Phosphorylation Of Microtubule-associated Proteins Research Articles

Studying Microtubule Dynamics in Human Neurons: Two-Dimensional Microtubule Tracing and Kymographs in iPSC- and SH-SY5Y-Derived Neurons for Tau Research.

The study of microtubule (MT) dynamics is essential for the understanding of cellular transport, cell polarity, axon formation, and other neurodevelopmental mechanisms. All these processes rely on the constant transition between assembly and disassembly of tubulin polymers to/from MTs, known as dynamic instability. This process is well-regulated, among others, by phosphorylation of microtubule-associated proteins (MAP), including the Tau protein. Protein kinases, in particular the microtubule affinity regulating kinase (MARK), regulate the MT-Tau interaction, inducingTau dissociation by phosphorylation. Phosphorylated Tau dissociates from microtubules forming insoluble aggregates known as neurofibrillary tangles. These accumulations of hyperphosphorylated Tau in the neurons disrupt the physiological MT-based transport machinery within the cell and can potentially lead to the development of neurodegenerative disorders, such as Alzheimer's disease (AD) and related tauopathies. Further investigations on the MT cytoskeleton dynamics are essential as they may elucidate pathomechanisms of neurodegenerative diseases - particularly tauopathies - as well as fundamental neurodevelopmental processes.The study of thedynamic assembly and disassembly of the MT network requires live-cell imaging rather than conventional immunocytochemistry based on fixed samples. To investigate MT dynamics, we perform live-cell imaging of neurons transfected with a fluorescently tagged version of the microtubule plus-end tracking protein (+TIP) EB3. This protein associates with the growing ends of MTs and thus visualizes MT growth in real time. Our imaging analysis protocol allows the determination of quantity, orientation, and velocity of MT growth in the soma and neurites of transfected neurons,using ImageJ-based tracking software and kymographs. Furthermore, functional effects of Tau and MARK kinases on the MT cytoskeleton can be assessed by overexpression or downregulation experiments of the respective protein prior to the live imaging assay. We use two different human neuronal cell models, naive and differentiated SH-SY5Y neuroblastoma cells, and neurons derived from induced pluripotent stem cells (iPSCs), both of which have shown success as models to study Tau-related pathologies.This protocol describes an optimized method for analysis of microtubule dynamics using fluorescent tagged EB3 protein as microtubule plus end marker. In this chapter, we outline the process of neuronal transfection, live-cell imaging, and necessary time-lapse image analysis based on ImageJ in two human-derived neuronal systems, which are suitable for the analysis of Tau trafficking and sorting studies.

Specific phosphorylation of microtubule-associated protein 2c by extracellular signal–regulated kinase reduces interactions at its Pro-rich regions

Microtubule-associated protein 2 (MAP2) is an important neuronal target of extracellular signal–regulated kinase 2 (ERK2) involved in Raf signaling pathways, but mechanistic details of MAP2 phosphorylation are unclear. Here, we used NMR spectroscopy to quantitatively describe the kinetics of phosphorylation of individual serines and threonines in the embryonic MAP2 variant MAP2c. We carried out real-time monitoring of phosphorylation to discover major phosphorylation sites that were not identified in previous studies relying on specific antibodies. Our comparison with the phosphorylation of MAP2c by a model cyclin-dependent kinase CDK2 and with phosphorylation of the MAP2c homolog Tau revealed differences in phosphorylation profiles that explain specificity of regulation of biological functions of MAP2c and Tau. To probe the molecular basis of the regulatory effect of ERK2, we investigated the interactions of phosphorylated and unphosphorylated MAP2c by NMR with single-residue resolution. As ERK2 phosphorylates mostly outside the regions binding microtubules, we studied the binding of proteins other than tubulin, namely regulatory subunit RIIα of cAMP-dependent PKA, adapter protein Grb2, Src homology domain 3 of tyrosine kinases Fyn and Abl, and ERK2 itself. We found ERK2 phosphorylation interfered mostly with binding to proline-rich regions of MAP2c. Furthermore, our NMR experiments in SH-SY5Y neuroblastoma cell lysates showed that the kinetics of dephosphorylation are compatible with in-cell NMR studies and that residues targeted by ERK2 and PKA are efficiently phosphorylated in the cell lysates. Taken together, our results provide a deeper characterization of MAP2c phosphorylation and its effects on interactions with other proteins.

Paclitaxel Sensitivity of Ovarian Cancer Can be Enhanced by Knocking Down Pairs of Kinases that Regulate MAP4 Phosphorylation and Microtubule Stability.

Purpose: Most patients with ovarian cancer receive paclitaxel chemotherapy, but less than half respond. Pre-treatment microtubule stability correlates with paclitaxel response in ovarian cancer cell lines. Microtubule stability can be increased by depletion of individual kinases. As microtubule stability can be regulated by phosphorylation of microtubule-associated proteins (MAPs), we reasoned that depletion of pairs of kinases that regulate phosphorylation of MAPs could induce microtubule stabilization and paclitaxel sensitization.Experimental Design: Fourteen kinases known to regulate paclitaxel sensitivity were depleted individually in 12 well-characterized ovarian cancer cell lines before measuring proliferation in the presence or absence of paclitaxel. Similar studies were performed by depleting all possible pairs of kinases in six ovarian cancer cell lines. Pairs that enhanced paclitaxel sensitivity across multiple cell lines were studied in depth in cell culture and in two xenograft models.Results: Transfection of siRNA against 10 of the 14 kinases enhanced paclitaxel sensitivity in at least six of 12 cell lines. Dual knockdown of IKBKB/STK39 or EDN2/TBK1 enhanced paclitaxel sensitivity more than silencing single kinases. Sequential knockdown was superior to concurrent knockdown. Dual silencing of IKBKB/STK39 or EDN2/TBK1 stabilized microtubules by inhibiting phosphorylation of p38 and MAP4, inducing apoptosis and blocking cell cycle more effectively than silencing individual kinases. Knockdown of IKBKB/STK39 or EDN2/TBK1 enhanced paclitaxel sensitivity in two ovarian xenograft models.Conclusions: Sequential knockdown of dual kinases increased microtubule stability by decreasing p38-mediated phosphorylation of MAP4 and enhanced response to paclitaxel in ovarian cancer cell lines and xenografts, suggesting a strategy to improve primary therapy. Clin Cancer Res; 24(20); 5072-84. ©2018 AACR.

MARK3-mediated phosphorylation of ARHGEF2 couples microtubules to the actin cytoskeleton to establish cell polarity.

The PAR-1-MARK pathway controls cell polarity through the phosphorylation of microtubule-associated proteins. Rho-Rac guanine nucleotide exchange factor 2 (ARHGEF2), which activates Ras homolog family member A (RHOA), is anchored to the microtubule network and sequestered in an inhibited state through binding to dynein light chain Tctex-1 type 1 (DYNLT1). We showed in mammalian cells that liver kinase B1 (LKB1) activated the microtubule affinity-regulating kinase 3 (MARK3), which in turn phosphorylated ARHGEF2 at Ser151 This modification disrupted the interaction between ARHGEF2 and DYNLT1 by generating a 14-3-3 binding site in ARHGEF2, thus causing ARHGEF2 to dissociate from microtubules. Phosphorylation of ARHGEF2 by MARK3 stimulated RHOA activation and the formation of stress fibers and focal adhesions, and was required for organized cellular architecture in three-dimensional culture. Protein phosphatase 2A (PP2A) dephosphorylated Ser151 in ARHGEF2 to restore the inhibited state. Thus, we have identified a regulatory switch controlled by MARK3 that couples microtubules to the actin cytoskeleton to establish epithelial cell polarity through ARHGEF2.

Gene expression analysis of microtubule affinity-regulating kinase 2 in non-small cell lung cancer.

Lung cancer is the leading cause of cancer death worldwide, and has a five-year survival rate of 18% [1]. MARK2 is a serine/threonine-protein kinase, and is a key component in the phosphorylation of microtubule-associated proteins [2], [3]. A recent study published by Hubaux et al. found that microtubule affinity-regulating kinase 2 (MARK2) showed highly frequent DNA and RNA level disruption in lung cancer cell lines and independent non-small cell lung cancer (NSCLC) cohorts [4]. These alterations result in the acquisition of oncogenic properties in cell lines, such as increased viability and anchorage-independent growth. Furthermore, a microarray-based transcriptome analysis of three short hairpin RNA (shRNA)-mediated MARK2 knockdown lung adenocarcinoma cell lines (GEO#: GSE57966) revealed an association between MARK2 gene expression and cell cycle activation and DNA damage response. Here, we present a detailed description of transcriptome analysis to support the described role of MARK2 in promoting a malignant phenotype.

Tissue plasminogen activator regulates Purkinje neuron development and survival.

The cerebellar cortex is centrally involved in motor coordination and learning, and its sole output is provided by Purkinje neurons (PNs). Growth of PN dendrites and their major synaptic input from granule cell parallel fiber axons takes place almost entirely in the first several postnatal weeks. PNs are more vulnerable to cell death than most other neurons, but the mechanisms remain unclear. We find that the homozygous nervous (nr) mutant mouse's 10-fold-increased cerebellar tissue plasminogen activator (tPA), a part of the tPA/plasmin proteolytic system, influences several different molecular mechanisms, each regulating a key aspect of postnatal PN development, followed by selective PN necrosis, as follows. (i) Excess endogenous or exogenous tPA inhibits dendritic growth in vivo and in vitro by activating protein kinase Cγ and phosphorylation of microtubule-associated protein 2. (ii) tPA/plasmin proteolysis impairs parallel fiber-PN synaptogenesis by blocking brain-derived neurotrophic factor/tyrosine kinase receptor B signaling. (iii) Voltage-dependent anion channel 1 (a mitochondrial and plasma membrane protein) bound with kringle 5 (a peptide derived from the excess plasminogen) promotes pathological enlargement and rounding of PN mitochondria, reduces mitochondrial membrane potential, and damages plasma membranes. These abnormalities culminate in young nr PN necrosis that can be mimicked in wild-type PNs by exogenous tPA injection into cerebellum or prevented by endogenous tPA deletion in nr:tPA-knockout double mutants. In sum, excess tPA/plasmin, through separate downstream molecular mechanisms, regulates postnatal PN dendritogenesis, synaptogenesis, mitochondrial structure and function, and selective PN viability.

Monoubiquitination Promotes Calpain Cleavage of the Protein Phosphatase 2A (PP2A) Regulatory Subunit α4, Altering PP2A Stability and Microtubule-associated Protein Phosphorylation

Multiple neurodegenerative disorders are linked to aberrant phosphorylation of microtubule-associated proteins (MAPs). Protein phosphatase 2A (PP2A) is the major MAP phosphatase; however, little is known about its regulation at microtubules. α4 binds the PP2A catalytic subunit (PP2Ac) and the microtubule-associated E3 ubiquitin ligase MID1, and through unknown mechanisms can both reduce and enhance PP2Ac stability. We show MID1-dependent monoubiquitination of α4 triggers calpain-mediated cleavage and switches α4's activity from protective to destructive, resulting in increased Tau phosphorylation. This regulatory mechanism appears important in MAP-dependent pathologies as levels of cleaved α4 are decreased in Opitz syndrome and increased in Alzheimer disease, disorders characterized by MAP hypophosphorylation and hyperphosphorylation, respectively. These findings indicate that regulated inter-domain cleavage controls the dual functions of α4, and dysregulation of α4 cleavage may contribute to Opitz syndrome and Alzheimer disease.

Abstract 17221: AMPK Regulation of Microtubule Dynamics in Cardiac Hypertrophy

Cardiomyocyte hypertrophy requires increased protein synthesis as well as remodeling/growth of the cytoskeletal networks necessary to support cell enlargement. AMPK is a metabolic sensor that couples cell growth to ATP levels. Whether AMPK regulates cardiomyocyte cytoskeletal dynamics and how this influences hypertrophy is not known. Here we used cultured neonatal cardiomyocytes and AMPK KO mice to examine the role of AMPK in cytoskeletal remodeling that occurs during hypertrophy. In cardiomyocytes exposed to hypertrophic stress (phenylephrine or constitutively active Raf), AMPK activation (AICAR 0.1-0.2 mM) or constitutively active AMPK had little effect on intermediate filaments or myofilaments, but did attenuate accumulation and stabilization of microtubules (AICAR reduced MT levels to 38% +/- 3.6 % and detyrosinated MTs to 14% +/- 11% of PE treated; p<.001). Importantly, AMPK reduction of microtubule stability occurred at low doses of AICAR that did not block mTOR/p70S6k signaling or translation, suggesting microtubule dynamics are an extremely sensitive target of AMPK. Interestingly, AMPK reduction of microtubules limited cardiomyocyte elongation, suggesting a mechanism by which AMPK influences cell morphology. In mice exposed to pressure overload (transverse aortic constriction;TAC), microtubule levels were exacerbated by AMPK alpha 2 KO as compared to WT mice (202 % +/- 42%; p=.047). Furthermore, microtubule levels exhibited a significant inverse correlation with LV ejection fraction (R 2 =.752; p<.001), suggesting microtubule densification may contribute to reduced contractile function in AMPK KO mice. Mechanistically, AMPK activation increased phosphorylation of Microtubule Associated Protein 4 (MAP4) at serine 768, which prevents its ability to stabilize microtubules. Phosphorylation of MAP4 Ser768 was reduced in AMPK KO mice, but increased in WT mice exposed to metformin, which also reduced MT stability. RNAi depletion of MAP4 in isolated cardiomyocytes further confirmed its important role in stabilizing microtubules during hypertrophy. Together, our data identify a novel protective role for AMPK in preventing aberrant stabilization and accumulation of cardiac microtubules in response to hypertrophic stress.

CAMP-Dependent Phosphorylation of Microtubule-Associated Protein-2 during Treatment of Sodium Valproate and Audiogenic Kindling

Administration of anticonvulsant sodium valproate alleviated audiogenic seizures in Krushinskii-Molodkina rats, which was accompanied by a decrease in cAMP-dependent phosphorylation of microtubule-associated protein MAP2 in the hippocampus ex vivo. In contrast, audiogenic kindling resulted in a marked increase in MAP2 phosphorylation at cAMP-dependent protein kinase-specific sites. These changes in the state of MAP2 phosphorylation providing restructuring of dendrites in response to specific influences modulate neuronal activity and are the important mechanisms of neuronal plasticity.

The Distribution of MAP-2 Phosphorylation in Cerebral Cortex of Long-Tailed Monkey Fetuses (Macaca fascicularis) in the Last Trimester of Gestation

Memories are storage in cholinoceptive cells, the cells which are enriched with microtubule-associated protein 2 (MAP-2) that localized in the neuronal dendrite and the cell bodies. Phosphorylation of MAP-2 may increase memory with reduce stability of dendrite by altered dendrite length and lead new side-branches of neuronal as a neuronal plasticity processes in cerebral cortex. The aim of this research is to study the distribution of MAP-2 phosphorylation neurons in cerebral cortex of long-tailed macaques in the third semester of gestational immunohistochemically using avidin biotin conjugated complex method. Neurons MAP-2 phosphorylation immunoreactive were located in dendrites and cell bodies, mostly in pyramidal neurons of cerebral cortex. Intensity of MAP-2 phosphorylation immunoreactivity in layer V were stronger than another layer and the neurons that very intensely stained were the pyramidal cells in frontal and parietal lobes, that was suggested that neurons in this areas more responsive to neuroplasticity. From the results we concluded that MAP-2 phosphorylation already distributed in the cerebral cortex of long-tailed macaque fetuses at the last trimester of gestation, mostly in the pyramidal cells of layer V that is suggested plays a role for preparation of memory formation. Keywords: fetus, long-tailed monkey, cerebral cortex, memory, MAP-2 phosphorylation

Molecular function of microtubule-associated protein 2 for filial imprinting in domestic chicks ( Gallus gallus domesticus)

RNA interference (RNAi)-mediated gene-silencing can be a tool for elucidating the role of genes in the neural basis of behavioral plasticity. Previously, we reported that exogenous DNA could be successfully delivered into newly-hatched chick brains via electroporation. Here, we used this in vivo gene-transfer technique and showed that transfected microRNA vectors preferentially silence exogenous DNA expression in neuronal cells. Using this system, the up-regulation of microtubule-associated protein 2 (MAP2) accompanying filial imprinting was suppressed in vivo, which impaired the filial imprinting in chicks. In addition, the phosphorylation of MAP2 was found to increase in parallel with filial imprinting, and lithium chloride, an inhibitor of glycogen synthase kinase 3 (GSK3), was found to impair filial imprinting. Our results suggest that the regulation of MAP2 expression and its phosphorylation are required for filial imprinting and may modify microtubule stability, thereby leading to cytoskeletal reorganization during imprinting. This in vivo RNAi-mediated gene-silencing system will facilitate the analysis of gene function in the living chick brain and provides further clues regarding the molecular mechanisms underpinning avian learning.

Compression Alters Kinase and Phosphatase Activity and Tau and MAP2 Phosphorylation Transiently while Inducing the Fast Adaptive Dendritic Remodeling of Underlying Cortical Neurons

In traumatic brain injury (TBI) there is often compression of the cerebral cortex. Using a rat epidural bead implantation model we found that mechanical compression distorted the dendrites of underlying cortical pyramidal neurons, and that the deformed dendrites regained straight morphology in 3 days. This was accompanied by a transient increase in the phosphorylation of microtubule-associated proteins (MAPs) at sites known to destabilize microtubules, including MAP2 from 30 min to 1 h, and tau from 10 min to 12 h following compression. Immunostaining confirmed that phosphorylated MAPs were concentrated at the somata and dendrites of compressed cortical pyramidal neurons. Enzymes regulating MAP phosphorylation were found to be simultaneously altered, including downregulation of protein phosphatase 2A, but not 2B, activity from 10 min to 1 day, and transient excitatory phosphorylation of extracellular signal-regulated protein kinase 1/2 and p38/mitogen-activated protein kinase. The temporal coincidence of these events suggests that alterations of phosphatase and kinase activity underlie MAP2 and tau phosphorylation, thus causing the compressed cortical neurons to remodel their dendrites, including the proximal segments. The rapid onset of these molecular changes demonstrates that compression causes cortical neurons to undergo active changes much early than expected. The large-scale structural changes that result can alter cortical function for an extended period of time.

Investigating the Regulation of Brain-specific Kinases 1 and 2 by Phosphorylation

Brain-specific kinases 1 and 2 (BRSK1/2) are AMP-activated protein kinase (AMPK)-related kinases that are highly expressed in mammalian forebrain. Studies using transgenic animal models have implicated a role for these kinases in the establishment of neuronal polarity. BRSK1 and BRSK2 are activated by phosphorylation of a threonine residue in the T-loop activation segment of the kinase domain. In vitro studies have demonstrated that LKB1, an upstream kinase in the AMPK cascade, can catalyze this phosphorylation. However, to date, a detailed comparative analysis of the molecular regulation of BRSK1/2 has not been undertaken. Here we present evidence that excludes another upstream kinase in the AMPK cascade, Ca(2+)/calmodulin-dependent protein kinase kinase beta, from a role in activating BRSK1/2. We show that equivalent mutations in the ubiquitin-associated domains of the BRSK isoforms produce differential effects on the activation of BRSK1 and BRSK2. Contrary to previous reports, activation of cAMP-dependent protein kinase does not affect BRSK1 or BRSK2 activity in mammalian cells. Furthermore, stimuli that activate AMPK had no effect on BRSK1/2. Finally, we provide evidence suggesting that protein phosphatase 2C is a likely candidate for catalyzing the dephosphorylation and inactivation of BRSK1/2.

Epinephrine Increases Phosphorylation of MAP-2c in Rat Pheochromocytoma Cells (PC12 Cells) via a Protein Kinase C- and Mitogen Activated Protein Kinase-Dependent Mechanism

Adrenoceptors mediate effects of endogenous catecholamines and have been shown to affect the neuronal development. Microtubule-associated protein-2 (MAP-2) is an important cytoskeleton protein whose phosphorylation in response to extracellular signal is involved in the regulation of neurite outgrowth and neuronal plasticity. The present study was designed to determine the effect of activation of adrenoceptor by epinephrine on MAP-2 phosphorylation in differentiation PC12 cells and, if so, to explore the mediating mechanism. We found that epinephrine could significantly increase the phosphorylation of MAP-2c at ser136 in a dose- and time-dependent manner in differentiated PC12 cells as well as microtubule arrays. Differentiated PC12 cells express alpha 2A-adrenoceptor, whose antagonists could block these mentioned effects of epinephrine, and clonidine which is the agonist of alpha 2-adrenoceptor could mimic the effect of epinephrine. Moreover phosphorylation of ERK and PKC was induced by epinephrine, and ERK and PKC specific inhibitors concentration-dependently prevented epinephrine-induced phosphorylation of MAP-2c at ser136. In addition, pretreatment of PC12 cells with epinephrine partly inhibited 30 microM nocodazole induced neurites retraction. These findings suggest that epinephrine induces phosphorylation of MAP-2c at ser136 through a alpha 2-adrenoceptor mediated, ERK/PKC-dependent signaling pathway, which may contribute to the stabilization of neurites.

Hepatocyte growth factor and c-Met promote dendritic maturation during hippocampal neuron differentiation via the Akt pathway

During central nervous system development, growth factors and their associated receptor protein tyrosine kinases regulate many neuronal functions such as neurite extension and dendrite maturation. Hepatocyte growth factor (HGF) and its receptor, c-Met, can promote formation of neurites and enhance elaboration of dendrites in mature neurons, but their effects on the early stages of dendrite maturation in hippocampal neurons and the signaling pathways by which they promote dendrite formation have not been studied. Exogenous HGF treatment effectively enhanced the phosphorylation and activation of c-Met in cultured hippocampal neurons at 4 days in vitro. HGF treatment increased the number of dendrites and promoted dendrite elongation in these neurons. Consistent with these results, HGF activated Akt, which phosphorylates glycogen synthase kinase-3β (GSK-3β) to inactivate it, and reduced phosphorylation of microtubule-associated protein 2 (MAP2), which can promote microtubule polymerization and dendrite elongation when dephosphorylated. Conversely, pharmacological inhibition of c-Met with its specific inhibitor, PHA-665752, or genetic knock-down of c-Met with short hairpin RNAs (shRNAs) suppressed HGF-induced phosphorylation of Akt and GSK-3β, increased phosphorylation of MAP2, and reduced dendrite number and length in cultured hippocampal neurons. Moreover, suppressing c-Met with PHA-665752 or by shRNA decreased MAP2 expression. Inhibiting Akt activity with the phosphoinositide-3-kinase inhibitor LY294002 or Akt inhibitor X suppressed HGF-induced phosphorylation of GSK-3β, increased MAP2 phosphorylation, and blocked the ability of HGF to enhance dendritic length. These observations indicate that HGF and c-Met can regulate the early stages of dendrite maturation via activation of the Akt/GSK-3β pathway.

Very-KIND, a KIND domain–containing RasGEF, controls dendrite growth by linking Ras small GTPases and MAP2

The regulation of cytoskeletal components in the dendritic shaft core is critical for dendrite elongation and branching. Here, we report that a brain-specific Ras guanine nucleotide exchange factor (RasGEF) carrying two kinase non-catalytic C-lobe domains (KINDs), very-KIND (v-KIND), regulates microtubule-associated protein 2 (MAP2). v-KIND is expressed in developing mouse brain, predominantly in the cerebellar granule cells. v-KIND not only activates Ras small GTPases via the C-terminal RasGEF domain, but also specifically binds to MAP2 via the second KIND domain (KIND2), leading to threonine phosphorylation of MAP2. v-KIND overexpression suppresses dendritic extension and branching of hippocampal neurons and cerebellar granule cells, whereas knockdown of endogenous v-KIND expression promotes dendrite growth. These findings suggest that v-KIND mediates a signaling pathway that links Ras and MAP2 to control dendrite growth.

Neurotoxic effects of dietary aluminium.

Neurochemical responses to chronic oral aluminium administration have been studied in rats. Aluminium (0.3%) was added to drinking water of adult rats for four weeks or longer and weanling rats were given aluminium for eight weeks. Selective cognitive impairment was demonstrated in the adult rats. Aluminium inhibited calcium flux and phosphoinositide metabolism, one product of which (inositol 1,4,5-trisphosphate) modulates intracellular calcium levels. In weanling rats aluminium decreased the in vivo concentration of inositol 1,4,5-trisphosphate in the hippocampus. An increase in cyclic AMP concentrations by 30-70% in various brain regions in adult and weanling rats was found. Aluminium enhanced agonist-stimulated but not basal cyclic AMP production in vitro. It was postulated that aluminium inhibits the GTPase activity of the stimulatory G protein, Gs, leading to prolonged activation of Gs after receptor stimulation and increased cyclic AMP production. Aluminium treatment also increased the phosphorylation of microtubule-associated protein 2 (MAP-2) and the 200 kDa neurofilament protein (NF-H) but several other phosphoproteins were unaffected. Concentrations of seven structural proteins--MAP-2, tau, NF-H, NF-M (150 kDa), NF-L (68 kDa), tubulin and spectrin--were measured in rat brain regions by immunoblot methods. MAP-2 was most consistently decreased. These studies show that chronic oral aluminium administration to rats has significant neurochemical consequences. Three sites of action are implicated: altered calcium homeostasis, enhanced cyclic AMP production, and changes in cytoskeletal protein phosphorylation states and concentrations.

Suppression of Tubulin Polymerization by the LKB1-Microtubule-associated Protein/Microtubule Affinity-regulating Kinase Signaling

LKB1, a tumor suppressor gene mutated in the Peutz-Jeghers syndrome, encodes a serine/threonine protein kinase. Recent biochemical studies have shown that LKB1 activates 14 AMP-activated protein kinase-related kinases including MARKs (microtubule-associated protein/microtubule affinity-regulating kinases) that regulate microtubule dynamics. Here we show in vitro that LKB1 phosphorylates and activates MARK2, which in turn phosphorylates microtubule-associated protein Tau at the KXGS motif and suppresses tubulin polymerization. In cells, forced expression of LKB1 suppresses microtubule regrowth, whereas LKB1 knockdown accelerates it. We further show that the phosphorylation of Tau by the LKB1-MARK signaling triggers proteasome-mediated degradation of Tau. These results indicate that LKB1 is involved in the regulation of microtubule dynamics through the activation of MARKs.

Doublecortin-like Kinase Controls Neurogenesis by Regulating Mitotic Spindles and M Phase Progression

The mechanisms controlling neurogenesis during brain development remain relatively unknown. Through a differential protein screen with developmental versus mature neural tissues, we identified a group of developmentally enriched microtubule-associated proteins (MAPs) including doublecortin-like kinase (DCLK), a protein that shares high homology with doublecortin (DCX). DCLK, but not DCX, is highly expressed in regions of active neurogenesis in the neocortex and cerebellum. Through a dynein-dependent mechanism, DCLK regulates the formation of bipolar mitotic spindles and the proper transition from prometaphase to metaphase during mitosis. In cultured cortical neural progenitors, DCLK RNAi Lentivirus disrupts the structure of mitotic spindles and the progression of M phase, causing an increase of cell-cycle exit index and an ectopic commitment to a neuronal fate. Furthermore, both DCLK gain and loss of function in vivo specifically promote a neuronal identity in neural progenitors. These data provide evidence that DCLK controls mitotic division by regulating spindle formation and also determines the fate of neural progenitors during cortical neurogenesis.

Constitutively Active Cytoplasmic c-Jun N-Terminal Kinase 1 Is a Dominant Regulator of Dendritic Architecture: Role of Microtubule-Associated Protein 2 as an Effector

Normal functioning of the nervous system requires precise regulation of dendritic shape and synaptic connectivity. Here, we report a severe impairment of dendritic structures in the cerebellum and motor cortex of c-Jun N-terminal kinase 1 (JNK1)-deficient mice. Using an unbiased screen for candidate mediators, we identify the dendrite-specific high-molecular-weight microtubule-associated protein 2 (MAP2) as a JNK substrate in the brain. We subsequently show that MAP2 is phosphorylated by JNK in intact cells and that MAP2 proline-rich domain phosphorylation is decreased in JNK1-/- brain. We developed compartment-targeted JNK inhibitors to define whether a functional relationship exists between the physiologically active, cytosolic pool of JNK and dendritic architecture. Using these, we demonstrate that cytosolic, but not nuclear, JNK determines dendritic length and arbor complexity in cultured neurons. Moreover, we confirm that MAP2-dependent process elongation is enhanced after activation of JNK. Using JNK1-/- neurons, we reveal a dominant role for JNK1 over ERK in regulating dendritic arborization, whereas ERK only regulates dendrite shape under conditions in which JNK activity is low (JNK1-/- neurons). These results reveal a novel antagonism between JNK and ERK, potentially providing a mechanism for fine-tuning the dendritic arbor. Together, these data suggest that JNK phosphorylation of MAP2 plays an important role in defining dendritic architecture in the brain.