubiquitin-dependent proteases USP7 and USP9x, subunits of protein phosphatase 2, and gamma-tubulin (Brajenovic et al. 2004). MARK4 and possibly other MARKs are subject to inhibitory polyubiquitination in vivo by unusual K29/K33-linked ubiquitin chains, which are regulated by USP9X (Al-Hakim et al. 2008). The interaction with 14-3-3 proteins appears to constitute a major function of MARKs (see below).
MARKs and Cell Morphology
MARKs and its orthologues in lower eukaryotes comprise protein kinases conserved from yeast to C. elegans and Drosophila to mammals, which are essential for cellular polarity, governing the establishment of the embryonic body axis and maintaining cell differentiation. The MARK/Par-1 kinase regulates the localization of structural PAR-proteins, and tightly regulated phosphorylation events provide a cellular regulatory mechanism governing important developmental decisions (Chen et al. 2006). In epithelial cells MARK2 is asymmetrically localized and knockdown or overexpression of inactive mutants perturbs the polarity in this cell line suggesting a conserved mechanism governing polarization from C. elegans embryos to mammalian cells (Cohen et al. 2007). Many of the effects of MARK on cell polarity are mediated by the MT system. In neurons, the phosphorylation of MAPs controls the binding to MTs, and bound MAPs stabilize the tubulin polymer, and their phosphorylation potentially provides a mechanism for regulating stability of MTs in a spatial and temporal fashion. MTs function as structural elements of cell morphology and as “tracks” for intracellular transport of vesicles and organelles (Matenia and Mandelkow 2009). Therefore, kinases that regulate MT stability may not only control cell shape and polarity but also cellular transport mediated by the interplay of MAPs and motor proteins. In neurons, MARK2 and possibly MARK4 play role in neurite outgrowth and maintenance of neuronal polarity, a process requiring dynamic instability of MTs (Yoshimura et al. 2010; Trinczek et al. 2004). In hippocampal neurons, MARK2 overexpression inhibits axon formation whereas RNAi-mediated MARK2 knockdown induces multiple axons (Chen et al. 2006). MARK4 is absent from neuronal progenitor cells, but upregulated during neuronal differentiation (Moroni et al. 2006). MARK2 is also involved in the control of neuronal migration through the phosphorylation of doublecortin, a MAP present in the leading process of migrating neurons, which has been implied in neuronal migration disorders (Sapir et al. 2008). MARKs also impact body axis specification and morphogenic signaling via modulation of the Wnt signaling pathway by the phosphorylation of dishevelled (Dsh) which acts at a branching point for developmental decisions (Elbert et al. 2006).
Substrates of MARKs
Microtubule-associated proteins: Several of the effects of MARKs on the microtubule system are mediated via the phosphorylation of MAPs on serine and threonine residues. All four MARK isoforms can phosphorylate Tau and the related MAP 2, MAP 2c, and MARK4 at the repeated Lys-Xaa-Gly-Ser motifs in the MT-binding domain (Drewes et al. 1998). MARKs also phosphorylate other components of the MT system including doublecortins and motor proteins (Sapir et al. 2008; Yoshimura et al. 2010).
14-3-3 binding proteins: 14-3-3 proteins are phosphoserine adaptor proteins binding to multiple clients with pleiotropic cellular functions, e.g., in protein translocation and vesicle trafficking. MARK3 was shown to phosphorylate 14-3-3 binding motifs on different substrates: the phosphatases PTPH1 and CDC25; the suppressor of Ras/MAPK signaling KSR; the desmosomal protein plakophilin-2, and histone deacetylases. The phosphorylation of these substrates by MARK induces the formation of a complex between the substrate protein and 14-3-3, and changes the cellular localization and thus the function of the substrate (Matenia and Mandelkow 2009). 14-3-3 proteins also interact directly with the MARK catalytic domain, enabling the kinase to target substrates to 14-3-3 and re-phosphorylate sites quickly after dephosphorylation and disruption of 14-3-3 binding (Muller et al. 2003). Alternatively, the spacer domain of MARK2 can be phosphorylated by aPKC to generate a 14-3-3 binding motif leading to relocalization and inactivation (Hurov and Piwnica-Worms 2007).
Role of MARKs in Disease
Helicobacter infection: Helicobacter pylori is a pathogen associated with gastritis, ulcerations, and gastric adenocarcinoma. The virulence factor CagA can bind MARK2, inhibit its aPKC-dependent activation, and delocalize it from the membrane, causing junctional and polarity defects in gastric epithelial cells which results in mucosal damage, inflammation, and carcinogenesis (Saadat et al. 2007).
Metabolic syndrome: MARK2 knockout mice display growth retardation and dysfunctions in fertility, immune homeostasis, and learning and memory, but are lean, insulin hypersensitive, resistant to high-fat-diet-induced weight gain, and hypermetabolic (Hurov et al. 2007). MARK3 appears to have a related but nonredundant function as knockout mice exhibit increased energy expenditure, reduced adiposity with unaltered glucose handling, but normal insulin sensitivity. These mice were protected against diet-induced obesity and showed slow weight gain with resistance to fatty liver, with improved glucose handling and decreased insulin secretion. The crossing of MARK2 null mice with MARK3 null mice revealed that at least one allele is necessary for embryonic survival (Lennerz et al. 2010). The animal model data indicate that MARKs are important regulators of glucose and lipid metabolism and may be valuable drug target for the treatment of metabolic syndrome.
MARK genes are conserved in lower eukaryotic organisms like yeast, nematodes, and fruit flies where their function has been extensively studied by genetic means. From these studies MARKs have emerged as key regulators of cell polarity. Humans express four paralogous MARK genes encoding structurally related but functionally nonredundant protein serine/threonine kinases. MARKs are regulated by activating and inactivating phosphorylation events on distinct residues located in the activation loop and outside the catalytic domain. Key regulators of these pathways are the kinases LKB1 and aPKC which themselves constitute major regulators of cell polarity. MARK activity is also regulated by a tightly regulated interplay of 14-3-3 protein binding and membrane localization mediated by the C-terminal KA1-domain. Biochemical studies have proposed different classes of microtubule-binding proteins as substrates which are likely to transduce the effect of MARKs on cytoskeletal rearrangements during cellular transport processes and cell morphology. In disease, a deregulation of MARK activity may contribute to the loss of cell polarity in the affected highly polarized cell types including neurons (in Alzheimer’s disease and other forms of dementia), gastric epithelial cells (in H. pylori-induced gastritis and in gastric cancer), and possibly in immune cell homeostasis.
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