周期蛋白依赖性蛋白激酶活性的调控

周期蛋白依赖性蛋白激酶活性的调控邱嵘（解放军兰州医学高等专科学校,兰州７３００２０）关键词周期蛋白依赖性蛋白激酶,周期蛋白,磷酸化真核细胞周期中细胞不同状态之间的转换主要是通过“检查点”（ｃｈｅｃｋｐｏｉｎｔ）控制的。“检查点”主要由两个蛋白家族组成...     

周期蛋白和周期蛋白依赖性激酶及相关激酶抑制剂在细胞周期进程中的调控机制研究进展

在细胞发育过程中,细胞周期起着至关重要的作用。细胞周期进程主要受细胞周期蛋白依赖性激酶(cyclin dependent kinase, CDK)、周期蛋白和内源性CDK抑制剂(cyclin-dependent kinase inhibitors,CKI)调控。其中,CDK是主要的细胞周期调节因子,可与周期蛋白结合形成周期蛋白-CDK复合物,从而使数百种底物磷酸化,调控分裂间期和有丝分裂进程。各类细胞周期蛋白的活性异常,可引起不受控制的癌细胞增殖,导致癌症的发生与发展。因此,了解CDK的活性变化情况、周期蛋白-CDK的组装以及CKI的作用,将有助于了解细胞周期进程中潜在的调控过程,为癌症与疾病的治疗和CKI治疗药物的研发提供基础。本文关注了CDK激活和灭活的关键事件,并总结了周期蛋白-CDK在特定时期及位置的调控过程,以及相关CKI治疗药物在癌症及疾病中的研究进展,最后简单阐述了细胞周期进程研究面临的问题和存在的挑战,以期为后续细胞周期进程的深入研究提供参考和思路。     

染色质蛋白激酶VRK1的生物学功能及其在癌症靶向治疗中的意义

牛痘相关激酶1(vaccinia-related kinase 1,VRK1)是一种核染色质丝苏氨酸蛋白激酶,在癌症中不发生基因突变,但在很多类型的肿瘤中表达上调并与不良预后相关。在细胞核内,VRK1可以磷酸化几种转录因子、组蛋白和涉及DNA损伤反应途径的蛋白质,还可以参与转录过程中组蛋白的乙酰化修饰,调节细胞周期、有丝分裂等过程促进细胞增殖,并且在DNA损伤修复中发挥至关重要的作用。在DNA损伤修复反应中,VRK1调控组蛋白乙酰化,介导DNA损伤反应的触发,进一步参与非同源末端连接DNA修复途径,还可以调控p53相关的DNA损伤修复过程。基于VRK1的以上生物学功能,癌组织中VRK1的高表达可以促进肿瘤细胞增殖、转移以及参与肿瘤细胞DNA修复过程。在癌症靶向治疗研究中,VRK1可以作为癌症合成致死性策略的选择靶点用于多种癌症的防治。     

Cdk-5过度表达对Tau蛋白磷酸化的影响

目的 实验旨在细胞水平研究cdk-5过度表达对微管相关蛋白tau的磷酸化的影响。方法 利用基因转染技术建立过度表达cdk-5的鼠成神经瘤细胞株(N2a细胞株),免疫沉淀及酶活性检测法检测cdk-5的酶活性,免疫荧光技术和免疫印迹技术检测细胞内tau蛋白的磷酸化状况。结果 在N2a细胞株转染组中,cdk-5表达增加,并使得抗体tau-1显色减弱,PHF-1显色增强,提示tau蛋白在Ser199/202,Ser396／404位点过度磷酸化。与此同时,cdk-5酶活性较未转染组提高3.5倍。结论 这些结果提示细胞水平的cdk-5的过度表达会导致cdk-5酶活性增加和tau蛋白过度磷酸化,而过度磷酸化的tau蛋白可能参与了AD的病理过程。     

ERM蛋白在微血管内皮细胞通透性调控中的研究进展

埃兹蛋白(Ezrin)/根蛋白(Radixin)/膜突蛋白(Moesin)(ERM)是细胞膜与胞内骨架的连接蛋白,具有高度同源性。细胞外刺激因子可通过多种信号通路磷酸化ERM蛋白,使细胞骨架重构,从而调控微血管内皮细胞通透性,在感染、炎症、代谢异常等病理过程中发挥作用。ERM功能调节的一个重要环节就是其羧基末端苏氨酸残基磷酸化后引起ERM构象的改变,暴露的羧基末端尾部的肌动蛋白(actin)-细胞骨架结合位点;故通过ERM的桥接作用,可将肌动蛋白微丝与细胞膜相连,使血管内皮细胞屏障功能发生变化。目前已知能使ERM磷酸化的激酶有蛋白激酶C(PKC)、促分裂原活化蛋白激酶(MAPK)、Rho相关激酶(ROCK),分别通过p38-MAPK、Rho/ROCK、PKC信号通路参与微血管内皮屏障功能的调控。本文旨在阐述ERM及其相关信号通路在微血管内皮细胞通透性调控中发挥的作用。     

细胞周期蛋白依赖性激酶2的功能及其抑制剂的研究

细胞周期蛋白依赖性激酶2(cyclin-dependent kinase 2,CDK2)是CDK家族中的重要成员之一.CDK2的表达或功能异常与多种疾病(如肿瘤、病毒复制与感染、免疫缺陷性疾病和雄性不育等)发生机制密切相关.CDK2抑制剂已成为抗肿瘤药物研发中的一个重要靶点.该文对CDK2在细胞周期调控、细胞增殖、细胞...     

高等植物细胞周期调控研究进展

高等植物的细胞周期（cell cycle)在其生长发育过程中受严格调控的,细胞周期的运转是基因有序表达的结果,并受的因素的影响,植物细胞周期研究近年来已取得的较大的进展,本文综述了近几年与植物细胞周期调控相关的细胞周期蛋白（cyclins),细胞周期蛋白依赖性激酶（CDKs)等内部调控因子及外源影响因素的研究进展。     

Structure of an activated DNA-PK and its implications for NHEJ

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Cell polarity protein Par3 complexes with DNA-PK via Ku70 and regulates DNA double-strand break repair

The author affiliations were mixed up in the previous published version. The third fund number of National Natural Science Foundation of China in the Acknowledgments was wrong, it should be ＂30270335＂. The Shanghai Municipal Council for Science and Technology （No.06DZ22032） was missed in the Acknowledgments. There are some labeling and production errors in Figure 2A, Figure 3B and 3C, Figure 5C, Figure 6B and 6E, Figure 7B and 7D.     

Multiple protein-protein interactions within the DNA-PK complex are mediated by the C-terminus of Ku 80

DNA double strand breaks (DSB) are among the most lethal forms of DNA damage and, in humans, are repaired predominantly by the non-homologous end joining (NHEJ) pathway. NHEJ is initiated by the Ku70/80 heterodimer binding free DNA termini and then recruiting the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) to form the catalytically active DNA-PK holoenzyme. The extreme C-terminus of Ku80 (Ku80CTD) has been shown to be important for in vitro stimulation of DNA-PK activity and NHEJ in vivo. To better define the mechanism by which the Ku80CTD elicits these activities, we assessed its functional and physical interactions with DNA-PKcs and Ku70/80. The results demonstrate that DNA-PKcs activity could not be complemented by addition of a Ku80CTD suggesting that the physical connection of the C-terminus to the DNA binding domain of Ku70/80 is required for DNA -PKcs activation. Analysis of protein-protein interactions revealed a low but measurable binding of the Ku80CTD for Ku70/80ΔC and for DNA-PKcs while dimer formation and the formation of higher ordered structures of the Ku80CTD was readily apparent. Ku has been shown to tether DNA termini possibly due to protein/protein interactions. Results demonstrate that the presence of the Ku80CTD stimulates this activity possibly through Ku80CTD/Ku80CTD interactions.     

Involvement of the ubiquitin pathway in decreasing Ku70 levels in response to drug-induced apoptosis

Ku70 plays an important role in DNA damage repair and prevention of cell death. Previously, we reported that apoptosis caused a decrease in cellular Ku70 levels. In this study, we analyzed the mechanism of how Ku70 levels decrease during drug-induced apoptosis. In HeLa cells, staurosporin (STS) caused a decrease in Ku70 levels without significantly affecting Ku70 mRNA levels. We found that Ku70 protein was highly ubiquitinated in various cell types, such as HeLa, HEK293T, Dami (a megakaryocytic cell line), endothelial, and rat kidney cells. An increase in ubiquitinated Ku70 protein was observed in apoptotic cells, and proteasome inhibitors attenuated the decrease in Ku70 levels in apoptotic cells. These results suggest that the ubiquitin-proteasome proteolytic pathway plays a role in decreasing Ku70 levels in apoptotic cells. Ku70 forms a heterodimer with Ku80, which is required for the DNA repair activity of Ku proteins. We also found that Ku80 levels decreased in apoptotic cells and that Ku80 is a target of ubiquitin. Ubiquitinated Ku70 was not found in the Ku70-Ku80 heterodimer, suggesting that modification by ubiquitin inhibits Ku heterodimer formation. We propose that the ubiquitin-dependent modification of Ku70 plays an important role in the control of cellular levels of Ku70.     

Changes in the level and distribution of Ku proteins during cellular senescence

Aging is associated with accumulation of genomic rearrangements consistent with aberrant repair of DNA breaks. We have shown previously that DNA repair by non-homologous end joining (NHEJ) becomes less efficient and more error-prone in senescent cells. Here, we show that the levels of Ku70 and Ku80 drop approximately twofold in replicatively senescent cells. Intracellular distribution of Ku also changes. In the young cells roughly half of Ku is located in the nucleus and half in the cytoplasm. In senescent cells the nuclear levels of Ku do not change, while the cytoplasmic Ku fraction disappears. Upon treatment with gamma-irradiation, in the young cells cytoplasmic Ku moved into the nuclear and membrane fractions, while no change in the Ku distribution occurred in senescent cells. Upon treatment with UVC Ku moved out of the nucleus in the young cells, while most Ku remained nuclear in senescent cells. This suggests that the nuclear Ku in senescent cells is unable to respond to DNA damage. We hypothesize that overall decline in Ku levels changes in Ku intracellular distribution, and the loss of appropriate response of Ku to DNA damage in senescent cells contribute to the decline of NHEJ and to age-related genomic instability.     

Identification of a highly potent and selective DNA-dependent protein kinase (DNA-PK) inhibitor (NU7441) by screening of chromenone libraries

A solution-phase multiple-parallel synthesis approach was employed for the preparation of 6-, 7- and 8-aryl-substituted chromenone libraries, which were screened as inhibitors of the DNA repair enzyme DNA-dependent protein kinase (DNA-PK). These studies resulted in the identification of 8-dibenzothiophen-4-yl-2-morpholin-4-yl-chromen-4-one (NU7441) as a highly potent and selective DNA-PK inhibitor (IC₅₀ = 14 nM), exhibiting ATP-competitive inhibition kinetics.     

Coordinated assembly of Ku and p460 subunits of the DNA-dependent protein kinase on DNA ends is necessary for XRCC4-ligase IV recruitment

Repair of DNA double-strand breaks by the non-homologous end-joining pathway (NHEJ) requires a minimal set of proteins including DNA-dependent protein kinase (DNA-PK), DNA-ligase IV and XRCC4 proteins. DNA-PK comprises Ku70/Ku80 heterodimer and the kinase subunit DNA-PKcs (p460). Here, by monitoring protein assembly from human nuclear cell extracts on DNA ends in vitro, we report that recruitment to DNA ends of the XRCC4-ligase IV complex responsible for the key ligation step is strictly dependent on the assembly of both the Ku and p460 components of DNA-PK to these ends. Based on co-immunoprecipitation experiments, we conclude that interactions of Ku and p460 with components of the XRCC4-ligase IV complex are mainly DNA-dependent. In addition, under p460 kinase permissive conditions, XRCC4 is detected at DNA ends in a phosphorylated form. This phosphorylation is DNA-PK-dependent. However, phosphorylation is dispensable for XRCC4-ligase IV loading to DNA ends since stable DNA-PK/XRCC4-ligase IV/DNA complexes are recovered in the presence of the kinase inhibitor wortmannin. These findings extend the current knowledge of the assembly of NHEJ repair proteins on DNA termini and substantiate the hypothesis of a scaffolding role of DNA-PK towards other components of the NHEJ DNA repair process.     

Ku70 is stabilized by increased cellular SUMO

Ku70 is a protein that finds itself at the heart of several important cellular processes. It is essential to the non-homologous end joining pathway as a part of the DNA-end binding complex, required for proper maintenance of telomeres and contributes to DNA damage recognition and regulation of apoptosis. Forces that regulate Ku70 are therefore likely to have large consequences on the physiologic state of the cell. We report here that transient expression of the small protein SUMO resulted in a dramatic increase in the abundance of Ku70. Surprisingly, the direct SUMOylation of Ku70 does not appear to be required for this effect. Rather, Ku70 appears to be stabilized through indirect effects on the rate of degradation. The same outcome was obtained by raising the expression of enzymes that promote SUMOylation. It is likely that many other proteins will be similarly regulated, providing a general control of cellular state.