scholarly journals Initial triggering of M-phase in starfish oocytes: a possible novel component of maturation-promoting factor besides cdc2 kinase.

1996 ◽  
Vol 132 (1) ◽  
pp. 125-135 ◽  
Author(s):  
E Okumura ◽  
T Sekiai ◽  
S Hisanaga ◽  
K Tachibana ◽  
T Kishimoto
Keyword(s):  
Kinase Activity ◽  
Cdc25 Phosphatase ◽  
Cdc2 Kinase ◽  
M Phase ◽  
Initial Activation ◽  
Independent Process ◽  
G2 Phase ◽  
Hormonal Stimulus ◽  
Cdc25 Protein ◽  
Maturation Promoting Factor

G2-phase-arrested immature starfish oocytes contain inactive cdc2 kinase and cdc25 phosphatase, and an inactivator for cdc2 kinase. In this system, we have studied how the regulatory balance is apped toward the initial activation of cdc2 kinase. During the hormone-dependent period (Guerrier, P., and M. Doree, 1975. Dev. Biol. 47:341-348), p34cdc2 and cdc25 protein are already converted, though not fully, to active forms, whereas the inactivators for cdc2 kinase and cdc25 phosphatase are able to exhibit their activities if the hormone were removed. We produced "triggered oocytes," in which due to a neutralizing anticdc25 antibody, the activation of cdc2 kinase is prevented out cdc25 protein is phosphorylated slightly after the maturation-inducing hormonal stimulus. In contrast to control immature oocytes, in triggered oocytes the injected cdc2 kinase is not inactivated, and accordingly the level of cdc2 kinase activity required for meiosis reinitiation is much less. These results imply the presence of a cdc2 kinase activity-independent process(es) that suppresses the inactivator for cdc2 kinase and initially phosphorylates cdc25 protein, although this process is reversible during the initial activation of cdc2 kinase. At the most initial triggering of M-phase, the cdc2 kinase activity-independent process might trip the switch leading to the initial activation of cdc2 kinase. Thereafter, in parallel, the cdc2 kinase-dependent feedback loops described by others may cause further increase in cdc2 kinase activity. We propose that a putative suppressor, which downregulates the inactivator for cdc2 kinase independently of nuclear components, might be a previously unrecognized component of maturation-promoting factor.

scholarly journals Periodic changes in phosphorylation of the Xenopus cdc25 phosphatase regulate its activity.

1992 ◽  
Vol 3 (8) ◽  
pp. 927-939 ◽  
Author(s):  
T Izumi ◽  
D H Walker ◽  
J L Maller
Keyword(s):  
Tyrosine Phosphatase ◽  
Mitotic Cell ◽  
Cdc25 Phosphatase ◽  
Mobility Shift ◽  
Cdc2 Kinase ◽  
Kinase Activation ◽  
Cell Cycles ◽  
M Phase ◽  
Egg Extracts ◽  
Cdc25 Protein

The cdc25 tyrosine phosphatase is known to activate cdc2 kinase in the G2/M transition by dephosphorylation of tyrosine 15. To determine how entry into M-phase in eukaryotic cells is controlled, we have investigated the regulation of the cdc25 protein in Xenopus eggs and oocytes. Two closely related Xenopus cdc25 genes have been cloned and sequenced and specific antibodies generated. The cdc25 phosphatase activity oscillates in both meiotic and mitotic cell cycles, being low in interphase and high in M-phase. Increased activity of cdc25 at M-phase is accompanied by increased phosphorylation that retards electrophoretic mobility in gels from 76 to 92 kDa. Treatment of cdc25 with either phosphatase 1 or phosphatase 2A removes phosphate from cdc25, reverses the mobility shift, and decreases its ability to activate cdc2 kinase. Furthermore, the addition of okadaic acid to egg extracts arrested in S-phase by aphidicolin causes phosphorylation and activation of the cdc25 protein before cyclin B/cdc2 kinase activation. These results demonstrate that the activity of the cdc25 phosphatase at the G2/M transition is directly regulated through changes in its phosphorylation state.

scholarly journals Mutant Caldesmon Lacking cdc2 Phosphorylation Sites Delays M-Phase Entry and Inhibits Cytokinesis

2001 ◽  
Vol 12 (1) ◽  
pp. 239-250 ◽  
Author(s):  
Shigeko Yamashiro ◽  
Hueylan Chern ◽  
Yoshihiko Yamakita ◽  
Fumio Matsumura
Keyword(s):  
Cho Cells ◽  
Kinase Activity ◽  
Chinese Hamster ◽  
Phosphorylation Sites ◽  
Wild Type ◽  
Cdc2 Kinase ◽  
Effective Inhibition ◽  
M Phase ◽  
Phase Progression ◽  
Phase Entry

Caldesmon is phosphorylated by cdc2 kinase during mitosis, resulting in the dissociation of caldesmon from microfilaments. To understand the physiological significance of phosphorylation, we generated a caldesmon mutant replacing all seven cdc2 phosphorylation sites with Ala, and examined effects of expression of the caldesmon mutant on M-phase progression. We found that microinjection of mutant caldesmon effectively blocked early cell division ofXenopus embryos. Similar, though less effective, inhibition of cytokinesis was observed with Chinese hamster ovary (CHO) cells microinjected with 7th mutant. When mutant caldesmon was introduced into CHO cells either by protein microinjection or by inducible expression, delay of M-phase entry was observed. Finally, we found that 7th mutant inhibited the disassembly of microfilaments during mitosis. Wild-type caldesmon, on the other hand, was much less potent in producing these three effects. Because mutant caldesmon did not inhibit cyclin B/cdc2 kinase activity, our results suggest that alterations in microfilament assembly caused by caldesmon phosphorylation are important for M-phase progression.

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 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 Mutant MyoD Lacking Cdc2 Phosphorylation Sites Delays M-Phase Entry

2004 ◽  
Vol 24 (4) ◽  
pp. 1809-1821 ◽  
Author(s):  
Lionel A. J. Tintignac ◽  
Valentina Sirri ◽  
Marie Pierre Leibovitch ◽  
Yann Lécluse ◽  
Maria Castedo ◽  
...  
Keyword(s):  
Transcriptional Activation ◽  
Kinase Activity ◽  
Expression Patterns ◽  
Cyclin B ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Cdc2 Kinase ◽  
Specific Expression ◽  
M Phase ◽  
Phase Entry

ABSTRACT The transcription factors MyoD and Myf-5 control myoblast identity and differentiation. MyoD and Myf-5 manifest opposite cell cycle-specific expression patterns. Here, we provide evidence that MyoD plays a pivotal role at the G2/M transition by controlling the expression of p21Waf1/Cip1 (p21), which is believed to regulate cyclin B-Cdc2 kinase activity in G2. In growing myoblasts, MyoD reaccumulates during G2 concomitantly with p21 before entry into mitosis; MyoD is phosphorylated on Ser5 and Ser200 by cyclin B-Cdc2, resulting in a decrease of its stability and down-regulation of both MyoD and p21. Inducible expression of a nonphosphorylable MyoD A5/A200 enhances the MyoD interaction with the coactivator P/CAF, thereby stimulating the transcriptional activation of a luciferase reporter gene placed under the control of the p21 promoter. MyoD A5/A200 causes sustained p21 expression, which inhibits cyclin B-Cdc2 kinase activity in G2 and delays M-phase entry. This G2 arrest is not observed in p21−/− cells. These results show that in cycling cells MyoD functions as a transcriptional activator of p21 and that MyoD phosphorylation is required for G2/M transition.

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 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.

scholarly journals cdc25 is one of the MPM-2 antigens involved in the activation of maturation-promoting factor.

1994 ◽  
Vol 5 (2) ◽  
pp. 135-145 ◽  
Author(s):  
J Kuang ◽  
C L Ashorn ◽  
M Gonzalez-Kuyvenhoven ◽  
J E Penkala
Keyword(s):  
Monoclonal Antibody ◽  
Xenopus Oocytes ◽  
Cdc2 Kinase ◽  
Kinase Activation ◽  
Positive Regulator ◽  
Egg Extract ◽  
M Phase ◽  
Important Regulator ◽  
Maturation Promoting Factor

MPM-2 antigens, a discrete set of phosphoproteins that contain similar phosphoepitopes recognized by the monoclonal antibody MPM-2, are phosphorylated during M-phase induction. Our previous studies suggested that certain MPM-2 antigens are involved in the appearance of maturation-promoting factor (MPF) activity. Because the central mitotic regulator cdc2 kinase has been shown to exhibit MPF activity, we explored the possibility that certain MPM-2 antigens are regulators of cdc2 kinase. We found that MPM-2 binding of its antigens would inhibit the autoamplification of cdc2 kinase in Xenopus oocytes and interfere with cyclin-activation of cdc2 kinase in Xenopus interphase egg extract. Immunodepletion of MPM-2 antigens from cyclin-induced M-phase egg extract caused the inactivation of cdc2 kinase, which was accompanied by an inhibitory phosphorylation of p34cdc2 on Thr 14 and Tyr 15, indicating that at least one MPM-2 antigen is a positive regulator of p34cdc2 dephosphorylation. We then showed that cdc25 from M-phase arrested egg extract is an MPM-2 antigen. These results suggest that phosphorylation of the epitope recognized by MPM-2 may be a crucial event in the activation of cdc25 and that the kinase(s) that phosphorylates this MPM-2 epitope may be an important regulator of cdc2 kinase activation.

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.

An okadaic acid-sensitive phosphatase negatively controls the cyclin degradation pathway in amphibian eggs

1991 ◽  
Vol 11 (2) ◽  
pp. 1171-1175
Author(s):  
T Lorca ◽  
D Fesquet ◽  
F Zindy ◽  
F Le Bouffant ◽  
M Cerruti ◽  
...  
Keyword(s):  
Okadaic Acid ◽  
Protease Activity ◽  
Degradation Pathway ◽  
Meiotic Metaphase ◽  
Factor Activity ◽  
Cdc2 Kinase ◽  
Phosphatase Inhibitor ◽  
M Phase ◽  
Maturation Promoting Factor

Inhibition of okadaic acid-sensitive phosphatases released the cyclin degradation pathway from its inhibited state in extracts prepared from unfertilized Xenopus eggs arrested at the second meiotic metaphase. It also switched on cyclin protease activity in a permanent fashion in interphase extracts prepared from activated eggs. Even after cdc2 kinase inactivation, microinjection of okadaic acid-treated interphase extracts pushed G2-arrested recipient oocytes into the M phase, suggesting that the phosphatase inhibitor stabilizes the activity of an unidentified factor which shares in common with cdc2 kinase the maturation-promoting factor activity.

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