scholarly journals Mitotic timing is differentially controlled by A- and B-type cyclins and by CDC6 associated with a bona fide CDK inhibitor Xic1 in Xenopus laevis cell-free extract

2021 ◽  
Vol 65 (7-8-9) ◽  
pp. 487-496
Author(s):  
Mohammed El Dika ◽  
Lisa Wechselberger ◽  
Bilal Djeghout ◽  
Djamel Eddine Benouareth ◽  
Mohammed El Dika ◽  
...  
Keyword(s):  
Xenopus Laevis ◽  
Kinase Activity ◽  
Cyclin A ◽  
Histone H1 ◽  
Cyclin B ◽  
Mitotic Kinase ◽  
M Phase ◽  
Bona Fide ◽  
Full Activation ◽  
Dependent Mechanism

The timing of the M-phase is precisely controlled by a CDC6-dependent mechanism inhibiting the mitotic histone H1 kinase. Here, we describe the differential regulation of the dynamics of this mitotic kinase activity by exogenous cyclin A or cyclin B in the Xenopus laevis cycling extracts. We show that the experimental increase in cyclin A modifies only the level of histone H1 kinase activity, while the cyclin B increase modifies two parameters: histone H1 kinase activity and the timing of its full activation, which is accelerated. On the other hand, the cyclin A depletion significantly delays full activation of histone H1 kinase. However, when CDC6 is added to such an extract, it inhibits cyclin B-associated histone H1 kinase, but does not modify the mitotic timing in the absence of cyclin A. Further, we show via p9 co-precipitation with Cyclin-Dependent Kinases (CDKs), that both CDC6 and the bona fide CDK1 inhibitor Xic1 associate with the mitotic CDKs. Finally, we show that the Xic1 temporarily separates from the mitotic CDKs complexes during the peak of histone H1 kinase activity. These data show the differential coordination of the M-phase progression by cyclin A- and cyclin B-dependent CDKs, confirm the critical role of the CDC6-dependent histone H1 kinase inhibition in this process, and show that CDC6 acts differentially through the cyclin B- and cyclin A-associated CDKs. This CDC6- and cyclins-dependent mechanism likely depends on the precisely regulated association of Xic1 with the mitotic CDKs complexes. We postulate that: i. the dissociation of Xic1 from the CDKs complexes allows the maximal activation of CDK1 during the M-phase, ii. the switch between cyclin A- and cyclin B-CDK inhibition upon M-phase initiation may be responsible for the diauxic growth of mitotic histone H1 kinase activity.

scholarly journals CDC6 regulates mitotic CDK1 via cyclin B and not cyclin A and acts through a bona fide CDK inhibitor Xic1

2020 ◽  
Author(s):  
Mohammed El Dika ◽  
Lisa Wechselberger ◽  
Bilal Djeghout ◽  
Djamel Eddine Benouareth ◽  
Krystyna Jęderka ◽  
...  
Keyword(s):  
Critical Role ◽  
Cyclin A ◽  
Cyclin B ◽  
Cyclin Dependent Kinases ◽  
Mitotic Kinase ◽  
M Phase ◽  
Bona Fide ◽  
Phase Progression ◽  
Dependent Mechanism

AbstractThe timing of the M-phase entry and its progression are precisely controlled by a CDC6-dependent mechanism that inhibits the major mitotic kinase CDK1, and, thus, regulates the dynamic of CDK1 during the M-phase. In this paper, we describe the differential regulation of the mitotic CDK1 dynamics by exogenous cyclin A or a non-degradable cyclin B added to the Xenopus laevis embryo cycling extracts. We showed that the variations in the level of cyclin B modify both CDK1 activity and the timing of the M-phase progression, while the cyclin A levels modify only CDK1 activity without changing the timing of the M-phase events. In consequence, CDC6 regulates the M-phase through endogenous cyclin B, but not cyclin A, which we demonstrated directly by the depletion of cyclin A, and the addition of CDC6 to the cycling extracts. Further, we showed, by p9 precipitation (p9 protein associates with Cyclin-Dependent Kinases, CDK), followed by the Western blotting that CDC6, and the bona fide CDK1 inhibitor Xic1, associate with CDK1 and/or another CDK present in Xenopus embryos, the CDK2. Finally, we demonstrated that the Xic1 temoprarily separates from the mitotic CDK complexes during the peak of CDK1 activity. These data show the differential coordination of the M-phase progression by CDK1/cyclin A and CDK1/cyclin B, confirm the critical role of the CDC6-dependent CDK1 inhibition in this process and show that CDC6 acts through the cyclin B- and not cyclin A/CDK complexes. This CDC6- and cyclin B-dependent mechanism may also depend on the precisely regulated association of Xic1 with the CDK complexes. We postulate that the dissociation of Xic1 from the CDK complexes allows the maximal activation of CDK1 during the M-phase.

scholarly journals Mitotic Effects of a Constitutively Active Mutant of the Xenopus Polo-Like Kinase Plx1

1999 ◽  
Vol 19 (12) ◽  
pp. 8625-8632 ◽  
Author(s):  
Yue-Wei Qian ◽  
Eleanor Erikson ◽  
James L. Maller
Keyword(s):  
Xenopus Laevis ◽  
Aspartic Acid ◽  
Kinase Activity ◽  
Spindle Assembly ◽  
Cyclin B ◽  
Interphase Nuclei ◽  
Aspartic Acid Residue ◽  
Early Embryos ◽  
M Phase ◽  
Egg Extracts

ABSTRACT During mitosis the Xenopus polo-like kinase 1 (Plx1) plays key roles in the activation of Cdc25C, in spindle assembly, and in cyclin B degradation. Previous work has shown that the activation of Plx1 requires phosphorylation on serine and threonine residues. In the present work, we demonstrate that replacement of Ser-128 or Thr-201 with a negatively charged aspartic acid residue (S128D or T201D) elevates Plx1 activity severalfold and that replacement of both Ser-128 and Thr-201 with Asp residues (S128D/T201D) increases Plx1 activity approximately 40-fold. Microinjection of mRNA encoding S128D/T201D Plx1 into Xenopus oocytes induced directly the activation of both Cdc25C and cyclin B-Cdc2. In egg extracts T201D Plx1 delayed the timing of deactivation of Cdc25C during exit from M phase and accelerated Cdc25C activation during entry into M phase. This supports the concept that Plx1 is a “trigger” kinase for the activation of Cdc25C during the G2/M transition. In addition, during anaphase T201D Plx1 reduced preferentially the degradation of cyclin B2 and delayed the reduction in Cdc2 histone H1 kinase activity. In early embryos S128D/T201D Plx1 resulted in arrest of cleavage and formation of multiple interphase nuclei. Consistent with these results, Plx1 was found to be localized on centrosomes at prophase, on spindles at metaphase, and at the midbody during cytokinesis. These results demonstrate that in Xenopus laevis activation of Plx1 is sufficient for the activation of Cdc25C at the initiation of mitosis and that inactivation of Plx1 is required for complete degradation of cyclin B2 after anaphase and completion of cytokinesis.

scholarly journals Elimination of cdc2 phosphorylation sites in the cdc25 phosphatase blocks initiation of M-phase.

1993 ◽  
Vol 4 (12) ◽  
pp. 1337-1350 ◽  
Author(s):  
T Izumi ◽  
J L Maller
Keyword(s):  
Recombinant Proteins ◽  
Kinase Activity ◽  
Cyclin B ◽  
Phosphorylation Sites ◽  
Cdc25 Phosphatase ◽  
M Phase ◽  
Full Activation ◽  
Periodic Changes

The cdc25 phosphatase is a mitotic inducer that activates p34cdc2 at the G2/M transition by dephosphorylation of Tyr15 in p34cdc2. cdc25 itself is also regulated through periodic changes in its phosphorylation state. To elucidate the mechanism for induction of mitosis, phosphorylation of cdc25 has been investigated using recombinant proteins. cdc25 is phosphorylated by both cyclin A/p34cdc2 and cyclin B/p34cdc2 at similar sets of multiple sites in vitro. This phosphorylation retards its electrophoretical mobility and activates its ability to increase cyclin B/p34cdc2 kinase activity three- to fourfold in vitro, as found for endogenous Xenopus cdc25 in M-phase extracts. The threonine and serine residues followed by proline that are conserved between Xenopus and human cdc25 have been mutated. Both the triple mutation of Thr48, Thr67, and Thr138 and the quintuple mutation of these three threonine residues plus Ser205 and Ser285, almost completely abolish the shift in electrophoretic mobility of cdc25 after incubation with M-phase extracts or phosphorylation by p34cdc2. These mutations inhibit the activation of cdc25 by phosphorylation with p34cdc2 by 70 and 90%, respectively. At physiological concentrations these mutants cannot activate cyclin B/p34cdc2 in cdc25-immunodepleted oocyte extracts, suggesting that a positive feed-back loop between cdc2 and cdc25 is necessary for the full activation of cyclin B/p34cdc2 that induces abrupt entry into mitosis in vivo.

Procaine-induced maturation of Xenopus oocytes is mediated by a transient activation of M-Phase promoting factor

Zygote ◽  
1997 ◽  
Vol 5 (1) ◽  
pp. 11-19 ◽  
Author(s):  
Stéphane Flament ◽  
Jean-François Bodart ◽  
Edith Browaeys ◽  
Marc Bertout ◽  
Arlette Rousseau ◽  
...  
Keyword(s):  
Xenopus Laevis ◽  
Xenopus Oocytes ◽  
Kinase Activity ◽  
Germinal Vesicle ◽  
Histone H1 ◽  
White Spot ◽  
Germinal Vesicle Breakdown ◽  
Transient Activation ◽  
M Phase ◽  
Spot Formation

SummaryWe have recently shown that the incubation of Xenopus laevis oocytes in procaine-containing solutions induced germinal vesicle breakdown without white spot formation and, in some cases, with the appearance of spindle and chromosomes in the cytoplasm. The present study was performed to determine whether M-phase promoting factor was involved in this unusual maturation. Procaine failed to induce maturation in the presence of 6-dimethylamino purine or roscovitine, which are both known to inhibit p34cdc2kinase. Histone H1 kinase activity was detected in procaine-treated oocytes but it was always lower than in progesterone-treated controls. A shift in p34cdc2 was observed in oocytes that had been exposed to procaine for 16h, but it was not detected in those exposed for 24h. Finally, cytoplasm transfer experiments demonstrated that the maturation promoting activity that occurred in oocytes incubated in procaine for 16h could induce maturation of recipient stage VI oocytes. This transferable activity was weaker than that from progesterone-treated controls since only 30% of the recipients underwent germinal vesicle breakdown and only a few spindles were observed, which were not always correctly located. Taken together these results demonstrate that M-phase promoting factor is involved in the procaine maturing effect despite some differences compared with progesterone-treated oocytes which might explain the particular type of maturation induced by this substance. The discovery of the mechanisms by which procaine is able to activate M-phase promoting factor might now help in the understanding of some steps in progesterone-induced maturation that have still to be elucidated.

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 SGK phosphorylates Cdc25 and Myt1 to trigger cyclin B–Cdk1 activation at the meiotic G2/M transition

2019 ◽  
Vol 218 (11) ◽  
pp. 3597-3611 ◽  
Author(s):  
Daisaku Hiraoka ◽  
Enako Hosoda ◽  
Kazuyoshi Chiba ◽  
Takeo Kishimoto
Keyword(s):  
Somatic Cell ◽  
Cyclin A ◽  
Pi3k Pathway ◽  
Cyclin B ◽  
Hormonal Stimulation ◽  
Oocyte Meiosis ◽  
M Phase ◽  
Mitosis And Meiosis ◽  
Cell Mitosis ◽  
Stimulation Of

The kinase cyclin B–Cdk1 complex is a master regulator of M-phase in both mitosis and meiosis. At the G2/M transition, cyclin B–Cdk1 activation is initiated by a trigger that reverses the balance of activities between Cdc25 and Wee1/Myt1 and is further accelerated by autoregulatory loops. In somatic cell mitosis, this trigger was recently proposed to be the cyclin A–Cdk1/Plk1 axis. However, in the oocyte meiotic G2/M transition, in which hormonal stimuli induce cyclin B–Cdk1 activation, cyclin A–Cdk1 is nonessential and hence the trigger remains elusive. Here, we show that SGK directly phosphorylates Cdc25 and Myt1 to trigger cyclin B–Cdk1 activation in starfish oocytes. Upon hormonal stimulation of the meiotic G2/M transition, SGK is activated by cooperation between the Gβγ-PI3K pathway and an unidentified pathway downstream of Gβγ, called the atypical Gβγ pathway. These findings identify the trigger in oocyte meiosis and provide insights into the role and activation of SGK.

scholarly journals Both cyclin A delta 60 and B delta 97 are stable and arrest cells in M-phase, but only cyclin B delta 97 turns on cyclin destruction.

1991 ◽  
Vol 10 (13) ◽  
pp. 4311-4320 ◽  
Author(s):  
F.C. Luca ◽  
E.K. Shibuya ◽  
C.E. Dohrmann ◽  
J.V. Ruderman
Keyword(s):  
Cyclin A ◽  
Cyclin B ◽  
M Phase

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 The requirements for protein synthesis and degradation, and the control of destruction of cyclins A and B in the meiotic and mitotic cell cycles of the clam embryo.

1992 ◽  
Vol 116 (3) ◽  
pp. 707-724 ◽  
Author(s):  
T Hunt ◽  
F C Luca ◽  
J V Ruderman
Keyword(s):  
Protein Synthesis ◽  
Cyclin A ◽  
Mitotic Cell ◽  
Mitotic Division ◽  
Cyclin B ◽  
Cell Cycles ◽  
Activated State ◽  
Inhibition Of Protein Synthesis ◽  
M Phase ◽  
Meiosis I

Fertilization of clam oocytes initiates a series of cell divisions, of which the first three--meiosis I, meiosis II, and the first mitotic division--are highly synchronous. After fertilization, protein synthesis is required for the successful completion of every division except meiosis I. When protein synthesis is inhibited, entry into meiosis I and the maintenance of M phase for the normal duration of meiosis occur normally, but the chromosomes fail to interact correctly with the spindle in meiosis II metaphase. By contrast, inhibition of protein synthesis immediately after completion of meiosis or mitosis stops cells entering the next mitosis. We describe the behavior of cyclins A and B in relation to these "points of no return." The cyclins are synthesized continuously and are rapidly destroyed shortly before the metaphase-anaphase transition of the mitotic cell cycles, with cyclin A being degraded in advance of cyclin B. Cyclin destruction normally occurs during a 5-min window in mitosis, but in the monopolar mitosis that occurs after parthenogenetic activation of clam oocytes, or when colchicine is added to fertilized eggs about to enter first mitosis, the destruction of cyclin B is strongly delayed, whereas proteolysis of cyclin A is maintained in an activated state for the duration of metaphase arrest. Under either of these abnormal conditions, inhibition of protein synthesis causes a premature return to interphase that correlates with the time when cyclin B disappears.

scholarly journals A complex containing p34cdc2 and cyclin B phosphorylates the nuclear lamin and disassembles nuclei of clam oocytes in vitro.

1991 ◽  
Vol 112 (4) ◽  
pp. 523-533 ◽  
Author(s):  
G Dessev ◽  
C Iovcheva-Dessev ◽  
J R Bischoff ◽  
D Beach ◽  
R Goldman
Keyword(s):  
Protein Kinase ◽  
Hela Cells ◽  
Kinase Activity ◽  
Nuclear Lamina ◽  
Cyclin B ◽  
Protein Kinase Activity ◽  
Nuclear Lamin ◽  
Single Protein ◽  
M Phase

Cell-free extracts prepared from activated clam oocytes contain factors which induce phosphorylation of the single 67-kD lamin (L67), disassemble clam oocyte nuclei, and cause chromosome condensation in vitro (Dessev, G., R. Palazzo, L. Rebhun, and R. Goldman. 1989. Dev. Biol. 131:469-504). To identify these factors, we have fractionated the oocyte extracts. The nuclear lamina disassembly (NLD) activity, together with a protein kinase activity specific for L67, appear as a single peak throughout a number of purification steps. This peak also contains p34cdc2, cyclin B, and histone H1-kinase activity, which are components of the M-phase promoting factor (MPF). The NLD/L67-kinase activity is depleted by exposure of this purified material to Sepharose conjugated to p13suc1, and is restored upon addition of a p34cdc2/p62 complex from HeLa cells. The latter complex phosphorylates L67 and induces NLD in the absence of other clam oocyte proteins. Our results suggest that a single protein kinase activity (p34cdc2-H1 kinase, identical with MPF) phosphorylates the lamin and is involved in the meiotic breakdown of the nuclear envelope in clam oocytes.

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