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

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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Involvement of protein phosphatases 1 and 2A in the control of M phase-promoting factor activity in starfish.

The Journal of Cell Biology ◽

10.1083/jcb.109.6.3347 ◽

1989 ◽

Vol 109 (6) ◽

pp. 3347-3354 ◽

Cited By ~ 78

Author(s):

A Picard ◽

J P Capony ◽

D L Brautigan ◽

M Dorée

Keyword(s):

Okadaic Acid ◽

Protein Phosphatases ◽

Germinal Vesicle ◽

Histone H1 ◽

Specific Inhibition ◽

Factor Activity ◽

Microtubule Network ◽

Germinal Vesicle Breakdown ◽

M Phase

Specific inhibition of types 1 and 2A protein phosphatases by microinjection of okadaic acid (OA) into starfish oocytes induced germinal vesicle breakdown and activation of M phase-promoting factor (MPF) and histone H1 kinase. The effects were evident in immature oocytes arrested at first meiotic prophase as well as in fully mature oocytes arrested at the pronucleus stage. In addition, MPF and histone H1 kinase were stabilized for several hours and protected from inactivation by inhibition of type 1 protein phosphatases with either OA or specific anti-phosphatase antibodies. Microinjection of okadaic acid was associated with unusual changes of the microtubule network, including the disappearance of spindles and extension of the cytoplasmic array of microtubules. MPF activation after OA injection was associated with dephosphorylation of phosphothreonine and phosphoserine residues in cdc2, showing that neither type 1 nor 2A protein phosphatases catalyzes these dephosphorylations. The effects of OA on MPF activation and inactivation appeared to involve the cyclin subunit. OA did not induce MPF activation in the absence of protein synthesis and it prevented degradation of cyclin. Therefore protein phosphatases types 1 and 2A appear to be involved in activation and inactivation of MPF involving mechanisms that operate after cyclin synthesis and before its degradation.

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Induction of M-phase entry of prophase-blocked mouse oocytes through microinjection of okadaic acid, a specific phosphatase inhibitor

Experimental Cell Research ◽

10.1016/0014-4827(91)90159-r ◽

1991 ◽

Vol 192 (1) ◽

pp. 75-81 ◽

Cited By ~ 59

Author(s):

Anne-Claude Gavin ◽

Yasumasa Tsukitani ◽

Sabine Schorderet-Slatkine

Keyword(s):

Okadaic Acid ◽

Mouse Oocytes ◽

Phosphatase Inhibitor ◽

M Phase ◽

Phase Entry

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

Journal of Cell Science ◽

10.1242/jcs.102.1.55 ◽

1992 ◽

Vol 102 (1) ◽

pp. 55-62 ◽

Cited By ~ 3

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.

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Okadaic acid mimics a nuclear component required for cyclin B-cdc2 kinase microinjection to drive starfish oocytes into M phase.

The Journal of Cell Biology ◽

10.1083/jcb.115.2.337 ◽

1991 ◽

Vol 115 (2) ◽

pp. 337-344 ◽

Cited By ~ 39

Author(s):

A Picard ◽

J C Labbé ◽

H Barakat ◽

J C Cavadore ◽

M Dorée

Keyword(s):

Okadaic Acid ◽

Xenopus Oocytes ◽

Cyclin B ◽

G2 Arrest ◽

Cdc2 Kinase ◽

Nuclear Envelope Breakdown ◽

M Phase ◽

Nuclear Component ◽

Amplification Loop ◽

Do So

G2-arrested oocytes contain cdc2 kinase as an inactive cyclin B-cdc2 complex. When a small amount of highly purified and active cdc2 kinase, prepared from starfish oocytes at first meiotic metaphase, is microinjected into Xenopus oocytes, it induces activation of the inactive endogenous complex and, as a consequence, drives the recipient oocytes into M phase. In contrast, the microinjected kinase undergoes rapid inactivation in starfish oocytes, which remain arrested at G2. Endogenous cdc2 kinase becomes activated in both nucleated and enucleated starfish oocytes injected with cytoplasm taken from maturing oocytes at the time of nuclear envelope breakdown, but only cytoplasm taken from nucleated oocytes becomes able thereafter to release second recipient oocytes from G2 arrest, and thus contains M phase-promoting factor (MPF) activity. Both nucleated and enucleated starfish oocytes produce MPF activity when type 2A phosphatase is blocked by okadaic acid. If type 2A phosphatase is only partially inhibited, neither nucleated nor enucleated oocytes produce MPF activity, although both do so if purified cdc2 kinase is subsequently injected as a primer to activate the endogenous kinase. The nucleus of starfish oocytes contains an inhibitor of type 2A phosphatase, but neither active nor inactive cdc2 kinase. Microinjection of the content of a nucleus into the cytoplasm of G2-arrested starfish oocytes activates endogenous cdc2 kinase, produces MPF activity, and drives the recipient oocytes into M phase. Together, these results show that the MPF amplification loop is controlled, both positively and negatively, by cdc2 kinase and type 2A phosphatase, respectively. Activation of the MPF amplification loop in starfish requires a nuclear component to inhibit type 2A phosphatase in cytoplasm.

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

Molecular Biology of the Cell ◽

10.1091/mbc.5.2.135 ◽

1994 ◽

Vol 5 (2) ◽

pp. 135-145 ◽

Cited By ~ 53

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.

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Mitotic cytosol inhibits invagination of coated pits in broken mitotic cells.

The Journal of Cell Biology ◽

10.1083/jcb.114.6.1159 ◽

1991 ◽

Vol 114 (6) ◽

pp. 1159-1166 ◽

Cited By ~ 39

Author(s):

M Pypaert ◽

D Mundy ◽

E Souter ◽

J C Labbé ◽

G Warren

Keyword(s):

Liquid Nitrogen ◽

Okadaic Acid ◽

Hela Cells ◽

Mammalian Cells ◽

A431 Cells ◽

Coated Pits ◽

Cdc2 Kinase ◽

Phosphatase Inhibitor ◽

Interphase Cells ◽

Mitotic Cells

Receptor-mediated endocytosis is inhibited during mitosis in mammalian cells and earlier work on A431 cells suggested that one of the sites inhibited was the invagination of coated pits (Pypaert, M., J. M. Lucocq, and G. Warren. 1987. Eur. J. Cell Biol. 45: 23-29). To explore this inhibition further, we have reproduced it in broken HeLa cells. Mitotic or interphase cells were broken by freeze-thawing in liquid nitrogen and warmed in the presence of mitotic or interphase cytosol. Using a morphological assay, we found invagination to be inhibited only when mitotic cells were incubated in mitotic cytosol. This inhibition was reversed by diluting the cytosol during the incubation. Reversal was sensitive to okadaic acid, a potent phosphatase inhibitor, showing that phosphorylation was involved in the inhibition of invagination. This was confirmed using purified cdc2 kinase which alone could partially substitute for mitotic cytosol.

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

The Journal of Cell Biology ◽

10.1083/jcb.132.1.125 ◽

1996 ◽

Vol 132 (1) ◽

pp. 125-135 ◽

Cited By ~ 41

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.

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