scholarly journals Cell cycle regulation of p34cdc2 kinase activity in Physarum polycephalum

1990 ◽  
Vol 96 (4) ◽  
pp. 683-689
Author(s):  
B. Ducommun ◽  
Y. Tollon ◽  
M. Gares ◽  
D. Beach ◽  
M. Wright
Keyword(s):  
Cell Cycle ◽  
Dna Synthesis ◽  
Physarum Polycephalum ◽  
Kinase Activity ◽  
Histone H1 ◽  
Early Event ◽  
Binding Property ◽  
Kinase Activation ◽  
Regulatory Pathways ◽  
Mitotic Kinase

The regulation of the mitotic histone H1 kinase activity has been analyzed during the naturally synchronous cell cycle of Physarum polycephalum plasmodia. The universal binding property of the p13suc1 Schizosaccharomyces pombe gene product was used to precipitate and assay the cdc2 histone H1 kinase activity. The kinase activity peaks at the beginning of metaphase and its decline, which requires protein synthesis, appears to be an early event during the metaphase process. Microtubular poisons, temperature shifts and DNA synthesis inhibitors were used to perturb cell cycle regulatory pathways and characterize their effects on cdc2 kinase activation. Our results suggest that the full activation of the mitotic kinase requires at least two successive triggering signals involving microtubular components and DNA synthesis.

scholarly journals Fission Yeast Rad26 Is a Regulatory Subunit of the Rad3 Checkpoint Kinase

2002 ◽  
Vol 13 (2) ◽  
pp. 480-492 ◽  
Author(s):  
Tom D. Wolkow ◽  
Tamar Enoch
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Arrest ◽  
Fission Yeast ◽  
Complex Analysis ◽  
Kinase Activity ◽  
Regulatory Subunit ◽  
Kinase Activation ◽  
Checkpoint Proteins ◽  
Cycle Arrest ◽  
Phosphoinositide 3 Kinase

Fission yeast Rad3 is a member of a family of phosphoinositide 3-kinase -related kinases required for the maintenance of genomic stability in all eukaryotic cells. In fission yeast, Rad3 regulates the cell cycle arrest and recovery activities associated with the G2/M checkpoint. We have developed an assay that directly measures Rad3 kinase activity in cells expressing physiological levels of the protein. Using the assay, we demonstrate directly that Rad3 kinase activity is stimulated by checkpoint signals. Of the five other G2/M checkpoint proteins (Hus1, Rad1, Rad9, Rad17, and Rad26), only Rad26 was required for Rad3 kinase activity. Because Rad26 has previously been shown to interact constitutively with Rad3, our results demonstrate that Rad26 is a regulatory subunit, and Rad3 is the catalytic subunit, of the Rad3/Rad26 kinase complex. Analysis of Rad26/Rad3 kinase activation in rad26.T12, a mutant that is proficient for cell cycle arrest, but defective in recovery, suggests that these two responses to checkpoint signals require quantitatively different levels of kinase activity from the Rad3/Rad26 complex.

scholarly journals Full activation of p34CDC28 histone H1 kinase activity is unable to promote entry into mitosis in checkpoint-arrested cells of the yeast Saccharomyces cerevisiae.

1993 ◽  
Vol 13 (6) ◽  
pp. 3744-3755 ◽  
Author(s):  
C S Stueland ◽  
D J Lew ◽  
M J Cismowski ◽  
S I Reed
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Kinase Activity ◽  
Budding Yeast ◽  
Regulatory Protein ◽  
Histone H1 ◽  
Yeast Saccharomyces Cerevisiae ◽  
Kinase Activation ◽  
Egg Extracts ◽  
Xenopus Egg Extracts ◽  
Yeast Homolog

In most cells, mitosis is dependent upon completion of DNA replication. The feedback mechanisms that prevent entry into mitosis by cells with damaged or incompletely replicated DNA have been termed checkpoint controls. Studies with the fission yeast Schizosaccharomyces pombe and Xenopus egg extracts have shown that checkpoint controls prevent activation of the master regulatory protein kinase, p34cdc2, that normally triggers entry into mitosis. This is achieved through inhibitory phosphorylation of the Tyr-15 residue of p34cdc2. However, studies with the budding yeast Saccharomyces cerevisiae have shown that phosphorylation of this residue is not essential for checkpoint controls to prevent mitosis. We have investigated the basis for checkpoint controls in this organism and show that these controls can prevent entry into mitosis even in cells which have fully activated the cyclin B (Clb)-associated forms of the budding yeast homolog of p34cdc2, p34CDC28, as assayed by histone H1 kinase activity. However, the active complexes in checkpoint-arrested cells are smaller than those in cycling cells, suggesting that assembly of mitosis-inducing complexes requires additional steps following histone H1 kinase activation.

Activation of p34cdc2 protein kinase activity in meiotic and mitotic cell cycles in mouse oocytes and embryos

Development ◽  
1991 ◽  
Vol 113 (3) ◽  
pp. 789-795 ◽  
Author(s):  
T. Choi ◽  
F. Aoki ◽  
M. Mori ◽  
M. Yamashita ◽  
Y. Nagahama ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Protein Kinase ◽  
Kinase Activity ◽  
Polar Body ◽  
Mitotic Cell ◽  
Histone H1 ◽  
Protein Kinase Activity ◽  
Mitotic Cell Cycle ◽  
Mouse Oocytes ◽  
First Polar Body

p34cdc2 protein kinase is a universal regulator of M-phase in eukaryotic cell cycle. To investigate the regulation of meiotic and mitotic cell cycle in mammals, we examined the changes in phosphorylation states of p34cdc2 and its histone H1 kinase activity in mouse oocytes and embryos. We showed that p34cdc2 has three different migrating bands (referred to as upper, middle and lower bands) on SDS-PAGE followed by immunoblotting with anti-PSTAIR antibody, and that the upper and middle bands are phosphorylated forms since these two bands shifted to the lower one by alkaline phosphatase treatment. In meiotic cell cycle, only germinal vesicle (GV) stage oocytes had the three forms. The phosphorylated forms decreased gradually in oocytes up to 2 h after isolation from follicles, and thereafter the phosphorylation states did not change significantly until metaphase II. However, the histone H1 kinase activity oscillated, being activated at the first and second metaphase in meiosis and inactivated at the time of the first polar body extrusion. These results suggest that changes in phosphorylation states of p34cdc2 triggered its activation at the first metaphase, but not inactivation and reactivation at the first and second metaphase, respectively. In mitotic cell cycle, phosphorylated forms appeared at 4 h after insemination, increased greatly just before metaphase, and were dephosphorylated in metaphase. Histone H1 kinase activity was high only at metaphase. This kinase activation is probably triggered by dephosphorylation of p34cdc2.

Microtubule and chromatin behavior follow MAP kinase activity but not MPF activity during meiosis in mouse oocytes

Development ◽  
1994 ◽  
Vol 120 (4) ◽  
pp. 1017-1025 ◽  
Author(s):  
M.H. Verlhac ◽  
J.Z. Kubiak ◽  
H.J. Clarke ◽  
B. Maro
Keyword(s):  
Cell Cycle ◽  
Myelin Basic Protein ◽  
Map Kinase ◽  
Kinase Activity ◽  
Basic Protein ◽  
Chromatin Condensation ◽  
Meiotic Maturation ◽  
Protein Kinase Activity ◽  
Microtubule Organization ◽  
Kinase Activation

Oocyte meiotic maturation is triggered by different stimuli (hormones, unknown signals through cell interactions) in different species. These stimuli indirectly lead to the activation of a major cell cycle regulating activity, the maturation promoting factor (MPF). Other factors, such as the product of the proto-oncogene c-mos or enzymes of the MAP kinase family, are also involved in the process of maturation. MAP kinase activation occurs during meiotic maturation in oocytes from different species with different kinetics. The relationships between MPF activation and MAP kinase activation have been well studied in species such as clam and Xenopus. In this paper, we study the precise timing of MAP kinase activation (as measured by phosphorylation of exogenous myelin basic protein and shifts in mobility of ERK 1 and ERK 2) versus MPF activation (as measured by phosphorylation of exogenous histone H1) during mouse oocyte maturation and, in parallel, morphological events such as changes in microtubule organization and chromatin condensation. We observed that MAP kinase activation was delayed after MPF activation and that this activity persisted throughout maturation whereas MPF activity dropped between the two meiotic metaphases. After parthenogenetic activation of ovulated eggs, MAP kinase inactivation was very slow compared to MPF inactivation. During the first mitotic cell cycle, a rise in myelin basic protein kinase activity at M-phase was observed but it was not related to MAP kinase activation. Furthermore, microtubules and chromatin remained in a metaphase-like state during the complete period of maturation (including the period between the two meiotic metaphases) and a few hours after activation.(ABSTRACT TRUNCATED AT 250 WORDS)

Some evidence for replication-transcription coupling in Physarum polycephalum

1975 ◽  
Vol 18 (1) ◽  
pp. 27-39
Author(s):  
H. Fouquet ◽  
R. Bohme ◽  
R. Wick ◽  
H.W. Sauer ◽  
K. Scheller
Keyword(s):  
Cell Cycle ◽  
Dna Synthesis ◽  
Dna Sequences ◽  
Physarum Polycephalum ◽  
Rna Synthesis ◽  
Selective Inhibition ◽  
S Phase ◽  
Dna Molecules ◽  
Uridine Incorporation ◽  
G2 Phase

Hydroxyurea, at concentrations of 40–60 mM, selectively and effectively blocked incorporation of thymidine into DNA. Inhibition occurred within 5–10 min of application of the agent when DNA synthesis was in progress, while the onset of replication at the beginning of S-phase and DNA synthesis in G2 phase were not affected. Uridine incorporation into TCA-precipitable material, in the presence of hydroxyurea, was significantly (up to 70%) inhibited in early S-phase of the cell cycle. Selective inhibition of RNA synthesis was confirmed for RNA separated into rRNA-rich and poly(A)-rich RNA fractions and analysed by the 2 kinds of DNA-RNA hybridization reactions. Uridine incorporation into poly (A) RNA was also inhibited under conditions where cycloheximide prevented maturation of nascent DNA molecules in early S-phase. We assume that chromatin which is replicating early DNA sequences may be a more competent template for transcription.

scholarly journals Autophosphorylation of Polo-like Kinase 4 and Its Role in Centriole Duplication

2010 ◽  
Vol 21 (4) ◽  
pp. 547-561 ◽  
Author(s):  
James E. Sillibourne ◽  
Frederik Tack ◽  
Nele Vloemans ◽  
An Boeckx ◽  
Sathiesan Thambirajah ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Kinase Activity ◽  
Proteasome Inhibition ◽  
Centrosome Duplication ◽  
Active Fraction ◽  
Kinase Activation ◽  
Centriole Duplication ◽  
M Phase ◽  
Mother Centriole ◽  
Daughter Centriole

Centrosome duplication occurs once every cell cycle in a strictly controlled manner. Polo-like kinase 4 (PLK4) is a key regulator of this process whose kinase activity is essential for centriole duplication. Here, we show that PLK4 autophosphorylation of serine S305 is a consequence of kinase activation and enables the active fraction to be identified in the cell. Active PLK4 is detectable on the replicating mother centriole in G1/S, with the proportion of active kinase increasing through interphase to reach a maximum in mitosis. Activation of PLK4 at the replicating daughter centriole is delayed until G2, but a level equivalent to the replicating mother centriole is achieved in M phase. Active PLK4 is regulated by the proteasome, because either proteasome inhibition or mutation of the degron motif of PLK4 results in the accumulation of S305-phosphorylated PLK4. Autophosphorylation probably plays a role in the process of centriole duplication, because mimicking S305 phosphorylation enhances the ability of overexpressed PLK4 to induce centriole amplification. Importantly, we show that S305-phosphorylated PLK4 is specifically sequestered at the centrosome contrary to the nonphosphorylated form. These data suggest that PLK4 activity is restricted to the centrosome to prevent aberrant centriole assembly and sustained kinase activity is required for centriole duplication.

scholarly journals Uncoupled cell cycle without mitosis induced by a protein kinase inhibitor, K-252a.

1991 ◽  
Vol 115 (5) ◽  
pp. 1275-1282 ◽  
Author(s):  
T Usui ◽  
M Yoshida ◽  
K Abe ◽  
H Osada ◽  
K Isono ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Protein Kinase ◽  
Dna Synthesis ◽  
Kinase Activity ◽  
Kinase Inhibitor ◽  
De Novo ◽  
S Phase ◽  
Continuous Treatment ◽  
Protein Kinase Activity

The staurosporine analogues, K-252a and RK-286C, were found to cause DNA re-replication in rat diploid fibroblasts (3Y1) without an intervening mitosis, producing tetraploid cells. Analysis of cells synchronized in early S phase in the presence of K-252a revealed that initiation of the second S phase required a lag period of 8 h after completion of the previous S phase. Reinitiation of DNA synthesis was inhibited by cycloheximide, actinomycin D, and serum deprivation, but not by Colcemid, suggesting that a functional G1 phase dependent on de novo synthesis of protein and RNA is essential for entry into the next S phase. In a src-transformed 3Y1 cell line, as well as other cell lines, giant cells containing polyploid nuclei with DNA contents of 16C to 32C were produced by continuous treatment with K-252a, indicating that the agent induced several rounds of the incomplete cell cycle without mitosis. Although the effective concentration of K-252a did not cause significant inhibition of affinity-purified p34cdc2 protein kinase activity in vitro, in vivo the full activation of p34cdc2 kinase during the G2/M was blocked by K-252a. On the other hand, the cyclic fluctuation of partially activated p34cdc2 kinase activity peaking in S phase still continued. These results suggest that a putative protein kinase(s) sensitive to K-252a plays an important role in the mechanism for preventing over-replication after completion of previous DNA synthesis. They also suggest that a periodic activation of p34cdc2 is required for S phases in the cell cycle without mitosis.

scholarly journals Involvement of Myc Activity in a G1/S-Promoting Mechanism Parallel to the pRb/E2F Pathway

2000 ◽  
Vol 20 (10) ◽  
pp. 3497-3509 ◽  
Author(s):  
Eric Santoni-Rugiu ◽  
Jacob Falck ◽  
Niels Mailand ◽  
Jiri Bartek ◽  
Jiri Lukas
Keyword(s):  
Dna Synthesis ◽  
Mammalian Cells ◽  
Kinase Activity ◽  
Cyclin E ◽  
Ectopic Expression ◽  
S Phase ◽  
Dominant Negative ◽  
Kinase Activation ◽  
Cdc25a Phosphatase ◽  
In Cells

ABSTRACT The retinoblastoma protein (pRb)/E2F pathway regulates commitment of mammalian cells to replicate DNA. On the other hand, mitogen-stimulated cells deprived of E2F activity can still maintain physiologically relevant levels of cyclin E-dependent kinase activity and gradually enter S phase, suggesting the existence of a DNA synthesis-inducing mechanism parallel to the pRb/E2F axis. Here we show that regulatable ectopic expression of cyclin E or transcriptionally active Myc can rapidly induce DNA synthesis in U2OS-derived cell lines whose E2F activity is blocked by a constitutively active pRb (pRbΔcdk) mutant. The effect of Myc is associated with Cdc25A phosphatase and cyclin E-CDK2 kinase activation and abolished by antagonizing Myc activity with the dominant-negative (dn) MadMyc chimera. Moreover, while abrogation of either endogenous E2F or Myc activity only delays and lowers DNA synthesis in synchronized U2OS cells or rat diploid fibroblasts, concomitant neutralization of both abolishes it. Whereas ectopic Myc and E2F1 rescue the G1/S delay caused by pRbΔcdk (or dnDP1) and MadMyc, respectively, cyclin E or Cdc25A can restore DNA replication even in cells concomitantly exposed to pRbΔcdk and MadMyc. However, coexpression of dnCDK2 neutralizes all of these rescuing effects. Finally, proper transcription of cyclin E and Cdc25A at the G1/S transition requires both Myc and E2F activities, and subthreshold levels of ectopic cyclin E and Cdc25A synergistically restore DNA synthesis in cells with silenced Myc and E2F activities. These results suggest that Myc controls a G1/S-promoting mechanism regulating cyclin E-CDK2 in parallel to the “classical” pRb/E2F pathway.

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