scholarly journals Cell cycle control by Xenopus p28Kix1, a developmentally regulated inhibitor of cyclin-dependent kinases.

1996 ◽  
Vol 7 (3) ◽  
pp. 457-469 ◽  
Author(s):  
W Shou ◽  
W G Dunphy
Keyword(s):  
Cell Cycle ◽  
Mammalian Cells ◽  
Cell Cycle Progression ◽  
Cyclin E ◽  
Cell Cycle Control ◽  
Cyclin A ◽  
Cyclin B ◽  
Nuclear Antigen ◽  
Cdk Inhibitors ◽  
Egg Extracts

We have isolated Xenopus p28Kix1, a member of the p21CIP1/p27KIP1/p57KIP2 family of cyclin-dependent kinase (Cdk) inhibitors. Members of this family negatively regulate cell cycle progression in mammalian cells by inhibiting the activities of Cdks. p28 shows significant sequence homology with p21, p27, and p57 in its N-terminal region, where the Cdk inhibition domain is known to reside. In contrast, the C-terminal domain of p28 is distinct from that of p21, p27, and p57. In co-immunoprecipitation experiments, p28 was found to be associated with Cdk2, cyclin E, and cyclin A, but not the Cdc2/cyclin B complex in Xenopus egg extracts. Xenopus p28 associates with the proliferating cell nuclear antigen, but with a substantially lower affinity than human p21. In kinase assays with recombinant Cdks, p28 inhibits pre-activated Cdk2/cyclin E and Cdk2/cyclin A, but not Cdc2/cyclin B. However, at high concentrations, p28 does prevent the activation of Cdc2/cyclin B by the Cdk-activating kinase. Consistent with the role of p28 as a Cdk inhibitor, recombinant p28 elicits an inhibition of both DNA replication and mitosis upon addition to egg extracts, indicating that it can regulate multiple cell cycle transitions. The level of p28 protein shows a dramatic developmental profile: it is low in Xenopus oocytes, eggs, and embryos up to stage 11, but increases approximately 100-fold between stages 12 and 13, and remains high thereafter. The induction of p28 expression temporally coincides with late gastrulation. Thus, although p28 may play only a limited role during the early embryonic cleavages, it may function later in development to establish a somatic type of cell cycle. Taken together, our results indicate that Xenopus p28 is a new member of the p21/p27/p57 class of Cdk inhibitors, and that it may play a role in developmental processes.

Association of cyclin-bound p34cdc2 with subcellular structures in xenopus eggs

1992 ◽  
Vol 102 (2) ◽  
pp. 285-297 ◽  
Author(s):  
D. Leiss ◽  
M.A. Felix ◽  
E. Karsenti
Keyword(s):  
Cell Cycle ◽  
Ribosomal Proteins ◽  
Cell Cycle Progression ◽  
Gradient Centrifugation ◽  
Preliminary Evidence ◽  
Embryonic Cell ◽  
Cytoskeletal Proteins ◽  
Cyclin B ◽  
Post Translational Modifications ◽  
Egg Extracts

Cell cycle progression is controlled by changes in kinase activity of homologs of the fission yeast protein p34cdc2. The p34cdc2 kinase is activated by its association with a cyclin subunit, followed by post-translational modifications. Here, we show that in Xenopus eggs stimulated to enter the early embryonic cell cycle by an electric shock, part of the p34cdc2 becomes associated with subcellular fractions as the eggs progress towards mitosis. This occurs as a result of cyclin accumulation because most of the B-type cyclins and some of the A-type cyclins are found in the particulate fraction. Moreover, as soon as cyclins are degraded, p34cdc2 is released in the soluble fraction. The p34cdc2-cyclin complex can be solubilised by 80 mM beta-glycerophosphate (in the standard MPF extraction buffer) or by high salt concentrations. The post-translational modifications leading to cdc2 kinase activation by cyclin occur in the insoluble form. Following fractionation of egg extracts by sucrose gradient centrifugation, the p34cdc2-cyclin B complex is found in several fractions, but especially in two discrete peaks. We present evidence that in the slow-sedimenting peak the p34cdc2-cyclin B complex is associated with the 60 S subunit of monoribosomes. It could be targeted in this fashion to substrates such as ribosomal proteins and maybe to cytoskeletal proteins, since ribosomes bind to microtubules and are present in the spindle. The p34cdc2-cyclin B complex is also found in a faster-migrating fraction containing various membranous structures, including Golgi stacks. Therefore, as observed by immunofluorescence in other systems, it seems that cyclin subunits target p34cdc2 to specific cellular sites and this is certainly important for its function. In addition, we present preliminary evidence suggesting that some component present in the ribosome-containing fraction is required for activation of the p34cdc2-cyclin B complex.

The Polo-like kinase Plx1 is a component of the MPF amplification loop at the G2/M-phase transition of the cell cycle in Xenopus eggs

1998 ◽  
Vol 111 (12) ◽  
pp. 1751-1757 ◽  
Author(s):  
A. Abrieu ◽  
T. Brassac ◽  
S. Galas ◽  
D. Fisher ◽  
J.C. Labbe ◽  
...  
Keyword(s):  
Phase Transition ◽  
Cell Cycle ◽  
Cyclin A ◽  
Cyclin B ◽  
Cdc2 Kinase ◽  
C Terminus ◽  
M Phase ◽  
Egg Extracts ◽  
Amplification Loop

We have investigated whether Plx1, a kinase recently shown to phosphorylate cdc25c in vitro, is required for activation of cdc25c at the G2/M-phase transition of the cell cycle in Xenopus. Using immunodepletion or the mere addition of an antibody against the C terminus of Plx1, which suppressed its activation (not its activity) at G2/M, we show that Plx1 activity is required for activation of cyclin B-cdc2 kinase in both interphase egg extracts receiving recombinant cyclin B, and cycling extracts that spontaneously oscillate between interphase and mitosis. Furthermore, a positive feedback loop allows cyclin B-cdc2 kinase to activate Plx1 at the G2/M-phase transition. In contrast, activation of cyclin A-cdc2 kinase does not require Plx1 activity, and cyclin A-cdc2 kinase fails to activate Plx1 and its consequence, cdc25c activation in cycling extracts.

CD4+CD25+ Regulatory T Cells from Cord Blood Have Functional and Molecular Properties of T Cell Anergy.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 316-316
Author(s):  
Lequn Li ◽  
Wayne R. Godfrey ◽  
Stephen B. Porter ◽  
Ying Ge ◽  
Carle H. June ◽  
...  
Keyword(s):  
Cell Cycle ◽  
T Cells ◽  
Regulatory T Cells ◽  
Cord Blood ◽  
Cell Lines ◽  
Cell Cycle Progression ◽  
Map Kinases ◽  
Cyclin E ◽  
Cyclin A ◽  
Cycle Progression

Abstract CD4+CD25+ regulatory T cells (Tr) are negative regulators of immune responses. Studies of human Tr are restricted by their small numbers in peripheral blood and their hypoproliferative state. A recently established method achieved in vitro expansion and generation of Tr cell lines (Godfrey et al; Blood 2004,104:453-61). This approach facilitates the evaluation of cultured Tr cells as a novel form of immunosuppressive therapy and provides a system for molecular analysis of Tr. Activation of Ras and MAP kinases is mandatory for IL-2 production, viability and cell cycle progression of T cells. In anergic T cells activation of these signaling events is impaired, whereas activation of Rap1 is retained. Subsequently, anergic cells have defective IL-2 production, impaired cell cycle progression, and increased susceptibility to apoptosis. In the current study, we sought to determine the signaling and biochemical properties of Tr. Human CD4+CD25+ (Tr) and control CD4+CD25− (Tc) cell lines were generated from human cord blood cells. We examined activation of Ras, Rap1 and MAP kinases as well as cell cycle progression and cell viability, in response to TCR/CD3-plus-CD28 mediated stimulation. Stimulation was done for 15 min, 2 and 16 hrs for assessment of signaling events or for 24, 48 and 72 hrs for assessment of cell cycle progression and viability. Although activation of Rap1 was not affected, activation of Ras was reduced in Tr as compared to Tc. Activation of JNK and Erk1/2 MAP kinases was also significantly impaired. Both Tr and Tc entered the cell cycle and expressed cyclin E and cyclin A at 24 and 48 hrs of culture. However, p27 was downregulated only in Tc and not in Tr and hyperphosphorylation of Rb, which is the hallmark of cell cycle progression, was detected only in the Tc and not in the Tr population. At 72 hrs of culture, expression of cyclin E and cyclin A was dramatically diminished in Tr whereas it remained unchanged in Tc. More strikingly, expression of p27 in Tr was increased to levels higher than background. Since Tr do not produce IL-2, we examined whether addition of exogenous IL-2 would downregulate p27 and rescue Tr from defective cell cycle progression, similarly to its effect on anergic cells. Addition of exogenous IL-2 resulted in decrease of p27, sustained increase of cyclin E and cyclin A and cell cycle progression. Besides inhibiting cell cycle progression, p27 also promotes apoptosis. Therefore, we examined whether Tr had a higher susceptibility to apoptosis. As determined by Annexin V staining, Tr had a high degree of apoptosis only at 72 hrs of culture, when p27 expression was highly upregulated. Exogenous IL-2 reversed both p27 upregulation and apoptosis. Addition of IL-2 to Tr, also resulted in sustained IL-2-receptor-mediated activation of Erk1/2 at levels equivalent to those of Tc. Thus Tr cells share many biochemical and molecular characteristics of anergy, including defective TCR/CD3-plus-CD28-mediated activation of Ras and MAP kinases, increased expression of p27, defective cell cycle progression and high susceptibility to apoptosis. Moreover, these results suggest that TCR/CD3-mediated and IL-2 receptor-mediated signals converge at the level of MAP kinases to determine the fate of Tr cells towards expansion or cell cycle arrest and subsequent apoptosis.

scholarly journals Cyclin E2, a Novel G1 Cyclin That Binds Cdk2 and Is Aberrantly Expressed in Human Cancers

1999 ◽  
Vol 19 (1) ◽  
pp. 612-622 ◽  
Author(s):  
Jean M. Gudas ◽  
Marc Payton ◽  
Sushil Thukral ◽  
Eddy Chen ◽  
Michael Bass ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Mammalian Cells ◽  
Cell Cycle Progression ◽  
Expressed Sequence Tag ◽  
Cyclin E ◽  
Cyclin E1 ◽  
Acid Protein ◽  
Cyclin E2 ◽  
Rate Limiting ◽  
Amino Acid Protein

ABSTRACT A novel cyclin gene was discovered by searching an expressed sequence tag database with a cyclin box profile. The human cyclin E2 gene encodes a 404-amino-acid protein that is most closely related to cyclin E. Cyclin E2 associates with Cdk2 in a functional kinase complex that is inhibited by both p27Kip1 and p21Cip1. The catalytic activity associated with cyclin E2 complexes is cell cycle regulated and peaks at the G1/S transition. Overexpression of cyclin E2 in mammalian cells accelerates G1, demonstrating that cyclin E2 may be rate limiting for G1 progression. Unlike cyclin E1, which is expressed in most proliferating normal and tumor cells, cyclin E2 levels were low to undetectable in nontransformed cells and increased significantly in tumor-derived cells. The discovery of a novel second cyclin E family member suggests that multiple unique cyclin E-CDK complexes regulate cell cycle progression.

scholarly journals Cell Cycle Dysregulation by Human Cytomegalovirus: Influence of the Cell Cycle Phase at the Time of Infection and Effects on Cyclin Transcription

1998 ◽  
Vol 72 (5) ◽  
pp. 3729-3741 ◽  
Author(s):  
Bryan S. Salvant ◽  
Elizabeth A. Fortunato ◽  
Deborah H. Spector
Keyword(s):  
Cell Cycle ◽  
Dna Synthesis ◽  
Cell Cycle Progression ◽  
Hcmv Infection ◽  
Cyclin A ◽  
S Phase ◽  
Cyclin B ◽  
Cycle Progression ◽  
Cycle Arrest ◽  
Time Of Infection

ABSTRACT Human cytomegalovirus (HCMV) infection inhibits cell cycle progression and alters the expression of cyclins E, A, and B (F. M. Jault, J.-M. Jault, F. Ruchti, E. A. Fortunato, C. Clark, J. Corbeil, D. D. Richman, and D. H. Spector, J. Virol. 69:6697–6704, 1995). In this study, we examined cell cycle progression, cyclin gene expression, and early viral events when the infection was initiated at different points in the cell cycle (G0, G1, and S). In all cases, infection led to cell cycle arrest. Cells infected in G0 or G1phase also showed a complete or partial absence, respectively, of cellular DNA synthesis at a time when DNA synthesis occurred in the corresponding mock-infected cells. In contrast, when cells were infected near or during S phase, many cells were able to pass through S phase and undergo mitosis prior to cell cycle arrest. S-phase infection also produced a delay in the appearance of the viral cytopathic effect and in the synthesis of immediate-early and early proteins. Labeling of cells with bromodeoxyuridine immediately prior to HCMV infection in S phase revealed that viral protein expression occurred primarily in cells which were not engaged in DNA synthesis at the time of infection. The viral-mediated induction of cyclin E, maintenance of cyclin-B protein levels, and inhibitory effects on the accumulation of cyclin A were not significantly affected when infection occurred during different phases of the cell cycle (G0, G1, and S). However, there was a delay in the observed inhibition of cyclin A in cells infected during S phase. This finding was in accord with the pattern of cell cycle progression and delay in viral gene expression associated with S-phase infection. Analysis of the mRNA revealed that the effects of the virus on cyclin E and cyclin A, but not on cyclin B, were primarily at the transcriptional level.

scholarly journals Cellular targets for activation by the E2F1 transcription factor include DNA synthesis- and G1/S-regulatory genes.

1995 ◽  
Vol 15 (8) ◽  
pp. 4215-4224 ◽  
Author(s):  
J DeGregori ◽  
T Kowalik ◽  
J R Nevins
Keyword(s):  
Transcription Factor ◽  
Cell Cycle Progression ◽  
Target Genes ◽  
Recombinant Adenovirus ◽  
Cyclin E ◽  
Cyclin A ◽  
S Phase ◽  
Nuclear Antigen ◽  
G1 Arrest ◽  
E2f1 Transcription Factor

Although a number of transfection experiments have suggested potential targets for the action of the E2F1 transcription factor, as is the case for many transcriptional regulatory proteins, the actual targets in their normal chromosomal environment have not been demonstrated. We have made use of a recombinant adenovirus containing the E2F1 cDNA to infect quiescent cells and then measure the activation of endogenous cellular genes as a consequence of E2F1 production. We find that many of the genes encoding S-phase-acting proteins previously suspected to be E2F targets, including DNA polymerase alpha, thymidylate synthase, proliferating cell nuclear antigen, and ribonucleotide reductase, are indeed induced by E2F1. Several other candidates, including the dihydrofolate reductase and thymidine kinase genes, were only minimally induced by E2F1. In addition to the S-phase genes, we also find that several genes believed to play regulatory roles in cell cycle progression, such as the cdc2, cyclin A, and B-myb genes, are also induced by E2F1. Moreover, the cyclin E gene is strongly induced by E2F1, thus defining an autoregulatory circuit since cyclin E-dependent kinase activity can stimulate E2F1 transcription, likely through the phosphorylation and inactivation of Rb and Rb family members. Finally, we also demonstrate that a G1 arrest brought about by gamma irradiation is overcome by the overexpression of E2F1 and that this coincides with the enhanced activation of key target genes, including the cyclin A and cyclin E genes.

Rapamycin-induced G1 arrest in cycling B-CLL cells is associated with reduced expression of cyclin D3, cyclin E, cyclin A, and survivin

Blood ◽  
2003 ◽  
Vol 101 (1) ◽  
pp. 278-285 ◽  
Author(s):  
Thomas Decker ◽  
Susanne Hipp ◽  
Ingo Ringshausen ◽  
Christian Bogner ◽  
Madlene Oelsner ◽  
...  
Keyword(s):  
Cell Cycle ◽  
B Cells ◽  
Cell Cycle Regulation ◽  
Cell Cycle Progression ◽  
Cyclin E ◽  
Cyclin A ◽  
Cyclin D3 ◽  
G1 Arrest ◽  
Cycle Progression ◽  
Cycle Regulation

Abstract In B-cell chronic lymphocytic leukemia (B-CLL), malignant cells seem to be arrested in the G0/early G1phase of the cell cycle, and defective apoptosis might be involved in disease progression. However, increasing evidence exists that B-CLL is more than a disease consisting of slowly accumulating resting B cells: a proliferating pool of cells has been described in lymph nodes and bone marrow and might feed the accumulating pool in the blood. Rapamycin has been reported to inhibit cell cycle progression in a variety of cell types, including human B cells, and has shown activity against a broad range of human tumor cell lines. Therefore, we investigated the ability of rapamycin to block cell cycle progression in proliferating B-CLL cells. We have recently demonstrated that stimulation with CpG-oligonucleotides and interleukin-2 provides a valuable model for studying cell cycle regulation in malignant B cells. In our present study, we demonstrated that rapamycin induced cell cycle arrest in proliferating B-CLL cells and inhibited phosphorylation of p70s6 kinase (p70s6k). In contrast to previous reports on nonmalignant B cells, the expression of the cell cycle inhibitor p27 was not changed in rapamycin-treated leukemic cells. Treatment with rapamycin prevented retinoblastoma protein (RB) phosphorylation in B-CLL cells without affecting the expression of cyclin D2, but cyclin D3 was no longer detectable in rapamycin-treated B-CLL cells. In addition, rapamycin treatment inhibited cyclin-dependent kinase 2 activity by preventing up-regulation of cyclin E and cyclin A. Interestingly, survivin, which is expressed in the proliferation centers of B-CLL patients in vivo, is not up-regulated in rapamycin-treated cells. Therefore, rapamycin interferes with the expression of many critical molecules for cell cycle regulation in cycling B-CLL cells. We conclude from our study that rapamycin might be an attractive substance for therapy for B-CLL patients by inducing a G1 arrest in proliferating tumor cells.

scholarly journals Golgi-associated RhoBTB3 targets Cyclin E for ubiquitylation and promotes cell cycle progression

2013 ◽  
Vol 203 (2) ◽  
pp. 233-250 ◽  
Author(s):  
Albert Lu ◽  
Suzanne R. Pfeffer
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Progression ◽  
Cyclin E ◽  
Cell Cycle Control ◽  
S Phase ◽  
Cycle Progression ◽  
Ubiquitin Ligase Complex ◽  
Golgi Fragmentation ◽  
Ring E3 ◽  
Normal Cell Cycle

Cyclin E regulates the cell cycle transition from G1 to S phase and is degraded before entry into G2 phase. Here we show that RhoBTB3, a Golgi-associated, Rho-related ATPase, regulates the S/G2 transition of the cell cycle by targeting Cyclin E for ubiquitylation. Depletion of RhoBTB3 arrested cells in S phase, triggered Golgi fragmentation, and elevated Cyclin E levels. On the Golgi, RhoBTB3 bound Cyclin E as part of a Cullin3 (CUL3)-dependent RING–E3 ubiquitin ligase complex comprised of RhoBTB3, CUL3, and RBX1. Golgi association of this complex was required for its ability to catalyze Cyclin E ubiquitylation and allow normal cell cycle progression. These experiments reveal a novel role for a Ras superfamily member in catalyzing Cyclin E turnover during S phase, as well as an unexpected, essential role for the Golgi as a ubiquitylation platform for cell cycle control.

scholarly journals Overexpression of Cyclin A Inhibits Augmentation of Recombinant Adeno-Associated Virus Transduction by the Adenovirus E4orf6 Protein

1999 ◽  
Vol 73 (12) ◽  
pp. 10010-10019 ◽  
Author(s):  
Mirta Grifman ◽  
Nancie N. Chen ◽  
Guang-ping Gao ◽  
Toni Cathomen ◽  
James M. Wilson ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Progression ◽  
Cell Cycle Control ◽  
Cyclin A ◽  
Cycle Progression ◽  
Adeno Associated Virus ◽  
P53 Expression ◽  
Reading Frame ◽  
E4 Region ◽  
Wide Range

ABSTRACT The 34-kDa product of adenovirus E4 region open reading frame 6 (E4orf6) dramatically enhances transduction by recombinant adeno-associated virus vectors (rAAV). This is achieved by promoting the conversion of incoming single-stranded viral genomes into transcriptionally competent duplex molecules. The molecular mechanism for enhancing second-strand synthesis is not fully understood. In this study, we analyzed the cellular consequences of E4orf6 expression and the requirements for efficient rAAV transduction mediated by E4orf6. Expression of E4orf6 in 293 cells led to an inhibition of cell cycle progression and an accumulation of cells in S phase. This was preceded by specific degradation of cyclin A and p53, while the levels of other proteins involved in cell cycle control remained unchanged. In addition, the kinase activity of cdc2 was inhibited. We further showed that p53 expression is not necessary or inhibitory for augmentation of rAAV transduction by E4orf6. However, overexpression of cyclin A inhibited E4orf6-mediated enhancement of rAAV transduction. A cyclin A mutant incapable of recruiting protein substrates for cdk2 was unable to inhibit E4orf6-mediated augmentation. In addition, we created an E4orf6 mutant that is selectively defective in rAAV augmentation of transduction. Based on these findings, we suggest that cyclin A degradation represents a viral mechanism to disrupt cell cycle progression, resulting in enhanced viral transduction. Understanding the cellular pathways used during transduction will increase the utility of rAAV vectors in a wide range of gene therapy applications.

scholarly journals Myc stimulates cell cycle progression through the activation of Cdk1 and phosphorylation of p27

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Lucía García-Gutiérrez ◽  
Gabriel Bretones ◽  
Ester Molina ◽  
Ignacio Arechaga ◽  
Catherine Symonds ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Progression ◽  
Human Cancer ◽  
Cyclin A ◽  
Cyclin B1 ◽  
Cdk Inhibitors ◽  
Synthetic Lethal Interaction ◽  
Cycle Progression ◽  
Synthetic Lethal ◽  
Murine Fibroblasts

AbstractCell cycle stimulation is a major transforming mechanism of Myc oncoprotein. This is achieved through at least three concomitant mechanisms: upregulation of cyclins and Cdks, downregulation of the Cdk inhibitors p15 and p21 and the degradation of p27. The Myc-p27 antagonism has been shown to be relevant in human cancer. To be degraded, p27 must be phosphorylated at Thr-187 to be recognized by Skp2, a component of the ubiquitination complex. We previously described that Myc induces Skp2 expression. Here we show that not only Cdk2 but Cdk1 phosphorylates p27 at the Thr-187. Moreover, Myc induced p27 degradation in murine fibroblasts through Cdk1 activation, which was achieved by Myc-dependent cyclin A and B induction. In the absence of Cdk2, p27 phosphorylation at Thr-187 was mainly carried out by cyclin A2-Cdk1 and cyclin B1-Cdk1. We also show that Cdk1 inhibition was enough for the synthetic lethal interaction with Myc. This result is relevant because Cdk1 is the only Cdk strictly required for cell cycle and the reported synthetic lethal interaction between Cdk1 and Myc.

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