Protein kinases and cell cycle control

Protein kinases play a central role in the regulation of the eukaryotic cell cycle. Recent research has concentrated on a particular family of protein kinases, the cyclin-dependent kinases (CDKs), and their involvement in regulating particular cell cycle transitions, such as the initiation of DNA synthesis (S phase) or of cell division (mitosis). One can think of these enzymes as the basic machinery of the cell; their activity is then modulated by proteins which transduce signals from the external environment, and by proteins that monitor the progress of events such as DNA replication or the formation of the mitotic spindle. This review will be structured so as to introduce the cyclin-CDK motif, outline which cyclin-CDKs are involved at different cell cycle stages, their direct regulation by other protein kinases and phosphatases, and lastly the importance of other protein kinases in the cell cycle.     

A plant-specific cyclin-dependent kinase is involved in the control of G2/M progression in plants

Cyclin-dependent kinases (CDKs) control the key transitions in the eukaryotic cell cycle. All the CDKs known to control G(2)/M progression in yeast and animals are distinguished by the characteristic PSTAIRE motif in their cyclin-binding domain and are closely related. Higher plants contain in addition a number of more divergent non-PSTAIRE CDKs with still obscure functions. We show that a plant-specific type of non-PSTAIRE CDKs is involved in the control of the G(2)/M progression. In synchronized tobacco BY-2 cells, the corresponding protein, accumulated in a cell cycle-regulated fashion, peaking at the G(2)/M transition. The associated histone H1 kinase activity reached a maximum in mitosis and required a yet unidentified subunit to be fully active. Down-regulation of the associated kinase activity in transgenic tobacco plants using a dominant-negative mutation delayed G(2)/M transition. These results provide the first evidence that non-PSTAIRE CDKs are involved in the control of the G(2)/M progression in plants.     

Cell cycle in the fucus zygote parallels a somatic cell cycle but displays a unique translational regulation of cyclin-dependent kinases

In eukaryotic cells, the basic machinery of cell cycle control is highly conserved. In particular, many cellular events during cell cycle progression are controlled by cyclin-dependent kinases (CDKs). The cell cycle in animal early embryos, however, differs substantially from that of somatic cells or yeasts. For example, cell cycle checkpoints that ensure that the sequence of cell cycle events is correct have been described in somatic cells and yeasts but are largely absent in embryonic cells. Furthermore, the regulation of CDKs is substantially different in the embryonic and somatic cells. In this study, we address the nature of the first cell cycle in the brown alga Fucus, which is evolutionarily distant from the model systems classically used for cell cycle studies in embryos. This cycle consists of well-defined G1, S, G2, and M phases. The purine derivative olomoucine inhibited CDKs activity in vivo and in vitro and induced different cell cycle arrests, including at the G1/S transition, suggesting that, as in somatic cells, CDKs tightly control cell cycle progression. The cell cycle of Fucus zygotes presented the other main features of a somatic cell cycle, such as a functional spindle assembly checkpoint that targets CDKs and the regulation of the early synthesis of two PSTAIRE CDKs, p32 and p34, and the associated histone H1 kinase activity as well as the regulation of CDKs by tyrosine phosphorylation. Surprisingly, the synthesis after fertilization of p32 and p34 was translationally regulated, a regulation not described previously for CDKs. Finally, our results suggest that the activation of mitotic CDKs relies on an autocatalytic amplification mechanism.     

Cell Cycle Regulation of DNA Replication Initiator Factor Dbf4p

The precise duplication of eukaryotic genetic material takes place once and only once per cell cycle and is dependent on the completion of the previous mitosis. Two evolutionarily conserved kinases, the cyclin B (Clb)/cyclin-dependent kinase (Cdk/Cdc28p) and Cdc7p along with its interacting factor Dbf4p, are required late in G1 to initiate DNA replication. We have determined that the levels of Dbf4p are cell cycle regulated. Dbf4p levels increase as cells begin S phase and remain high through late mitosis, after which they decline dramatically as cells begin the next cell cycle. We report that Dbf4p levels are sensitive to mutations in key components of the anaphase-promoting complex (APC). In addition, Dbf4p is modified in response to DNA damage, and this modification is dependent upon the DNA damage response pathway. We had previously shown that Dbf4p interacts with the M phase polo-like kinase Cdc5p, a key regulator of the APC late in mitosis. These results further link the actions of the initiator protein, Dbf4p, to the completion of mitosis and suggest possible roles for Dbf4p during progression through mitosis.     

Sex and the eukaryotic cell cycle is consistent with a viral ancestry for the eukaryotic nucleus

The origin of the eukaryotic cell cycle, including mitosis, meiosis, and sex are as yet unresolved aspects of the evolution of the eukaryotes. The wide phylogenetic distribution of both mitosis and meiosis suggest that these processes are integrally related to the origin of the earliest eukaryotic cells. According to the viral eukaryogenesis (VE) hypothesis, the eukaryotes are a composite of three phylogenetically unrelated organisms: a viral lysogen that evolved into the nucleus, an archaeal cell that evolved into the eukaryotic cytoplasm, and an alpha-proteobacterium that evolved into the mitochondria. In the extended VE hypothesis presented here, the eukaryotic cell cycle arises as a consequence of the derivation of the nucleus from a lysogenic DNA virus.     

Identification and cloning of Lmairk, a member of the Aurora/Ipl1p protein kinase family, from the human protozoan parasite Leishmania.

Lmairk, a gene encoding a member of the Aurora/Ipl1p family of protein kinases (AIRK), was cloned from the protozoan parasite Leishmania major. Aurora kinases are key enzymes involved in the regulation of normal chromosome segregation during mitosis and cytokenesis of eukaryotic cells. This single-copy gene located on L. major chromosome 28 encodes a 301 amino acid polypeptide. All 11 conserved eukaryotic protein kinase catalytic subdomains are present and the proposed AIRK signature sequence was identified in the activation loop between subdomains VII and VIII. Lmairk is expressed, as an approximately 2.4 kb message, in at least three different species of Leishmania. This report represents the first identification of an AIRK from the trypanosomatid family of early divergent eukaryotes.     

Involvement of cyclin-dependent kinase CDK1/CDC28 in regulation of cell cycle

Cyclin-dependent kinases (CDKs) are a family of enzymes essential for the progression of the cells through the cell cycle in eukaryotes. Moreover, genetic stability-maintaining processes, such as check-point control and DNA repair, require the phosphorylation of a wide variety of target substrates by CDK. In budding yeast Saccharomyces cerevisiae, the key role in the cell cycle progression is played by CDK1/CDC28 kinase. This enzyme is the most thoroughly investigated. In this review the involvement of CDC28 kinase in regulation of the cell cycle is discussed in the light of newly obtained data.     

Cdc2-kinases,cyclins, and the switch from proliferation to polyploidization

Summary Almost all organisms, from protists to humans, and from algae to orchids, display somatic polyploidy, including polyteny. In insects and higher plants, nearly all normal, differentiated cells are polyploid, corresponding to the majority of living matter. So far, no universal mechanism controlling the switch from proliferation to polyploidization has been proposed. However, recent progress in understanding regulation of the mitotic cell cycle by protein kinases and cyclins allows some unifying ideas which can be experimentally tested to be put forward. The key events are the abolishment of the dependence of DNA replication on mitosis, and changes in the expression and activity of the complexes formed by cyclin-dependent kinases and cyclins. In addition, repression of further cell cycle control genes may allow underreplication of DNA, characteristic of endo-cycles in many insects and angiosperms. Change to a different checkpoint may be responsible for gene amplification. The switch in cell cycle control is developmentally regulated by signal transduction cascades, which are briefly discussed. Polyploidy is also known from many cancers, where genetic and metabolic disturbances lead to a similar switch to that in normal cells. The related literature is reviewed and some possible lines of future research are suggested.Abbreviations CAK p34^cdc2-activating kinase - cdc2 cell division cycle gene inSchizosaccharomyces pombe (fission yeast), named cdk1 in mammals - CDKs cyclin-dependent kinases - cdk2 S-phase specific CDK gene in higher organisms - MAP kinase mitogen-activated protein kinase - MAPs microtubule-associated proteins - MPF maturation (or mitosis) promoting factor - p34^cdc2 mitosis specific protein kinase     

The activity of total histone H1 kinases is related to growth and commitment points while the p13suc1-bound kinase activity relates to mitoses in the alga Scenedesmus quadricauda

Under optimal growing conditions, synchronous cultures of the alga Scenedesmus quadricauda underwent three DNA replications and three mitoses during one cell cycle. This resulted in eight daughter cells. By different illumination regimes and temporal addition of cycloheximide, cell divisions resulting in two, four or eight daughter cells per cycle were obtained. The selected cell cycle patterns differed in timing of commitment points, the number of nuclear divisions, and their positioning in the cell cycle. These distinct cell cycle patterns allowed to assess the correlation of histone H1 kinase activity with commitment points and mitoses. The activity of the histone H1 kinases was assayed in cellular protein extracts and after affinity purification using the p13^suc1 protein. The main peaks of kinase activity in the cellular extract were found to correlate with the commitment points. Small histone H1 kinase activity peaks were also found which preceded the nuclear division. Contrary to the histone H1 kinase activity of cellular extracts, the p13^suc1-bound kinase activity preceded the nuclear division, whilst its activity was negligible at the commitment points. Being able to manipulate the timing of commitment points and cell division by manipulating experimental conditions, we could precisely match the commitment points to an as yet unidentified histone H1 kinase activity and mitosis to p13^suc1-bound CDK activity during a particular cell division pattern with overlapping cycles. This provides molecular evidence, that local activation of CDKs regulates distinct events of the cell cycle.     

A Novel Member of the Cyclin-Dependent Kinase Family in Paramecium tetraurelia

Passage through the cell cycle in eukaryotes requires the successive activation of different cyclin-dependent protein kinases. Here, we describe the identification and characterization of a novel class of cyclin-dependent protein kinase, termed Cdk2, in the ciliate Paramecium tetraurelia. It is 301 amino acids long, 7 amino acids shorter than Cdk1, the CDK that is associated with macronuclear DNA synthesis. All the catalytic domains typical of protein kinases can be located within the sequence and putative regulatory phosphorylation sites equivalent to Thr14, Tyr15, and Thr161 in human CDK1 are also conserved. The 'PSTAIRE' region characteristic of most CDKs is perfectly conserved. Cdk2 shares only 48% homology to Cdk1 at the amino acid level, suggesting that the evolutionary separation of Cdk1 and Cdk2 is ancient, and implying that they have different roles in cell cycle regulation. Like Cdk1, Cdk2 does not bind to yeast p13suc1, even though it has better conservation of p13suc1 binding sites than Cdk1 does. The Cdk2 protein level is relatively constant throughout the vegetative cell cycle. Cdk2 exhibits kinase activity towards bovine histone H1 in vitro with the maximal level late in the cell cycle, suggesting it may be involved in the regulation of cytokinesis. Our results further support the view that an analogue of the cyclin-dependent kinase cell cycle regulatory system like that of yeast and higher eukaryotic cells operates in Paramecium and that a family of cyclin-dependent kinases may control different aspects of the Paramecium cell cycle.     

The substrates of the cdc2 kinase.

The eukaryotic cell cycle is characterized by two major events, DNA replication (S phase) and mitosis (M phase). According to the current paradigm of the cell cycle as a cdc2 cycle, both of these events are driven by serine-threonine specific protein kinases encoded by functional homologs of the fission yeast cdc2 gene. To understand how cdc2 kinases function, it is necessary to identify their physiological substrates and to determine how phosphorylation of these substrates promotes cell cycle progression. Definitive information about substrates relevant to early stages of the cell cycle (G1 and S phases) remains scarce, but several likely physiological targets of the mitotic cdc2 kinase have recently been identified. Current evidence indicates that cdc2 kinase may trigger entry of cells into mitosis not only by initiating important regulatory pathways but also by direct phosphorylation of abundant structural proteins.     

Overexpression of active Aurora-C kinase results in cell transformation and tumour formation

Aurora kinases belong to a conserved family of serine/threonine kinases key regulators of cell cycle progression. Aurora-A and Aurora-B are expressed in somatic cells and involved mainly in mitosis while Aurora-C is expressed during spermatogenesis and oogenesis and is involved in meiosis. Aurora-C is hardly detectable in normal somatic cells. However all three kinases are overexpressed in many cancer lines. Aurora-A possesses an oncogenic activity while Aurora-B does not. Here we investigated whether Aurora-C possesses such an oncogenic activity. We report that overexpression of Aurora-C induces abnormal cell division resulting in centrosome amplification and multinucleation in both transiently transfected cells and in stable cell lines. Only stable NIH3T3 cell clones overexpressing active Aurora-C formed foci of colonies when grown on soft agar, indicating that a gain of Aurora-C activity is sufficient to transform cells. Furthermore, we reported that NIH-3T3 stable cell lines overexpressing Aurora-C induced tumour formation when injected into nude mice, demonstrating the oncogenic activity of enzymatically active Aurora kinase C. Interestingly enough tumor aggressiveness was positively correlated with the quantity of active kinase, making Aurora-C a potential anti-cancer therapeutic target.     

蛋白激酶在卵母细胞减数分裂和受精中的作用

脊椎动物卵母细胞的减数分裂和受精过程受到多种蛋白激酶的调节。近年来对于卵母细胞成熟、活化和受精的分子机制研究取得了长足进步 ,发现促成熟因子 (MPF)和促分裂原活化蛋白激酶 (MAPK)是调节卵母细胞细胞周期的关键分子 ,二者的激活和失活导致了减数分裂的恢复、阻滞和完成。许多蛋白激酶通过调节MPF和MAPK活性来影响减数分裂。Polo like激酶活化MPF ,Mos激活MAPK而启动成熟分裂并维持中期阻滞。CaMKII通过泛素途径灭活MPF使卵突破MII期阻滞。另外 ,p90 rsk作为MAPK的下游分子参与减数分裂调节 ,蛋白激酶C(PKC)诱导皮质颗粒排放并抑制MAPK激活 ,酪氨酸蛋白激酶家族成员介导受精诱发的Ca2 释放。这些蛋白激酶的协同作用推动了卵母细胞正常的成熟与受精     

极光(aurora)激酶在细胞有丝分裂和肿瘤形成中的重要功能

极光激酶（aurora kinases）是负责调控细胞有丝分裂的一类重要的丝氨酸／苏氨酸激酶。在不同的模式生物中,极光激酶各家族成员的结构和功能都高度保守。近年来,随着极光激酶相关研究的不断深入,人们逐渐认识到极光激酶在细胞有丝分裂以及肿瘤形成中的重要功能。在细胞有丝分裂中,极光激酶参与了诸如中心体成熟分离、纺锤体组装和维持、染色体分离以及胞质分裂等多个事件。异常表达的极光激酶往往会导致细胞在有丝分裂的过程中出现大量的异常现象。此外,极光激酶还参与了肿瘤形成的过程,已经发现一些靶向作用于极光的小分子具有显著的抑癌作用。本文围绕哺乳动物的三种极光激酶,重点讨论了它们在细胞有丝分裂中的动态定位、生物学功能以及时空上的调节方式,并分析了异常表达的极光激酶参与肿瘤形成的可能途径,提出了肿瘤治疗的新思路。     

Depletion of a Cks homolog in C. elegans embryos uncovers a post-metaphase role in both meiosis and mitosis

In eukaryotic cells, the key regulators of cell-cycle transitions are the cyclin-dependent kinases (CDKs). The best studied CDK is a component of the M-phase promoting factor (MPF), which promotes entry into and progression through meiosis and mitosis. One of the enduring mysteries of the MPF complex has been the role of Cks/Suc1, a highly conserved member of the cell-cycle machinery in eukaryotes [1,2]. Cks has been proposed to be involved in activation of MPF [3], general interactions of MPF with its mitotic substrates [4] and/or inactivation of MPF [5,6]. We identified two Cks homologs in the genome of Caenorhabditis elegans and used RNA-mediated interference (RNAi) to investigate their roles in development. Whereas cks-2(RNAi) embryos display no apparent defects, cks-1(RNAi) embryos display defects in both meiosis and mitosis. Specifically, cks-1(RNAi) embryos fail to exit M phase properly. We propose that CKS-1 has an essential role in the inactivation of MPF during early C. elegans embryogenesis.     

OSD1 Promotes Meiotic Progression via APC/C Inhibition and Forms a Regulatory Network with TDM and CYCA1;2/TAM

Cell cycle control is modified at meiosis compared to mitosis, because two divisions follow a single DNA replication event. Cyclin-dependent kinases (CDKs) promote progression through both meiosis and mitosis, and a central regulator of their activity is the APC/C (Anaphase Promoting Complex/Cyclosome) that is especially required for exit from mitosis. We have shown previously that OSD1 is involved in entry into both meiosis I and meiosis II in Arabidopsis thaliana; however, the molecular mechanism by which OSD1 controls these transitions has remained unclear. Here we show that OSD1 promotes meiotic progression through APC/C inhibition. Next, we explored the functional relationships between OSD1 and the genes known to control meiotic cell cycle transitions in Arabidopsis. Like osd1, cyca1;2/tam mutation leads to a premature exit from meiosis after the first division, while tdm mutants perform an aberrant third meiotic division after normal meiosis I and II. Remarkably, while tdm is epistatic to tam, osd1 is epistatic to tdm. We further show that the expression of a non-destructible CYCA1;2/TAM provokes, like tdm, the entry into a third meiotic division. Finally, we show that CYCA1;2/TAM forms an active complex with CDKA;1 that can phosphorylate OSD1 in vitro. We thus propose that a functional network composed of OSD1, CYCA1;2/TAM, and TDM controls three key steps of meiotic progression, in which OSD1 is a meiotic APC/C inhibitor.     

A family of human cdc2-related protein kinases.

The p34cdc2 protein kinase is known to regulate important transitions in the eukaryotic cell cycle. We have identified 10 human protein kinases based on their structural relation to p34cdc2. Seven of these kinases are novel and the products of five share greater than 50% amino acid sequence identity with p34cdc2. The seven novel genes are broadly expressed in human cell lines and tissues with each displaying some cell type or tissue specificity. The cdk3 gene, like cdc2 and cdk2, can complement cdc28 mutants of Saccharomyces cerevisiae, suggesting that all three of these protein kinases can play roles in the regulation of the mammalian cell cycle. The identification of a large family of cdc2-related kinases opens the possibility of combinatorial regulation of the cell cycle together with the emerging large family of cyclins.     

Functional specialization of chordate CDK1 paralogs during oogenic meiosis

Cyclin-dependent kinases (CDKs) are central regulators of eukaryotic cell cycle progression. In contrast to interphase CDKs, the mitotic phase CDK1 is the only CDK capable of driving the entire cell cycle and it can do so from yeast to mammals. Interestingly, plants and the marine chordate, Oikopleura dioica, possess paralogs of the highly conserved CDK1 regulator. However, whereas in plants the 2 CDK1 paralogs replace interphase CDK functions, O. dioica has a full complement of interphase CDKs in addition to its 5 odCDK1 paralogs. Here we show specific sub-functionalization of odCDK1 paralogs during oogenesis. Differential spatiotemporal dynamics of the odCDK1a, d and e paralogs and the meiotic polo-like kinase 1 (Plk1) and aurora kinase determine the subset of meiotic nuclei in prophase I arrest that will seed growing oocytes and complete meiosis. Whereas we find odCDK1e to be non-essential, knockdown of the odCDK1a paralog resulted in the spawning of non-viable oocytes of reduced size. Knockdown of odCDK1d also resulted in the spawning of non-viable oocytes. In this case, the oocytes were of normal size, but were unable to extrude polar bodies upon exposure to sperm, because they were unable to resume meiosis from prophase I arrest, a classical function of the sole CDK1 during meiosis in other organisms. Thus, we reveal specific sub-functionalization of CDK1 paralogs, during the meiotic oogenic program.