scholarly journals A 62-kD protein required for mitotic progression is associated with the mitotic apparatus during M-phase and with the nucleus during interphase.

1992 ◽  
Vol 119 (4) ◽  
pp. 843-854 ◽  
Author(s):  
J A Johnston ◽  
R D Sloboda
Keyword(s):  
Cell Cycle ◽  
Nuclear Envelope ◽  
Cellular Protein ◽  
Immunogold Electron Microscopy ◽  
Mitotic Apparatus ◽  
Surf Clam ◽  
Calcium Calmodulin Dependent ◽  
Protein Pool ◽  
Mitotic Cyclin

A protein of 62 kD is a substrate of a calcium/calmodulin-dependent protein kinase, and both proteins copurify with isolated mitotic apparatuses (Dinsmore, J. H., and R. D. Sloboda. 1988. Cell. 53:769-780). Phosphorylation of the 62-kD protein increases after fertilization; maximum incorporation of phosphate occurs during late metaphase and anaphase and correlates directly with microtubule disassembly as determined by in vitro experiments with isolated mitotic apparatuses. Because 62-kD protein phosphorylation occurs in a pattern similar to the accumulation of the mitotic cyclin proteins, experiments were performed to determine the relationship between cyclin and the 62-kD protein. Continuous labeling of marine embryos with [35S]methionine, as well as immunoblots of marine embryo proteins using specific antibodies, were used to identify both cyclin and the 62-kD protein. These results clearly demonstrate that the 62-kD protein is distinct from cyclin and, unlike cyclin, is a constant member of the cellular protein pool during the first two cell cycles in sea urchin and surf clam embryos. Similar results were obtained using immunofluorescence microscopy of intact eggs and embryos. In addition, immunogold electron microscopy reveals that the 62-kD protein associates with the microtubules of the mitotic apparatus in dividing cells. Interestingly, the protein changes its subcellular distribution with respect to microtubules during the cell cycle. Specifically, during mitosis the 62-kD protein associates with the mitotic apparatus; before nuclear envelope breakdown, however, the 62-kD protein is confined to the nucleus. After anaphase, the 62-kD protein returns to the nucleus, where it resides until nuclear envelope disassembly of the next cell cycle.

Microtubule-entrained kinase activities associated with the cortical cytoskeleton during cytokinesis

1997 ◽  
Vol 110 (12) ◽  
pp. 1373-1386 ◽  
Author(s):  
G.R. Walker ◽  
C.B. Shuster ◽  
D.R. Burgess
Keyword(s):  
Cell Cycle ◽  
Protein Kinase ◽  
Protein Phosphorylation ◽  
Immunoblot Analysis ◽  
Negative Regulator ◽  
Mitogen Activated Protein Kinase ◽  
Mitotic Apparatus ◽  
Cortical Cytoskeleton

Research over the past few years has demonstrated the central role of protein phosphorylation in regulating mitosis and the cell cycle. However, little is known about how the mechanisms regulating the entry into mitosis contribute to the positional and temporal regulation of the actomyosin-based contractile ring formed during cytokinesis. Recent studies implicate p34cdc2 as a negative regulator of myosin II activity, suggesting a link between the mitotic cycle and cytokinesis. In an effort to study the relationship between protein phosphorylation and cytokinesis, we examined the in vivo and in vitro phosphorylation of actin-associated cortical cytoskeletal (CSK) proteins in an isolated model of the sea urchin egg cortex. Examination of cortices derived from eggs or zygotes labeled with 32P-orthophosphate reveals a number of cortex-associated phosphorylated proteins, including polypeptides of 20, 43 and 66 kDa. These three major phosphoproteins are also detected when isolated cortices are incubated with [32P]ATP in vitro, suggesting that the kinases that phosphorylate these substrates are also specifically associated with the cortex. The kinase activities in vivo and in vitro are stimulated by fertilization and display cell cycle-dependent activities. Gel autophosphorylation assays, kinase assays and immunoblot analysis reveal the presence of p34cdc2 as well as members of the mitogen-activated protein kinase family, whose activities in the CSK peak at cell division. Nocodazole, which inhibits microtubule formation and thus blocks cytokinesis, significantly delays the time of peak cortical protein phosphorylation as well as the peak in whole-cell histone H1 kinase activity. These results suggest that a key element regulating cortical contraction during cytokinesis is the timing of protein kinase activities associated with the cortical cytoskeleton that is in turn regulated by the mitotic apparatus.

scholarly journals Quantitative Characterization of a Mitotic Cyclin Threshold Regulating Exit from Mitosis

2005 ◽  
Vol 16 (5) ◽  
pp. 2129-2138 ◽  
Author(s):  
Frederick R. Cross ◽  
Lea Schroeder ◽  
Martin Kruse ◽  
Katherine C. Chen
Keyword(s):  
Cell Cycle ◽  
Budding Yeast ◽  
Cell Cycle Control ◽  
Predictive Ability ◽  
Eukaryotic Cell ◽  
Model Predictions ◽  
Mitotic Cyclin ◽  
Constitutive Overexpression

Regulation of cyclin abundance is central to eukaryotic cell cycle control. Strong overexpression of mitotic cyclins is known to lock the system in mitosis, but the quantitative behavior of the control system as this threshold is approached has only been characterized in the in vitro Xenopus extract system. Here, we quantitate the threshold for mitotic block in budding yeast caused by constitutive overexpression of the mitotic cyclin Clb2. Near this threshold, the system displays marked loss of robustness, in that loss or even heterozygosity for some regulators becomes deleterious or lethal, even though complete loss of these regulators is tolerated at normal cyclin expression levels. Recently, we presented a quantitative kinetic model of the budding yeast cell cycle. Here, we use this model to generate biochemical predictions for Clb2 levels, asynchronous as well as through the cell cycle, as the Clb2 overexpression threshold is approached. The model predictions compare well with biochemical data, even though no data of this type were available during model generation. The loss of robustness of the Clb2 overexpressing system is also predicted by the model. These results provide strong confirmation of the model's predictive ability.

scholarly journals Human cyclins A and B1 are differentially located in the cell and undergo cell cycle-dependent nuclear transport.

1991 ◽  
Vol 115 (1) ◽  
pp. 1-17 ◽  
Author(s):  
J Pines ◽  
T Hunter
Keyword(s):  
Cell Cycle ◽  
Human Fibroblasts ◽  
Nuclear Lamina ◽  
Cyclin A ◽  
Cyclin B1 ◽  
Cell Fractionation ◽  
Mitotic Apparatus ◽  
Epithelial Tumor ◽  
Mitotic Cyclin ◽  
Cycle Dependent

We have used immunofluorescence staining to study the subcellular distribution of cyclin A and B1 during the somatic cell cycle. In both primary human fibroblasts and in epithelial tumor cells, we find that cyclin A is predominantly nuclear from S phase onwards. Cyclin A may associated with condensing chromosomes in prophase, but is not associated with condensed chromosomes in metaphase. By contrast, cyclin B1 accumulates in the cytoplasm of interphase cells and only enters the nucleus at the beginning of mitosis, before nuclear lamina breakdown. In mitotic cells, cyclin B1 associates with condensed chromosomes in prophase and metaphase, and with the mitotic apparatus. Cyclin A is degraded during metaphase and cyclin B1 is precipitously destroyed at the metaphase----anaphase transition. Cell fractionation and immunoprecipitation studies showed that both cyclin A and cyclin B1 are associated with PSTAIRE-containing proteins. The nuclear, but not the cytoplasmic form, of cyclin A is associated with a 33-kD PSTAIRE-containing protein. Cyclin B1 is associated with p34cdc2 in the cytoplasm. Thus we propose that the different localization of cyclin A and cyclin B1 in the cell cycle could be the means by which the two types of mitotic cyclin confer substrate specificity upon their associated PSTAIRE-containing protein kinase subunit.

Repetitive sperm-induced Ca2+ transients in mouse oocytes are cell cycle dependent

Development ◽  
1995 ◽  
Vol 121 (10) ◽  
pp. 3259-3266 ◽  
Author(s):  
K.T. Jones ◽  
J. Carroll ◽  
J.A. Merriman ◽  
D.G. Whittingham ◽  
T. Kono
Keyword(s):  
Cell Cycle ◽  
Nuclear Envelope ◽  
Dependent Manner ◽  
Nuclear Envelope Breakdown ◽  
Mouse Oocytes ◽  
A Cell ◽  
Mature Mouse ◽  
Cycle Dependent ◽  
Stage 1

Mature mouse oocytes are arrested at metaphase of the second meiotic division. Completion of meiosis and a block to polyspermy is caused by a series of repetitive Ca2+ transients triggered by the sperm at fertilization. These Ca2+ transients have been widely reported to last for a number of hours but when, or why, they cease is not known. Here we show that Ca2+ transients cease during entry into interphase, at the time when pronuclei are forming. In fertilized oocytes arrested at metaphase using colcemid, Ca2+ transients continued for as long as measurements were made, up to 18 hours after fertilization. Therefore sperm is able to induce Ca2+ transients during metaphase but not during interphase. In addition metaphase II oocytes, but not pronuclear stage 1-cell embryos showed highly repetitive Ca2+ oscillations in response to microinjection of inositol trisphosphate. This was explored further by treating in vitro maturing oocytes at metaphase I for 4–5 hours with cycloheximide, which induced nuclear progression to interphase (nucleus formation) and subsequent re-entry to metaphase (nuclear envelope breakdown). Fertilization of cycloheximide-treated oocytes revealed that continuous Ca2+ oscillations in response to sperm were observed after nuclear envelope breakdown but not during interphase. However interphase oocytes were able to generate Ca2+ transients in response to thimerosal. This data suggests that the ability of the sperm to trigger repetitive Ca2+ transients in oocytes is modulated in a cell cycle-dependent manner.

Two proteins that cycle asynchronously between centrosomes and nuclear structures: Drosophila CP60 and CP190

1997 ◽  
Vol 110 (14) ◽  
pp. 1573-1583 ◽  
Author(s):  
K. Oegema ◽  
W.F. Marshall ◽  
J.W. Sedat ◽  
B.M. Alberts
Keyword(s):  
Cell Cycle ◽  
Nuclear Envelope ◽  
Wide Field ◽  
Dependent Manner ◽  
Independent Manner ◽  
Nuclear Envelope Breakdown ◽  
Nuclear Structures ◽  
Dynamic Structures ◽  
Cycle Dependent

Both the nucleus and the centrosome are complex, dynamic structures whose architectures undergo cell cycle-specific rearrangements. CP190 and CP60 are two Drosophila proteins of unknown function that shuttle between centrosomes and nuclei in a cell cycle-dependent manner. These two proteins are associated in vitro, and localize to centrosomes in a microtubule independent manner. We injected fluorescently labeled, bacterially expressed CP190 and CP60 into living Drosophila embryos and followed their behavior during the rapid syncytial blastoderm divisions (nuclear cycles 10–13). Using quantitative 3-D wide-field fluorescence microscopy, we show that CP190 and CP60 cycle between nuclei and centrosomes asynchronously with the accumulation of CP190 leading that of CP60 both at centrosomes and in nuclei. During interphase, CP190 is found in nuclei. Immediately following nuclear envelope breakdown, CP190 localizes to centrosomes where it remains until telophase, thereafter accumulating in reforming nuclei. Unlike CP190, CP60 accumulates at centrosomes primarily during anaphase, where it remains into early interphase. During nuclear cycles 10 and 11, CP60 accumulates in nuclei simultaneous with nuclear envelope breakdown, suggesting that CP60 binds to an unknown nuclear structure that persists into mitosis. During nuclear cycles 12 and 13, CP60 accumulates gradually in nuclei during interphase, reaching peak levels just before nuclear envelope breakdown. Once in the nucleus, both CP190 and CP60 appear to form fibrous intranuclear networks that remain coherent even after nuclear envelope breakdown. The CP190 and CP60 networks do not co-localize extensively with each other or with DNA. This work provides direct evidence, in living cells, of a coherent protein network that may represent a nuclear skeleton.

Analysis of the PI3/Akt/Survivin Pathway in Acute Myelogenous Leukemia.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2645-2645 ◽  
Author(s):  
Josefina Serrano ◽  
Juana Serrano-Lopez ◽  
Joaquin Sanchez Garcia ◽  
Concepcion Herrera ◽  
Antonio P. Torres Gomez
Keyword(s):  
Cell Cycle ◽  
Cell Lines ◽  
Conflicts Of Interest ◽  
Myelogenous Leukemia ◽  
Akt Pathway ◽  
Cycle Phase ◽  
Mitotic Apparatus ◽  
Npm1 Mutation ◽  
Apoptosis Protein

Abstract Abstract 2645 Poster Board II-621 BACKGROUND AND AIMS: The PI3/Akt pathway has been implicated in the pathogenesis of a wide variety of cancers including Acute Myeloid Leukemia (AML). Activated Akt is known to function as an essential survival factor by inhibiting apoptosis through its ability to phosphorylate several targets including Bad, FoxO transcription factors, Raf-1, caspase-9 and inhibitor of apoptosis protein family (IAPs). Survivin is a member of IAP regulating both apoptosis and cell cycle progression. Survivin binds to several structural components of the mitotic apparatus and can block apoptosis by inhibiting caspases 9, 3 and 7. Recently the importance of PI3/Akt/survivin pathway in solid neoplasias such as breast or prostate cancer has been highlighted but its role in AML remains unknown. In this work we analyzed the PI3/Akt/survivin pathway in human AML. METHODS: Bone marrow samples obtained at diagnosis of 68 AML consecutive patients and K562, MV4-11 and HL-60 cell lines were included. Patients Median age was 62 years (range 8–89). Median leukocyte count was 10.3×109/L (range 0.8–285). FAB subtypes were: M0=8, M1=20, M2=13, M3=8, M4=11, M5=7 and M6=1 with the following cytogenetic findings: t(15;17)=8, t(8;21)=2, complex karyotype=8, 11q23=2, normal karyotype=36 and others=12. There were 11.3% patients with FLT3-ITD and 16.9% with NPM1 mutation. Cytoplasmic and nuclear Proteins were harvested with Q-proteome cell compartment (Qiaqen) and protein concentration assayed using Protein Assay Kit (Bio-Rad). Total Akt, Akt-pSer473 and survivin proteins were detected by Western Blot and were visualized by enhanced chemiluminescence (ECL-Plus, GE Healthcare) in Chemigenius-2 and quantified using Gene-Tools software. Cell cycle analysis was assessed by double Hoechst 33342- Pyronin Y staining and flow cytometry in FACSvantage. Inhibition experiments were done using Ly294002 at 25 μM, and Wortmaninn at 250 nM for 12 hours. RESULTS: In our series p-Ser473Akt was detected in 56% of AML marrow samples (with high levels expression in 27%) and in all cell lines. In cytoplasmic protein extracts, Survivin WT and 2B isoform were detected in 45% and 45.2% of patients respectively. All three leukemic cell lines showed only Survivin 2B expression. Interestingly, there was strong statistical correlation between the levels of p-Ser473Akt with cytoplasmic Survivin (P=.01). Inhibitors of PI3K/Akt pathway LY294002 and Wortmaninn both decreased in vitro p-Ser473Akt expression but only the irreversible action of Wortmaninn caused a marked dowregulation of cytoplasmic survivin. Meaningfully, lack of cytoplasmic Survivin was associated with an increased proportion of cells in Go cell cycle phase (11.1 % vs. 3.6%, P=.04). Moreover, cytoplasmic Survivin WT localization and high p-Ser473 Akt levels, were both significantly correlated with less unfavourable FAB leukemia subtype and cytogenetic risk (P<.01), more CR achievement with one induction cycle (P<.01), less relapse rate (P=.01) and less mortality rate (P=.03). CONCLUSIONS: Survivin cytoplasmic expression is regulated by PI3-kinase/Akt pathway in AML. The activation of PI3/Akt/survivin pathway is associated with an increased proliferative status and our series suggest that this finding could be associated with a more favorable outcome. Financial support: This study was supported by a grant of Conserjeria de Salud, Junta de Andalucia 2006/0355. J. Serrano López is a post-doc fellow from Fundación Española de Hematologíıa y Hemoterapia Disclosures: No relevant conflicts of interest to declare.

scholarly journals Posttranslational Regulation of NF-YA Modulates NF-Y Transcriptional Activity

2008 ◽  
Vol 19 (12) ◽  
pp. 5203-5213 ◽  
Author(s):  
Isabella Manni ◽  
Giuseppina Caretti ◽  
Simona Artuso ◽  
Aymone Gurtner ◽  
Velia Emiliozzi ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Progression ◽  
Transcriptional Activity ◽  
Regulatory Subunit ◽  
Posttranslational Regulation ◽  
Wild Type ◽  
Cycle Progression ◽  
Mitotic Cyclin ◽  
Wild Type Form

NF-Y binds to CCAAT motifs in the promoter region of a variety of genes involved in cell cycle progression. The NF-Y complex comprises three subunits, NF-YA, -YB, and -YC, all required for DNA binding. Expression of NF-YA fluctuates during the cell cycle and is down-regulated in postmitotic cells, indicating its role as the regulatory subunit of the complex. Control of NF-YA accumulation is posttranscriptional, NF-YA mRNA being relatively constant. Here we show that the levels of NF-YA protein are regulated posttranslationally by ubiquitylation and acetylation. A NF-YA protein carrying four mutated lysines in the C-terminal domain is more stable than the wild-type form, indicating that these lysines are ubiquitylated Two of the lysines are acetylated in vitro by p300, suggesting a competition between ubiquitylation and acetylation of overlapping residues. Interestingly, overexpression of a degradation-resistant NF-YA protein leads to sustained expression of mitotic cyclin complexes and increased cell proliferation, indicating that a tight regulation of NF-YA levels contributes to regulate NF-Y activity.

Multisite Phosphorylation of NIPA at G2/M.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3348-3348
Author(s):  
Anna L. Illert ◽  
Florian Bassermann ◽  
Christine von Klitzing ◽  
Petra Seipel ◽  
Stephan W. Morris ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Control ◽  
E3 Ligase ◽  
Nuclear Interaction ◽  
Cyclin B1 ◽  
Ubiquitin E3 Ligase ◽  
Mitotic Cyclin ◽  
F Box Protein

Abstract The regulated oscillation of protein expression is an essential mechanism of cell cycle control. The SCF class of E3 ubiquitin ligases is involved in this process by targeting cell cycle regulatory proteins for degradation by the proteasome, with the F-Box subunit of the SCF specifically recruiting a given substrate to the SCF core. We previously reported the cloning of NIPA (Nuclear Interaction Partner of ALK) in complex with constitutively active oncogenic fusions of ALK, which contributes to the development of lymphomas and sarcomas. Subsequently we characterized NIPA as a F-Box protein (FBP) that defines an oscillating ubiquitin E3 ligase. The SCFNIPA complex targets nuclear cyclin B1 for ubiquitination in interphase while phosphorylation of NIPA in late G2 phase and mitosis inactivates the complex to allow for accumulation of cyclin B1. Here, we identify the region of NIPA that mediates binding to its substrate cyclin B1. In addition to the recently described serine residue 354, we specify 2 new residues, Ser-359 and Ser-395, implicated in the phosphorylation process at G2M within this region. Moreover, we found cyclin B1/Cdk1 to phosphorylate NIPA at Ser-395 in mitosis. Mutation of both Ser-359 and Ser-395 impaired effective inactivation of the SCFNIPA complex, resulting in reduced levels of mitotic cyclin B1. Furthermore, we aimed to identify the kinases involved in the initial phosphorylation of Ser-345. Therefore, we tested a panel of different kinases active at the G2M transition such as GSK3?, Casein kinase 2, PLK-1 and Erk1. Effective in vitro phosphorylation of NIPA could only be demonstrated with Erk-1. Moreover, we demonstrate an interaction of Erk-1 and NIPA at G2M but not in interphase cells. Binding of Erk-1 and NIPA led to phosphorylation at Ser-354 in vivo and could be blocked by the MEK-1/MEK-2 inhibitor PD98059. Together these data suggest a process of sequential phosphorylation where NIPA is initially phosphorylated by Erk-1 leading to the dissociation of NIPA from the SCF core complex. Once Ser-354 is phosphorylated, cyclin B1/CDK1 amplifies phosphorylation of NIPA, thus contributing to the regulation of its own abundance in early mitosis. In ALK positive lymphomas enhanced phosphorylation of NIPA at Ser 354 can be observed. We demonstrate that NPM-ALK leads to the activation of Erk-1, thereby phosphorylating and inactivating the SCFNIPA E3 ligase. Inactivation of SCFNIPA may have an important impact on the cell cycle turnover of lymphoma cells and thus for the pathogenesis of NPM-ALK induced lymphomas.

scholarly journals Cell Cycle–regulated Proteolysis of Mitotic Target Proteins

1999 ◽  
Vol 10 (11) ◽  
pp. 3927-3941 ◽  
Author(s):  
Holger Bastians ◽  
Leana M. Topper ◽  
Gary L. Gorbsky ◽  
Joan V. Ruderman
Keyword(s):  
Cell Cycle ◽  
Metaphase Plate ◽  
S Phase ◽  
Dominant Negative ◽  
Anaphase Promoting Complex ◽  
Anaphase Onset ◽  
Mitotic Cyclin ◽  
Ubiquitin Conjugating Enzyme

The ubiquitin-dependent proteolysis of mitotic cyclin B, which is catalyzed by the anaphase-promoting complex/cyclosome (APC/C) and ubiquitin-conjugating enzyme H10 (UbcH10), begins around the time of the metaphase–anaphase transition and continues through G1 phase of the next cell cycle. We have used cell-free systems from mammalian somatic cells collected at different cell cycle stages (G0, G1, S, G2, and M) to investigate the regulated degradation of four targets of the mitotic destruction machinery: cyclins A and B, geminin H (an inhibitor of S phase identified in Xenopus), and Cut2p (an inhibitor of anaphase onset identified in fission yeast). All four are degraded by G1 extracts but not by extracts of S phase cells. Maintenance of destruction during G1 requires the activity of a PP2A-like phosphatase. Destruction of each target is dependent on the presence of an N-terminal destruction box motif, is accelerated by additional wild-type UbcH10 and is blocked by dominant negative UbcH10. Destruction of each is terminated by a dominant activity that appears in nuclei near the start of S phase. Previous work indicates that the APC/C–dependent destruction of anaphase inhibitors is activated after chromosome alignment at the metaphase plate. In support of this, we show that addition of dominant negative UbcH10 to G1 extracts blocks destruction of the yeast anaphase inhibitor Cut2p in vitro, and injection of dominant negative UbcH10 blocks anaphase onset in vivo. Finally, we report that injection of dominant negative Ubc3/Cdc34, whose role in G1–S control is well established and has been implicated in kinetochore function during mitosis in yeast, dramatically interferes with congression of chromosomes to the metaphase plate. These results demonstrate that the regulated ubiquitination and destruction of critical mitotic proteins is highly conserved from yeast to humans.

Sign in / Sign up

Export Citation Format

Share Document