Inactivation of the checkpoint kinase Cds1 is dependent on cyclin B-Cdc2 kinase activation at the meiotic G2/M-phase transition in Xenopus oocytes

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.

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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

Journal of Cell Science ◽

10.1242/jcs.111.12.1751 ◽

1998 ◽

Vol 111 (12) ◽

pp. 1751-1757 ◽

Cited By ~ 16

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.

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Plk is an M-phase-specific protein kinase and interacts with a kinesin-like protein, CHO1/MKLP-1.

Molecular and Cellular Biology ◽

10.1128/mcb.15.12.7143 ◽

1995 ◽

Vol 15 (12) ◽

pp. 7143-7151 ◽

Cited By ~ 181

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.

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

Molecular and Cellular Biology ◽

10.1128/mcb.11.8.3860-3867.1991 ◽

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.

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Cyclin B/cdc2 Induces c-Mos Stability by Direct Phosphorylation in Xenopus Oocytes

Molecular Biology of the Cell ◽

10.1091/mbc.12.9.2660 ◽

2001 ◽

Vol 12 (9) ◽

pp. 2660-2671 ◽

Cited By ~ 39

Author(s):

Anna Castro ◽

Marion Peter ◽

Laura Magnaghi-Jaulin ◽

Suzanne Vigneron ◽

Simon Galas ◽

...

Keyword(s):

Xenopus Oocytes ◽

Phosphorylation Site ◽

Cyclin B ◽

Mobility Shift ◽

Cdc2 Kinase ◽

Phosphopeptide Analysis ◽

Vertebrate Eggs ◽

The One

The c-Mos proto-oncogene product plays an essential role during meiotic divisions in vertebrate eggs. In Xenopus, it is required for progression of oocyte maturation and meiotic arrest of unfertilized eggs. Its degradation after fertilization is essential to early embryogenesis. In this study we investigated the mechanisms involved in c-Mos degradation. We present in vivo evidence for ubiquitin-dependent degradation of c-Mos in activated eggs. We found that c-Mos degradation is not directly dependent on the anaphase-promoting factor activator Fizzy/cdc20 but requires cyclin degradation. We demonstrate that cyclin B/cdc2 controls in vivo c-Mos phosphorylation and stabilization. Moreover, we show that cyclin B/cdc2 is capable of directly phosphorylating c-Mos in vitro, inducing a similar mobility shift to the one observed in vivo. Tryptic phosphopeptide analysis revealed a practically identical in vivo and in vitro phosphopeptide map and allowed identification of serine-3 as the largely preferential phosphorylation site as previously described ( Freeman et al., 1992 ). Altogether, these results demonstrate that, in vivo, stability of c-Mos is directly regulated by cyclin B/cdc2 kinase activity.

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

Molecular and Cellular Biology ◽

10.1128/mcb.11.8.3860 ◽

1991 ◽

Vol 11 (8) ◽

pp. 3860-3867 ◽

Cited By ~ 48

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

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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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Active cyclin B-cdc2 kinase does not inhibit DNA replication and cannot drive prematurely fertilized sea urchin eggs into mitosis

Journal of Cell Science ◽

10.1242/jcs.108.7.2693 ◽

1995 ◽

Vol 108 (7) ◽

pp. 2693-2703 ◽

Cited By ~ 8

Author(s):

A.M. Geneviere-Garrigues ◽

A. Barakat ◽

M. Doree ◽

J.L. Moreau ◽

A. Picard

Keyword(s):

Dna Replication ◽

Sea Urchin ◽

S Phase ◽

Cyclin B ◽

G1 Arrest ◽

Cdc2 Kinase ◽

Kinase Activation ◽

M Phase ◽

Set Up ◽

The One

Feedback mechanisms preventing M phase occurrence before S phase completion are assumed to depend on inhibition of cyclin B-cdc2 kinase activation by unreplicated DNA. In sea urchin, fertilization stimulates protein synthesis and releases eggs from G1 arrest. We found that in the one-cell sea urchin embryo cyclin B-cdc2 kinase undergoes partial activation before S phase, reaching in S phase a level that is sufficient for G2-M phase transition. S phase entry is not inhibited by this level of cyclin B-dependent kinase activity. Inhibition of DNA replication by aphidicolin suppresses nuclear envelope breakdown, yet it does not prevent the microtubule array from being converted from its interphasic to its mitotic state. Moreover, mitotic cytoplasmic events occur at the same time in control and aphidicolin-treated embryos. Thus unreplicated DNA only prevents mitotic nuclear, not cytoplasmic, events from occurring prematurely. These results together show that the inhibition of cyclin B-cdc2 kinase activation is probably not the only mechanism that prevents mitotic nuclear events from occurring as long as DNA replication has not been completed. In contrast, cytoplasmic mitotic events seem to be controlled by a timing mechanism independent of DNA replication, set up at fertilization, that prevents premature opening of a window for mitotic events.

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Mitogen-activated protein kinase activation down-regulates a mechanism that inactivates cyclin B-cdc2 kinase in G2-arrested oocytes.

Molecular Biology of the Cell ◽

10.1091/mbc.8.2.249 ◽

1997 ◽

Vol 8 (2) ◽

pp. 249-261 ◽

Cited By ~ 25

Author(s):

A Abrieu ◽

M Dorée ◽

A Picard

Keyword(s):

Map Kinase ◽

Xenopus Oocytes ◽

Cyclin B ◽

Mitogen Activated Protein Kinase ◽

Cdc2 Kinase ◽

Hormonal Stimulation ◽

Germinal Vesicle Breakdown ◽

Kinase Activation ◽

Map Kinase Phosphatase ◽

Mitogen Activated Protein

The G2 arrest of oocytes from frogs, clams, and starfish requires that preformed cyclin B-cdc2 complexes [prematuration-promoting factor (MPF)] be kept in an inactive form that is largely due to inhibitory phosphorylation of this pre-MPF. We have investigated the role of mitogen-activated protein (MAP) kinase in the activation of this pre-MPF. The cytoplasm of both frog and starfish oocytes contains an activity that can rapidly inactivate injected MPF. When the MAP kinase of G2-arrested starfish or Xenopus oocytes was prematurely activated by microinjection of c-mos or Ste-11 delta N fusion proteins, the rate and extent of MPF inactivation was much reduced. Both effects were suppressed by expression of the specific MAP kinase phosphatase Pyst 1. These results show that MAP kinase down-regulates a mechanism that inactivates cyclin B-cdc2 kinase in Xenopus oocytes. In starfish oocytes, however, MAP kinase activation occurs only after germinal vesicle breakdown, much after MPF activation. In this case, down-regulation of the cyclin B-cdc2 inhibiting pathway is a sensitive response to hormonal stimulation that does not require MAP kinase activation.

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Overexpression of IRS-4 Correlates with Procaspase 3 Levels in Tumoural Tissue of Patients with Colorectal Cancer

Journal of Oncology ◽

10.1155/2018/3812581 ◽

2018 ◽

Vol 2018 ◽

pp. 1-14

Author(s):

Patricia Sanmartín-Salinas ◽

Luis G. Guijarro

Keyword(s):

Colorectal Cancer ◽

Caspase 3 ◽

Cultured Cells ◽

Cyclin Dependent Kinase ◽

Kinase Activation ◽

Tnm System ◽

Protein Levels ◽

Receptor Signalling ◽

T Values

We reported that insulin receptor substrate 4 (IRS-4) levels increased in tissue from colorectal cancer (CRC) patients and promoted retinoblastoma-cyclin-dependent kinase activation. The aim of the present study was to evaluate the effect of IRS-4 on IGF-1 receptor pathway and its impact on procaspase 3 and PARP expression in RKO and HepG2 cancer cell lines. The results obtained in vitro were compared with those obtained from biopsies of patients with CRC (n = 18), tubulovillous adenomas (TA) (n = 2) and in matched adjacent normal colorectal (MANC) tissue (n = 20). IRS-4 overexpression in cultured cells induced the overactivation of IGF-1/BRK/AKT/GSK-3/β-catenin/cyclin D1 pathways, which led to increased expression of procaspase 3 and PARP protein levels. Studies carried out on CRC and TA tissues revealed the overactivation of the IGF-1 receptor signalling pathway, as well as the overexpression of procaspase 3 and PARP in tumoural tissue with respect to MANC tissue. The upregulation of IRS-4 in tumoural samples correlated significantly with the increase in pIGF-1 receptor (Tyr 1165/1166) (r = 0.84; p < 0.0001), procaspase 3 (r = 0. 77; p < 0. 0005) and PARP (r = 0. 89; p < 0. 0005). Similarly, we observed an increase in the proteolysis of procaspase 3 in tumoural tissue with respect to MANC tissue, which correlated significantly with the degradation of PARP (r = 0.86; p < 0.0001), p53 (r = 0.84; p < 0.0001), and GSK-3 (r = 0.78; p < 0.0001). The stratification of patient samples using the TNM system revealed that procaspase 3 and caspase 3 increased gradually with T values, which suggests their involvement in the size and local invasion of primary tumours. Taken together, our findings suggest that IRS-4 overexpression promotes the activation of the IGF-1 receptor pathway, which leads to the increase in procaspase 3 levels in CRC.

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