E2F-4 switches from p130 to p107 and pRB in response to cell cycle reentry.

The E2F transcription factor couples the coordinate expression of cell cycle proteins to their appropriate transition points. Its activity is controlled by the cell cycle regulators pRB, p107, and p130. These bind to E2F at defined but distinct stages of the cell cycle. Using specific antisera, we have identified the DP and E2F components of each of these species. Although present at very different levels, DP-1 and DP-2 are evenly distributed among each of these complexes. In contrast, the individual E2Fs have distinctly different binding profiles. Consistent with previous studies, E2F-1, E2F-2, and E2F-3 bind specifically to the retinoblastoma protein. In each case, their expression and DNA binding activity are restricted to post-G1/S fractions. Surprisingly, E2F-1 and E2F-3 make unequal contributions to the pRB-associated and free E2F activity, suggesting that these proteins perform different cell cycle functions. Most significantly, this study showed E2F-4 accounts for the vast majority of the endogenous E2F activity. In arrested cells, E2F-4 is sequestered by the p130 protein. However, as the cells pass the G1-to-S transition, the levels of pRB and p107 increase and E2F-4 now associates with both of these regulators. Despite this, a considerable amount of E2F-4 exists as free E2F. In G1 cells, this accounts for almost all of the free activity. Once the cells enter S phase, free E2F is composed of an equal mixture of E2F-4 and E2F-1.

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Separation of v-Src-induced mitogenesis and morphological transformation by inhibition of AP-1.

Molecular Biology of the Cell ◽

10.1091/mbc.5.11.1177 ◽

1994 ◽

Vol 5 (11) ◽

pp. 1177-1184 ◽

Cited By ~ 5

Author(s):

M C Frame ◽

K Simpson ◽

V J Fincham ◽

D H Crouch

Keyword(s):

Cell Cycle ◽

Leucine Zipper ◽

Binding Activity ◽

Temperature Sensitive ◽

Morphological Transformation ◽

Mobility Shift ◽

Dna Binding Activity ◽

Cell Cycle Reentry ◽

Gel Mobility Shift ◽

Basic Region

v-Src activity results in both morphological transformation and reentry of quiescent chick embryo fibroblasts (CEF) into cell cycle. We have previously used temperature-sensitive v-Src mutants to show that enhanced activity of cellular AP-1 in the first few hours after activation of v-Src invariably precedes the biological consequences. Here we have investigated whether the early activation of AP-1 is essential for any or all of the v-Src responses by using a mutant c-Fos that comprises the leucine zipper and a disrupted basic region. Expression of the c-Fos mutant partially reduced cellular AP-1 activity in exponentially growing cells. However, in CEF that had been made quiescent by serum deprivation, v-Src-induced stimulation of AP-1 DNA binding activity was substantially reduced. In addition, quiescent CEF stably transfected with this mutant show an impaired mitogenic response to v-Src, indicating that the AP-1 stimulation is a necessary prerequisite for cell-cycle reentry. The ability of v-Src to morphologically transform quiescent CEF was not impaired by the inhibition of AP-1 stimulation, indicating that the mitogenic and morphological consequences of v-Src have distinguishable biochemical mediators. Focal adhesion kinase, a recently identified determinant of cell morphology, undergoes a gel mobility shift, characteristic of its hyperphosphorylated state, in response to v-Src activation in cells expressing the inhibitory AP-1 protein. This provides further evidence that the pathways that regulate morphological transformation are independent of AP-1.

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p53 transactivation and protein accumulation are independently regulated by UV light in different phases of the cell cycle.

Molecular and Cellular Biology ◽

10.1128/mcb.17.6.3074 ◽

1997 ◽

Vol 17 (6) ◽

pp. 3074-3080 ◽

Cited By ~ 17

Author(s):

T Haapajärvi ◽

K Pitkänen ◽

M Tsubari ◽

M Laiho

Keyword(s):

Cell Cycle ◽

Dna Binding ◽

Target Genes ◽

Binding Activity ◽

S Phase ◽

Protein Stabilization ◽

Cycle Phase ◽

Protein Accumulation ◽

Dna Binding Activity ◽

G1 Cells

DNA damage-induced activation of the p53 tumor suppressor gene is suggested to be central in the cellular damage response pathway. In this study, we analyzed the responses of p53 to UVC radiation in synchronized mouse fibroblasts in terms of p53 accumulation, transcriptional activation, and sequence-specific DNA-binding activity. UVC was found to induce accumulation of p53 cell cycle dependently in G1/S- and S-phase cells but not in G0 or G1 cells. In contrast, p53 transcriptional activity and its target genes, p21 and GADD45, were stimulated by UVC in G0 and G1 cells in the absence of detectable p53 protein. The accumulation of p53 and increased p21 and GADD45 expression were replication dependent in S-phase cells. Interestingly, sequence-specific p53 DNA-binding activity was stimulated also replication independently in S phase, though the effect was not conveyed to stimulation of p53 target genes, suggesting that additional events are required for p53-stimulated gene expression. The results show that opposed to the cell cycle dependence of p53 accumulation, the UVC-mediated transactivation by p53 is independent of the cell cycle phase and protein stabilization.

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Biochemical and Initial Structural Characterization of the Monocot Chimeric Jacalin OsJAC1

International Journal of Molecular Sciences ◽

10.3390/ijms22115639 ◽

2021 ◽

Vol 22 (11) ◽

pp. 5639

Author(s):

Nikolai Huwa ◽

Oliver H. Weiergräber ◽

Christian Kirsch ◽

Ulrich Schaffrath ◽

Thomas Classen

Keyword(s):

Physiological Role ◽

Basic Function ◽

Binding Activity ◽

Carbohydrate Binding ◽

Lectin Domain ◽

Reducing Conditions ◽

Transition Points ◽

The Individual ◽

High Yields ◽

Dirigent Domain

The monocot chimeric jacalin OsJAC1 from Oryza sativa consists of a dirigent and a jacalin-related lectin domain. The corresponding gene is expressed in response to different abiotic and biotic stimuli. However, there is a lack of knowledge about the basic function of the individual domains and their contribution to the physiological role of the entire protein. In this study, we have established a heterologous expression in Escherichia coli with high yields for the full-length protein OsJAC1 as well as its individual domains. Our findings showed that the secondary structure of both domains is dominated by β-strand elements. Under reducing conditions, the native protein displayed clearly visible transition points of thermal unfolding at 59 and 85 °C, which could be attributed to the lectin and the dirigent domain, respectively. Our study identified a single carbohydrate-binding site for each domain with different specificities towards mannose and glucose (jacalin domain), and galactose moieties (dirigent domain), respectively. The recognition of different carbohydrates might explain the ability of OsJAC1 to respond to different abiotic and biotic factors. This is the first report of specific carbohydrate-binding activity of a DIR domain, shedding new light on its function in the context of this monocot chimeric jacalin.

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Trans-repressor activity of nuclear glycosaminoglycans on Fos and Jun/AP-1 oncoprotein-mediated transcription.

The Journal of Cell Biology ◽

10.1083/jcb.116.1.31 ◽

1992 ◽

Vol 116 (1) ◽

pp. 31-42 ◽

Cited By ~ 85

Author(s):

S J Busch ◽

G A Martin ◽

R L Barnhart ◽

M Mano ◽

A D Cardin ◽

...

Keyword(s):

Gene Expression ◽

Cell Cycle ◽

Enhanced Recovery ◽

Present Report ◽

Binding Activity ◽

Heparin Binding ◽

Dna Binding Activity ◽

Cycle Arrest ◽

Specific Mrnas ◽

F9 Teratocarcinoma

Heparin blocks the phorbol ester-induced progression of nontransformed cells through the G0/G1 phase (Wright, T.C., L.A. Pukac, J.J. Castellot, M.J. Karnovsky, R.A. Levine, H.-Y. Kim-Park, and J. Campisi. 1989. Proc. Natl. Acad. Sci. USA. 86: 3199-3203) or G1 to S phase (Reilly, C. F., M. S. Kindy, K. E. Brown, R. D. Rosenberg, and G. E Sonenshein. 1989. J. Biol. Chem. 264:6990-6995) of the cell cycle. Cell cycle arrest was associated with decreased levels of stage-specific mRNAs suggesting transcriptional regulation of cell growth. In the present report, we show that heparin selectively repressed TPA-inducible AP-1-mediated gene expression. Heparin-induced trans-repression was observed in primary vascular smooth muscle cells, as well as in the transformed HeLa cell line and in nondifferentiated F9 teratocarcinoma cells. Inhibition of AP-1-mediated trans-activation occurred with heparin and pentosan polysulfate but not with chondroitin sulfate A or C. Heparin-binding peptides or heparitinase I addition to nuclear lysates of heparin-treated cells allowed enhanced recovery of endogenous AP-1-specific DNA binding activity. We propose a model in which nuclear glycosaminoglycans play a trans-regulatory role in altering the patterns of inducible gene expression.

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Silencing of the DEK gene induces apoptosis and senescence in CaSki cervical carcinoma cells via the up-regulation of NF-κB p65

Bioscience Reports ◽

10.1042/bsr20100141 ◽

2012 ◽

Vol 32 (3) ◽

pp. 323-332 ◽

Cited By ~ 20

Author(s):

Kuiran Liu ◽

Tianda Feng ◽

Jie Liu ◽

Ming Zhong ◽

Shulan Zhang

Keyword(s):

Cell Cycle ◽

Nuclear Translocation ◽

Annexin V ◽

Nuclear Factor Κb ◽

Binding Activity ◽

Cell Senescence ◽

Stable Cell Line ◽

Reverse Transcription Pcr ◽

Dna Binding Activity ◽

Nuclear Extracts

The human DEK proto-oncogene has been found to play an important role in autoimmune disease, viral infection and human carcinogenesis. Although it is transcriptionally up-regulated in cervical cancer, its intracellular function and regulation is still unexplored. In the present study, DEK and IκBα [inhibitor of NF-κB (nuclear factor κB) α] shRNAs (short hairpin RNAs) were constructed and transfected into CaSki cells using Lipofectamine™. The stable cell line CaSki–DEK was obtained after G418 selection. CaSki–IκB cells were observed at 48 h after psiRNA-IκB transfection. The inhibitory efficiency of shRNAs were detected by RT (reverse transcription)–PCR and Western blot analysis. The proliferation activity of cells were measured using an MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide] assay, cell apoptosis was measured using an Annexin V/PI (propidium iodide) kit, the cell cycle was analysed by flow cytometry and cell senescence was detected using senescence β-galactosidase staining. The intracellular expression of NF-κB p65 protein was studied by cytochemistry. The expression levels of NF-κB p65, p50, c-Rel, IκBα and phospho-IκBα protein were analysed by immunoblotting in whole-cell lysates, cytosolic fractions and nuclear extracts. The protein expression and activity of p38 and JNK (c-Jun N-terminal kinase) were also assayed. In addition, the NF-κB p65 DNA-binding activity was measured by ELISA. Following the silencing of DEK and IκBα, cell proliferation was inhibited, apoptosis was increased, the cell cycle was blocked in the G0/G1-phase with a corresponding decrease in the G2/M-phase, and cell senescence was induced. All of these effects may be related to the up-regulation of NF-κB p65 expression and its nuclear translocation.

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Evidence for DNA binding activity of numatrin (B23), a cell cycle-regulated nuclear matrix protein

Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression ◽

10.1016/0167-4781(90)90196-9 ◽

1990 ◽

Vol 1087 (2) ◽

pp. 127-136 ◽

Cited By ~ 29

Author(s):

Nili Feuerstein ◽

James J. Mond ◽

Paul R. Kinchington ◽

Robert Hickey ◽

Marja-Liisa Karjalainen Lindsberg ◽

...

Keyword(s):

Cell Cycle ◽

Dna Binding ◽

Nuclear Matrix ◽

Matrix Protein ◽

Binding Activity ◽

Nuclear Matrix Protein ◽

Dna Binding Activity ◽

A Cell

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Constitutive c-Myb amino-terminal phosphorylation and DNA binding activity uncoupled during entry and passage through the cell cycle

Oncogene ◽

10.1038/sj.onc.1204345 ◽

2001 ◽

Vol 20 (14) ◽

pp. 1784-1792 ◽

Cited By ~ 14

Author(s):

Alina Cures ◽

Colin House ◽

Chie Kanei-Ishii ◽

Bruce Kemp ◽

Robert G Ramsay

Keyword(s):

Cell Cycle ◽

Dna Binding ◽

Binding Activity ◽

Amino Terminal ◽

Dna Binding Activity

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Phosphorylation Controls Ikaros's Ability To Negatively Regulate the G1-S Transition

Molecular and Cellular Biology ◽

10.1128/mcb.24.7.2797-2807.2004 ◽

2004 ◽

Vol 24 (7) ◽

pp. 2797-2807 ◽

Cited By ~ 71

Author(s):

Pablo Gómez-del Arco ◽

Kazushige Maki ◽

Katia Georgopoulos

Keyword(s):

Cell Cycle ◽

Dna Binding ◽

Cell Cycle Progression ◽

Rapid Development ◽

Binding Activity ◽

Casein Kinase ◽

Cycle Progression ◽

Dna Binding Activity ◽

Increased Activity ◽

Inactivating Mutations

ABSTRACT Ikaros is a key regulator of lymphocyte proliferative responses. Inactivating mutations in Ikaros cause antigen-mediated lymphocyte hyperproliferation and the rapid development of leukemia and lymphoma. Here we show that Ikaros's ability to negatively regulate the G1-S transition can be modulated by phosphorylation of a serine/threonine-rich conserved region (p1) in exon 8. Ikaros phosphorylation in p1 is induced during the G1-S transition. Mutations that prevent phosphorylation in p1 increase Ikaros's ability to impede cell cycle progression and its affinity for DNA. Casein kinase II, whose increased activity in lymphocytes leads to transformation, is a key player in Ikaros p1 phosphorylation. We thus propose that Ikaros's activity as a regulator of the G1-S transition is controlled by phosphorylation in response to signaling events that downmodulate its DNA binding activity.

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Imatinib Induced Re-Activation of FoxO3 Transcription Factor in CML Is Responsible for the Induction of a Quiescent Status of CD34 Leukaemic Progenitor Cells.

Blood ◽

10.1182/blood.v112.11.1090.1090 ◽

2008 ◽

Vol 112 (11) ◽

pp. 1090-1090

Author(s):

Daniela Cilloni ◽

Cristina Panuzzo ◽

Francesca Messa ◽

Francesca Arruga ◽

Enrico Bracco ◽

...

Keyword(s):

Cell Cycle ◽

Cell Growth ◽

Dna Binding ◽

Progenitor Cells ◽

Binding Activity ◽

Mrna Levels ◽

Dna Binding Activity ◽

Cd34 Cells

Abstract The FoxO family of transcription factors is regulated by PI3K/Akt induced phosphorylation resulting in nuclear exclusion and degradation. Nuclear FoxO transcribes proapoptotic molecules and cell cycle inhibitors. In CML cells the TK activity of Bcr-Abl leads to the abnormal activation of downstream effectors including PI3K/Akt. The aim of this study was to investigate the role of FoxO3 in Bcr-Abl induced apoptotic arrest and cell growth and the effect of imatinib (IM) induced re-activation of FoxO3 activity in CML progenitor cells. BM cells were collected from 52 CML patients and 20 healthy donors. The expression level of FoxO3 was tested by RQ-PCR. The protein amount and localization was analyzed by Western blot and immunofluorescence, DNA binding activity was measured by EMSA. In addition, FoxO3 was analyzed in CML primary cells and CD34+ cells after IM incubation. Cell cycle and the expression levels of CD47, which has been demonstrated to increased during progression through the cell cycle and stem cell mobilization, was measured by FACS in CD34+ cell population. In addition K562 cells was transfected with pECE-FoxO3 to clarify FoxO3 effects on cell growth and apoptosis. Finally we used our already set up model of Drosophila melanogaster (Dm) transgenic for human Bcr-Abl to study the pathway leading to FoxO3 inactivation. We found that, despite either FoxO3 mRNA levels or protein amount are similar in CML cells compared to controls, FoxO3 protein is equally distributed in the nucleus and cytoplasm in controls but it is completely cytoplasmatic in CML cells and it enters the nucleus during in vivo IM treatment or in vitro IM incubation. Additionally, FoxO3 DNA binding activity in CML patients is completely absent at diagnosis and reappears after IM treatment. Moreover FoxO3 overexpression in transfected cells results into a 49±9 % reduction of proliferation which was further reduced of 75±5 % after IM incubation. Furthermore, we demonstrated that IM incubation results into the reactivation of FoxO3 in Ph+ CD34+ cells inducing quiescence into this population as demonstrated by the comparison of cell cycle kinetics and by a decreased expression of CD47. Finally, the progeny obtained from the crossbreeding of Bcr-Abl flies and flies transgenic for FoxO showed a rescue of FoxO phenotype demonstrating that FoxO inactivation is Bcr-Abl mediated. Overall, these in vitro and in vivo experiments suggest that FoxO3 is inactivated in CML cells and its delocalization is mainly dependant from Bcr-Abl activity. The antiproliferative activity of IM may be mediated by FoxO3 re-localization. On the other side, FoxO3 re-activation induced by IM results into a quiescence of Bcr-Abl CD34+ progenitor cells, which raises a hypothesis that FoxO3 could play a role in IM resistance. This investigation was conducted by CML Correlative Studies Network (CCSN), TOPS, which is sponsored by Novartis Oncology

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