scholarly journals The Ubc3 (Cdc34) ubiquitin-conjugating enzyme is ubiquitinated and phosphorylated in vivo.

1994 ◽  
Vol 14 (5) ◽  
pp. 3022-3029 ◽  
Author(s):  
M G Goebl ◽  
L Goetsch ◽  
B Byers
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Progression ◽  
Protein S ◽  
S Phase ◽  
Cycle Progression ◽  
Endogenous Protein ◽  
Ubiquitin Conjugating Enzyme ◽  
Conjugating Enzyme

The transition from G1 to S phase of the cell cycle in Saccharomyces cerevisiae requires the activity of the Ubc3 (Cdc34) ubiquitin-conjugating enzyme. S. cerevisiae cells lacking a functional UBC3 (CDC34) gene are able to execute the Start function that initiates the cell cycle but fail to form a mitotic spindle or enter S phase. The Ubc3 (Cdc34) enzyme has previously been shown to catalyze the attachment of multiple ubiquitin molecules to model substrates, suggesting that the role of this enzyme in cell cycle progression depends on its targeting an endogenous protein(s) for degradation. In this report, we demonstrate that the Ubc3 (Cdc34) protein is itself a substrate for both ubiquitination and phosphorylation. Immunochemical localization of the gene product to the nucleus renders it likely that the relevant substrates similarly reside within the nucleus.

scholarly journals Cdc53p acts in concert with Cdc4p and Cdc34p to control the G1-to-S-phase transition and identifies a conserved family of proteins.

1996 ◽  
Vol 16 (12) ◽  
pp. 6634-6643 ◽  
Author(s):  
N Mathias ◽  
S L Johnson ◽  
M Winey ◽  
A E Adams ◽  
L Goetsch ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Progression ◽  
Large Family ◽  
S Phase ◽  
Genetic Interactions ◽  
Cycle Progression ◽  
Cyclin Dependent Kinases ◽  
Ubiquitin Conjugating Enzyme ◽  
Regulation Of Cell Cycle

Regulation of cell cycle progression occurs in part through the targeted degradation of both activating and inhibitory subunits of the cyclin-dependent kinases. During G1, CDC4, encoding a WD-40 repeat protein, and CDC34, encoding a ubiquitin-conjugating enzyme, are involved in the destruction of these regulators. Here we describe evidence indicating that CDC53 also is involved in this process. Mutations in CDC53 cause a phenotype indistinguishable from those of cdc4 and cdc34 mutations, numerous genetic interactions are seen between these genes, and the encoded proteins are found physically associated in vivo. Cdc53p defines a large family of proteins found in yeasts, nematodes, and humans whose molecular functions are uncharacterized. These results suggest a role for this family of proteins in regulating cell cycle proliferation through protein degradation.

scholarly journals Role of AKT/mTORC1 pathway in pancreatic β-cell proliferation

2012 ◽  
pp. 235-243 ◽  
Author(s):  
Norman Balcazar Morales ◽  
Cecilia Aguilar de Plata
Keyword(s):  
Cell Cycle ◽  
Growth Factors ◽  
Cell Cycle Progression ◽  
Cell Mass ◽  
Pancreatic Β Cell ◽  
Β Cell ◽  
Cycle Progression ◽  
Mtorc1 Signaling

Growth factors, insulin signaling and nutrients are important regulators of β-cell mass and function. The events linking these signals to regulation of β-cell mass are not completely understood. Recent findings indicate that mTOR pathway integrates signals from growth factors and nutrients with transcription, translation, cell size, cytoskeleton remodeling and mitochondrial metabolism. mTOR is a part of two distinct complexes; mTORC1 and mTORC2. The mammalian TORC1 is sensitive to rapamycin and contains Raptor, deptor, PRAS40 and the G protein β-subunit-like protein (GβL). mTORC1 activates key regulators of protein translation; ribosomal S6 kinase (S6K) and eukaryote initiation factor 4E-binding protein 1. This review summarizes current findings about the role of AKT/mTORC1 signaling in regulation of pancreatic β cell mass and proliferation. mTORC1 is a major regulator of β-cell cycle progression by modulation of cyclins D2, D3 and cdk4/cyclin D activity. These studies uncovered key novel pathways controlling cell cycle progression in β-cells in vivo. This information can be used to develop alternative approaches to expand β-cell mass in vivo and in vitro without the risk of oncogenic transformation. The acquisition of such knowledge is critical for the design of improved therapeutic strategies for the treatment and cure of diabetes as well as to understand the effects of mTOR inhibitors in β-cell function.

scholarly journals Activation of the G2/M-Specific Gene CLB2 Requires Multiple Cell Cycle Signals

2007 ◽  
Vol 27 (23) ◽  
pp. 8364-8373 ◽  
Author(s):  
J. Veis ◽  
H. Klug ◽  
M. Koranda ◽  
G. Ammerer
Keyword(s):  
Cell Cycle ◽  
Transcriptional Activation ◽  
Cell Cycle Progression ◽  
S Phase ◽  
Specific Gene ◽  
Histone H4 ◽  
Cycle Progression ◽  
Histone H4 Acetylation ◽  
Multiple Cell

ABSTRACT In budding yeast (Saccharomyces cerevisiae), the periodic expression of the G2/M-specific gene CLB2 depends on a DNA binding complex that mediates its repression during G1 and activation from the S phase to the exit of mitosis. The switch from low to high expression levels depends on the transcriptional activator Ndd1. We show that the inactivation of the Sin3 histone deacetylase complex bypasses the essential role of Ndd1 in cell cycle progression. Sin3 and its catalytic subunit Rpd3 associate with the CLB2 promoter during the G1 phase of the cell cycle. Both proteins dissociate from the promoter at the onset of the S phase and reassociate during G2 phase. Sin3 removal coincides with a transient increase in histone H4 acetylation followed by the expulsion of at least one nucleosome from the promoter region. Whereas the first step depends on Cdc28/Cln1 activity, Ndd1 function is required for the second step. Since the removal of Sin3 is independent of Ndd1 recruitment and Cdc28/Clb activity it represents a unique regulatory step which is distinct from transcriptional activation.

scholarly journals The p110 Isoform of the CDP/Cux Transcription Factor Accelerates Entry into S Phase

2006 ◽  
Vol 26 (6) ◽  
pp. 2441-2455 ◽  
Author(s):  
Laurent Sansregret ◽  
Brigitte Goulet ◽  
Ryoko Harada ◽  
Brian Wilson ◽  
Lam Leduy ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Transcription Factor ◽  
Cell Cycle Progression ◽  
S Phase ◽  
Proteolytic Processing ◽  
Experimental Conditions ◽  
Division Rate ◽  
Cycle Progression ◽  
Processing Event

ABSTRACT The CDP/Cux transcription factor was previously found to acquire distinct DNA binding and transcriptional properties following a proteolytic processing event that takes place at the G1/S transition of the cell cycle. In the present study, we have investigated the role of the CDP/Cux processed isoform, p110, in cell cycle progression. Populations of cells stably expressing p110 CDP/Cux displayed a faster division rate and reached higher saturation density than control cells carrying the empty vector. p110 CDP/Cux cells reached the next S phase faster than control cells under various experimental conditions: following cell synchronization in G0 by growth factor deprivation, synchronization in S phase by double thymidine block treatment, or enrichment in G2 by centrifugal elutriation. In each case, duration of the G1 phase was shortened by 2 to 4 h. Gene inactivation confirmed the role of CDP/Cux as an accelerator of cell cycle progression, since mouse embryo fibroblasts obtained from Cutl1z/z mutant mice displayed a longer G1 phase and proliferated more slowly than their wild-type counterparts. The delay to enter S phase persisted following immortalization by the 3T3 protocol and transformation with H-RasV12. Moreover, CDP/Cux inactivation hindered both the formation of foci on a monolayer and tumor growth in mice. At the molecular level, expression of both cyclin E2 and A2 was increased in the presence of p110 CDP/Cux and decreased in its absence. Overall, these results establish that p110 CDP/Cux functions as a cell cycle regulator that accelerates entry into S phase.

scholarly journals Association of human ubiquitin-conjugating enzyme CDC34 with the mitotic spindle in anaphase

2000 ◽  
Vol 113 (10) ◽  
pp. 1687-1694 ◽  
Author(s):  
F. Reymond ◽  
C. Wirbelauer ◽  
W. Krek
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Mitotic Spindle ◽  
Mammalian Cells ◽  
Cell Cycle Progression ◽  
Intracellular Localization ◽  
Cycle Progression ◽  
Ubiquitin Conjugating Enzyme ◽  
Regulation Of Cell Cycle ◽  
Conjugating Enzyme

Present in organisms ranging from yeast to man, homologues of the Saccharomyces cerevisiae ubiquitin-conjugating enzyme CDC34 have been shown to play important roles in the regulation of cell cycle progression and checkpoint function. Here we analyze the expression and intracellular localization of endogenous CDC34 during mammalian cell cycle progression. We find that CDC34 protein is constitutively expressed during all stages of the cell cycle. Immunofluorescence experiments reveal that during interphase, endogenous CDC34 is localized to distinct speckles in both the nucleus and the cytoplasm. The presence of CDC34 in these compartments has also been established by biochemical fractionation experiments. Interestingly, nuclear localization depends on the presence of specific carboxy-terminal CDC34 sequences that have previously been shown to be required for CDC34's cell cycle function in Saccharomyces cerevisiae. Finally, we find that in anaphase and not during early stages of mitosis, CDC34 colocalizes with (beta)-tubulin at the mitotic spindle, implying that it may contribute to spindle function at later stages of mitosis. Taken together, these results support a model in which CDC34 ubiquitin-conjugating enzyme functions in the regulation of nuclear and cytoplasmic activities as well as in the process of chromosome segregation at the onset of anaphase in mammalian cells.

IGFBP7 Gene Promoting Cell Proliferation in Acute Myeloid Leukemia Through Activation of AKT3 and CCND1

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1447-1447
Author(s):  
Shaoyan Hu ◽  
Shui-yan Wu ◽  
Jian-nong Cen ◽  
Jian Pan ◽  
Xiaofei Qi ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Myeloid Leukemia ◽  
Cell Cycle Progression ◽  
K562 Cells ◽  
S Phase ◽  
G1 Phase ◽  
Rt Pcr ◽  
Cycle Progression ◽  
Childhood Aml

Abstract Abstract 1447 Insulin-like growth factor binding protein 7 (IGFBP7) has been ascribed properties of both tumor suppressor and enhancer of cell proliferation. In solid tumors the important role of IGFBP7 as a tumor suppressor was revealed in several studies. In acute T-lymphoblastic leukemia (T-ALL), high IGFBP7 expression is associated with a more immature phenotype of early T-ALL, inferior survival, and predicts primary chemotherapy resistance. In a separate study, IGFBP7 acts as a positive regulator of ALL and bone marrow stromal cells growth, and significantly enhances in-vitro resistance to asparaginase. Higher IGFBP7 mRNA levels were associated with lower leukemia-free survival (P=0.003) in precursor B-cell Ph negative ALL patients (n=147) treated with a contemporary polychemotherapy protocol. In acute myeloid leukemia, the role of IGFBP7 is largely unknown. In our previous published study [Hu et al, 2011], we demonstrated that IGFBP7 overexpressed in majority of childhood AML (n=66) at diagnosis and upon relapsed, but not at remission stage. We now further explore its mechanism in promoting AML cells proliferation. Compared with control, transfection of full length IGFBP7 in K562 cells [V-BP7] resulted in 23% increased of proliferation in 48 hours. Cell cycle analysis by flow cytometry showed decreased G0/G1 phase and increased S phase in V-BP7 comparing with control, suggesting enhanced cell cycle progression. While transfection of IGFPB7 siRNA produced an opposite effect of reducing the cell growth in K562 cells. In consistent with the nature of a secretory protein, the extracellular IGFBP7 level in the condition media from v-BP7 was significantly higher than that from vector control or parental K562 cells measured by ELISA. Incubation parental K562 cells in V-BP7 derived conditioned medium resulted in significant growth enhancement. Gene expression profiling (GEP) was performed on V-BP7 in contrast to parental K562 cells. Genes which were up-regulated or down-regulated more than 2 folds were regarded as significant difference. Among 10 verified genes, AKT3 showed the highest extent of up-regulation and IGFBP7 siRNA transfection reduced its expression. Cyclin D1 (CCND1) expression was also significantly up-regulated and validated by RT-PCR and Western blot. V-BP7 treated with an AKT inhibitor (Triciribine) at 2.5μM for 72 hours showed 50% reduction of cell viability. The cell cycle analysis indicated that triciribine reversed cell cycle progression in V-BP7, by increasing cells in G0/G1 phase and reducing cells in S phase. Western blot demonstrated that both phospho-AKT3 and CCND1 were down regulated after treatment with triciribine. Using real time RT-PCR, we further identified that IGFBP7 and AKT3 expression were significantly correlated (p=0.001; r=0.255) in 39 newly diagnosed childhood AML. Conclusions IGFBP7 aberrantly overexpressed in majority of childhood AML. IGFBP7 promotes proliferation of K562 cells and AML via overexpression/activation of AKT3 and CCND1. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Blocking the transcription factor E2F/DP by dominant-negative mutants in a normal breast epithelial cell line efficiently inhibits apoptosis and induces tumor growth in SCID mice.

1996 ◽  
Vol 183 (3) ◽  
pp. 1205-1213 ◽  
Author(s):  
R C Bargou ◽  
C Wagener ◽  
K Bommert ◽  
W Arnold ◽  
P T Daniel ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Transcription Factor ◽  
Tumor Growth ◽  
Cell Cycle Progression ◽  
S Phase ◽  
Dominant Negative ◽  
Serum Starvation ◽  
Cycle Progression ◽  
Transcription Factor E2f

The transcription factor E2F is regulated during the cell cycle through interactions with the product of the retinoblastoma susceptibility gene and related proteins. It is thought that E2F-mediated gene regulation at the G1/S boundary and during S phase may be one of the rate-limiting steps in cell proliferation. It was reported that in vivo overexpression of E2F-1 in fibroblasts induces S phase entry and leads to apoptosis. This observation suggests that E2F plays a role in both cell cycle regulation and apoptosis. To further understand the role of E2F in cell cycle progression, cell death, and tumor development, we have blocked endogenous E2F activity in HBL-100 cells, derived from nonmalignant human breast epithelium, using dominant-negative mutants under the control of a tetracycline-dependent expression system. We have shown here that induction of dominant-negative mutants led to strong downregulation of transiently transfected E2F-dependent chloramphenicol acetyl transferase reporter constructs and of endogenous c-myc, which has been described as a target gene of the transcription factor E2F/DP. In addition, we have shown that blocking of E2F could efficiently protect from apoptosis induced by serum starvation within a period of 10 d, whereas control cells started to die after 24 h. Surprisingly, blocking of E2F did not alter the rate of proliferation or of DNA synthesis of these cells; this finding indicates that cell-cycle progression could be driven in an E2F-independent manner. In addition, we have been able to show that blocking of endogenous E2F in HBL-100 cells led to rapid induction of tumor growth in severe combined immunodeficiency mice. No tumor growth could be observed in mice that received mock-transfected clones or tetracycline to block expression of the E2F mutant constructs in vivo. Thus, it appears that E2F has a potential tumor-suppressive function under certain circumstances. Furthermore, we provide evidence that dysregulation of apoptosis may be an important step in tumorigenesis.

scholarly journals A ubiquitin-conjugating enzyme in fission yeast that is essential for the onset of anaphase in mitosis.

1997 ◽  
Vol 17 (6) ◽  
pp. 3388-3397 ◽  
Author(s):  
F Osaka ◽  
H Seino ◽  
T Seno ◽  
F Yamao
Keyword(s):  
Fission Yeast ◽  
Cell Cycle Progression ◽  
Protein S ◽  
Growth Defect ◽  
Restrictive Temperature ◽  
Checkpoint Pathway ◽  
Mitotic Cyclin ◽  
Ubiquitin Conjugating Enzyme ◽  
Conjugating Enzyme

A cDNA encoding a ubiquitin-conjugating enzyme designated UbcP4 in fission yeast was isolated. Disruption of its genomic gene revealed that it was essential for cell viability. In vivo depletion of the UbcP4 protein demonstrated that it was necessary for cell cycle progression at two phases, G2/M and metaphase/anaphase transitions. The G2 arrest of UbcP4-depleted cells was dependent upon chk1, which mediates checkpoint pathway. UbcP4-depleted cells arrested at metaphase had condensed chromosomes but were defective in separation. However, septum formation and cytokinesis were not restrained during the metaphase arrest. Overexpression of UbcP4 specifically rescued the growth defect of cut9ts cells at a restrictive temperature. cut9 encodes a component of the anaphase-promoting complex (APC) which is required for chromosome segregation at anaphase and moreover is defined as cyclin-specific ubiquitin ligase. Cdc13, a mitotic cyclin in fission yeast, was accumulated in the UbcP4-depleted cells. These results strongly suggested that UbcP4 is a ubiquitin-conjugating enzyme working in conjunction with APC and mediates the ubiquitin pathway for degradation of "sister chromatid holding protein(s)" at the onset of anaphase and possibly of mitotic cyclin at the exit of mitosis.

scholarly journals The Drosophila F-box protein dSkp2 regulates cell proliferation by targeting Dacapo for degradation

2013 ◽  
Vol 24 (11) ◽  
pp. 1676-1687 ◽  
Author(s):  
Wen Dui ◽  
Bin Wei ◽  
Feng He ◽  
Wei Lu ◽  
Changqing Li ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Progression ◽  
Functional Relationship ◽  
Kinase Inhibitors ◽  
Cycle Progression ◽  
Cell Doubling Time ◽  
F Box Protein ◽  
Negative Factors

Cell cycle progression is controlled by a complex regulatory network consisting of interacting positive and negative factors. In humans, the positive regulator Skp2, an F-box protein, has been a subject of intense investigation in part because of its oncogenic activity. By contrast, the molecular and developmental functions of its Drosophila homologue, dSkp2, are poorly understood. Here we investigate the role of dSkp2 by focusing on its functional relationship with Dacapo (Dap), the Drosophila homologue of the cyclin-dependent kinase inhibitors p21cip1/p27kip1/p57kip2. We show that dSkp2 interacts physically with Dap and has a role in targeting Dap for ubiquitination and proteasome-mediated degradation. We present evidence that dSkp2 regulates cell cycle progression by antagonizing Dap in vivo. dSkp2 knockdown reduces cell density in the wing by prolonging the cell doubling time. In addition, the wing phenotype caused by dSkp2 knockdown resembles that caused by dap overexpression and can be partially suppressed by reducing the gene dose of dap. Our study thus documents a conserved functional relationship between dSkp2 and Dap in their control of cell cycle progression, suggesting the possibility of using Drosophila as a model system to study Skp2-mediated tumorigenesis.

scholarly journals CUL4B promotes replication licensing by up-regulating the CDK2–CDC6 cascade

2013 ◽  
Vol 200 (6) ◽  
pp. 743-756 ◽  
Author(s):  
Yongxin Zou ◽  
Jun Mi ◽  
Wenxing Wang ◽  
Juanjuan Lu ◽  
Wei Zhao ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Progression ◽  
Positive Regulation ◽  
Cycle Progression ◽  
Developmental Abnormalities ◽  
Replication Complex ◽  
Cellular Processes ◽  
Cell Cycle Reentry

Cullin-RING ubiquitin ligases (CRLs) participate in the regulation of diverse cellular processes including cell cycle progression. Mutations in the X-linked CUL4B, a member of the cullin family, cause mental retardation and other developmental abnormalities in humans. Cells that are deficient in CUL4B are severely selected against in vivo in heterozygotes. Here we report a role of CUL4B in the regulation of replication licensing. Strikingly, CDC6, the licensing factor in replication, was positively regulated by CUL4B and contributed to the loading of MCM2 to chromatin. The positive regulation of CDC6 by CUL4B depends on CDK2, which phosphorylates CDC6, protecting it from APCCDH1-mediated degradation. Thus, aside being required for cell cycle reentry from quiescence, CDK2 also contributes to pre-replication complex assembly in G1 phase of cycling cells. Interestingly, the up-regulation of CDK2 by CUL4B is achieved via the repression of miR-372 and miR-373, which target CDK2. Our findings thus establish a CUL4B–CDK2–CDC6 cascade in the regulation of DNA replication licensing.

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