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.

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 Regulation of Cdc2/Cyclin B Activation in Xenopus Egg Extracts via Inhibitory Phosphorylation of Cdc25C Phosphatase by Ca2+/Calmodium-dependent Kinase II

2003 ◽  
Vol 14 (10) ◽  
pp. 4003-4014 ◽  
Author(s):  
James R. A. Hutchins ◽  
Dina Dikovskaya ◽  
Paul R. Clarke
Keyword(s):  
Cell Cycle ◽  
Protein Kinase ◽  
Kinase Activity ◽  
Embryonic Cell ◽  
Cyclin B ◽  
Checkpoint Kinases ◽  
M Phase ◽  
Egg Extracts ◽  
Inhibitory Site

Activation of Cdc2/cyclin B kinase and entry into mitosis requires dephosphorylation of inhibitory sites on Cdc2 by Cdc25 phosphatase. In vertebrates, Cdc25C is inhibited by phosphorylation at a single site targeted by the checkpoint kinases Chk1 and Cds1/Chk2 in response to DNA damage or replication arrest. In Xenopus early embryos, the inhibitory site on Cdc25C (S287) is also phosphorylated by a distinct protein kinase that may determine the intrinsic timing of the cell cycle. We show that S287-kinase activity is repressed in extracts of unfertilized Xenopus eggs arrested in M phase but is rapidly stimulated upon release into interphase by addition of Ca2+, which mimics fertilization. S287-kinase activity is not dependent on cyclin B degradation or inactivation of Cdc2/cyclin B kinase, indicating a direct mechanism of activation by Ca2+. Indeed, inhibitor studies identify the predominant S287-kinase as Ca2+/calmodulin-dependent protein kinase II (CaMKII). CaMKII phosphorylates Cdc25C efficiently on S287 in vitro and, like Chk1, is inhibited by 7-hydroxystaurosporine (UCN-01) and debromohymenialdisine, compounds that abrogate G2 arrest in somatic cells. CaMKII delays Cdc2/cyclin B activation via phosphorylation of Cdc25C at S287 in egg extracts, indicating that this pathway regulates the timing of mitosis during the early embryonic cell cycle.

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.

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.

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 Plk is a functional homolog of Saccharomyces cerevisiae Cdc5, and elevated Plk activity induces multiple septation structures.

1997 ◽  
Vol 17 (6) ◽  
pp. 3408-3417 ◽  
Author(s):  
K S Lee ◽  
R L Erikson
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Cell Division ◽  
Specific Activity ◽  
Ectopic Expression ◽  
Sf9 Cells ◽  
Wild Type ◽  
C Terminus ◽  
M Phase

Plk is a mammalian serine/threonine protein kinase whose activity peaks at the onset of M phase. It is closely related to other mammalian kinases, Snk, Fnk, and Prk, as well as to Xenopus laevis Plx1, Drosophila melanogaster polo, Schizosaccharomyces pombe Plo1, and Saccharomyces cerevisiae Cdc5. The M phase of the cell cycle is a highly coordinated process which insures the equipartition of genetic and cellular materials during cell division. To enable understanding of the function of Plk during M phase progression, various Plk mutants were generated and expressed in Sf9 cells and budding yeast. In vitro kinase assays with Plk immunoprecipitates prepared from Sf9 cells indicate that Glu206 and Thr210 play equally important roles for Plk activity and that replacement of Thr210 with a negatively charged residue elevates Plk specific activity. Ectopic expression of wild-type Plk (Plk WT) complements the cell division defect associated with the cdc5-1 mutation in S. cerevisiae. The degree of complementation correlates closely with the Plk activity measured in vitro, as it is enhanced by a mutationally activated Plk, T210D, but is not observed with the inactive forms K82M, D194N, and D194R. In a CDC5 wild-type background, expression of Plk WT or T210D, but not of inactive forms, induced a sharp accumulation of cells in G1. Consistent with elevated Plk activity, this phenomenon was enhanced by the C-terminally deleted forms WT deltaC and T210D deltaC. Expression of T210D also induced a class of cells with unusually elongated buds which developed multiple septal structures. This was not observed with the C-terminally deleted form T210D deltaC, however. It appears that the C terminus of Plk is not required for the observed cell cycle influence but may be important for polarized cell growth and septal structure formation.

scholarly journals Activation of p34cdc2 kinase by cyclin A.

1991 ◽  
Vol 113 (3) ◽  
pp. 507-514 ◽  
Author(s):  
L M Roy ◽  
K I Swenson ◽  
D H Walker ◽  
B G Gabrielli ◽  
R S Li ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Expression System ◽  
Kinetic Studies ◽  
Baculovirus Expression System ◽  
Cyclin A ◽  
Maximal Activity ◽  
Cdc2 Kinase ◽  
Kinase Activation ◽  
Egg Extracts ◽  
Maximal Induction

Functional clam cyclin A and B proteins have been produced using a baculovirus expression system. Both cyclin A and B can induce meiosis I and meiosis II in Xenopus in the absence of protein synthesis. Half-maximal induction occurs at 50 nM for cyclin A and 250 nM for cyclin B. Addition of 25 nM cyclin A to activated Xenopus egg extracts arrested in the cell cycle by treatment with RNase or emetine activates cdc2 kinase to the normal metaphase level and stimulates one oscillatory cell cycle. High levels of cyclin A cause marked hyperactivation of cdc2 kinase and a stable arrest at the metaphase point in the cell cycle. Kinetic studies demonstrate the concentration of cyclin A added does not affect the 10 min lag period required for kinase activation or the timing of maximal activity, but does control the rate of deactivation of cdc2 kinase during exit from mitosis. In addition, exogenous clam cyclin A inhibits the degradation of both A- and B-type endogenous Xenopus cyclins. These results define a system for investigating the biochemistry and regulation of cdc2 kinase activation by cyclin A.

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 Activation of the p42 Mitogen-activated Protein Kinase Pathway Inhibits Cdc2 Activation and Entry into M-Phase in CyclingXenopus Egg Extracts

1998 ◽  
Vol 9 (2) ◽  
pp. 451-467 ◽  
Author(s):  
John C. Bitangcol ◽  
Andrew S.-S. Chau ◽  
Ellamae Stadnick ◽  
Manfred J. Lohka ◽  
Bryan Dicken ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Protein Kinase ◽  
Specific Inhibitor ◽  
Mapk Signaling ◽  
Cyclin B ◽  
Mitogen Activated Protein Kinase ◽  
M Phase ◽  
Egg Extracts ◽  
Mitogen Activated Protein ◽  
Xenopus Egg Extracts

We have added constitutively active MAP kinase/ERK kinase (MEK), an activator of the mitogen-activated protein kinase (MAPK) signaling pathway, to cycling Xenopus egg extracts at various times during the cell cycle. p42MAPK activation during entry into M-phase arrested the cell cycle in metaphase, as has been shown previously. Unexpectedly, p42MAPK activation during interphase inhibited entry into M-phase. In these interphase-arrested extracts, H1 kinase activity remained low, Cdc2 was tyrosine phosphorylated, and nuclei continued to enlarge. The interphase arrest was overcome by recombinant cyclin B. In other experiments, p42MAPK activation by MEK or by Mos inhibited Cdc2 activation by cyclin B. PD098059, a specific inhibitor of MEK, blocked the effects of MEK(QP) and Mos. Mos-induced activation of p42MAPK did not inhibit DNA replication. These results indicate that, in addition to the established role of p42MAPK activation in M-phase arrest, the inappropriate activation of p42MAPK during interphase prevents normal entry into M-phase.

Concerted Roles of Cyclin A, cdc25+ Mitotic Inducer, and Type 2A Phosphatase in Activating the Cyclin B/cdc2 Protein Kinase at the G2/M Phase Transition

1991 ◽  
Vol 56 (0) ◽  
pp. 503-513 ◽  
Author(s):  
A. Devault ◽  
J.-C. Cavadore ◽  
D. Fesquet ◽  
J.-C. Labbe ◽  
T. Lorca ◽  
...  
Keyword(s):  
Phase Transition ◽  
Protein Kinase ◽  
Cyclin A ◽  
Cyclin B ◽  
M Phase
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