Active cyclin B-cdc2 kinase does not inhibit DNA replication and cannot drive prematurely fertilized sea urchin eggs into mitosis

1995 ◽  
Vol 108 (7) ◽  
pp. 2693-2703 ◽  
Author(s):  
A.M. Geneviere-Garrigues ◽  
A. Barakat ◽  
M. Doree ◽  
J.L. Moreau ◽  
A. Picard
Keyword(s):  
Dna Replication ◽  
Sea Urchin ◽  
S Phase ◽  
Cyclin B ◽  
G1 Arrest ◽  
Cdc2 Kinase ◽  
Kinase Activation ◽  
M Phase ◽  
Set Up ◽  
The One

Feedback mechanisms preventing M phase occurrence before S phase completion are assumed to depend on inhibition of cyclin B-cdc2 kinase activation by unreplicated DNA. In sea urchin, fertilization stimulates protein synthesis and releases eggs from G1 arrest. We found that in the one-cell sea urchin embryo cyclin B-cdc2 kinase undergoes partial activation before S phase, reaching in S phase a level that is sufficient for G2-M phase transition. S phase entry is not inhibited by this level of cyclin B-dependent kinase activity. Inhibition of DNA replication by aphidicolin suppresses nuclear envelope breakdown, yet it does not prevent the microtubule array from being converted from its interphasic to its mitotic state. Moreover, mitotic cytoplasmic events occur at the same time in control and aphidicolin-treated embryos. Thus unreplicated DNA only prevents mitotic nuclear, not cytoplasmic, events from occurring prematurely. These results together show that the inhibition of cyclin B-cdc2 kinase activation is probably not the only mechanism that prevents mitotic nuclear events from occurring as long as DNA replication has not been completed. In contrast, cytoplasmic mitotic events seem to be controlled by a timing mechanism independent of DNA replication, set up at fertilization, that prevents premature opening of a window for mitotic events.

Inhibiting proteasome activity causes overreplication of DNA and blocks entry into mitosis in sea urchin embryos

2000 ◽  
Vol 113 (15) ◽  
pp. 2659-2670 ◽  
Author(s):  
H. Kawahara ◽  
R. Philipova ◽  
H. Yokosawa ◽  
R. Patel ◽  
K. Tanaka ◽  
...  
Keyword(s):  
Sea Urchin ◽  
Embryonic Cell ◽  
S Phase ◽  
Cdc2 Kinase ◽  
Replication Checkpoint ◽  
Kinase Activation ◽  
Peptide Aldehydes ◽  
M Phase ◽  
Dna Replication Checkpoint ◽  
P34 Cdc2

The proteasome has been shown to be involved in exit from mitosis by bringing about destruction of mitotic cyclins. Here, we present evidence that the proteasome is also required for proper completion of S phase and for entry into mitosis in the sea urchin embryonic cleavage cycle. A series of structurally related peptide-aldehydes prevent nuclear envelope breakdown in their order of inhibitory efficacies against the proteasome. Their efficacies in blocking exit from S phase and exit from mitosis correlate well, indicating that the proteasome is involved at both these steps. Mitotic histone HI kinase activation and tyrosine dephosphorylation of p34(cdc2) kinase are blocked by inhibition of the proteasome, indicating that the proteasome plays an important role in the pathway that leads to embryonic p34(cdc2)kinase activation. Arrested embryos continued to incorporate [(3)H]thymidine and characteristically developed large nuclei. Pre-mitotic arrest can be overcome by treatment with caffeine, a manoeuvre that is known to override the DNA replication checkpoint. These data demonstrate that the proteasome is involved in the control of termination of S phase and consequently in the initiation of M phase of the first embryonic cell cycle.

scholarly journals Cig2, a B-type cyclin, promotes the onset of S in Schizosaccharomyces pombe.

1996 ◽  
Vol 16 (4) ◽  
pp. 1527-1533 ◽  
Author(s):  
O Mondesert ◽  
C H McGowan ◽  
P Russell
Keyword(s):  
Dna Replication ◽  
Schizosaccharomyces Pombe ◽  
Double Mutant ◽  
Kinase Activity ◽  
Nitrogen Starvation ◽  
Gradual Increase ◽  
S Phase ◽  
G1 Arrest ◽  
Cyclin Dependent Kinases ◽  
M Phase

Cdc2, a catalytic subunit of cyclin-dependent kinases, is required for both the G1-to-S and G2-to-M transitions in the fission yeast Schizosaccharomyces pombe. Cdc13, a B-type cyclin, is required for the M-phase induction function of Cd2. Two additional B-type cyclins, Cig1 and Cig2, have been identified in S. pombe, but none of the B-type cyclins are individually required for the onset of S. We report that Cdc13 is important for DNA replication in a strain lacking Cig2. Unlike deltacdc13 cells, double-mutant deltacdc13 deltacig2 cells are defective in undergoing multiple rounds of DNA replication. The conclusion that Cig2 promotes S is further supported by the finding that Cig2 protein and Cig2-associated kinase activity appear soon after the completion of M and peak during S, as well as the observation that S is delayed in deltacig2 cells as they recover from a G1 arrest induced by nitrogen starvation. These studies indicate that Cig2 is the primary S-phase-promoting cyclin in S. pombe but that Cdc13 can effectively substitute for Cig2 in deltacig2 cells. These observations also suggest that the gradual increase in the activity of Cdc2-Cdc13 kinase can be sufficient for the correct temporal ordering of S and M phases in deltacig2 cells.

scholarly journals Phosphorylation of p21 in G2/M Promotes Cyclin B-Cdc2 Kinase Activity

2005 ◽  
Vol 25 (8) ◽  
pp. 3364-3387 ◽  
Author(s):  
Bipin C. Dash ◽  
Wafik S. El-Deiry
Keyword(s):  
Kinase Activity ◽  
Cyclin B1 ◽  
S Phase ◽  
Cdk Inhibitor ◽  
Wild Type ◽  
Cdc2 Kinase ◽  
Dependent Protein Kinase ◽  
Kinase Activation ◽  
M Phase ◽  
Dependent Protein

ABSTRACT Little is known about the posttranslational control of the cyclin-dependent protein kinase (CDK) inhibitor p21. We describe here a transient phosphorylation of p21 in the G2/M phase. G2/M-phosphorylated p21 is short-lived relative to hypophosphorylated p21. p21 becomes nuclear during S phase, prior to its phosphorylation by CDK2. S126-phosphorylated cyclin B1 binds to T57-phosphorylated p21. Cdc2 kinase activation is delayed in p21-deficient cells due to delayed association between Cdc2 and cyclin B1. Cyclin B1-Cdc2 kinase activity and G2/M progression in p21−/− cells are restored after reexpression of wild-type but not T57A mutant p21. The cyclin B1 S126A mutant exhibits reduced Cdc2 binding and has low kinase activity. Phosphorylated p21 binds to cyclin B1 when Cdc2 is phosphorylated on Y15 and associates poorly with the complex. Dephosphorylation on Y15 and phosphorylation on T161 promotes Cdc2 binding to the p21-cyclin B1 complex, which becomes activated as a kinase. Thus, hyperphosphorylated p21 activates the Cdc2 kinase in the G2/M transition.

Inactivation of the checkpoint kinase Cds1 is dependent on cyclin B-Cdc2 kinase activation at the meiotic G2/M-phase transition in Xenopus oocytes

2001 ◽  
Vol 114 (18) ◽  
pp. 3397-3406
Author(s):  
Tetsuya Gotoh ◽  
Keita Ohsumi ◽  
Tomoko Matsui ◽  
Haruhiko Takisawa ◽  
Takeo Kishimoto
Keyword(s):  
Phase Transition ◽  
Xenopus Oocytes ◽  
Cyclin B ◽  
Cdc2 Kinase ◽  
Kinase Activation ◽  
Immature Oocytes ◽  
Protein Levels ◽  
M Phase ◽  
G2 Phase

Checkpoint controls ensure chromosomal integrity through the cell cycle. Chk1 and Cds1/Chk2 are effector kinases in the G2-phase checkpoint activated by damaged or unreplicated DNA, and they prevent entry into M-phase through inhibition of cyclin B-Cdc2 kinase activation. However, little is known about how the effector kinases are regulated when the checkpoint is attenuated. Recent studies indicate that Chk1 is also involved in the physiological G2-phase arrest of immature Xenopus oocytes via direct phosphorylation and inhibition of Cdc25C, the activator of cyclin B-Cdc2 kinase. Bearing in mind the overlapping functions of Chk1 and Cds1, here we have studied the involvement of Xenopus Cds1 (XCds1) in the G2/M-phase transition of immature oocytes and the regulation of its activity during this period. Protein levels of XCds1 remained constant throughout oocyte maturation and early embryonic development. The levels of XCds1 kinase activity were high in immature oocytes and decreased at the meiotic G2/M-phase transition. Consistently, when overexpressed in immature oocytes, wild-type, but not kinase-deficient, XCds1 significantly delayed entry into M-phase after progesterone treatment. The inactivation of XCds1 depended on the activation of cyclin B-Cdc2 kinase, but not MAP kinase. Although XCds1 was not directly inactivated by cyclin B-Cdc2 kinase in vitro, XCds1 was inactivated by overexpression of cyclin B, which induces the activation of cyclin B-Cdc2 kinase without progesterone. Thus, the present study is the first indication of Cds1 activity in cells that are physiologically arrested at G2-phase, and of its downregulation at entry into M-phase.

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.

scholarly journals Determination of initiation of DNA replication before and after nuclear formation in Xenopus egg cell free extracts.

1993 ◽  
Vol 123 (6) ◽  
pp. 1321-1331 ◽  
Author(s):  
Y Kubota ◽  
H Takisawa
Keyword(s):  
Dna Replication ◽  
Specific Activity ◽  
S Phase ◽  
Egg Cell ◽  
Sperm Dna ◽  
Before And After ◽  
M Phase ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Initiation Of Dna Replication

Xenopus egg extracts prepared before and after egg activation retain M- and S-phase specific activity, respectively. Staurosporine, a potent inhibitor of protein kinase, converted M-phase extracts into interphase-like extracts that were capable of forming nuclei upon the addition of sperm DNA. The nuclei formed in the staurosporine treated M-phase extract were incapable of replicating DNA, and they were unable to initiate replication upon the addition of S-phase extracts. Furthermore, replication was inhibited when the staurosporine-treated M-phase extract was added in excess to the staurosporine-treated S-phase extract before the addition of DNA. The membrane-depleted S-phase extract supported neither nuclear formation nor replication; however, preincubation of sperm DNA with these extracts allowed them to form replication-competent nuclei upon the addition of excess staurosporine-treated M-phase extract. These results demonstrate that positive factors in the S-phase extracts determined the initiation of DNA replication before nuclear formation, although these factors were unable to initiate replication after nuclear formation.

scholarly journals Plk is an M-phase-specific protein kinase and interacts with a kinesin-like protein, CHO1/MKLP-1.

1995 ◽  
Vol 15 (12) ◽  
pp. 7143-7151 ◽  
Author(s):  
K S Lee ◽  
Y L Yuan ◽  
R Kuriyama ◽  
R L Erikson
Keyword(s):  
Protein Kinase ◽  
Kinase Activity ◽  
Maximum Level ◽  
Specific Protein ◽  
Cyclin B ◽  
Cdc2 Kinase ◽  
M Phase ◽  
Nih 3T3

PLK (STPK13) encodes a murine protein kinase closely related to those encoded by the Drosophila melanogaster polo gene and the Saccharomyces cerevisiae CDC5 gene, which are required for normal mitotic and meiotic divisions. Affinity-purified antibody generated against the C-terminal 13 amino acids of Plk specifically recognizes a single polypeptide of 66 kDa in MELC, NIH 3T3, and HeLa cellular extracts. The expression levels of both poly(A)+ PLK mRNA and its encoded protein are most abundant about 17 h after serum stimulation of NIH 3T3 cells. Plk protein begins to accumulate at the S/G2 boundary and reaches the maximum level at the G2/M boundary in continuously cycling cells. Concurrent with cyclin B-associated cdc2 kinase activity, Plk kinase activity sharply peaks at the onset of mitosis. Plk enzymatic activity gradually decreases as M phase proceeds but persists longer than cyclin B-associated cdc2 kinase activity. Plk is localized to the area surrounding the chromosomes in prometaphase, appears condensed as several discrete bands along the spindle axis at the interzone in anaphase, and finally concentrates at the midbody during telophase and cytokinesis. Plk and CHO1/mitotic kinesin-like protein 1 (MKLP-1), which induces microtubule bundling and antiparallel movement in vitro, are colocalized during late M phase. In addition, CHO1/MKLP-1 appears to interact with Plk in vivo and to be phosphorylated by Plk-associated kinase activity in vitro.

scholarly journals Endoreduplication in Maize Endosperm: An Approach for Increasing Crop Productivity

2000 ◽  
Author(s):  
Gideon Grafi ◽  
Brian Larkins
Keyword(s):  
Cell Cycle ◽  
Dna Synthesis ◽  
Storage Protein ◽  
Molecular Mechanisms ◽  
S Phase ◽  
Cyclin B ◽  
Regulatory Subunit ◽  
Maize Endosperm ◽  
M Phase

The focus of this research project is to investigate the role of endoreduplication in maize endosperm development and the extent to which this process contributes to high levels of starch and storage protein synthesis. Although endoreduplication has been widely observed in many cells and tissues, especially those with high levels of metabolic activity, the molecular mechanisms through which the cell cycle is altered to produce consecutive cycles of S-phase without an intervening M-phase are unknown. Our previous research has shown that changes in the expression of several cell cycle regulatory genes coincide with the onset of endoreduplication. During this process, there is a sharp reduction in the activity of the mitotic cyclin-dependent kinase (CDK) and activation of the S-phase CDK. It appears the M-phase CDK is stable, but its activity is blocked by a proteinaceous inhibitor. Coincidentally, the S-phase checkpoint protein, retinoblastoma (ZmRb), becomes phosphorylated, presumably releasing an E2F-type transcriptional regulator which promotes the expression of genes responsible for DNA synthesis. To investigate the role of these cell cycle proteins in endoreduplication, we have created transgenic maize plants that express various genes in an endosperm-specific manner using a storage protein (g-zein) promoter. During the first year of the grant, we constructed point mutations of the maize M-phase kinase, p34cdc2. One alteration replaced aspartic acid at position 146 with asparagine (p3630-CdcD146N), while another changed threonine 161 to alanine (p3630-CdcT161A). These mutations abolish the activity of the CDK. We hypothesized that expression of the mutant forms of p34cdc2 in endoreduplicating endosperm, compared to a control p34cdc2, would lead to extra cycles of DNA synthesis. We also fused the gene encoding the regulatory subunit of the M- phase kinase, cyclin B, under the g-zein promoter. Normally, cyclin B is expected to be destroyed prior to the onset of endoreduplication. By producing high levels of this protein in developing endosperm, we hypothesized that the M-phase would be extended, potentially reducing the number of cycles of endoreduplication. Finally, we genetically engineered the wheat dwarf virus RepA protein for endosperm-specific expression. RepA binds to the maize retinoblastoma protein and presumably releases E2F-like transcription factors that activate DNA synthesis. We anticipated that inactivation of ZmRb by RepA would lead to additional cycles of DNA synthesis.

scholarly journals Activation of the Xenopus cyclin degradation machinery by full-length cyclin A

1996 ◽  
Vol 109 (5) ◽  
pp. 1071-1079 ◽  
Author(s):  
C. Jones ◽  
C. Smythe
Keyword(s):  
Protein Synthesis ◽  
Dna Replication ◽  
Kinase Activity ◽  
Cyclin A ◽  
Cell Types ◽  
Cyclin B1 ◽  
Full Length ◽  
Cyclin B ◽  
Cdc2 Kinase ◽  
Nuclear Envelope Breakdown

The entry into mitosis is dependent on the activation of mitotic forms of cdc2 kinase. In many cell types, cyclin A-associated kinase activity peaks just prior to that of cyclin B, although the precise role of cyclin A-associated kinase in the entry into mitosis is still unclear. Previous work has suggested that while cyclin B is capable of triggering cyclin destruction in Xenopus cell-free systems, cyclin A-associated kinase is not able to support this function. Here we have expressed a full-length human cyclin A in Escherichia coli and purified the protein to homogeneity by virtue of an N-terminal histidine tag. We have found that when added to Xenopus cell-free extracts free of cyclin B and incapable of protein synthesis, the temporal pattern of cyclin A-associated cdc2 kinase activity showed distinct differences that were dependent on the concentration of cyclin A added. When cyclin A was added to a concentration that generated levels of cdc2 kinase activity capable of inducing nuclear envelope breakdown, the histone H1 kinase activity profile was bi-phasic, consisting of an activation phase followed by an inactivation phase. Inactivation was found to be due to cyclin destruction, which was prevented by mos protein. Cyclin destruction was followed by nuclear reassembly and an additional round of DNA replication, indicating that there is no protein synthesis requirement for DNA replication in this embryonic system. It has been suggested that the evolutionary recruitment of cyclin A into an S phase function may have necessitated the loss of an original mitotic ability to activate the cyclin destruction pathway. The results presented here indicate that cyclin A has not lost the ability to activate its own destruction and that cyclin A-mediated activation of the cyclin destruction pathway permitted destruction of cyclin B1 as well as cyclin A, indicating that there are not distinct cyclin A and cyclin B destruction pathways. Thus the ordered progression of the cell cycle requires the careful titration of cyclin. A concentration in order to avoid activation of the cyclin destruction pathway before sufficient active cyclin B/cdc2 kinase has accumulated.

scholarly journals The dynamics of replication licensing in live Caenorhabditis elegans embryos

2012 ◽  
Vol 196 (2) ◽  
pp. 233-246 ◽  
Author(s):  
Remi Sonneville ◽  
Matthieu Querenet ◽  
Ashley Craig ◽  
Anton Gartner ◽  
J. Julian Blow
Keyword(s):  
Caenorhabditis Elegans ◽  
Dna Replication ◽  
Dna Binding ◽  
Feedback Loop ◽  
Origin Recognition Complex ◽  
S Phase ◽  
Chromosomal Dna ◽  
M Phase ◽  
Prereplicative Complex

Accurate DNA replication requires proper regulation of replication licensing, which entails loading MCM-2–7 onto replication origins. In this paper, we provide the first comprehensive view of replication licensing in vivo, using video microscopy of Caenorhabditis elegans embryos. As expected, MCM-2–7 loading in late M phase depended on the prereplicative complex (pre-RC) proteins: origin recognition complex (ORC), CDC-6, and CDT-1. However, many features we observed have not been described before: GFP–ORC-1 bound chromatin independently of ORC-2–5, and CDC-6 bound chromatin independently of ORC, whereas CDT-1 and MCM-2–7 DNA binding was interdependent. MCM-3 chromatin loading was irreversible, but CDC-6 and ORC turned over rapidly, consistent with ORC/CDC-6 loading multiple MCM-2–7 complexes. MCM-2–7 chromatin loading further reduced ORC and CDC-6 DNA binding. This dynamic behavior creates a feedback loop allowing ORC/CDC-6 to repeatedly load MCM-2–7 and distribute licensed origins along chromosomal DNA. During S phase, ORC and CDC-6 were excluded from nuclei, and DNA was overreplicated in export-defective cells. Thus, nucleocytoplasmic compartmentalization of licensing factors ensures that DNA replication occurs only once.

Sign in / Sign up

Export Citation Format

Share Document