高等真核细胞蛋白质磷酸化与细胞周期G1向S期过渡的调控

真核生物细胞周期运行涉及三个时期过渡(Phase transitions)的调控;1.G_0向G_1期过渡,2.G_1向S期过渡,3.G_2向M期过渡等的调控。近年来,对调控G_1向S期过渡的生化事件的研究结果表明,转录和转录后水平上的调控机制在其中起重要作用。通过磷酸化与去磷酸化而参与G_1向S期过渡调节的蛋白主要有:pRB(retinoblastoma gene product,视网膜母细胞瘤基因蛋白),p107(pRB related pro-tein,pRB相关蛋白),p53,RPA(replicati一     

Survivin在细胞分裂中的作用

Survivin是凋亡蛋白抑制因子家族的一个新成员,它主要在细胞周期G2/M期表达,并有两种不同的亚细胞定位。Survivin与有丝分裂细胞周期的启动有关,并在有丝分裂中的中心体装配、纺锤体检验点的监控、胞质分裂等过程中发挥作用。Survivin在减数分裂中也有一定的作用,目前研究甚少。Survivin发挥功能的首要前提是通过主要的有丝分裂激酶p34cdc2-cyclinB1使Thr34磷酸化,其表达水平受很多因素的调节。     

有丝分裂期间蛋白质的翻译后修饰及其功能

有丝分裂期间蛋白质的翻译后修饰对于有丝分裂顺利完成以及细胞功能发挥具有重要的调控作用。常见的修饰类型包括磷酸化修饰、糖基化修饰、SUMO化修饰、乙酰化修饰、甲基化修饰。这些翻译后修饰可以维持染色体结构、促进后期染色体分离、协助末期核膜重新形成。本文对有丝分裂过程中相关蛋白质翻译后修饰的最新类型和功能进行了系统总结,以期能为肿瘤基础研究提供新的方向。     

Bora在细胞功能调控中的作用和机制研究进展

Aurora蛋白激酶A及Polo样蛋白激酶1（PLK在）作为重要的细胞周期调节蛋白可参与调控纺锤体组装、有丝分裂等细胞进程,但其激活机制及在有丝分裂中的作用机制仍然不是很清楚。Bora作为Aurora蛋白激酶A的结合蛋白,在果蝇和脊椎动物中功能高度保守,其主要通过结合Aurora蛋白激酶A从而调节Aurora蛋白激酶A的活性、促进PLK1的磷酸化、调节纺锤体的组装以及调控细胞周期进程等。随着对Bora研究的深入,人们对AuroraA和PLK1的激活机制以及Bora、Aurora蛋白激酶A、PLK1三者对细胞的调控也有了进一步的认识。主要综述Bora在细胞功能调控中的作用和研究机制。     

dbcAMP通过Cdc25B蛋白调控小鼠1-细胞期受精卵发育

为了研究PKA激活剂dbcAMP通过调控小鼠Cdc25B蛋白S149和S321位点磷酸化状态影响 小鼠1-细胞期受精卵的发育,将质粒pBSK-Cdc25B-WT、pBSK-Cdc25B-S149A、pBSK- Cdc25B-S321A和pBSK-Cdc25B-S149A/S321A体外转录成mRNA;显微注射入S期受精卵中 ,在2 mmol/L dbcAMP的M16培养基中培养,观察其对受精卵发育、MPF活性及CDC2- pTyr15磷酸化状态的影响. 结果显示,在有dbcAMP存在时,各组受精卵卵裂时间延迟 ,但Cdc25B-S/A mRNAs注射组受精卵卵裂率明显高于Cdc25B-WT mRNA注射组,MPF 活性提前达到高峰;CDC2-pTyr15磷酸化状态和MPF活性变化相一致. 因此,在小鼠1- 细胞期受精卵有丝分裂过程中,PKA对小鼠Cdc25B蛋白S149位点与S321位点的磷酸化 修饰是控制受精卵G2/M转换的重要方式.     

磷蛋白组的研究技术及其进展

真核细胞中蛋白质磷酸化是一个重要事件。真核细胞利用可逆的蛋白磷酸化来控制许多细胞过程包括信号转换、基因表达、细胞周期等。磷蛋白组的研究涉及磷蛋白的分离和鉴定 ,磷酸化残基定位和定量分析。由于蛋白质磷酸化是一个动态过程 ,在细胞中磷蛋白含量低 ,磷酸化位点可变 ,且磷酸肽的质谱信号常常会受到抑制 ,所以磷蛋白的分析存在更多的困难。本文介绍了国内外在磷酸蛋白的分离鉴定及定量分析方面的研究技术以及进展情况。目前 ,质谱仍然是核心的鉴定技术 ,寻找更好富集方法是最大的挑战。定量蛋白组学是对蛋白质的差异表达进行精确的定量分析。目前还不存在一种独立的方法可以完成磷蛋白的分离、鉴定 ,以及磷酸位点的定位和定量分析。随着样品分离技术和相关仪器的发展 ,磷酸蛋白快速、准确、全面分析鉴定将能够实现。     

磷蛋白组的研究技术及其进展

真核细胞中蛋白质磷酸化是一个重要事件。真核细胞利用可逆的蛋白磷酸化来控制许多细胞过程包括信号转换、基因表达、细胞周期等。磷蛋白组的研究涉及磷蛋白的分离和鉴定,磷酸化残基定位和定量分析。由于蛋白质磷酸化是一个动态过程,在细胞中磷蛋白含量低,磷酸化位点可变,且磷酸肽的质谱信号常常会受到抑制,所以磷蛋白的分析存在更多的困难。本文介绍了国内外在磷酸蛋白的分离鉴定及定量分析方面的研究技术以及进展情况。目前,质谱仍然是核心的鉴定技术,寻找更好富集方法是最大的挑战。定量蛋白组学是对蛋白质的差异表达进行精确的定量分析。目前还不存在一种独立的方法可以完成磷蛋白的分离、鉴定,以及磷酸位点的定位和定量分析。随着样品分离技术和相关仪器的发展,磷酸蛋白快速、准确、全面分析鉴定将能够实现。     

Cdc25B在小鼠受精卵的表达和定位

为阐明细胞分裂周期(Cdc)25B调控小鼠受精卵发育的机制,利用Western印迹检测小鼠受精卵各时期Cdc25B的表达及Cdc2-Tyr15的磷酸化状态。利用间接免疫荧光技术观察Cdc25B在小鼠受精卵的定位。构建pEGFP-Cdc25B融合表达载体并显微注射到受精卵中,观察Cdc25B在受精卵M期的定位变化。结果表明Cdc25B在G1和S期被磷酸化,在G2和M期去磷酸化。Cdc2-Tyr15在G1和S期处于磷酸化状态,G2期只检测到Cdc2-Tyr15轻微的磷酸化信号,M期未检测到任何Cdc2-Tyr15的磷酸化信号。Cdc25B在G1期定位于细胞质和细胞核中,S和G2期定位于细胞质的皮质部分,M期由细胞质转向核区。证明Cdc25B核输出后激活有丝分裂促进因子,从而启动小鼠受精卵的有丝分裂。     

6-溴异香兰素BVAN08诱发肝癌细胞HepG2纺锤体损伤和有丝分裂灾变死亡

研究香兰素衍生物中的6.溴异香兰素(BVAN08)对细胞纺锤体结构的影响及诱发灾变死亡的相关机制,为开发该化合物为新的抗癌药物提供理论依据.通过光学显微镜观察BvAN08作用后细胞形态学变化,流式细胞术检测细胞周期,纺锤体功能检测点实验和原位免疫荧光杂交实验分析细胞有丝分裂进程和纺锤体结构.western印记检测BVAN08作用后相关蛋白质的变化.结果表明20～60 μmol/L BVAN08作用后,HepG2细胞变圆不再贴壁生长、随后脱落死亡,具有浓度依赖性量效关系;明显诱导细胞G2/M期阻滞、导致细胞有丝分裂指数升高,并出现大量的非二倍体和多倍体细胞;破坏细胞纺锤体的结构,多中心体细胞显著增加;该化合物促使细胞周期转录调节因子FoxM1及其下游靶分子细胞周期蛋白B1和CdK1的降解、阻止有丝分裂过程而导致有丝分裂灾变死亡.研究揭示BVAN08通过破坏纺锤体结构、诱发M期阻滞,导致细胞有丝分裂灾变死亡,FoxM1失活可能参与其作用机制.     

癌症高表达蛋白——Hecl在纺锤体组装检查点中的作用

细胞增殖依赖于细胞分裂前染色体的复制及随后的姐妹染色单体分离到达两极。纺锤体组装检查点具有确保染色体信息传递保真性的作用,检查点的缺失可能导致染色体的分离异常和肿瘤形成。癌症高表达蛋白(Hecl)通过与调控G2／M期的蛋白间的相互作用而在染色体的分离中发挥重要作用。Hecl与Nuf2的复合物,在G2／M期与动粒相结合,Hecl的缺失将导致严重的染色体分离错误。Hecl具有召集Mpsl和Mad1／Mad2复合物结合到动粒上的作用,这种结合可以激活纺锤体组装检查点途径中非常重要的APC^cdc20途径。但是Hecl、Mpsl、Madl三者之间的相互作用仍未明了。Hecl还可以通过与26S蛋白酶复合物的不同亚基结合调控其功能。Hecl是一种丝氨酸磷酸化蛋白,其磷酸化是由Nek2在G2／M期完成的。     

Probing microtubule organizing centres with MPM-2 in dividing cells of higher plants using immunofluorescence and immunogold techniques

Summary The localization in higher plant cells of phosphorylated proteins recognized by the monoclonal antibody MPM-2 was investigated, with particular attention to putative microtubule organizing centres (MTOCs). Immunofluorescence and immunogold electron microscopy showed that MPM-2 did not localize with most putative MTOCs in cells and protoplasts of the gymnospermPicea glauca and in cells of the angiospermVicia faba. The distribution of phosphoproteins detected by MPM-2 was similar during mitosis in both species. At late interphase and early prophase MPM-2 preferentially labelled nucleoli and the region around the condensing chromosomes but not the cytoplasm. General labelling of the cytoplasm followed dissolution of the nuclear envelope and by prometaphase centromeres stained strongly. At metaphase and very early anaphase kinetochores stained strongly by immunofluorescence but only weakly using immunogold; spindle microtubules (MTs) showed little staining. Kinetochore staining disappeared during anaphase and by telophase centromeres and loose regions of chromatin in reforming nuclei were labelled. Treatment with the anti-microtubular drug amiprophosmethyl (APM) showed that the phosphorylation/dephosphorylation cycle detected by MPM-2 proceeded independently of the mitotic spindle. Staining of centromeres/kinetochores with MPM-2 suggests that phosphorylation and dephosphorylation of this region of mitotic chromosomes may be involved in chromosome organization, chromatid separation and MT nucleation and/or attachment.Abbreviations APM amiprophos-methyl - DAPI 4,6-diamidino-2-phenylindole - EGTA ethylene glycol-bis(-aminoethyl ether) - FITC fluorescein isothiocyanate - MT microtubule - MTOC microtubule organizing centre - MtSB microtubule stabilizing buffer - PBS phosphate buffered saline - PBSB phosphate buffered saline with bovine serum albumin - PIPES piperazine-N,N-bis (2-ethanesulfonic acid) - PPB preprophase band - SPB spindle pole body - TRITC tetramethylrhodamine isothiocyanate     

Mos-induced p42 mitogen-activated protein kinase activation stabilizes M-phase in Xenopus egg extracts after cyclin destruction

Previously, we have shown that the addition of a constitutively-active mitogen-activated protein kinase kinase protein (MAPKK = MEK) to cycling Xenopus egg extracts activates the p42MAPK pathway, leading to a G2 or M-phase cell cycle arrest. The stage of the arrest depends on the timing of p42MAPK activation. If p42MAPK is activated prior to M-phase, or after exit from M-phase, the extract is arrested in G2. If p42MAPK is activated during entry into M-phase, the extract is arrested in M-phase. In this study, we show that the addition of recombinant Mos protein (which directly phosphorylates and activates MEK) to cycling egg extracts has the same effect as those described for MEK. The addition of Mos to the extract at the start of incubation leads to a G2 arrest with large interphase nuclei with intact nuclear envelopes. If Mos is added at later times, however, the activation of p42MAPK leads to an M-phase arrest with condensed chromosomes and mitotic arrays of microtubules. Moreover, the extent of M-phase specific phosphorylations is shown by the sustained presence of phosphoproteins that are detected by the monoclonal antibody MPM-2. Unexpectedly, in certain M-phase arrested extracts, histone H1 kinase activity levels reach a peak on entry into M-phase but then fall abruptly to interphase levels. When these extracts are analyzed by immunoblotting, Cyclin B2 is destroyed in those samples containing low maturation promoting factor activity (MPF, cyclin B/Cdc2), yet chromosomes remain condensed with associated mitotic arrays of microtubules and M-phase-specific phosphorylations are sustained. These results suggest that although MPF is required for entry into M-phase, once established, M-phase can be maintained by the p42MAPK pathway after the proteolysis of mitotic cyclins.     

Cell cycle control of higher-order chromatin assembly around naked DNA in vitro.

We have developed an in vitro system in which higher-order chromatin structures are assembled around naked DNAs in a cell cycle-dependent manner. Membrane-free soluble extracts specific to interphase and mitotic states were prepared from Xenopus eggs. When high molecular weight DNA is incubated with interphase extracts, fluffy chromatin-like structures are assembled. In contrast, mitotic extracts produce highly condensed chromosome-like structures. Immunofluorescence studies show that a monoclonal antibody MPM-2, which recognizes a class of mitosis-specific phosphoproteins, stains the "core" or "axis" of condensed mitotic chromatin but not interphase chromatin. By adding mitotic extracts, interphase chromatin structures are synchronously converted into the condensed state. The increasingly condensed state of chromatin correlates with the appearance and structural rearrangements of the MPM-2-stained structures. These results suggest that mitosis-specific phosphoproteins recognized by MPM-2 may be directly involved in the assembly of the chromosome scaffold-like structures and chromatin condensation. Although both extracts promote nucleosome assembly at the same rate, topoisomerase II (topo II) activity is four to five times higher in mitotic extracts compared with interphase extracts. The addition of a topo II inhibitor VM-26 into mitotic assembly mixtures disturbs the organization of the MPM-2-stained structures and affects the final stage of chromatin condensation. This in vitro system should be useful for identifying cis- and trans-acting elements responsible for higher-order chromatin assembly and its structural changes in the cell cycle.     

The mitosis-specific monoclonal antibody MPM-2 recognizes phosphoproteins associated with the nuclear envelope in Chlamydomonas reinhardtii cells.

The monoclonal antibody MPM-2 recognizes a family of phosphorylated proteins present in mitotic cells. In a number of organisms it stains nuclei and also cytoskeletal structures which contain or organize tubulin. In mitotic Chlamydomonas reinhardtii cells MPM-2 reacts with phosphoproteins associated with the nuclear envelope (NE). Staining of the NE region appears in preprophase, reaches a maximum intensity in metaphase/anaphase and disappears rapidly in telophase. Localized hyperphosphorylation of the anterior NE region is apparent in many cells throughout mitosis. The distribution and timing of MPM-2 labeling suggests that in Chlamydomonas MPM-2 may be interacting with lamin-like phosphoproteins.     

Distribution of cytoskeletal proteins sharing a conserved phosphorylated epitope

A group of antigens related by their reactivity with monoclonal antibodies MPM-1 and MPM-2 appear as cells enter mitosis. These antibodies bind to a phosphorylated epitope on certain proteins, and therefore the antigens are presumed to be a group of phosphoproteins. A subset of these proteins has been shown previously to be components of mitotic microtubule organizing centers in PtK1 cells. We present here evidence that the mitosis-specific appearance of these phosphoproteins is a phenomenon common to all eukaryotic cells. The MPM reactive phosphoproteins were localized to mitotic spindle poles regardless of whether the spindle formed in the cytoplasm after nuclear envelope breakdown (open mitosis) or within the nucleus (closed mitosis). This reactivity was not dependent upon the presence of centrioles at the spindle poles. Proteins that contained the phosphorylated epitope were not, however, restricted to mitotic cells. Cells of neuronal derivation and flagellated cells showed specific localization of MPM antibody to the microtubule network and basal bodies respectively. On immunoblots, the MPM antibody reacted with brain MAP-1 among a number of other phosphoproteins. The identification of microtubule-associated protein (MAP)-1 correlates with the localization of the antibody to microtubules of neuroblastoma cells. These results suggest, that different phosphoprotein molecules detected by the MPM antibody may be specific for different mitotic microtubule organizing centers, basal bodies, and other specialized cytoskeletal structures; and the presence of a related phosphorylated domain on these proteins may be important for their proper function and/or interaction with microtubules.     

cdc25 is one of the MPM-2 antigens involved in the activation of maturation-promoting factor.

MPM-2 antigens, a discrete set of phosphoproteins that contain similar phosphoepitopes recognized by the monoclonal antibody MPM-2, are phosphorylated during M-phase induction. Our previous studies suggested that certain MPM-2 antigens are involved in the appearance of maturation-promoting factor (MPF) activity. Because the central mitotic regulator cdc2 kinase has been shown to exhibit MPF activity, we explored the possibility that certain MPM-2 antigens are regulators of cdc2 kinase. We found that MPM-2 binding of its antigens would inhibit the autoamplification of cdc2 kinase in Xenopus oocytes and interfere with cyclin-activation of cdc2 kinase in Xenopus interphase egg extract. Immunodepletion of MPM-2 antigens from cyclin-induced M-phase egg extract caused the inactivation of cdc2 kinase, which was accompanied by an inhibitory phosphorylation of p34cdc2 on Thr 14 and Tyr 15, indicating that at least one MPM-2 antigen is a positive regulator of p34cdc2 dephosphorylation. We then showed that cdc25 from M-phase arrested egg extract is an MPM-2 antigen. These results suggest that phosphorylation of the epitope recognized by MPM-2 may be a crucial event in the activation of cdc25 and that the kinase(s) that phosphorylates this MPM-2 epitope may be an important regulator of cdc2 kinase activation.     

Localization of MPM-2 recognized phosphoproteins and tubulin during cell cycle progression in synchronized Vicia faba root meristem cells.

MPM-2 antibody reacts with a subset of mitotic phosphoproteins. We followed localization of MPM-2 immunoreactive material and localization of microtubules during cell cycle progression in a highly synchronous population of Vicia faba root meristem cells and isolated nuclei. The MPM-2 antibody labelling showed significant cell cycle dependence. MPM-2 nuclear reactivity was weak and homogeneous in G1 and S phase of the cell cycle and became stronger and heterogeneous during G2, resembling staining of the nuclear matrix, with maximum staining at the G2/M interface. Similarly the staining intensity of nucleoli increased from late G1 phase to nucleoli dispersion in early prophase. During mitosis MPM-2 immunoreactivity was associated with spindle configurations and the brightest signal was localized in kinetochores from prophase to metaphase.     

Distribution of phosphorylated spindle-associated proteins in the diatom Stephanopyxis turris

Mitotic spindles isolated from the diatom Stephanopyxis turris become thiophosphorylated in the presence of ATP gamma S at specific locations within the mitotic apparatus, resulting in a stimulation of ATP-dependent spindle elongation in vitro. Here, using indirect immunofluorescence, we compare the staining pattern of an antibody against thiophosphorylated proteins to that of MPM-2, an antibody against mitosis-specific phosphoproteins, in isolated spindles. Both antibodies label spindle poles, kinetochores, and the midzone. Neither antibody exhibits reduced labeling in salt-extracted spindles, although prior salt extraction inhibits thiophosphorylation in ATP gamma S. Furthermore, both antibodies recognize a 205 kd band on immunoblots of spindle extracts. Microtubule-organizing centers and mitotic spindles label brightly with the MPM-2 antibody in intact cells. These results show that functional mitotic spindles isolated from S. turris are phosphorylated both in vivo and in vitro. We discuss the possible role of phosphorylated cytoskeletal proteins in the control of mitotic spindle function.     

Increased cell cycle-dependent staining of plant cells by the antibody MPM-2 correlates with preprophase band formation

Cytoplasmic microtubules of animal cells catastrophically depolymerize upon entry into mitosis but in higher plants there is a longer transition during which cortical microtubules form an increasingly narrow preprophase band, and the chromatin gradually condenses. Progression towards mitosis in onion root tip cells was analysed using a CCD camera and image processing to quantify fluorescence staining by the monoclonal antibody MPM-2, which recognizes mitotic phosphoproteins in a range of eukaryotic cells. MPM-2 fluorescence, which was predominantly nuclear, was categorized relative to the stage of the DNA cycle (using DAPI), and to the microtubule cycle (using anti-tubulin) in individual cells. Cells with the characteristic interphase cortical microtubule arrays had a bimodal distribution of DAPI fluorescence, indicating that some were in G1 (2C DNA) whilst the double value suggested the others to be in G2 (4C). There was no difference in MPM-2 fluorescence between 2C and 4C cells possessing the cortical array in which microtubules were evenly distributed. However, in 4C cells possessing a preprophase band MPM-2 values doubled; this relationship applied not only to tight PPBs but to early, broad PPBs in which the individual microtubules could still be distinguished. Since alkaline phosphatase abolished MPM-2 reactivity it is concluded that mitotic phosphoproteins do not necessarily begin to accumulate in G2 per se , but during that part of G2 when the preprophase band first becomes recognizable as a distinct entity.     

Spindle-pole organization during early mouse development

Spindle-pole organization during early mouse development was examined using a variety of immunological reagents that recognize centrosomal components. Spindle poles of unfertilized eggs and blastocysts were found to react positively with two antisera (centrin and NRS-01), whereas poles of activated eggs and early cleavage-stage embryos were negative when treated with the same sera. In contrast, a third antiserum (5051) showed positive spindle-pole staining throughout the preimplantation stages of development. Two monoclonal antibodies (MPM-1 and MPM-2) that are known to react with mitotic phosphoproteins were also used in this study. Both antibodies stained the cytoplasm of mitotic cells with extremely high intensity. In addition, MPM-2 was found to stain spindle poles. These results suggest that organizational changes in the spindle pole are occurring during early mouse development. Embryos homozygous for a recessive lethal mutation known as oligosyndactyly (Os) were also treated with the reagents described above. This mutation results in a metaphase arrest at the blastocyst stage with intact spindles being present. Spindle poles were observed in Os homozygous mutants stained with centrin, NRS-01, and 5051. However, when Os mutants were stained with the MPM monoclonal antibodies, about half of the mitotic cells completely lacked the dramatic cytoplasmic staining. This observation is in contrast to that observed for wild-type embryos, where greater than 95% of mitotic cells showed positive cytoplasmic staining.