A role for glycogen synthase kinase-3beta in the mammalian circadian clock

The Drosophila shaggy gene product is a mammalian glycogen synthase kinase-3beta (GSK-3beta) homologue that contributes to the circadian clock of the Drosophila through TIMELESS phosphorylation, and it regulates nuclear translocation of the PERIOD/TIMELESS heterodimer. We found that mammalian GSK-3beta is expressed in the suprachiasmatic nucleus and liver of mice and that GSK-3beta phosphorylation exhibits robust circadian oscillation. Rhythmic GSK-3beta phosphorylation is also observed in serum-shocked NIH3T3 cells. Exposing serum-shocked NIH3T3 cells to lithium chloride, a specific inhibitor of GSK-3beta, increases GSK-3beta phosphorylation and delays the phase of rhythmic clock gene expression. On the other hand, GSK-3beta overexpression advances the phase of clock gene expression. We also found that GSK-3beta interacts with PERIOD2 (PER2) in vitro and in vivo. Recombinant GSK-3beta can phosphorylate PER2 in vitro. GSK-3beta promotes the nuclear translocation of PER2 in COS1 cells. The present data suggest that GSK-3beta plays important roles in mammalian circadian clock.     

The E3 ubiquitin ligase CTRIP controls CLOCK levels and PERIOD oscillations in Drosophila

In the Drosophila circadian clock, the CLOCK/CYCLE complex activates the period and timeless genes that negatively feedback on CLOCK/CYCLE activity. The 24-h pace of this cycle depends on the stability of the clock proteins. RING-domain E3 ubiquitin ligases have been shown to destabilize PERIOD or TIMELESS. Here we identify a clock function for the circadian trip (ctrip) gene, which encodes a HECT-domain E3 ubiquitin ligase. ctrip expression in the brain is mostly restricted to clock neurons and its downregulation leads to long-period activity rhythms in constant darkness. This altered behaviour is associated with high CLOCK levels and persistence of phosphorylated PERIOD during the subjective day. The control of CLOCK protein levels does not require PERIOD. Thus, CTRIP seems to regulate the pace of the oscillator by controlling the stability of both the activator and the repressor of the feedback loop.     

Short-period mutations of per affect a double-time-dependent step in the Drosophila circadian clock

Circadian (24 hour) PERIOD (PER) protein oscillation is dependent on the double-time (dbt) gene, a casein kinase Ivarepsilon homolog [1-3]. Without dbt activity, hypophosphorylated PER proteins over-accumulate, indicating that dbt is required for PER phosphorylation and turnover [3,4]. There is evidence of a similar role for casein kinase Ivarepsilon in the mammalian circadian clock [5,6]. We have isolated a new dbt allele, dbt(ar), which causes arrhythmic locomotor activity in homozygous viable adults, as well as molecular arrhythmicity, with constitutively high levels of PER proteins, and low levels of TIMELESS (TIM) proteins. Short-period mutations of per, but not of tim, restore rhythmicity to dbt(ar) flies. This suppression is accompanied by a restoration of PER protein oscillations. Our results suggest that short-period per mutations, and mutations of dbt, affect the same molecular step that controls nuclear PER turnover. We conclude that, in wild-type flies, the previously defined PER'short domain' [7,8] may regulate the activity of DBT on PER.     

Developmental profiles of PERIOD and DOUBLETIME in Drosophila melanogaster ovary

The clock protein PERIOD (PER) displays circadian cycles of accumulation, phosphorylation, nuclear translocation and degradation in Drosophila melanogaster clock cells. One exception to this pattern is in follicular cells enclosing previtellogenic ovarian egg chambers. In these cells, PER remains high and cytoplasmic at all times of day. Genetic evidence suggest that PER and its clock partner TIMELESS (TIM) interact in these cells, yet, they do not translocate to the nucleus. Here, we investigated the levels and subcellular localization of PER in older vitellogenic follicles. Cytoplasmic PER levels decreased in the follicular cells at the onset of vitellogenesis (stage 9). Interestingly, PER was observed in the nuclei of some follicular cells at this stage. PER signal disappeared in more advanced (stage 10) vitellogenic follicles. Since the phosphorylation state of PER is critical for the progression of circadian cycle, we investigated the status of PER phosphorylation in the ovary and the expression patterns of DOUBLETIME (DBT), a kinase known to affect PER in the clock cells. DBT was absent in previtellogenic follicular cells, but present in the cytoplasm of some stage 9 follicular cells. DBT was not distributed uniformly but was present in patches of adjacent cells, in a pattern resembling PER distribution at the same stage. Our data suggest that the absence of dbt expression in the follicular cells of previtellogenic egg chambers may be related to stable and cytoplasmic expression of PER in these cells. Onset of dbt expression in vitellogenic follicles coincides with nuclear localization of PER protein.     

Glycogen synthase kinase-3beta is tyrosine-phosphorylated by MEK1 in human skin fibroblasts

Glycogen synthase kinase-3beta (GSK-3beta) can be associated with several proteins in cell. We analyzed the immunoprecipitates by an anti-GSK-3beta antibody from cell lysate of human fibroblasts and found that this protein was co-precipitated with mitogen-activated protein kinase kinase (MEK1/2). U0126, a MEK1/2 inhibitor, inhibited tyrosine phosphorylation of GSK-3beta, suggesting that MEK1/2 was involved in the phosphorylation of Tyr(216) in GSK-3beta. In vitro kinase assay was carried out using a recombinant human active MEK1 and we found that GSK-3beta was phosphorylated on Tyr(216) by this kinase in a dose- and time-dependent manner. Further, the pretreatment of fibroblasts with U0126 inhibited serum-induced nuclear translocation of GSK-3beta. These results suggested that MEK1/2 induces tyrosine phosphorylation of GSK-3beta and this cellular event might induce nuclear translocation of GSK-3beta. This is the first report to suggest that MEK1/2 phosphorylates not only ERK1/2 but also GSK-3beta.     

Setting clock speed in mammals: the CK1 epsilon tau mutation in mice accelerates circadian pacemakers by selectively destabilizing PERIOD proteins

The intrinsic period of circadian clocks is their defining adaptive property. To identify the biochemical mechanisms whereby casein kinase1 (CK1) determines circadian period in mammals, we created mouse null and tau mutants of Ck1 epsilon. Circadian period lengthened in CK1epsilon-/-, whereas CK1epsilon(tau/tau) shortened circadian period of behavior in vivo and suprachiasmatic nucleus firing rates in vitro, by accelerating PERIOD-dependent molecular feedback loops. CK1epsilon(tau/tau) also accelerated molecular oscillations in peripheral tissues, revealing its global role in circadian pacemaking. CK1epsilon(tau) acted by promoting degradation of both nuclear and cytoplasmic PERIOD, but not CRYPTOCHROME, proteins. Together, these whole-animal and biochemical studies explain how tau, as a gain-of-function mutation, acts at a specific circadian phase to promote degradation of PERIOD proteins and thereby accelerate the mammalian clockwork in brain and periphery.     

糖尿病大鼠脑GSK-3与PP-2A失调诱导tau蛋白过度磷酸化

探讨胰岛素缺乏的糖尿病大鼠皮层糖原合酶激酶-3(GSK-3)及蛋白磷酯酶-2A(PP-2A)变化及其对tau蛋白磷酸化的作用.用链脲佐菌素(streptozotocin,STZ)建立胰岛素缺乏的糖尿病大鼠模型,用放射性配体结合实验检测了GSK-3和PP-2A的活性,蛋白质印迹检测了tau蛋白的磷酸化水平及PP-2A的表达.结果提示:在糖尿病大鼠皮层,GSK-3活性升高,PP-2A活性及表达降低,tau蛋白在Ser198/Ser199/Ser202和Ser396/Ser404位点磷酸化.应用GSK-3的选择性抑制剂Li2CO3后,GSK-3活性降低,PP-2A活性及表达恢复,tau蛋白在Ser198/Ser199/Ser202和Ser396/Ser404位点磷酸化水平降低.研究提示:糖尿病大鼠皮层GSK-3升高可能抑制PP-2A的活性,升高的GSK-3和降低的PP-2A协同促进tau蛋白的磷酸化.     

Ectopic CRYPTOCHROME renders TIM light sensitive in the Drosophila ovary

The period (per) and timeless (tim) genes play a central role in the Drosophila circadian clock mechanism. PERIOD (PER) and TIMELESS (TIM) proteins periodically accumulate in the nuclei of pace-making cells in the fly brain and many cells in peripheral organs. In contrast, TIM and PER in the ovarian follicle cells remain cytoplasmic and do not show daily oscillations in their levels. Moreover, TIM is not light sensitive in the ovary, while it is highly sensitive to this input in circadian tissues. The mechanism underlying this intriguing difference is addressed here. It is demonstrated that the circadian photoreceptor CRYPTOCHROME (CRY) is not expressed in ovarian tissues. Remarkably, ectopic cry expression in the ovary is sufficient to cause degradation of TIM after exposure to light. In addition, PER levels are reduced in response to light when CRY is present, as observed in circadian cells. Hence, CRY is the key component of the light input pathway missing in the ovary. However, the factors regulating PER and TIM levels downstream of light/cry action appear to be present in this non-circadian organ.     

Circadian proteins in the regulation of cell cycle and genotoxic stress responses

The mammalian circadian system has been implicated in the regulation of the genotoxic stress response of an organism; however, the underlying molecular mechanisms are not well understood. Recent data suggest that, in addition to circadian variations in the expression of genes involved in genotoxic stress responses, core circadian proteins PERIOD1 (PER1) and TIMELESS (TIM) interact with components of the cell cycle checkpoint system, such as ataxia telangiectasia mutated (ATM)-checkpoint kinase 2 (Chk2) and ataxia telangiectasia and Rad3-related (ATR)-Chk1, and are necessary for activation of Chk1 and Chk2 by DNA damage. Moreover, in complex with its recently identified partner, TIM-interacting protein (TIPIN), TIM interacts with components of the DNA replication system to regulate DNA replication processes under both normal and stress conditions. These discoveries shed new light on the role of core circadian proteins in various cellular and physiological processes.     

GSK-3在阿尔茨海默病样细胞骨架蛋白过度磷酸化中的作用(英)

神经原纤维缠结是阿尔茨海默病（Alzheimer disease, AD）的特征性病理改变.蛋白激酶和蛋白磷酸酯酶失衡可导致骨架蛋白的异常过度磷酸化,而异常过度磷酸化的tau 和神经丝 (neurofilament, NF) 是神经原纤维缠结的组成部分.在众多激酶中,糖原合酶激酶-3（glycogen synthase kinase-3,GSK-3）可能是AD神经退行性变起重要作用.为深入探讨GSK-3在AD样神经退行性变中的作用,以磷酯酰肌醇三磷酸激酶（phosphatidylinositol 3-kinase,PI3K）的特异性抑制剂渥曼青霉素（wortmannin,WT）处理野生型鼠成神经瘤细胞株（wild type mouse neuroblastoma cell lines, N2a wt）,系统观察WT处理N2a wt不同时间点（1 h、3 h、6 h）细胞代谢率、细胞形态、细胞骨架蛋白tau和NF的磷酸化状态改变以及细胞的命运,并分析了GSK-3活性与上述参数改变之间的相关性.结果发现：1 μmol/L WT处理细胞1 h,GSK-3活性与未经WT处理的对照组相比明显增高,并伴有Ser9磷酸化的GSK-3水平的降低; NF磷酸化程度增强,tau在Ser198/Ser199/Ser202位点的磷酸化增强. 1 μmol/L WT处理细胞3 h,GSK-3活性与对照组和处理1 h 组相比明显下降,NF磷酸化程度较1 h降低,但仍高于正常水平.1 μmol/L WT处理细胞6 h,细胞形态、GSK-3活性、Ser9磷酸化形式的GSK-3β的表达、NF磷酸化程度与对照组相比均无明显改变.WT呈剂量依赖性降低细胞代谢率.1 μmol/L WT处理细胞1 h和3 h导致细胞变圆,突起变短甚至消失.1 μmol/L WT处理细胞1 h,用TUNEL法和电子显微镜技术未观察到细胞凋亡.研究结果提示：在N2a细胞中过度激活GSK-3可导致神经细丝和tau蛋白的AD样过度磷酸化,从而引起神经细胞的AD样退行性变.     

Anisomycin过度激活丝裂原活化蛋白激酶使tau发生过度磷酸化

阿尔茨海默病(Alzheimer's disease,AD)的病理特征之一是神经元内存在神经原纤维缠结(neurofibrillary tangles,NFTs),后者是由过度磷酸化的微管相关蛋白tau形成的双股螺旋细丝(paired helical filaments,PHFs)构成.为了探讨丝裂原活化蛋白激酶(mitogen-activated protein kinase,MAPK)在微管相关蛋白tau磷酸化中的作用及机制,本实验用0.1 μg/mL、0.2 μg/mL和0.4μg/mL三种不同浓度的MAPK激动剂anisomycin处理小鼠成神经瘤细胞株(mouse neuroblastoma cells,N2a),检测MAPK活性的变化及其与tau蛋白多个AD相关位点过度磷酸化的关系,并检测糖原合酶激酶-3(glycogen synthase kinase-3,GSK-3)和蛋白激酶A(protein kinase A,PKA)的活性变化.结果显示,anisomycin以剂量依赖的方式激活MAPK活性,但免疫印迹结果显示tau蛋白的Ser-198/199/202位点和Ser-396/404位点的过度磷酸化只在anisomycin浓度为0.4 μg/mL时出现,三种浓度的anisomycin均未引起tau蛋白Ser-214位点磷酸化的改变;同时,GSK-3活性在anisomycin为0.1 μg/mL时没有明显变化,当anisomycin浓度升高到0.2 μg/mL和0.4 μg/mL时出现明显增高,而PKA的活性没有明显的改变.使用GSK-3的特异性抑制剂氯化锂(LiCl)则完全阻断MAPK被过度激活导致的tau蛋白磷酸化水平的增高,而同时MAPK活性不受影响.以上结果提示:过度激活MAPK可以导致tau蛋白Ser-198/199/202和Ser-396/404位点过度磷酸化,其机制可能涉及MAPK激活GSK-3的间接作用.     

Functional studies of shaggy/glycogen synthase kinase 3 phosphorylation sites in Drosophila melanogaster

Early studies of glycogen synthase kinase 3 (GSK-3) in mammalian systems focused on its pivotal role in glycogen metabolism and insulin-mediated signaling. It is now recognized that GSK-3 is central to a number of diverse signaling systems. Here, we show that the major form of the kinase Shaggy (Sgg), the GSK-3 fly ortholog, is negatively regulated during insulin-like/phosphatidylinositol 3-kinase (PI3K) signaling in vivo. Since genetic studies of Drosophila melanogaster had previously shown that Wingless (Wg) signaling also acts to antagonize Sgg, we investigate how the kinase might integrate, or else discriminate, signaling inputs by Wg and insulin. Using Drosophila cell line assays, we found, in contrast to previous reports, that Wg induces accumulation of its transducer Armadillo (Arm)/beta-catenin without significant alteration of global Sgg-specific activity. In agreement with a previous study using human GSK-3beta, Wg did not cause phosphorylation changes of the Ser9 or Tyr214 regulatory phosphorylated sites of Sgg. Conversely, as shown in mammalian systems, insulin-induced inhibition of Sgg-specific activity by phosphorylation at the N-terminal pseudosubstrate site (Ser9) did not induce Arm/beta-catenin accumulation, showing selectivity in response to the different signaling pathways. Interestingly, a minigene bearing a Ser9-to-Ala change rescued mutant sgg without causing abnormal development, suggesting that the regulation of Sgg via the inhibitory pseudosubstrate domain is dispensable for many aspects of its function. Our studies of Drosophila show that Wg and insulin or PI3K pathways do not converge on Sgg but that they exhibit cross-regulatory interactions.