Control of cell cycle progression by phosphorylation of cyclin-dependent kinase (CDK) substrates

The eukaryotic cell cycle is a fundamental evolutionarily conserved process that regulates cell division from simple unicellular organisms, such as yeast, through to higher multicellular organisms, such as humans. The cell cycle comprises several phases, including the S-phase (DNA synthesis phase) and M-phase (mitotic phase). During S-phase, the genetic material is replicated, and is then segregated into two identical daughter cells following mitotic M-phase and cytokinesis. The S- and M-phases are separated by two gap phases (G1 and G2) that govern the readiness of cells to enter S- or M-phase. Genetic and biochemical studies demonstrate that cell division in eukaryotes is mediated by CDKs (cyclin-dependent kinases). Active CDKs comprise a protein kinase subunit whose catalytic activity is dependent on association with a regulatory cyclin subunit. Cell-cycle-stage-dependent accumulation and proteolytic degradation of different cyclin subunits regulates their association with CDKs to control different stages of cell division. CDKs promote cell cycle progression by phosphorylating critical downstream substrates to alter their activity. Here, we will review some of the well-characterized CDK substrates to provide mechanistic insights into how these kinases control different stages of cell division.

Context dependent splicing functions of Bud31/Ycr063w define its role in budding and cell cycle progression

The yeast Bud31 protein, a Prp19 complex (NTC) member, aids spliceosome assembly and thus promotes efficient pre-mRNA splicing. The bud31 null cells show mild budding abnormalities at optimal growth temperatures and, at higher temperatures, have growth defects with aberrant budding. Here we have assessed cell cycle transitions which require Bud31. We find Bud31 facilitates passage through G1-S regulatory point (Start) but is not needed for G2-M transition or for exit from mitosis. To co-relate Bud31 functions in cell division with splicing, we studied the splicing status of transcripts that encode proteins involved in budding. We find Bud31 promotes efficient splicing of only some of these pre-mRNAs, for example, ARP2 and SRC1. Wild type cells have a long and a short isoform of SRC1 mRNA and protein, out of which the shorter mRNA splice variant is predominant. bud31 Delta cells show inefficient SRC1 splicing and entirely lack the shorter SRC1 spliced mRNA isoform. Yeast PRP17, another NTC sub-complex member, is also required for G1-S and G2-M cell cycle transitions. We examined genetic interactions between BUD31 and PRP17. While both factors were needed for efficient cell cycle dependent gene expression, our data indicate that distinct pre-mRNAs depend on each of these non-essential splicing factors.

A Proteomic Study on Cell Cycle Progression of Endothelium Exposed to Tumor Conditioned Medium and the Possible Role of Cyclin D-1/E

This study was designed to comprehensively analyze the differential expression of proteins from human umbilical vein endothelial cells (HUVECs) exposed to tumor conditioned medium (TCM) and to identify the key regulator in the cell cycle progression. The HUVECs were exposed to TCM from breast carcinoma cell line MDA-MB-231, then their cell cycle distribution was measured by flow cytometer (FCM). The role of protein in cell cycle progression was detected via two-dimensional polyacrylamide gel electrophoresis (2-DE) and western blotting. Following the stimulation of TCM, HUVECs showed a more cells in the S phase than did the negative control group (ECGF-free medium with 20% FBS), but the HUVECs' level was similar to the positive control group (medium with 25 mug/ml ECGF and 20% FBS). Increased expression of cyclin D-1/E and some changes in other related proteins occurred after incubation with TCM. From our results, we can conclude that breast carcinoma cell line MDA-MB-231 may secrete soluble pro-angiogenic factors that induce the HUVEC angiogenic switch, during which the expression of cell cycle regulator cyclin D-1/E increases and related proteins play an important role in this process.

Ca(2+) signals coordinate zygotic polarization and cell cycle progression in the brown alga Fucus serratus

Zygotes of the fucoid brown algae provide excellent models for addressing fundamental questions about zygotic symmetry breaking. Although the acquisition of polarity is tightly coordinated with the timing and orientation of the first asymmetric division-with zygotes having to pass through a G1/S-phase checkpoint before the polarization axis can be fixed -the mechanisms behind the interdependence of polarization and cell cycle progression remain unclear. In this study, we combine in vivo Ca(2+) imaging, single cell monitoring of S-phase progression and multivariate analysis of high-throughput intracellular Ca(2+) buffer loading to demonstrate that Ca(2+) signals coordinate polarization and cell cycle progression in the Fucus serratus zygote. Consistent with earlier studies on this organism, and in contrast to animal models, we observe no fast Ca(2+) wave following fertilization. Rather, we show distinct slow localized Ca(2+) elevations associated with both fertilization and S-phase progression, and we show that both S-phase and zygotic polarization are dependent on pre-S-phase Ca(2+) increases. Surprisingly, this Ca(2+) requirement cannot be explained by co-dependence on a single G1/ S-phase checkpoint, as S phase and zygotic polarization are differentially sensitive to pre-S-phase Ca(2+) elevations and can be uncoupled. Furthermore, subsequent cell cycle progression through M phase is independent of localized actin polymerization and zygotic polarization. This absence of a morphogenesis checkpoint, together with the observed Ca(2+)dependences of S phase and polarization, show that the regulation of zygotic division in the brown algae differs from that in other eukaryotic model systems, such as yeast and Drosophila.

A proteome-wide strategy reveals a novel mechanism of control of cell cycle progression through modulation of cyclin mRNA stability

La quantité de données générée dans le cadre d'étude à grande échelle du réseau d'interaction protéine-protéine dépasse notre capacité à les analyser et à comprendre leur sens; d'une part, par leur complexité et leur volume, et d'un autre part, par la qualité du jeu de donnée produit qui semble bondé de faux positifs et de faux négatifs. Cette dissertation décrit une nouvelle méthode de criblage des interactions physique entre protéines à haut débit chez Saccharomyces cerevisiae, la complémentation de fragments protéiques (PCA). Cette approche est accomplie dans des cellules intactes dans les conditions natives des protéines; sous leur promoteur endogène et dans le respect des contextes de modifications post-traductionnelles et de localisations subcellulaires. Une application biologique de cette méthode a permis de démontrer la capacité de ce système rapporteur à répondre aux questions d'adaptation cellulaire à des stress, comme la famine en nutriments et un traitement à une drogue. Dans le premier chapitre de cette dissertation, nous avons présenté un criblage des paires d'interactions entre les protéines résultant des quelques 6000 cadres de lecture de Saccharomyces cerevisiae. Nous avons identifié 2770 interactions entre 1124 protéines. Nous avons estimé la qualité de notre criblage en le comparant à d'autres banques d'interaction. Nous avons réalisé que la majorité de nos interactions sont nouvelles, alors que le chevauchement avec les données des autres méthodes est large. Nous avons pris cette opportunité pour caractériser les facteurs déterminants dans la détection d'une interaction par PCA. Nous avons remarqué que notre approche est sous une contrainte stérique provenant de la nécessité des fragments rapporteurs à pouvoir se rejoindre dans l'espace cellulaire afin de récupérer l'activité observable de la sonde d'interaction. L'intégration de nos résultats aux connaissances des dynamiques de régulations génétiques et des modifications protéiques nous dirigera vers une meilleure compréhension des processus cellulaires complexes orchestrés aux niveaux moléculaires et structuraux dans les cellules vivantes. Nous avons appliqué notre méthode aux réarrangements dynamiques opérant durant l'adaptation de la cellule à des stress, comme la famine en nutriments et le traitement à une drogue. Cette investigation fait le détail de notre second chapitre. Nous avons déterminé de cette manière que l'équilibre entre les formes phosphorylées et déphosphorylées de l'arginine méthyltransférase de Saccharomyces cerevisiae, Hmt1, régulait du même coup sont assemblage en hexamère et son activité enzymatique. L'activité d'Hmt1 a directement un impact dans la progression du cycle cellulaire durant un stress, stabilisant les transcrits de CLB2 et permettant la synthèse de Cln3p. Nous avons utilisé notre criblage afin de déterminer les régulateurs de la phosphorylation d'Hmt1 dans un contexte de traitement à la rapamycin, un inhibiteur de la kinase cible de la rapamycin (TOR). Nous avons identifié la sous-unité catalytique de la phosphatase PP2a, Pph22, activé par l'inhibition de la kinase TOR et la kinase Dbf2, activé durant l'entrée en mitose de la cellule, comme la phosphatase et la kinase responsable de la modification d'Hmt1 et de ses fonctions de régulations dans le cycle cellulaire. Cette approche peut être généralisée afin d'identifier et de lier mécanistiquement les gènes, incluant ceux n'ayant aucune fonction connue, à tout processus cellulaire, comme les mécanismes régulant l'ARNm.

Effects of 15-deoxy-Delta(12, 14) prostaglandin J(2) and ciglitazone on human cancer cell cycle progression and death: The role of PPAR gamma

The role of PPAR-gamma in ciglitazone and 15-d PGJ(2)-induced apoptosis and cell cycle arrest of Jurkat (before and after PPAR gamma gene silencing), U937 (express high levels of PPAR gamma) and HeLa (that express very low levels of PPAR gamma) cells was investigated. PPAR gamma gene silencing, per se, induced a G2/M cell arrest, loss of membrane integrity and DNA fragmentation of Jurkat cells, indicating that PPAR gamma is important for this cell survival and proliferation. Ciglitazone-induced apoptosis was abolished after knockdown of PPAR gamma suggesting a PPAR gamma-dependent pro-apoptotic effect. However, ciglitazone treatment was toxic for U937 and HeLa cells regardless of the presence of PPAR gamma. This treatment did not change the cell cycle distribution corroborating with a PPAR gamma-independent mechanism. On the other hand, 15-d PGJ(2) induced apoptosis of the three cancer cell lines regardless of the expression of PPAR gamma. These results suggest that PPAR gamma plays an important role for death of malignant T lymphocytes (Jurkat cells) and PPAR gamma agonists exert their effects through PPAR gamma-dependent and -independent mechanisms depending on the drug and the cell type. (C) 2007 Elsevier B.V. All rights reserved.

Gamma-linolenic acid inhibits both tumour cell cycle progression and angiogenesis in the orthotopic C6 glioma model through changes in VEGF, Flt1, ERK1/2, MMP2, cyclin D1, pRb, p53 and p27 protein expression

Background: Gamma-linolenic acid is a known inhibitor of tumour cell proliferation and migration in both in vitro and in vivo conditions. The aim of the present study was to determine the mechanisms by which gamma-linolenic acid (GLA) osmotic pump infusion alters glioma cell proliferation, and whether it affects cell cycle control and angiogenesis in the C6 glioma in vivo. Methods: Established C6 rat gliomas were treated for 14 days with 5 mM GLA in CSF or CSF alone. Tumour size was estimated, microvessel density (MVD) counted and protein and mRNA expression measured by immunohistochemistry, western blotting and RT-PCR. Results: GLA caused a significant decrease in tumour size (75 +/- 8.8%) and reduced MVD by 44 +/- 5.4%. These changes were associated with reduced expression of vascular endothelial growth factor (VEGF) (71 +/- 16%) and the VEGF receptor Flt1 (57 +/- 5.8%) but not Flk1. Expression of ERK1/2 was also reduced by 27 +/- 7.7% and 31 +/- 8.7% respectively. mRNA expression of matrix metalloproteinase-2 (MMP2) was reduced by 35 +/- 6.8% and zymography showed MMP2 proteolytic activity was reduced by 32 +/- 8.5%. GLA altered the expression of several proteins involved in cell cycle control. pRb protein expression was decreased (62 +/- 18%) while E2F1 remained unchanged. Cyclin D1 protein expression was increased by 42 +/- 12% in the presence of GLA. The cyclin dependent kinase inhibitors p21 and p27 responded differently to GLA, p27 expression was increased (27 +/- 7.3%) while p21 remained unchanged. The expression of p53 was increased (44 +/- 16%) by GLA. Finally, the BrdU incorporation studies found a significant inhibition (32 +/- 11%) of BrdU incorporation into the tumour in vivo. Conclusion: Overall the findings reported in the present study lend further support to the potential of GLA as an inhibitor of glioma cell proliferation in vivo and show it has direct effects upon cell cycle control and angiogenesis. These effects involve changes in protein expression of VEGF, Flt1, ERK1, ERK2, MMP2, Cyclin D1, pRb, p53 and p27. Combination therapy using drugs with other, complementary targets and GLA could lead to gains in treatment efficacy in this notoriously difficult to treat tumour.

A Robust Structural PGN Model for Control of Cell-Cycle Progression Stabilized by Negative Feedbacks

The cell division cycle comprises a sequence of phenomena controlled by a stable and robust genetic network. We applied a probabilistic genetic network (PGN) to construct a hypothetical model with a dynamical behavior displaying the degree of robustness typical of the biological cell cycle. The structure of our PGN model was inspired in well-established biological facts such as the existence of integrator subsystems, negative and positive feedback loops, and redundant signaling pathways. Our model represents genes interactions as stochastic processes and presents strong robustness in the presence of moderate noise and parameters fluctuations. A recently published deterministic yeast cell-cycle model does not perform as well as our PGN model, even upon moderate noise conditions. In addition, self stimulatory mechanisms can give our PGN model the possibility of having a pacemaker activity similar to the observed in the oscillatory embryonic cell cycle.

New DAG-dependent mechanisms modulate cell cycle progression

Through the years, several studies reported the involvement of nuclear lipid signalling as highly connected with cell cycle progression. Indeed, nuclear Phosphatidylinositol-4,5-Biphosphate (PIP2) hydrolisis mediated by Phospholipases C (PLC), which leads to production of the second messengers Diacylglycerol (DAG) and Inositol-1,4,5-Triphosphate (IP3), is a fundamental event for both G1/S and G2/M checkpoints. In particular, we found that nuclear DAG production was mediated by PLCbeta1, enzyme mainly localized in the nucleus of K562 human erythroleukemia cells. This event triggered the activation and nuclear translocation of PKCalpha, which, in turn, resulted able to affect cell cycle via modulation of Cyclin D3 and Cyclin B1, two important enzymes for G1/S transition and G2/M progression respectively.

miR-34a and miR-15a/16 are co-regulated in non-small cell lung cancer and control cell cycle progression in a synergistic and Rb-dependent manner

microRNAs (miRNAs) are small non-coding RNAs that are frequently involved in carcinogenesis. Although many miRNAs form part of integrated networks, little information is available how they interact with each other to control cellular processes. miR-34a and miR-15a/16 are functionally related; they share common targets and control similar processes including G1-S cell cycle progression and apoptosis. The aim of this study was to investigate the combined action of miR-34a and miR-15a/16 in non-small cell lung cancer (NSCLC) cells.

Perturbation of phosphatidylethanolamine synthesis affects mitochondrial morphology and cell-cycle progression in procyclic-form Trypanosoma brucei

Phosphatidylethanolamine (PE) and phosphatidylcholine (PC) are the two major constituents of eukaryotic cell membranes. In the protist Trypanosoma brucei, PE and PC are synthesized exclusively via the Kennedy pathway. To determine which organelles or processes are most sensitive to a disruption of normal phospholipid levels, the cellular consequences of a decrease in the levels of PE or PC, respectively, were studied following RNAi knock-down of four enzymes of the Kennedy pathway. RNAi against ethanolamine-phosphate cytidylyltransferase (ET) disrupted mitochondrial morphology and ultrastructure. Electron microscopy revealed alterations of inner mitochondrial membrane morphology, defined by a loss of disk-like cristae. Despite the structural changes in the mitochondrion, the cells maintained oxidative phosphorylation. Our results indicate that the inner membrane morphology of T. brucei procyclic forms is highly sensitive to a decrease of PE levels, as a change in the ultrastructure of the mitochondrion is the earliest phenotype observed after RNAi knock-down of ET. Interference with phospholipid synthesis also impaired normal cell-cycle progression. ET RNAi led to an accumulation of multinucleate cells. In contrast, RNAi against choline-/ethanolamine phosphotransferase, which affected PC as well as PE levels, caused a cell division phenotype characterized by non-division of the nucleus and production of zoids.

ARTEMIS INTERACTS WITH THE CUL4A UBIQUITIN E3 LIGASE COMPLEX AND REGULATES THE CELL CYCLE PROGRESSION

Artemis, a member of the SNM1 gene family, is one of the six known components of the non-homologous end joining pathway. It is a multifunctional phospho-protein that has been shown to be modified by the phosphatidylinositol 3-kinases (PIKs) DNA-PKcs, ATM and ATR in response to a variety of cellular stresses. Artemis has important roles in V(D)J recombination, DNA double strand breaks repair and damage-induced cell-cycle checkpoint regulation. The detailed mechanism by which Artemis mediates its functions in these cellular pathways needs to be further elucidated. My work presented here demonstrates a new function for Artemis in cell cycle regulation as a component of Cullin-based E3 ligase complex. I show that Artemis interacts with Cul4A-DDB1 ligase complex via a direct interaction with the substrate-specific receptor DDB2, and deletion mapping analysis shows that part of the Snm1 domain of Artemis is responsible for this interaction. Additionally, Artemis also interacts with p27, a substrate of Cul4A-DDB1 complex, and both DDB2 and Artemis are required for the degradation of p27 mediated by this complex. Furthermore, I show that the regulation of p27 by Artemis and DDB2 is critical for cell cycle progression in normally proliferating cells and in response to serum withdrawal. Finally, I provide evidence showing that Artemis may be also a part of other Cullin-based E3 ligase complexes, and it has a role in controlling p27 levels in response to different cellular stress, such as UV irradiation. These findings suggest a novel pathway to regulate p27 protein level and define a new function for Artemis as an effector of Cullin-based E3-ligase mediated ubiquitylation, and thus, a cell cycle regulator in proliferating cells.

c-{\it mos\/} RNA expression in somatic cells and its physiological significance in cell cycle progression

The c-mos proto-oncogene, which is expressed at relatively high levels in male and female germ cells, plays a key role in oocyte meiotic maturation. The c-mos gene product in oocytes (p39$\sp{\rm c-mos}$) is necessary and sufficient to initiate meiosis. p39$\sp{\rm c-mos}$ is also an essential component of the cytostatic factor, which is responsible for arresting vertebrate oocytes at the second meiotic metaphase by stabilizing the maturation promoting factor (MPF). MPF is a universal regulator of both meiosis and mitosis. Much less is understood about c-mos expression and function in somatic cells. In addition to gonadal tissues, c-Mos has been detected in some somatic tissues and non-germ cell lines including NIH 3T3 cells as a protein termed p43$\sp{\rm c-mos}$. Since c-mos RNA transcripts were not previously detected in this cell line by Northern blot or S1 protection analyses, a search was made for c-mos RNA in NIH 3T3 cells. c-mos transcripts were detected using the highly sensitive RNA-PCR method and RNase protection assays. Furthermore, cell cycle analyses indicated that expression of c-mos RNA is tightly controlled in a cell cycle dependent manner with highest levels of transcripts (approximately 5 copies/cell) during the G2 phase.^ In order to determine the physiological significance of c-mos RNA expression in somatic cells, antisense mos was placed under the control of an inducible promoter and introduced into either NIH 3T3 cells or C2 cells. It was found that a basal level of expression of antisense mos resulted in interference with mitotic progression and growth arrest. Several nuclear abnormalities were observed, especially the appearance of binucleated and multinucleated cells as well as the extrusion of microvesicles containing cellular material. These results indicate that antisense mos expression results in a block in cytokinesis. In summary, these results establish that c-mos expression is not restricted to germ cells, but instead indicate that c-mos RNA expression occurs during the G2 stage of the cell cycle. Furthermore, these studies demonstrate that the c-mos proto-oncogene plays an important role in cell cycle progression. As in meiosis, c-mos may have a similar but not identical function in regulating cell cycle events in somatic cells, particularly in controlling mitotic progression via activation/stabilization of MPF. ^

Identity of adenylyl cyclase isoform determines the rate of cell cycle progression in NIH 3T3 cells

Cell cycle progression is regulated by cAMP in several cell types. Cellular cAMP levels depend on the activity of different adenylyl cyclases (ACs), which have varied signal-receiving capabilities. The role of individual ACs in regulating proliferative responses was investigated. Native NIH 3T3 cells contain AC6, an isoform that is inhibited by a variety of signals. Proliferation of exogenous AC6-expressing cells was the same as in control cells. In contrast, expression of AC2, an isoform stimulated by protein kinase C (PKC), resulted in inhibition of cell cycle progression and increased doubling time. In AC2-expressing cells, platelet-derived growth factor (PDGF) elevated cAMP levels in a PKC-dependent manner. PDGF stimulation of mitogen-activated protein kinases 1 and 2 (MAPK 1,2), DNA synthesis, and cyclin D1 expression was reduced in AC2-expressing cells as compared with control cells. Dominant negative protein kinase A relieved the AC2 inhibition of PDGF-induced DNA synthesis. Expression of AC2 also blocked H-ras-induced transformation of NIH 3T3 cells. These observations indicate that, because AC2 is stimulated by PKC, it can be activated by PDGF concurrently with the stimulation of MAPK 1,2. The elevation in cAMP results in inhibition of signal flow from the PDGF receptor to MAPK 1,2 and a significant reduction in the proliferative response to PDGF. Thus, the molecular identity and signal receiving capability of the AC isoforms in a cell could be important for proliferative homeostasis.