Ser289 phosphorylation activates both DAPK1 and DAPK2 but in response to different intracellular signaling pathways

DAPK1 and DAPK2 are calmodulin (CaM)-regulated protein kinases that share a high degree of homology in their catalytic and CaM regulatory domains. Both kinases function as tumor suppressors, and both have been implicated in autophagy regulation. Over the years, common regulatory mechanisms for the two kinases as well as kinase-specific ones have been identified. In a recent work, we revealed that DAPK2 is phosphorylated on Ser289 by the metabolic sensor AMPK, and that this phosphorylation enhances DAPK2 catalytic activity. Notably, Ser289 is conserved between DAPK1 and DAPK2, and was previously found to be phosphorylated in DAPK1 by RSK. Intriguingly, Ser289 phosphorylation was conversely reported to inhibit the pro-apoptotic activity of DAPK1 in cells. However, as the direct effect of this phosphorylation on DAPK1 catalytic activity was not tested, indirect effects were not excluded. Here, we compared Ser289 phosphorylation of the two kinases in the same cells and found that the intracellular signaling pathways that lead to Ser289 phosphorylation are mutually-exclusive and different for each kinase. In addition, we found that Ser289 phosphorylation in fact enhances DAPK1 catalytic activity, similar to the effect on DAPK2. Thus, Ser289 phosphorylation activates both DAPK1 and DAPK2, but in response to different intracellular signaling pathways.     

Regulation by thyroid hormone of microtubule assembly and neuronal differentiation

In this review we examine successively: 1) the major effects of thyroid hormone deficiency seen during brain development with special emphasis on the changes in neuronal morphology and migration occurring postnatally in the cerebellum. 2) The effects of this hormone on microtubule assembly during neurite outgrowth and acquisition of neuronal polarity. 3) The changes in expression of the different tubulin isoforms occurring during development in the normal and hypothyroid rat brain. 4) The regulation by thyroid hormone of the transition occurring during development between the juvenile and adult microtubule-associated protein Tau.Special issue dedicated to Dr. Louis Sokoloff.     

Dynamic regulation of GEF-H1 localization at microtubules by Par1b/MARK2

Par1b/MARK2 is a serine/threonine kinase that plays key roles in the development of cell polarity, but its precise mechanism of action remains unknown. Here we report that GEF-H1, a guanine nucleotide exchange factor for Rho-family small GTPases, is a novel substrate for Par1b. GEF-H1 directly associates with microtubules via its N-terminal C1 domain, which is known to regulate the activity of GEF-H1. Ectopically expressed GEF-H1 markedly promotes stabilization of microtubules, resulting in acetylation of microtubules. We find that Par1b phosphorylates GEF-H1 at three serine residues conserved in vertebrates and releases GEF-H1 from microtubules, which abrogates stabilization and acetylation of microtubules induced by GEF-H1 overexpression. The alanine mutant for the three phosphorylation sites (3SA) of GEF-H1 strongly induces stabilization and acetylation of microtubules, which was resistant to Par1b. Time-lapse imaging analyses reveal that GFP-fused GEF-H1 dynamically moved on microtubules from one protrusion to another, whereas the 3SA mutant was static. These data suggest that Par1b-phosphorylation regulates turnover of GEF-H1 localization by regulating its interaction with microtubules, which may contribute to cell polarization.     

Death-associated protein kinase 1 has a critical role in aberrant tau protein regulation and function

The presence of tangles composed of phosphorylated tau is one of the neuropathological hallmarks of Alzheimer''s disease (AD). Tau, a microtubule (MT)-associated protein, accumulates in AD potentially as a result of posttranslational modifications, such as hyperphosphorylation and conformational changes. However, it has not been fully understood how tau accumulation and phosphorylation are deregulated. In the present study, we identified a novel role of death-associated protein kinase 1 (DAPK1) in the regulation of the tau protein. We found that hippocampal DAPK1 expression is markedly increased in the brains of AD patients compared with age-matched normal subjects. DAPK1 overexpression increased tau protein stability and phosphorylation at multiple AD-related sites. In contrast, inhibition of DAPK1 by overexpression of a DAPK1 kinase-deficient mutant or by genetic knockout significantly decreased tau protein stability and abolished its phosphorylation in cell cultures and in mice. Mechanistically, DAPK1-enhanced tau protein stability was mediated by Ser71 phosphorylation of Pin1, a prolyl isomerase known to regulate tau protein stability, phosphorylation, and tau-related pathologies. In addition, inhibition of DAPK1 kinase activity significantly increased the assembly of MTs and accelerated nerve growth factor-mediated neurite outgrowth. Given that DAPK1 has been genetically linked to late onset AD, these results suggest that DAPK1 is a novel regulator of tau protein abundance, and that DAPK1 upregulation might contribute to tau-related pathologies in AD. Therefore, we offer that DAPK1 might be a novel therapeutic target for treating human AD and other tau-related pathologies.     

Sodium arsenite-induced DAPK promoter hypermethylation and autophagy via ERK1/2 phosphorylation in human uroepithelial cells

Arsenic compounds or arsenicals are well-known toxic and carcinogenic agents. The toxic effects of arsenic that are of most concern to humans are those that occur from chronic, low-level exposure, and are associated with various human malignancies, including skin, lung and bladder cancers. In addition, arsenic could induce cell death, including apoptosis or autophagy in malignant cells. Previously, we have demonstrated that arsenite can induce autophagy and death-associated protein kinase (DAPK) promoter hypermethylation in the SV-40 immortalized human uroepithelial cell line (SV-HUC-1). However, the underlying mechanism of arsenite-induced autophagy is still unclear. In the present study, we demonstrate that arsenite can activate the extracellular signaling-regulated protein kinase 1/2 (ERK1/2) signaling pathway after treatment in SV-HUC-1 cells by using immunocytochemistry and Western blotting. In addition, our results also show an increase of autophagosomes was produced in arsenite-treated SV-HUC-1 cells by using electron microscopy. We found that, by incrementally increasing the dosages, microtubule-associated protein light chain 3B (LC3B) and Beclin-1 are important regulators for the formation of autophagosomes, in a dose-dependent manner. When the cells were pretreated with inhibitors 5-aza-CdR or U0126 for 24 h, the effect of arsenite on ERK1/2, LC3B, Beclin-1 and DAPK proteins expression is suppressed. Furthermore, our results support the notion that arsenite can induce the ERK1/2 signaling pathway to stimulate autophagy and DAPK promoter hypermethylation in human uroepithelial SV-HUC-1 cells. These findings may contribute to a better understanding of the carcinogenesis of arsenite.     

口腔白斑抑癌基因DAPK和TIG1高甲基化表达研究

摘要 目的：探讨抑癌基因DAPK、TIG1高甲基化在口腔白斑中表达状态及其对口腔癌发生发展中的作用。方法：取77例口腔白斑、32例口腔鳞癌、32份正常口腔黏膜组织,用实时定量甲基化特异性PCR技术检测组织中DAPK、TIG1高甲基化表达并进行统计学分析。结果：DAPK在口腔鳞癌组织中高甲基化表达率为46.9%,表达量为（0.0728±0.1617）,明显高于其在口腔白斑组织（19.5%,0.0070±0.0172）和口腔正常组织（18.8%,0.0021±0.0050）中的表达,差异有统计学意义（P<0.05）。DAPK高甲基化表达与口腔白斑组织上皮异常增生程度相关,上皮增生高风险组相对于低风险组DAPK高甲基化表达风险增加（OR,1.013;95% CI,1.004-1.023;P=0.004）。TIG1高甲基化在正常组织中未表达,在口腔鳞癌组织和口腔白斑组织表达为（28.1%,0.0174±0.0440）和（27.3%,0.0035±0.0076）,与正常组织相比具有统计学意义（P<0.05）。结论：抑癌基因 DAPK、TIG1高甲基化有望成为口腔黏膜癌变早期标志物。     

The toxicity of tau in Alzheimer disease: turnover, targets and potential therapeutics

It has been almost 25 years since the initial discovery that tau was the primary component of the neurofibrillary tangles (NFTs) in Alzheimer disease (AD) brain. Although AD is defined by both β-amyloid (Aβ) pathology (Aβ plaques) and tau pathology (NFTs), whether or not tau played a critical role in disease pathogenesis was a subject of discussion for many years. However, given the increasing evidence that pathological forms of tau can compromise neuronal function and that tau is likely an important mediator of Aβ toxicity, there is a growing awareness that tau is a central player in AD pathogenesis. In this review we begin with a brief history of tau, then provide an overview of pathological forms of tau, followed by a discussion of the differential degradation of tau by either the proteasome or autophagy and possible mechanisms by which pathological forms of tau may exert their toxicity. We conclude by discussing possible avenues for therapeutic intervention based on these emerging themes of tau's role in AD.     

Pin1 inhibition activates cyclin D and produces neurodegenerative pathology

Abnormal cell cycle events are increasingly becoming important attributes of neurodegenerative pathology. Pin1 is a crucial target of neurodegeneration in relation to its functions regarding these abnormal cell cycle events in neurons. Pin1 is majorly involved in many aspects of cell cycle regulation and it has also been suggested to have a neuroprotective function against neurodegenerative pathologies. Oxidative dysregulation of Pin1 affects not only normal tau regulation, eventually causing tangle formation, but also cell cycle regulation in neurons. Presence of cell cycle proteins has been shown in many neurodegenerative diseases. Importantly, many of these proteins have physical interactions with Pin1. Hence, understanding Pin1's role in abnormal cell cycle re-entry is critical in terms of finding new approaches for the future therapeutic options treating neurodegenerative pathologies. Here, we show that inhibition of Pin1 by its selective inhibitor juglone leads to up-regulation of cyclinD1, phospho-tau, and caspase 3, producing apoptosis in cultured rat hippocampal neurons. We also observed axonal retraction with a change in sub-cellular localizations of cyclins. Therefore, Pin1 dysregulation, in relation to its role in cell cycle regulation in neurons, may have profound effects in the progression of neurodegenerative pathology, making it a possible crucial target behind many neurodegenerative diseases.     

Microtubule affinity–regulating kinase 4 with an Alzheimer's disease-related mutation promotes tau accumulation and exacerbates neurodegeneration

Accumulation of the microtubule-associated protein tau is associated with Alzheimer''s disease (AD). In AD brain, tau is abnormally phosphorylated at many sites, and phosphorylation at Ser-262 and Ser-356 plays critical roles in tau accumulation and toxicity. Microtubule affinity–regulating kinase 4 (MARK4) phosphorylates tau at those sites, and a double de novo mutation in the linker region of MARK4, ΔG316E317D, is associated with an elevated risk of AD. However, it remains unclear how this mutation affects phosphorylation, aggregation, and accumulation of tau and tau-induced neurodegeneration. Here, we report that MARK4^ΔG316E317D increases the abundance of highly phosphorylated, insoluble tau species and exacerbates neurodegeneration via Ser-262/356–dependent and –independent mechanisms. Using transgenic Drosophila expressing human MARK4 (MARK4^wt) or a mutant version of MARK4 (MARK4^ΔG316E317D), we found that coexpression of MARK4^wt and MARK4^ΔG316E317D increased total tau levels and enhanced tau-induced neurodegeneration and that MARK4^ΔG316E317D had more potent effects than MARK4^wt. Interestingly, the in vitro kinase activities of MARK4^wt and MARK4^ΔG316E317D were similar. When tau phosphorylation at Ser-262 and Ser-356 was blocked by alanine substitutions, MARK4^wt did not promote tau accumulation or exacerbate neurodegeneration, whereas coexpression of MARK4^ΔG316E317D did. Both MARK4^wt and MARK4^ΔG316E317D increased the levels of oligomeric forms of tau; however, only MARK4^ΔG316E317D further increased the detergent insolubility of tau in vivo. Together, these findings suggest that MARK4^ΔG316E317D increases tau levels and exacerbates tau toxicity via a novel gain-of-function mechanism and that modification in this region of MARK4 may affect disease pathogenesis.     

Overexpression of tau protein in COS-1 cells results in the stabilization of centrosome-independent microtubules and extension of cytoplasmic processes

COS-1 cells were transfected with cDNAs coding for different human tau isoforms. Expressed tau isoforms bind to cellular microtubulesin vivo, preferentially at the distal regions of microtubules nucleated by the centrosome, leading to their stabilization. Eventually, tau-coated microtubules without any association with the centrosome were observed. A major difference between tau isoforms containing three tubulin-binding motifs and tau isoforms containing four tubulin-binding motifs is the greater ability of the latter in inducing the formation of long cytoplasmic processes.     

DAPK1、hTERT在口腔鳞癌中的表达及其临床意义研究

目的:探讨凋亡相关蛋白激酶1(DAPK1)、端粒酶催化亚单位(hTERT)在口腔鳞状细胞癌(OSCC)中的表达,并探讨其临床意义。方法:应用免疫组织化学方法检测DAPK1、hTERT在93例OSCC组织及10例癌旁正常组织中的表达,并分析其与OSCC临床病理参数之间的关系及其在OSCC浸润前沿中的作用。结果:癌旁正常组织中DAPK1的表达显著高于OSCC组织中DAPK1的表达,且高、中、低分化OSCC组织中DAPK1的表达比较均有显著差异(P0.05),OSCC浸润前沿组织中DAPK1的表达低于非前沿部分(P0.05),而DAPK1的表达与OSCC浸润前沿的IFG总分无显著相关性(P0.05)。癌旁正常组织中hTERT的表达显著低于OSCC组织中hTERT的表达(P0.05),且高、中、低分化OSCC组织中hTERT的表达比较均有显著差异(P0.05);OSCC浸润前沿组织中hTERT的表达高于非前沿部分(P0.05),且与OSCC浸润前沿的IFG总分有关(P0.05)。DAPK1、hTERT的表达均与OSCC患者的性别、年龄、淋巴结转移无显著相关性(P0.05)。结论:口腔鳞状细胞癌组织,特别是其前沿组织中DAPK1的表达显著下调和hTERT的表达明显上调,可能通过阻碍口腔鳞状细胞癌细胞凋亡,共同促进口腔鳞状细胞癌的生长、分化和浸润。     

MEK1/2 is a critical regulator of microtubule assembly and spindle organization during rat oocyte meiotic maturation

MEK (MAPK kinase) is an upstream protein kinase of MAPK in the MOS/MEK/MAPK/p90rsk signaling pathway. We previously reported the function and regulation of MAPK during rat oocyte maturation. In this study, we further investigated the localization and possible roles of MEK1/2. First, immunofluorescent staining revealed that p-MEK1/2 was restricted to the germinal vesicle (GV). After germinal vesicle breakdown (GVBD), p-MEK1/2 condensed in the vicinity of chromosomes and then translocated to the spindle poles at metaphase I, while spindle microtubules stained faintly. When the oocyte went through anaphase I and telophase I, p-MEK1/2 disappeared from spindle poles and became associated with the midbody. By metaphase II, p-MEK1/2 was again localized to the spindle poles. Second, p-MEK1/2 was localized to the centers of cytoplasmic microtubule asters induced by taxol. Third, p-MEK1/2 co-localized with gamma-tubulin in microtubule-organizing centers (MTOCs). Forth, treatment with U0126, a non-competitive MEK1/2 inhibitor, did not affect germinal vesicle breakdown, but caused chromosome mis-alignment in all MI oocytes examined and abnormal spindle organization as well as small cytoplasmic spindle-like structure formation in MII oocytes. Finally, U0126 reduced the number of cytoplasmic asters induced by taxol. Our data suggest that MEK1/2 has regulatory functions in microtubule assembly and spindle organization during rat oocyte meiotic maturation.     

Tau-tubulin kinase 1 (TTBK1), a neuron-specific tau kinase candidate, is involved in tau phosphorylation and aggregation

Neurofibrillary tangles, which are major pathological hallmarks of Alzheimer's disease (AD), are composed of paired helical filaments (PHFs) containing hyperphosphorylated tau. Specific kinases regulate tau phosphorylation and are closely linked to the pathogenesis of AD. We have characterized a human tau-tubulin kinase 1 (TTBK1) gene located on chromosome 6p21.1. TTBK1 is a serine/threonine/tyrosine kinase that is conserved among species and belongs to the casein kinase 1 superfamily. It is specifically expressed in the brain, especially in the cytoplasm of cortical and hippocampal neurons. TTBK1 phosphorylates tau proteins in both a Mg2+- and a Mn2+-dependent manner. Phosphopeptide mapping and immunoblotting analysis confirmed a direct tau phosphorylation by TTBK1 at Ser198, Ser199, Ser202 and Ser422, which are also phosphorylated in PHFs. TTBK1 also induces tau aggregation in human neuronal cells in a dose-dependent manner. We conclude that TTBK1 is a neuron-specific dual kinase involved in tau phosphorylation at AD-related sites and is also associated with tau aggregation.     

Suppressive effects of the peel of Citrus kawachiensis (Kawachi Bankan) on astroglial activation,tau phosphorylation,and inhibition of neurogenesis in the hippocampus of type 2 diabetic db/db mice

We previously reported that the dried peel powder of Citrus kawachiensis exerted anti-inflammatory effects in the brain in several animal models. Hyperglycemia induces inflammation and oxidative stress and causes massive damage in the brain; therefore, we herein examined the anti-inflammatory and other effects of the dried peel powder of C. kawachiensis in the streptozotocin-induced hyperglycemia mice model and in the type 2 diabetic db/db mice model. The C. kawachiensis administration inhibited microglial activation in the hippocampus in the streptozotocin-injected mice. Moreover, The C. kawachiensis treatment inhibited astroglial activation in the hippocampus and the hyperphosphorylation of tau at 231 of threonine and 396 of serine in hippocampal neurons, and also relieved the suppression of neurogenesis in the dentate gyrus of the hippocampus in the db/db mice. It was suggested that the dried peel powder of C. kawachiensis exerts anti-inflammatory and neuroprotective effects in the brain.     

IGF-1对冈田酸所致细胞损伤和Tau蛋白过度磷酸化的保护作用

目的:探讨胰岛素样生长因子-1(IGF-1)对冈田酸(OA)诱导的细胞损伤和tau蛋白过度磷酸化的保护作用。方法:模型组以OA40nmol/L作用于SH-SY5Y细胞24h;IGF-1预处理组分别以100、200和400ng/mlIGF-1预处理2h,再加入OA作用24h。倒置显微镜观察细胞形态学变化;MTT法检测细胞活力;Hoechst染色和分光光度法检测Caspase-3活化程度观察细胞损伤;蛋白免疫印迹法检测tau蛋白磷酸化程度。结果:与模型组比较,IGF-1预处理组细胞形态改善,细胞活力增强,Caspase-3活化程度降低,且磷酸化tau蛋白(Ser396)水平下降。结论:IGF-1可能通过抑制tau蛋白过度磷酸化对OA诱导的细胞损伤具有保护作用。     

Defects in axonal elongation and neuronal migration in mice with disrupted tau and map1b genes

Tau and MAP1B are the main members of neuronal microtubule-associated proteins (MAPs), the functions of which have remained obscure because of a putative functional redundancy (Harada, A., K. Oguchi, S. Okabe, J. Kuno, S. Terada, T. Ohshima, R. Sato-Yoshitake, Y. Takei, T. Noda, and N. Hirokawa. 1994. Nature. 369:488-491; Takei, Y., S. Kondo, A. Harada, S. Inomata, T. Noda, and N. Hirokawa. 1997. J. Cell Biol. 137:1615-1626). To unmask the role of these proteins, we generated double-knockout mice with disrupted tau and map1b genes and compared their phenotypes with those of single-knockout mice. In the analysis of mice with a genetic background of predominantly C57Bl/6J, a hypoplastic commissural axon tract and disorganized neuronal layering were observed in the brains of the tau+/+map1b-/- mice. These phenotypes are markedly more severe in tau-/-map1b-/- double mutants, indicating that tau and MAP1B act in a synergistic fashion. Primary cultures of hippocampal neurons from tau-/-map1b-/- mice showed inhibited axonal elongation. In these cells, a generation of new axons via bundling of microtubules at the neck of the growth cones appeared to be disturbed. Cultured cerebellar neurons from tau-/-map1b-/- mice showed delayed neuronal migration concomitant with suppressed neurite elongation. These findings indicate the cooperative functions of tau and MAP1B in vivo in axonal elongation and neuronal migration as regulators of microtubule organization.     

Phosphorylation of tau at serine 416 by Ca2+/calmodulin-dependent protein kinase II in neuronal soma in brain

It is well known that tau is a good in vitro substrate for Ca2+/calmodulin-dependent protein kinase II (CaM kinase II). However, it is not clear at present whether CaM kinase II phosphorylates tau in vivo or not. Serine 416, numbered according to the longest human tau isoform, has been reported to be one of the major phosphorylation sites by CaM kinase II in vitro. In this study, we produced a specific antibody against tau phosphorylated at serine 416 (PS416-tau). Immunoblot analysis revealed that the antibody reacted with tau in the rat brain extract which was prepared in the presence of protein phosphatase inhibitors. Developmental study indicated that serine 416 was strongly phosphorylated at early developmental stages in rat brain. We examined the localization of PS416-tau in primary cultured hippocampal neurons and the immortalized GnRH neurons (GT1-7 cells), which were stably transfected with CaM kinase IIalpha cDNA. Immunostaining of these cells indicated that tau was phosphorylated mainly in neuronal soma. Interestingly, tau in neuronal soma in Alzheimer's disease (AD) brain was strongly immunostained by the antibody. These results suggest that CaM kinase II is involved in the accumulation of tau in neuronal soma in AD brain.     

Neurodegeneration risk factor,EIF2AK3 (PERK), influences tau protein aggregation

Tauopathies are neurodegenerative diseases caused by pathologic misfolded tau protein aggregation in the nervous system. Population studies implicate EIF2AK3 (eukaryotic translation initiation factor 2 alpha kinase 3), better known as PERK (protein kinase R-like endoplasmic reticulum kinase), as a genetic risk factor in several tauopathies. PERK is a key regulator of intracellular proteostatic mechanisms—unfolded protein response and integrated stress response. Previous studies found that tauopathy-associated PERK variants encoded functional hypomorphs with reduced signaling in vitro. But, it remained unclear how altered PERK activity led to tauopathy. Here, we chemically or genetically modulated PERK signaling in cell culture models of tau aggregation and found that PERK pathway activation prevented tau aggregation, whereas inhibition exacerbated tau aggregation. In primary tauopathy patient brain tissues, we found that reduced PERK signaling correlated with increased tau neuropathology. We found that tauopathy-associated PERK variants targeted the endoplasmic reticulum luminal domain; and two of these variants damaged hydrogen bond formation. Our studies support that PERK activity protects against tau aggregation and pathology. This may explain why people carrying hypomorphic PERK variants have increased risk for developing tauopathies. Finally, our studies identify small-molecule augmentation of PERK signaling as an attractive therapeutic strategy to treat tauopathies by preventing tau pathology.     

Death-associated protein kinase 1 mediates Aβ42 aggregation-induced neuronal apoptosis and tau dysregulation in Alzheimer's disease

The aggregation of amyloid-β (Aβ) peptides into oligomers and fibrils is a key pathological feature of Alzheimer''s disease (AD). An increasing amount of evidence suggests that oligomeric Aβ might be the major culprit responsible for various neuropathological changes in AD. Death-associated protein kinase 1 (DAPK1) is abnormally elevated in brains of AD patients and plays an important role in modulating tau homeostasis by regulating prolyl isomerase Pin1 phosphorylation. However, it remains elusive whether and how Aβ species influence the function of DAPK1, and whether this may further affect the function and phosphorylation of tau in neurons. Herein, we demonstrated that Aβ aggregates (both oligomers and fibrils) prepared from synthetic Aβ42 peptides were able to upregulate DAPK1 protein levels and thereby its function through heat shock protein 90 (HSP90)-mediated protein stabilization. DAPK1 activation not only caused neuronal apoptosis, but also phosphorylated Pin1 at the Ser71 residue, leading to tau accumulation and phosphorylation at multiple AD-related sites in primary neurons. Both DAPK1 knockout (KO) and the application of a specific DAPK1 inhibitor could effectively protect primary neurons against Aβ aggregate-induced cell death and tau dysregulation, corroborating the critical role of DAPK1 in mediating Aβ aggregation-induced neuronal damage. Our study suggests a mechanistic link between Aβ oligomerization and tau hyperphosphorylation mediated by DAPK1, and supports the role of DAPK1 as a promising target for early intervention in AD.