scholarly journals Protein kinase B/Akt is essential for the insulin- but not progesterone-stimulated resumption of meiosis in Xenopus oocytes

2003 ◽  
Vol 369 (2) ◽  
pp. 227-238 ◽  
Author(s):  
Carsten B. ANDERSEN ◽  
Hiroshi SAKAUE ◽  
Taku NEDACHI ◽  
Kristina S. KOVACINA ◽  
Carol CLAYBERGER ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Cell Cycle Progression ◽  
Time Course ◽  
Xenopus Oocytes ◽  
Northern Blotting ◽  
Dominant Negative ◽  
Cycle Progression ◽  
Reverse Transcription Pcr ◽  
Rapid Activation ◽  
Stimulation Of

In the present study, we have characterized the Xenopus Akt expressed in oocytes from the African clawed frog Xenopus laevis and tested whether its activity is required for the insulin- and progesterone-stimulated resumption of meiosis. A cDNA encoding the Xenopus Akt was isolated and sequenced, and its expression in the Xenopus oocyte was confirmed by reverse transcription PCR and Northern blotting. Using phosphospecific antibodies and enzyme assays, a large and rapid activation of the Xenopus Akt was observed upon insulin stimulation of the oocytes. In contrast, progesterone caused a modest activation of this kinase with a slower time course. To test whether the activation of Akt was required in the stimulation of the resumption of meiosis, we have utilized two independent approaches: a functional dominant negative Akt mutant and an inhibitory monoclonal antibody. Both the mutant Akt, as well as the inhibitory monoclonal antibody, completely blocked the insulin-stimulated resumption of meiosis. In contrast, both treatments only partially inhibited (by approx. 30%) the progesterone-stimulated resumption of meiosis when submaximal doses of this hormone were utilized. These data demonstrate a crucial role for Akt in the insulin-stimulated cell cycle progression of Xenopus oocytes, whereas Akt may have an ancillary function in progesterone signalling.

The transcription factor E2F-1 mediates the autoregulation of RB gene expression

1994 ◽  
Vol 14 (1) ◽  
pp. 299-309
Author(s):  
B Shan ◽  
C Y Chang ◽  
D Jones ◽  
W H Lee
Keyword(s):  
Promoter Region ◽  
Promoter Activity ◽  
Cell Cycle Progression ◽  
Gene Mutations ◽  
Suppressor Gene ◽  
Recognition Sequence ◽  
Cycle Progression ◽  
Rb Gene ◽  
Transcription Factor E2f ◽  
Stimulation Of

The retinoblastoma (RB) gene is the prototype tumor suppressor gene. Mutations in this gene are often associated with the occurrence of various tumors. Several mutations have been found in the promoter region of the gene, suggesting that inappropriate transcriptional regulation of the RB gene contributes to tumorigenesis. Sequence analysis of the RB promoter has revealed a potential E2F recognition site within a region critical for RB gene transcription. By using the cloned E2F-1 gene, here we report that (i) RB expression is negatively regulated by its own gene product, (ii) E2F-1 binds specifically to an E2F recognition sequence in the RB promoter and transactivates the RB promoter, (iii) overexpression of RB suppresses E2F-1-mediated stimulation of RB promoter activity, and (iv) the expression of the RB gene is paralleled by the expression of the E2F-1 gene during cell cycle progression. These results demonstrate that expression of RB is negatively autoregulated through E2F-1.

scholarly journals A Dominant-Negative PPARγMutant Promotes Cell Cycle Progression and Cell Growth in Vascular Smooth Muscle Cells

PPAR Research ◽  
2009 ◽  
Vol 2009 ◽  
pp. 1-10 ◽  
Author(s):  
Joey Z. Liu ◽  
Christopher J. Lyon ◽  
Willa A. Hsueh ◽  
Ronald E. Law
Keyword(s):  
Cell Cycle ◽  
Cell Proliferation ◽  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Cell Cycle Progression ◽  
Muscle Cells ◽  
Dominant Negative ◽  
Wild Type ◽  
Cycle Progression ◽  
Positive Regulators

PPARγligands have been shown to have antiproliferative effects on many cell types. We herein report that a synthetic dominant-negative (DN) PPARγmutant functions like a growth factor to promote cell cycle progression and cell proliferation in human coronary artery smooth muscle cells (CASMCs). In quiescent CASMCs, adenovirus-expressed DN-PPARγpromoted G1→S cell cycle progression, enhanced BrdU incorporation, and increased cell proliferation. DN-PPARγexpression also markedly enhanced positive regulators of the cell cycle, increasing Rb and CDC2 phosphorylation and the expression of cyclin A, B1, D1, and MCM7. Conversely, overexpression of wild-type (WT) or constitutively-active (CA) PPARγinhibited cell cycle progression and the activity and expression of positive regulators of the cell cycle. DN-PPARγexpression, however, did not up-regulate positive cell cycle regulators in PPARγ-deficient cells, strongly suggesting that DN-PPARγeffects on cell cycle result from blocking the function of endogenous wild-type PPARγ. DN-PPARγexpression enhanced phosphorylation of ERK MAPKs. Furthermore, the ERK specific-inhibitor PD98059 blocked DN-PPARγ-induced phosphorylation of Rb and expression of cyclin A and MCM7. Our data thus suggest that DN-PPARγpromotes cell cycle progression and cell growth in CASMCs by modulating fundamental cell cycle regulatory proteins and MAPK mitogenic signaling pathways in vascular smooth muscle cells (VSMCs).

scholarly journals Dominant Negative Alleles ofSEC10Reveal Distinct Domains Involved in Secretion and Morphogenesis in Yeast

1998 ◽  
Vol 9 (7) ◽  
pp. 1725-1739 ◽  
Author(s):  
Dagmar Roth ◽  
Wei Guo ◽  
Peter Novick
Keyword(s):  
Plasma Membrane ◽  
Cell Cycle Progression ◽  
Secretory Pathway ◽  
Dominant Negative ◽  
Secretory Vesicles ◽  
Cycle Progression ◽  
Vesicle Docking ◽  
Amino Terminal ◽  
Exocyst Complex ◽  
Carboxy Terminal

The accurate targeting of secretory vesicles to distinct sites on the plasma membrane is necessary to achieve polarized growth and to establish specialized domains at the surface of eukaryotic cells. Members of a protein complex required for exocytosis, the exocyst, have been localized to regions of active secretion in the budding yeastSaccharomyces cerevisiae where they may function to specify sites on the plasma membrane for vesicle docking and fusion. In this study we have addressed the function of one member of the exocyst complex, Sec10p. We have identified two functional domains of Sec10p that act in a dominant-negative manner to inhibit cell growth upon overexpression. Phenotypic and biochemical analysis of the dominant-negative mutants points to a bifunctional role for Sec10p. One domain, consisting of the amino-terminal two-thirds of Sec10p directly interacts with Sec15p, another exocyst component. Overexpression of this domain displaces the full-length Sec10 from the exocyst complex, resulting in a block in exocytosis and an accumulation of secretory vesicles. The carboxy-terminal domain of Sec10p does not interact with other members of the exocyst complex and expression of this domain does not cause a secretory defect. Rather, this mutant results in the formation of elongated cells, suggesting that the second domain of Sec10p is required for morphogenesis, perhaps regulating the reorientation of the secretory pathway from the tip of the emerging daughter cell toward the mother–daughter connection during cell cycle progression.

scholarly journals Chloroplast division checkpoint in eukaryotic algae

2016 ◽  
Vol 113 (47) ◽  
pp. E7629-E7638 ◽  
Author(s):  
Nobuko Sumiya ◽  
Takayuki Fujiwara ◽  
Atsuko Era ◽  
Shin-ya Miyagishima
Keyword(s):  
Cell Cycle ◽  
Host Cell ◽  
Cell Cycle Progression ◽  
Chloroplast Division ◽  
Dominant Negative ◽  
Cyanidioschyzon Merolae ◽  
Temperature Sensitive ◽  
Specific Expression ◽  
Cycle Progression ◽  
Division Site

Chloroplasts evolved from a cyanobacterial endosymbiont. It is believed that the synchronization of endosymbiotic and host cell division, as is commonly seen in existing algae, was a critical step in establishing the permanent organelle. Algal cells typically contain one or only a small number of chloroplasts that divide once per host cell cycle. This division is based partly on the S-phase–specific expression of nucleus-encoded proteins that constitute the chloroplast-division machinery. In this study, using the red alga Cyanidioschyzon merolae, we show that cell-cycle progression is arrested at the prophase when chloroplast division is blocked before the formation of the chloroplast-division machinery by the overexpression of Filamenting temperature-sensitive (Fts) Z2-1 (Fts72-1), but the cell cycle progresses when chloroplast division is blocked during division-site constriction by the overexpression of either FtsZ2-1 or a dominant-negative form of dynamin-related protein 5B (DRP5B). In the cells arrested in the prophase, the increase in the cyclin B level and the migration of cyclin-dependent kinase B (CDKB) were blocked. These results suggest that chloroplast division restricts host cell-cycle progression so that the cell cycle progresses to the metaphase only when chloroplast division has commenced. Thus, chloroplast division and host cell-cycle progression are synchronized by an interactive restriction that takes place between the nucleus and the chloroplast. In addition, we observed a similar pattern of cell-cycle arrest upon the blockage of chloroplast division in the glaucophyte alga Cyanophora paradoxa, raising the possibility that the chloroplast division checkpoint contributed to the establishment of the permanent organelle.

scholarly journals Expression of dominant-negative mutant DP-1 blocks cell cycle progression in G1.

1996 ◽  
Vol 16 (7) ◽  
pp. 3698-3706 ◽  
Author(s):  
C L Wu ◽  
M Classon ◽  
N Dyson ◽  
E Harlow
Keyword(s):  
Cell Cycle ◽  
Dna Binding ◽  
Cell Cycle Progression ◽  
S Phase ◽  
Dominant Negative ◽  
Functional Domains ◽  
Dominant Negative Mutant ◽  
G1 Arrest ◽  
Wild Type ◽  
Cycle Progression

Unregulated expression of the transcription factor E2F promotes the G1-to-S phase transition in cultured mammalian cells. However, there has been no direct evidence for an E2F requirement in this process. To demonstrate that E2F is obligatory for cell cycle progression, we attempted to inactivate E2F by overexpressing dominant-negative forms of one of its heterodimeric partners, DP-1. We dissected the functional domains of DP-1 and separated the region that facilitate heterodimer DNA binding from the E2F dimerization domain. Various DP-1 mutants were introduced into cells via transfection, and the cell cycle profile of the transfected cells was analyzed by flow cytometry. Expression of wild-type DP-1 or DP-1 mutants that bind to both DNA and E2F drove cells into S phase. In contrast, DP-1 mutants that retained E2F binding but lost DNA binding arrested cells in the G1 phase of the cell cycle. The DP-1 mutants that were unable to bind DNA resulted in transcriptionally inactive E2F complexes, suggesting that the G1 arrest is caused by formation of defective E2F heterodimers. Furthermore, the G1 arrest instigated by these DP-1 mutants could be rescued by coexpression of wild-type E2F or DP protein. These experiments define functional domains of DP and demonstrate a requirement for active E2F complexes in cell cycle progression.

scholarly journals Transforming ras proteins accelerate hormone-induced maturation and stimulate cyclic AMP phosphodiesterase in Xenopus oocytes.

1990 ◽  
Vol 10 (4) ◽  
pp. 1689-1696 ◽  
Author(s):  
S E Sadler ◽  
J L Maller ◽  
J B Gibbs
Keyword(s):  
Enzyme Activity ◽  
Time Course ◽  
Xenopus Oocytes ◽  
Insulin Treatment ◽  
Ras Oncogene ◽  
Ras Proteins ◽  
Phosphodiesterase Activity ◽  
Oncogene Products ◽  
Stimulation Of

Transforming Harvey (Ha) ras oncogene products accelerated the time course of Xenopus oocyte maturation induced by insulin, insulinlike growth factor 1, or progesterone. The transforming constructs, [Val-12]Ha p21 and [Val-12, Thr-59]Ha p21, displayed equal potency and efficacy in their abilities to accelerate the growth peptide-induced response. Normal Ha p21 was only 60% as powerful and one-fifth as potent as the mutants containing valine in the 12 position. In contrast, two nontransforming constructs, [Val-12, Ala-35, Leu-36, Thr-59]Ha p21 and [Val-12, Thr-59]Ha(term-174) p21, had no effect on the time course of hormone-induced maturation. Effects of the transforming ras proteins on hormone-induced maturation correlated with their abilities to stimulate in vivo phosphodiesterase activity measured after microinjection of 200 microM cyclic [3H] AMP. When p21 injection followed 90 min of insulin treatment, there was no increase in phosphodiesterase activity over that measured after hormone treatment or p21 injection alone, but additive effects of p21 and insulin on enzyme activity were observed during the first 90 min of insulin treatment. Even though normal Ha p21 and transforming [Val-12, Thr-59]Ha p21 stimulated oocyte phosphodiesterase to equal levels when coinjected with substrate at the initiation of the in vivo assay, the transforming protein elicited a more sustained stimulation of enzyme activity. These results suggest that stimulation of a cyclic AMP phosphodiesterase activity associated with insulin-induced maturation is involved in the growth-promoting actions of ras oncogene products in Xenopus oocytes.

scholarly journals BCL-2 Is Phosphorylated and Inactivated by an ASK1/Jun N-Terminal Protein Kinase Pathway Normally Activated at G2/M

1999 ◽  
Vol 19 (12) ◽  
pp. 8469-8478 ◽  
Author(s):  
Kazuhito Yamamoto ◽  
Hidenori Ichijo ◽  
Stanley J. Korsmeyer
Keyword(s):  
Cell Cycle ◽  
Protein Kinase ◽  
Cell Cycle Progression ◽  
Peptide Mapping ◽  
Dominant Negative ◽  
Terminal Protein ◽  
Cycle Progression ◽  
M Phase ◽  
Death Signals

ABSTRACT Multiple signal transduction pathways are capable of modifying BCL-2 family members to reset susceptibility to apoptosis. We used two-dimensional peptide mapping and sequencing to identify three residues (Ser70, Ser87, and Thr69) within the unstructured loop of BCL-2 that were phosphorylated in response to microtubule-damaging agents, which also arrest cells at G2/M. Changing these sites to alanine conferred more antiapoptotic activity on BCL-2 following physiologic death signals as well as paclitaxel, indicating that phosphorylation is inactivating. An examination of cycling cells enriched by elutriation for distinct phases of the cell cycle revealed that BCL-2 was phosphorylated at the G2/M phase of the cell cycle. G2/M-phase cells proved more susceptible to death signals, and phosphorylation of BCL-2 appeared to be responsible, as a Ser70Ala substitution restored resistance to apoptosis. We noted that ASK1 and JNK1 were normally activated at G2/M phase, and JNK was capable of phosphorylating BCL-2. Expression of a series of wild-type and dominant-negative kinases indicated an ASK1/Jun N-terminal protein kinase 1 (JNK1) pathway phosphorylated BCL-2 in vivo. Moreover, the combination of dominant negative ASK1, (dnASK1), dnMKK7, and dnJNK1 inhibited paclitaxel-induced BCL-2 phosphorylation. Thus, stress response kinases phosphorylate BCL-2 during cell cycle progression as a normal physiologic process to inactivate BCL-2 at G2/M.

scholarly journals The jun and fos protein families are both required for cell cycle progression in fibroblasts.

1991 ◽  
Vol 11 (9) ◽  
pp. 4466-4472 ◽  
Author(s):  
K Kovary ◽  
R Bravo
Keyword(s):  
Cell Cycle ◽  
Transcription Factors ◽  
Dna Synthesis ◽  
Cell Cycle Progression ◽  
S Phase ◽  
3T3 Cells ◽  
Cycle Progression ◽  
Serum Stimulation ◽  
Fos Protein ◽  
Stimulation Of

The expression of different members of the Jun and Fos families of transcription factors is rapidly induced following serum stimulation of quiescent fibroblasts. To determine whether these proteins are required for cell cycle progression, we microinjected affinity-purified antibodies directed against c-Fos, FosB, Fra-1, c-Jun, JunB, and JunD, and antibodies that recognize either the Fos or the Jun family of proteins, into Swiss 3T3 cells and determined their effects in cell cycle progression by monitoring DNA synthesis. We found that microinjection of anti-Fos and anti-Jun family antibodies efficiently blocked the entrance to the S phase of serum-stimulated or asynchronously growing cells. However, the antibodies against single members of the Fos family only partially inhibited DNA synthesis. In contrast, all three Jun antibodies prevented DNA synthesis more effectively than did any of the anti-Fos antibodies.

Cyclin-Dependent Kinase Phosphorylation of RUNX1/AML1 on Three Sites Increases Trans-Activation Potency and Stimulates Cell Proliferation.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3351-3351
Author(s):  
Alan D. Friedman ◽  
Linsheng Zhang ◽  
Florence Bernardin Fried
Keyword(s):  
Cell Cycle ◽  
Cell Proliferation ◽  
Aspartic Acid ◽  
Cell Cycle Progression ◽  
Hematopoietic Cell ◽  
Cyclin D3 ◽  
Cycle Progression ◽  
Inhibition Of Proliferation ◽  
Cdk Phosphorylation ◽  
Stimulation Of

RUNX1/AML1 regulates lineage-specific genes during hematopoiesis and also stimulates G1 cell cycle progression. CBFβ-SMMHC or AML1-ETO dominantly inhibit RUNX1 and slow G1 progression in hematopoietic cell lines or in murine or human marrow progenitors, cdk4, cyclin D2, or c-Myc overcome inhibition of proliferation by these CBF oncoproteins, exogenous RUNX1 stimulates G1 progression, and stimulation of G1 via deletion of p16INK4a or expression of E7 cooperates with CBFβ-SMMHC or TEL-AML1 to induce acute leukemia in mice. Induction of cdk4 or cyclin D3 transcription may underlie stimulation of G1 progression by RUNX1. Remarkably, the C. elegans ortholog of RUNX1, RNT-1, also stimulates G1 progression and couples stem cell proliferation with differentiation. Not only does RUNX1 regulate cell cycle progression, but in addition RUNX1 levels increase as hematopoietic cells progress from G1 to S and from S to G2/M. Within RUNX1, S48, S303, and S424 fit the cdk phosphorylation consensus, (S/T)PX(R/K). Phosphorylation of RUNX1 by cyclin dependent kinases on serine 303 was shown to mediate destabilization of RUNX1 in G2/M. We now find that S48 and S424 are also phosphorylated by cdk1 or cdk6. S48, S303, or S424 phosphopeptide antiserum that we developed specifically recognized kinased GST-RUNX1 and interacted with RUNX1 expressed in 293T cells or in the Ba/F3 hematopoietic cell line. S48 phosphorylation of RUNX1 paralleled total RUNX1 levels during cell cycle progression, S303 was more effectively phosphorylated in G2/M, and S424 in G1. Single, double, and triple mutation to alanine or to the partially phosphomimetic aspartic acid progressively diminished or increased trans-activation, such that the tripleA mutant activated a RUNX1 reporter 5-fold less potently than the tripleD mutant. Aspartic acid does not perfectly mimic serine phosphorylation, as illustrated by the much greater affinity of our antisera for wild-type RUNX1 versus RUNX1(tripleD), suggesting that the biologic effect of RUNX1 cdk phosphorylation is even more significant. The p300 co-activator retained interaction with the tripleA variant. The tripleD RUNX1 mutant rescued Ba/F3 cells from inhibition of proliferation by CBFβ-SMMHC more effectively than the tripleA mutant. Cdk phosphorylation of RUNX1 on three sites increases its ability to active transcription and to stimulate proliferation, potentially coupling entry of stem/progenitors into cycle with induction of genes required for hematopoietic lineage progression, such as those encoding myeloperoxidase, neutrophil elastase, the M-CSF receptor, and PU.1.

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