HSP70-2 is required for CDC2 kinase activity in meiosis I of mouse spermatocytes

Development ◽  
1997 ◽  
Vol 124 (15) ◽  
pp. 3007-3014 ◽  
Author(s):  
D. Zhu ◽  
D.J. Dix ◽  
E.M. Eddy
Keyword(s):  
Complex Formation ◽  
Molecular Chaperone ◽  
Kinase Activity ◽  
Cyclin B1 ◽  
Meiotic Cell ◽  
Cdc2 Kinase ◽  
M Phase ◽  
Pachytene Spermatocytes ◽  
Meiosis I

Cyclin B-dependent CDC2 kinase activity has a key role in triggering the G2/M-phase transition during the mitotic and meiotic cell cycles. The Hsp70-2 gene is expressed only in spermatogenic cells at a significant level. In Hsp70-2 gene knock-out (Hsp70-2(−/−)) mice, primary spermatocytes fail to complete meiosis I, suggesting a link between HSP70-2 heat-shock protein and CDC2 kinase activity during this phase of spermatogenesis. Members of the HSP70 protein family are molecular chaperones that mediate protein de novo folding, translocation and multimer assembly. This study used immunoprecipitation-coupled western blot and in vitro reconstitution experiments to show that HSP70-2 interacts with CDC2 in the mouse testis, appears to be a molecular chaperone for CDC2, and is required for CDC2/cyclin B1 complex formation. Previous studies reported that most CDC2 kinase activity in the mouse testis is present in pachytene spermatocytes. Although CDC2 kinase activity for histone H1 was present in the testis of wild-type mice, it was nearly absent from the testis of Hsp70-2(−/−) mice, probably due to defective CDC2/cyclin B1 complex formation. Furthermore, addition of HSP70-2 to freshly prepared extracts of testis from Hsp70-2(−/−) mice not only restored CDC2/cyclin B1 complex formation but also reconstituted CDC2 kinase activity in vitro. It appears that one cause of failure to complete meiosis I during spermatogenesis in Hsp70-2(−/−) mice is disruption of CDC2/cyclin B1 assembly in pachytene spermatocytes, thereby preventing development of the CDC2 kinase activity required to trigger G2/M-phase transition. These studies provide novel in vivo evidence for a link between an HSP70 molecular chaperone and CDC2 kinase activity essential for the meiotic cell cycle in spermatogenesis.

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.

Phosphorylation of Xenopus cyclins B1 and B2 is not required for cell cycle transitions

1991 ◽  
Vol 11 (8) ◽  
pp. 3860-3867
Author(s):  
T Izumi ◽  
J L Maller
Keyword(s):  
Map Kinase ◽  
Cyclin B1 ◽  
Site Directed Mutagenesis ◽  
Cyclin B ◽  
Phosphorylation Sites ◽  
Cdc2 Kinase ◽  
M Phase ◽  
Egg Extracts ◽  
Xenopus Egg Extracts

The cdc2 kinase and B-type cyclins are known to be components of maturation- or M-phase-promoting factor (MPF). Phosphorylation of cyclin B has been reported previously and may regulate entry into and exit from mitosis and meiosis. To investigate the role of cyclin B phosphorylation, we replaced putative cdc2 kinase phosphorylation sites in Xenopus cyclins B1 and B2 by using oligonucleotide site-directed mutagenesis. We found that Ser-90 of cyclin B2 and Ser-94 or Ser-96 of cyclin B1 are the main phosphorylation sites both in functional Xenopus egg extracts and after phosphorylation with purified MPF in vitro. Microtubule-associated protein (MAP) kinase from Xenopus eggs phosphorylated cyclin B1 significantly at Ser-94 or Ser-96, whereas it was largely inactive against cyclin B2. The substitutions that ablated phosphorylation at these sites, however, resulted in no functional differences between mutant and wild-type cyclin, as judged by the kinetics of M-phase degradation, induction of mitosis in egg extracts, or induction of oocyte maturation. These results indicate that the phosphorylation of Xenopus B-type cyclins by cdc2 kinase or MAP kinase is not required for the hallmark functions of cyclin.

scholarly journals Phosphorylation and activation of the Xenopus Cdc25 phosphatase in the absence of Cdc2 and Cdk2 kinase activity.

1995 ◽  
Vol 6 (2) ◽  
pp. 215-226 ◽  
Author(s):  
T Izumi ◽  
J L Maller
Keyword(s):  
Kinase Activity ◽  
Cdc2 Kinase ◽  
Nuclear Envelope Breakdown ◽  
Dual Specificity ◽  
M Phase ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Xenopus Egg Extracts

The M-phase inducer, Cdc25C, is a dual-specificity phosphatase that directly phosphorylates and activates the cyclin B/Cdc2 kinase complex, leading to initiation of mitosis. Cdc25 itself is activated at the G2/M transition by phosphorylation on serine and threonine residues. Previously, it was demonstrated that Cdc2 kinase is capable of phosphorylating and activating Cdc25, suggesting the existence of a positive feedback loop. In the present study, kinases other than Cdc2 that can phosphorylate and activate Cdc25 were investigated. Cdc25 was found to be phosphorylated and activated by cyclin A/Cdk2 and cyclin E/Cdk2 in vitro. However, in interphase Xenopus egg extracts with no detectable Cdc2 and Cdk2, treatment with the phosphatase inhibitor microcystin activated a distinct kinase that could phosphorylate and activate Cdc25. Microcystin also induced other mitotic phenomena such as chromosome condensation and nuclear envelope breakdown in extracts containing less than 5% of the mitotic level of Cdc2 kinase activity. These findings implicate a kinase other than Cdc2 and Cdk2 that may initially activate Cdc25 in vivo and suggest that this kinase may also phosphorylate M-phase substrates even in the absence of Cdc2 kinase.

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.

scholarly journals Phosphorylation of Xenopus cyclins B1 and B2 is not required for cell cycle transitions.

1991 ◽  
Vol 11 (8) ◽  
pp. 3860-3867 ◽  
Author(s):  
T Izumi ◽  
J L Maller
Keyword(s):  
Map Kinase ◽  
Cyclin B1 ◽  
Site Directed Mutagenesis ◽  
Cyclin B ◽  
Phosphorylation Sites ◽  
Cdc2 Kinase ◽  
M Phase ◽  
Egg Extracts ◽  
Xenopus Egg Extracts

The cdc2 kinase and B-type cyclins are known to be components of maturation- or M-phase-promoting factor (MPF). Phosphorylation of cyclin B has been reported previously and may regulate entry into and exit from mitosis and meiosis. To investigate the role of cyclin B phosphorylation, we replaced putative cdc2 kinase phosphorylation sites in Xenopus cyclins B1 and B2 by using oligonucleotide site-directed mutagenesis. We found that Ser-90 of cyclin B2 and Ser-94 or Ser-96 of cyclin B1 are the main phosphorylation sites both in functional Xenopus egg extracts and after phosphorylation with purified MPF in vitro. Microtubule-associated protein (MAP) kinase from Xenopus eggs phosphorylated cyclin B1 significantly at Ser-94 or Ser-96, whereas it was largely inactive against cyclin B2. The substitutions that ablated phosphorylation at these sites, however, resulted in no functional differences between mutant and wild-type cyclin, as judged by the kinetics of M-phase degradation, induction of mitosis in egg extracts, or induction of oocyte maturation. These results indicate that the phosphorylation of Xenopus B-type cyclins by cdc2 kinase or MAP kinase is not required for the hallmark functions of cyclin.

Cyclin synthesis controls the progression of meiotic maturation in mouse oocytes

Development ◽  
1998 ◽  
Vol 125 (24) ◽  
pp. 4989-4997 ◽  
Author(s):  
Z. Polanski ◽  
E. Ledan ◽  
S. Brunet ◽  
S. Louvet ◽  
M.H. Verlhac ◽  
...  
Keyword(s):  
Messenger Rna ◽  
Kinase Activity ◽  
Polar Body ◽  
Germinal Vesicle ◽  
Cyclin B1 ◽  
Meiotic Maturation ◽  
Cdc2 Kinase ◽  
First Polar Body ◽  
M Phase ◽  
Kinetics Of

To study the mechanisms involved in the progression of meiotic maturation in the mouse, we used oocytes from two strains of mice, CBA/Kw and KE, which differ greatly in the rate at which they undergo meiotic maturation. CBA/Kw oocytes extrude the first polar body about 7 hours after breakdown of the germinal vesicle (GVBD), whilst the oocytes from KE mice take approximately 3–4 hours longer. In both strains, the kinetics of spindle formation are comparable. While the kinetics of MAP kinase activity are very similar in both strains (although slightly faster in CBA/Kw), the rise of cdc2 kinase activity is very rapid in CBA/Kw oocytes and slow and diphasic in KE oocytes. When protein synthesis is inhibited, the activity of the cdc2 kinase starts to rise but arrests shortly after GVBD with a slightly higher level in CBA/Kw oocytes, which may correspond to the presence of a larger pool of cyclin B1 in prophase CBA/Kw oocytes. After GVBD, the rate of cyclin B1 synthesis is higher in CBA/Kw than in KE oocytes, whilst the overall level of protein synthesis and the amount of messenger RNA coding for cyclin B1 are identical in oocytes from both strains. The injection of cyclin B1 messenger RNA in KE oocytes increased the H1 kinase activity and sped up first polar body extrusion. Finally, analysis of the rate of maturation in hybrids obtained after fusion of nuclear and cytoplasmic fragments of oocytes from both strains suggests that both the germinal vesicle and the cytoplasm contain factor(s) influencing the length of the first meiotic M phase. These results demonstrate that the rate of cyclin B1 synthesis controls the length of the first meiotic M phase and that a nuclear factor able to speed up cyclin B synthesis is present in CBA/Kw oocytes.

Activation of bovine oocytes by protein synthesis inhibitors: new findings on the role of MPF/MAPKs†

2021 ◽  
Author(s):  
Cecilia Valencia ◽  
Felipe Alonso Pérez ◽  
Carola Matus ◽  
Ricardo Felmer ◽  
María Elena Arias
Keyword(s):  
Protein Synthesis ◽  
Kinase Activity ◽  
Cyclin B1 ◽  
Protein Synthesis Inhibitors ◽  
Vitro Fertilization ◽  
New Findings ◽  
Bovine Oocytes ◽  
Dependent Pathway

Abstract The present study evaluated the mechanism by which protein synthesis inhibitors activate bovine oocytes. The aim was to analyze the dynamics of MPF and MAPKs. MII oocytes were activated with ionomycin (Io), ionomycin+anisomycin (ANY) and ionomycin+cycloheximide (CHX) and by in vitro fertilization (IVF). The expression of cyclin B1, p-CDK1, p-ERK1/2, p-JNK, and p-P38 were evaluated by immunodetection and the kinase activity of ERK1/2 was measured by enzyme assay. Evaluations at 1, 4, and 15 hours postactivation (hpa) showed that the expression of cyclin B1 was not modified by the treatments. ANY inactivated MPF by p-CDK1Thr14-Tyr15 at 4 hpa (P < 0.05), CHX increased pre-MPF (p-CDK1Thr161 and p-CDK1Thr14-Tyr15) at 1 hpa and IVF increased p-CDK1Thr14-Tyr15 at 17 hours postfertilization (hpf) (P < 0.05). ANY and CHX reduced the levels of p-ERK1/2 at 4 hpa (P < 0.05) and its activity at 4 and 1 hpa, respectively (P < 0.05). Meanwhile, IVF increased p-ERK1/2 at 6 hpf (P < 0.05); however, its kinase activity decreased at 6 hpf (P < 0.05). p-JNK in ANY, CHX, and IVF oocytes decreased at 4 hpa (P < 0.05). p-P38 was only observed at 1 hpa, with no differences between treatments. In conclusion, activation of bovine oocytes by ANY, CHX, and IVF inactivates MPF by CDK1-dependent specific phosphorylation without cyclin B1 degradation. ANY or CHX promoted this inactivation, which seemed to be more delayed in the physiological activation (IVF). Both inhibitors modulated MPF activity via an ERK1/2-independent pathway, whereas IVF activated the bovine oocytes via an ERK1/2-dependent pathway. Finally, ANY does not activate the JNK and P38 kinase pathways.

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.

Effects of cycloheximide treatment and interval between fusion and activation on in vitro development of pig nuclear transfer embryos

2002 ◽  
Vol 14 (4) ◽  
pp. 191 ◽  
Author(s):  
M. A. Martinez-Diaz ◽  
K. Ikeda ◽  
Y. Takahashi
Keyword(s):  
Nuclear Transfer ◽  
Kinase Activity ◽  
Short Interval ◽  
Cumulus Cells ◽  
Cleavage Rate ◽  
In Vitro Development ◽  
M Phase ◽  
Cytostatic Factor ◽  
Vitro Development

The effects of cycloheximide (CHX) treatment and the interval between fusion and activation on the development of pig nuclear transfer (NT) embryos constructed with enucleated oocytes and serum-starved granulosa/cumulus cells were examined. One group of couplets was fused and activated simultaneously (FAS) by a single electrical pulse (activation pulse). Another three groups of couplets were fused electricaly 1.5, 2.5 or 4.5 h before being subjected to the activation pulse (FBA). Each group was divided into two subgroups and incubated with or without CHX. The NT embryos treated with CHX showed a high and stable cleavage rate, regardless of the interval between fusion and activation; however, development to blastocysts was improved only when the NT embryos were subjected to FAS with CHX. These results indicate that CHX-sensitive events occurring shortly after FAS may be responsible for the development to blastocysts. Fusion pulse rarely activated M II oocytes, but rapidly dropped the p34cdc2 kinase activity in NT embryos. A pronucleus-like structure was observed 2-2.5 h after the activation pulse with CHX in NT embryos of both the FAS and FBA groups. Therefore, successive inactivation of M-phase promoting factor and cytostatic factor at a certain short interval may also play an important role in the development of NT embryos.

Huanglian, A Chinese Herbal Extract, Inhibits Cell Growth by Suppressing the Expression of Cyclin B1 and Inhibiting CDC2 Kinase Activity in Human Cancer Cells

2000 ◽  
Vol 58 (6) ◽  
pp. 1287-1293 ◽  
Author(s):  
Xiao-Kui Li ◽  
Monica Motwani ◽  
William Tong ◽  
William Bornmann ◽  
Gary K. Schwartz
Keyword(s):  
Cell Growth ◽  
Cancer Cells ◽  
Kinase Activity ◽  
Human Cancer ◽  
Cyclin B1 ◽  
Herbal Extract ◽  
Cdc2 Kinase ◽  
Human Cancer Cells ◽  
Chinese Herbal
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