Cyclin A-cdc2 kinase does not trigger but delays cyclin degradation in interphase extracts of amphibian eggs

1992 ◽  
Vol 102 (1) ◽  
pp. 55-62 ◽  
Author(s):  
T. Lorca ◽  
J.C. Labbe ◽  
A. Devault ◽  
D. Fesquet ◽  
U. Strausfeld ◽  
...  
Keyword(s):  
Cyclin A ◽  
Inhibitory Effect ◽  
Degradation Pathway ◽  
Full Length ◽  
Cyclin B ◽  
Cdc2 Kinase ◽  
Turn On ◽  
Complete Condensation ◽  
Maturation Promoting Factor

Purified cyclin B-cdc2 kinase has been shown previously to trigger cyclin degradation in interphase frog extracts by initiating a cascade of reactions that includes cyclin ubiquitinylation and ends with proteolysis. However, cyclin A-cdc2 kinase was not assayed in these early experiments. Here we have shown that full-length recombinant human cyclin A failed to induce cyclin degradation when it was added to frog extracts free of cyclin B, although it formed an active kinase complex with Xenopus cdc2. A highly purified kinase complex containing a truncated human cyclin A and starfish cdc2 also failed to switch on the cyclin degradation pathway. In contrast, both recombinant cyclin B and highly purified cyclin B-cdc2 kinase readily triggered degradation of both cyclins B and A in frog extracts. Whilst free cyclin A had no inhibitory effect, cyclin A-cdc2 kinase delayed degradation of both cyclins A and B induced by cyclin B-cdc2 kinase. The finding that cyclin A-cdc2 kinase cannot turn on, and even delays, cyclin destruction may be essential to prevent premature inactivation of MPF (maturation-promoting factor) before complete condensation of chromosomes and formation of the metaphase spindle.

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.

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 Cyclin A potentiates maturation-promoting factor activation in the early Xenopus embryo via inhibition of the tyrosine kinase that phosphorylates cdc2.

1992 ◽  
Vol 118 (5) ◽  
pp. 1109-1120 ◽  
Author(s):  
A Devault ◽  
D Fesquet ◽  
J C Cavadore ◽  
A M Garrigues ◽  
J C Labbé ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Early Development ◽  
Kinase Activity ◽  
Active Form ◽  
Cyclin A ◽  
Cyclin B ◽  
Lag Phase ◽  
Inhibitory Mechanism ◽  
Cdc2 Kinase ◽  
Histone Kinase

We have produced human cyclin A in Escherichia coli and investigated how it generates H1 kistone kinase activity when added to cyclin-free extracts prepared from parthenogenetically activated Xenopus eggs. Cyclin A was found to form a major complex with cdc2, and to bind cdk2/Eg1 only poorly. No lag phase was detected between the time when cyclin A was added and the time when H1 histone kinase activity was produced in frog extracts, even in the presence of 2 mM vanadate, which blocks cdc25 activity. Essentially identical results were obtained using extracts prepared from starfish oocytes. We conclude that formation of an active cyclin A-cdc2 kinase during early development escapes an inhibitory mechanism that delays formation of an active cyclin B-cdc2 kinase. This inhibitory mechanism involves phosphorylation of cdc2 on tyrosine 15. Okadaic acid (OA) activated cyclin B-cdc2 kinase and strongly reduced tyrosine phosphorylation of cyclin B-associated cdc2, even in the presence of vanadate. 6-dimethylamino-purine, a reported inhibitor of serine-threonine kinases, suppressed OA-dependent activation of cyclin B-cdc2 complexes. This indicates that the kinase(s) which phosphorylate(s) cdc2 on inhibitory sites can be inactivated by a phosphorylation event, itself antagonized by an OA-sensitive, most likely type 2A phosphatase. We also found that cyclin B- or cyclin A-cdc2 kinases can induce or accelerate conversion of the cyclin B-cdc2 complex from an inactive into an active kinase. Cyclin B-associated cdc2 does not undergo detectable phosphorylation on tyrosine in egg extracts containing active cyclin A-cdc2 kinase, even in the presence of vanadate. We propose that the active cyclin A-cdc2 kinase generated without a lag phase from neo-synthesized cyclin A and cdc2 may cause a rapid switch in the equilibrium of cyclin B-cdc2 complexes to the tyrosine-dephosphorylated and active form of cdc2 during early development, owing to strong inhibition of the cdc2-specific tyrosine kinase(s). This may explain why early cell cycles are so rapid in many species.

Behavior of the Components of Maturation-Promoting Factor, cdc2 Kinase and Cyclin B, during Oocyte Maturation of Goldfish

1993 ◽  
Vol 160 (1) ◽  
pp. 99-107 ◽  
Author(s):  
Yoshinao Katsu ◽  
Masakane Yamashita ◽  
Hiroko Kajiura ◽  
Yoshitaka Nagahama
Keyword(s):  
Oocyte Maturation ◽  
Cyclin B ◽  
Cdc2 Kinase ◽  
Maturation Promoting Factor

scholarly journals In vitro fusion of endocytic vesicles is inhibited by cyclin A-cdc2 kinase.

1993 ◽  
Vol 4 (5) ◽  
pp. 541-553 ◽  
Author(s):  
P G Woodman ◽  
J P Adamczewski ◽  
T Hunt ◽  
G Warren
Keyword(s):  
Indirect Effect ◽  
Mammalian Cells ◽  
Cyclin A ◽  
Vesicle Fusion ◽  
Cyclin B ◽  
Endocytic Vesicle ◽  
Cdc2 Kinase ◽  
Histone Kinase ◽  
Endocytic Vesicles

Receptor-mediated endocytosis and recycling are inhibited in mitotic mammalian cells, and previous studies have shown that inhibition of endocytic vesicle fusion in vitro occurs via cyclin B-cdc2 kinase. To test for the ability of cyclin A-cdc2 kinase to inhibit endocytic vesicle fusion, we employed recombinant cyclin A proteins. Addition of cyclin A to interphase extracts activated a histone kinase and markedly reduced the efficiency of endocytic vesicle fusion. By a number of criteria, inhibition of fusion was shown to be due to the action of cyclin A, via the mitosis-specific cdc2 kinase, and not an indirect effect through cyclin B. Two-stage incubations were used to demonstrate that at least one target of cyclin A-cdc2 kinase is a cytosolic component of the fusion apparatus. Reconstitution experiments showed that this component was also modified in mitotic cytosols and was unaffected by N-ethyl maleimide treatment.

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.

scholarly journals 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.

scholarly journals MAP kinase does not inactivate, but rather prevents the cyclin degradation pathway from being turned on in Xenopus egg extracts

1996 ◽  
Vol 109 (1) ◽  
pp. 239-246 ◽  
Author(s):  
A. Abrieu ◽  
T. Lorca ◽  
J.C. Labbe ◽  
N. Morin ◽  
S. Keyse ◽  
...  
Keyword(s):  
Map Kinase ◽  
Degradation Pathway ◽  
Cdc2 Kinase ◽  
Vitro Assay ◽  
Kinase Activation ◽  
Egg Extracts ◽  
Dependent Protein ◽  
Xenopus Egg ◽  
Xenopus Egg Extracts

Unfertilized frog eggs arrest at the second meiotic metaphase, due to cytostatic activity of the c-mos proto-oncogene (CSF). MAP kinase has been proposed to mediate CSF activity in suppressing cyclin degradation. Using an in vitro assay to generate CSF activity, and recombinant CL 100 phosphatase to inactivate MAP kinase, we confirm that the c-mos proto-oncogene blocks cyclin degradation through MAP kinase activation. We further show that for MAP kinase to suppress cyclin degradation, it must be activated before cyclin B-cdc2 kinase has effectively promoted cyclin degradation. Thus MAP kinase does not inactivate, but rather prevents the cyclin degradation pathway from being turned on. Using a constitutively active mutant of Ca2+/calmodulin dependent protein kinase II, which mediates the effects of Ca2+ at fertilization, we further show that the kinase can activate cyclin degradation in the presence of both MPF and the c-mos proto-oncogene without inactivating MAP kinase.

Delayed cyclin A and B1 degradation in non-transformed mammalian cells

1995 ◽  
Vol 108 (7) ◽  
pp. 2599-2608 ◽  
Author(s):  
F. Girard ◽  
A. Fernandez ◽  
N. Lamb
Keyword(s):  
Mammalian Cells ◽  
Marine Invertebrates ◽  
Cyclin A ◽  
Cdc2 Kinase ◽  
Marked Contrast ◽  
Anaphase Onset ◽  
Significant Difference ◽  
Primary Fibroblasts ◽  
Transformed Cell Lines ◽  
Derivatives Of

Cyclins A and B are known to exhibit significant differences in their function, cellular distribution and timing of degradation at mitosis. On the basis of observations in marine invertebrates and Xenopus, it was proposed that cyclin destruction triggers cdc2 kinase inactivation and anaphase onset. However, this model has recently been questioned, both in Xenopus and in budding yeast. In this report, we present evidence for delayed degradation of both cyclins A and B1 in non-transformed mammalian cells. Indeed, by means of indirect immunofluorescence and confocal microscopy, we show that cyclins A and B1 are present up to anaphase in REF52, Hs68, human primary fibroblasts and NRK epithelial cells. In marked contrast, cyclin A is shown to be degraded within metaphase and cyclin B just at the transition to anaphase in HeLa and two transformed cell lines, derivatives of normal NRK and REF52. These results further support the notion that cyclin destruction might be not correlated with anaphase onset in normal cells and highlight a significant difference in the fate of mitotic cyclins between transformed and non-transformed cells.

The interplay between cyclin-B-Cdc2 kinase (MPF) and MAP kinase during maturation of oocytes

2001 ◽  
Vol 114 (2) ◽  
pp. 257-267 ◽  
Author(s):  
A. Abrieu ◽  
M. Doree ◽  
D. Fisher
Keyword(s):  
Cell Cycle ◽  
Map Kinase ◽  
Cell Cycle Progression ◽  
Mitotic Cell ◽  
Model Systems ◽  
Cyclin B ◽  
Cdc2 Kinase ◽  
Meiotic Chromosomes ◽  
Map Kinase Cascade ◽  
Kinase Cascade

Throughout oocyte maturation, and subsequently during the first mitotic cell cycle, the MAP kinase cascade and cyclin-B-Cdc2 kinase are associated with the control of cell cycle progression. Many roles have been directly or indirectly attributed to MAP kinase and its influence on cyclin-B-Cdc2 kinase in different model systems; yet a principle theme does not emerge from the published literature, some of which is apparently contradictory. Interplay between these two kinases affects the major events of meiotic maturation throughout the animal kingdom, including the suppression of DNA replication, the segregation of meiotic chromosomes, and the prevention of parthenogenetic activation. Central to many of these events appears to be the control by MAP kinase of cyclin translation and degradation.

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