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At least 19 recordsLinked to original sources

PubMed1992

Inhibition of membrane fusion in vitro via cyclin B but not cyclin A.

It is now clear that complexes of cdc2 kinase with "mitotic" cyclins regulate the transition between the G2 phase of the cell cycle and mitosis and that membrane traffic in mammalian cells is arrested during mitosis. Using a cell-free assay, we have previously reported that the fusion of early endosomes is, in fact, inhibited via the cdc2 kinase (Tuomikoski, T., Felix, M.-A., Dorée, M., and Gruenberg, J. (1989) Nature 342, 942-945). In the present paper, we show that this in vitro inhibition occurs efficiently only when the kinase activity is specifically evoked by a cyclin of the B-type but not by cyclins of the A-type. In addition, high resolution two-dimensional gel analysis revealed that the kinases associated with A- and B-type cyclins exhibit different substrate preferences. These data suggest that the complexes of the cdc2 kinase with different cyclins may control specific events of the cell cycle.

Animals↗

PubMed1992

Cyclin B in fish oocytes: its cDNA and amino acid sequences, appearance during maturation, and induction of p34cdc2 activation.

Under the influence of maturation-inducing hormone (MIH) secreted from follicle cells, oocyte maturation is finally triggered by maturation-promoting factor (MPF), which consists of a homolog of the cdc2+ gene product of fission yeast (p34cdc2) and cyclin B. Two species of cyclin B clones were isolated from a cDNA library constructed from mature goldfish oocytes. Sequence comparisons revealed that these two clones are highly homologous (95%) and were found to be similar to Xenopus cyclin B1. Using monoclonal antibodies against Escherichia coli-produced goldfish cyclin B and the PSTAIR sequence of p34cdc2, we examined the levels of cyclin B and p34cdc2 proteins during goldfish oocyte maturation induced in vitro by 17 alpha, 20 beta-dihydroxy-4-pregnen-3-one (17 alpha, 20 beta-DP), a natural MIH in fish. Protein p34cdc2 was found in immature oocyte extracts and did not remarkably change during oocyte maturation. Cyclin B was not detected in immature oocyte extracts and appeared when oocytes underwent germinal vesicle breakdown. Cyclin B that appeared during oocyte maturation was labelled with [35S]methionine, indicating its de novo synthesis. Introduction of E. coli-produced cyclin B into immature oocyte extracts induced p34cdc2 (MPF) activation. Although the possibility that immature goldfish oocytes contain an insoluble cyclin B is not completely excluded, these results strongly suggest that 17 alpha, 20 beta-DP induces oocytes to synthesize cyclin B, which in turn activates preexisting p34cdc2, forming active MPF.

Amino Acid Sequence↗

PubMed1992

3' non-translated sequences in Drosophila cyclin B transcripts direct posterior pole accumulation late in oogenesis and peri-nuclear association in syncytial embryos.

We have characterised forms of the Drosophila cyclin B transcript that differ as a result of a splicing event which removes a nucleotide segment from the 3' untranslated region. In oogenesis, both cyclin A RNA and a shorter form of the cyclin B transcript are seen in the cells of the germarium that are undergoing mitosis. The shorter cyclin B transcript alone is then detectable in the presumptive oocyte until stages 7-8 of oogenesis. Both cyclin A RNA and a longer form of the cyclin B RNA are then synthesised in the nurse cells during stages 9-11, to be deposited in the oocyte during stages 11-12. These transcripts become evenly distributed throughout the oocyte cytoplasm but, in addition, those of cyclin B become concentrated at the posterior pole. Examination of the distributions of RNAs transcribed from chimeric cyclin genes indicates that sequences in the 3' untranslated region of the larger cyclin B RNA are required both for it to become concentrated at the posterior pole and to direct those transcripts in the body of the syncytial embryo to their peri-nuclear localisation. These sequences are disrupted by the splicing event which generates smaller cyclin B transcripts.

Animals↗

PubMed1992

Relocation and distinct subcellular localization of p34cdc2-cyclin B complex at meiosis reinitiation in starfish oocytes.

M phase promoting factor (MPF) is a major element controlling entry into the M phase of the eukaryotic cell cycle. MPF is composed of two subunits, p34cdc2 and cyclin B. Using indirect immunofluorescence staining with specific antibody against starfish cyclin B, we monitored the dynamics of the subcellular distribution of MPF during meiosis reinitiation in starfish oocytes. We found that all of the cyclin B is already associated with p34cdc2 in immature oocytes arrested at the G2/M border and that this inactive complex is present exclusively in the cytoplasm. After its activation, part of the p34cdc2-cyclin B complex moves into the germinal vesicle before nuclear envelope breakdown, independently of either microtubules or actin filaments. Thereafter, some part of the complex accumulates in the nucleolus and condensed chromosomes. Another portion of the complex accumulates on meiotic asters and spindles, while the rest is still present throughout the cytoplasm. As these patterns of localization are detected in the detergent-extracted oocytes, we propose at least four distinct subcellular states of the p34cdc2-cyclin B complex: freely soluble, microtubule-associated, detergent-resistant cytoskeleton-associated and chromosome-associated. Thus, in addition to the intramolecular modification of p34cdc2-cyclin B complex, its intracellular relocation plays a key role in promoting the M phase.

Animals↗

PubMed1992

Cyclin-B homologs in Saccharomyces cerevisiae function in S phase and in G2.

We have cloned four cyclin-B homologs from Saccharomyces cerevisiae, CLB1-CLB4, using the polymerase chain reaction and low stringency hybridization approaches. These genes form two classes based on sequence relatedness: CLB1 and CLB2 show highest homology to the Schizosaccharomyces pombe cyclin-B homolog cdc13 involved in the initiation of mitosis, whereas CLB3 and CLB4 are more highly related to the S. pombe cyclin-B homolog cig1, which appears to play a role in G1 or S phase. CLB1 and CLB2 mRNA levels peak late in the cell cycle, whereas CLB3 and CLB4 are expressed earlier in the cell cycle but peak later than the G1-specific cyclin, CLN1. Analysis of null mutations suggested that the CLB genes exhibit some degree of redundancy, but clb1,2 and clb2,3 cells were inviable. Using clb1,2,3,4 cells rescued by conditional overproduction of CLB1, we showed that the CLB genes perform an essential role at the G2/M-phase transition, and also a role in S phase. CLB genes also appear to share a role in the assembly and maintenance of the mitotic spindle. Taken together, these analyses suggest that CLB1 and CLB2 are crucial for mitotic induction, whereas CLB3 and CLB4 might participate additionally in DNA replication and spindle assembly.

Amino Acid Sequence↗

PubMed1992

Cyclin B is associated with centrosomes in Drosophila mitotic cells.

We have studied by way of confocal laser scanning microscopy the subcellular localization of cyclin B in Drosophila-cultured cells and report here evidence that a part of the cyclin B cell pool is closely associated with the centrosome. This cyclin B centrosomal signal is strong in prophase and metaphase but disappears during anaphase. Moreover, the signal is absent in the acentriolar Drosophila cell line 1182-4. These results put forward additional arguments suggesting that the centrosome plays an important role in the control of the cell cycle.

Animals↗

PubMed1992

Inactivation of the p34cdc2-cyclin B complex by the human WEE1 tyrosine kinase.

Entry into mitosis in Schizosaccharomyces pombe is negatively regulated by the wee1+ gene, which encodes a protein kinase with serine-, theonine-, and tyrosine-phosphorylating activities. The wee1+ kinase negatively regulates mitosis by phosphorylating p34cdc2 on tyrosine 15, thereby inactivating the p34cdc2-cyclin B complex. The human homolog of the wee1+ gene (WEE1Hu) was overproduced in bacteria and assayed in an in vitro system. Unlike its fission yeast homolog, the product of the WEE1Hu gene encoded a tyrosine-specific protein kinase. The human WEE1 kinase phosphorylated the p34cdc2-cyclin B complex on tyrosine 15 but not on threonine 14 in vitro and inactivated the p34cdc2-cyclin B kinase. This inhibition was reversed by the human Cdc25C protein, which catalyzed the dephosphorylation of p34cdc2. These results indicate that the product of the WEE1Hu gene directly regulates the p34cdc2-cyclin B complex in human cells and that a kinase other than that encoded by WEE1Hu phosphorylates p34cdc2 on threonine 14.

Base Sequence↗

PubMed1992

CLB5: a novel B cyclin from budding yeast with a role in S phase.

Budding yeast strains have three CLN genes, which have limited cyclin homology. At least one of the three is required for cell cycle START. Four B cyclins are known in yeast; two have been shown to function in mitosis. We have discovered a fifth B-cyclin gene, called CLB5, which when cloned on a CEN plasmid can rescue strains deleted for all three CLN genes. CLB5 transcript abundance peaks in G1, coincident with the CLN2 transcript but earlier than the CLB2 transcript. CLB5 deletion does not cause lethality, either alone or in combination with other CLN or CLB deletions. However, strains deleted for CLB5 require more time to complete S phase, suggesting that CLB5 promotes some step in DNA synthesis. CLB5 is the only yeast cyclin whose deletion lengthens S phase. CLB5 may also have some role in promoting the G1/S transition, because cln1 cln2 strains require both CLN3 and CLB5 for viability on glycerol media and cln1,2,3- strains require CLB5 for rescue by the Drosophila melanogaster cdc2 gene. In conjunction with cln1,2,3- rescue by CLB5 overexpression and the coincident transcriptional regulation of CLB5 and CLN2, these observations are suggestive of partial functional redundancy between CLB5 and CLN genes.

Amino Acid Sequence↗

PubMed1992

Oscillation of MPF is accompanied by periodic association between cdc25 and cdc2-cyclin B.

Activation of maturation-promoting factor at the onset of mitosis requires the tyrosine dephosphorylation of one of its components, the cdc2 protein kinase. cdc25 is the specific tyrosine phosphatase that activates cdc2. We find that Xenopus oocytes contain a relative of cdc25, p72. In Xenopus embryos the abundance of p72 does not oscillate during the cell cycle. However, p72 directly associates with cdc2-cyclin B in a cell cycle-dependent manner, reaching a peak at M phase. The M phase kinase that associates with p72 is catalytically active. These results suggest that the mechanism by which cdc25 triggers cdc2 activation involves a periodic physical association between cdc25 and the cyclin B-cdc2 complex and also that mitotic control can be affected by mechanisms other than transcriptional regulation of the cdc25 gene.

Animals↗

PubMed1992

Control of microtubule dynamics and length by cyclin A- and cyclin B-dependent kinases in Xenopus egg extracts.

In eukaryotic cells, the onset of mitosis involves cyclin molecules which interact with proteins of the cdc2 family to produce active kinases. In vertebrate cells, cyclin A dependent kinases become active in S- and pro-phases, whereas a cyclin B-dependent kinase is mostly active in metaphase. It has recently been shown that, when added to Xenopus egg extracts, bacterially produced A- and B-type cyclins associate predominantly with the same kinase catalytic subunit, namely p34cdc2, and induce its histone H1 kinase activity with different kinetics. Here, we show that in the same cell free system, both the addition of cyclin A and cyclin B changes microtubule behavior. However, the cyclin A-dependent kinase does not induce a dramatic shortening of centrosome-nucleated microtubules whereas the cyclin B-dependent kinase does, as previously reported. Analysis of the parameters of microtubule dynamics by fluorescence video microscopy shows that the dramatic shortening induced by the cyclin B-dependent kinase is correlated with a several fold increase in catastrophe frequency, an effect not observed with the cyclin A-dependent kinase. Using a simple mathematical model, we show how the length distributions of centrosome-nucleated microtubules relate to the four parameters that describe microtubule dynamics. These four parameters define a threshold between unlimited microtubule growth and the establishment of steady-state dynamics, which implies that well defined steady-state length distributions can be produced by regulating precisely the respective values of the dynamical parameters. Moreover, the dynamical model predicts that increasing catastrophe frequency is more efficient than decreasing the rescue frequency to reduce the average steady state length of microtubules. These theoretical results are quantitatively confirmed by the experimental data.

Animals↗

PubMed1992

Cyclin A- and cyclin B-dependent protein kinases are regulated by different mechanisms in Xenopus egg extracts.

Cyclins are proteins which are synthesized and degraded in a cell cycle-dependent fashion and form integral regulatory subunits of protein kinase complexes involved in the regulation of the cell cycle. The best known catalytic subunit of a cyclin-dependent protein kinase complex is p34cdc2. In the cell, cyclins A and B are synthesized at different stages of the cell cycle and induce protein kinase activation with different kinetics. The kinetics of activation can be reproduced and studied in extracts of Xenopus eggs to which bacterially produced cyclins are added. In this paper we report that in egg extracts, both cyclin A and cyclin B associate with and activate the same catalytic subunit, p34cdc2. In addition, cyclin A binds a less abundant p33 protein kinase related to p34cdc2, the product of the cdk2/Eg1 gene. When complexed to cyclin B, p34cdc2 is subject to transient inhibition by tyrosine phosphorylation, producing a lag between the addition of cyclin and kinase activation. In contrast, p34cdc2 is only weakly tyrosine phosphorylated when bound to cyclin A and activates rapidly. This finding shows that a given kinase catalytic subunit can be regulated in a different manner depending on the nature of the regulatory subunit to which it binds. Tyrosine phosphorylation of p34cdc2 when complexed to cyclin B provides an inhibitory check on the activation of the M phase inducing protein kinase, allowing the coupling of processes such as DNA replication to the onset of metaphase. Our results suggest that, at least in the early Xenopus embryo, cyclin A-dependent protein kinases may not be subject to this checkpoint and are regulated primarily at the level of cyclin translation.

Amino Acid Sequence↗

PubMed1992

Effects of ionizing radiation on cyclin expression in HeLa cells.

The levels of cyclin B mRNA and protein rise rapidly in G2 + M phase, and fall at the end of mitosis. The studies described here were initiated to determine the effects of ionizing radiation on the level of cyclin B bearing in mind that the division delay induced by ionizing radiation might be influenced by the expression of cyclin B. After irradiation in S phase, the cyclin B mRNA in HeLa cells was measured as the cells proceeded through the cell cycle. Instead of the usual rise, after irradiation cyclin B mRNA levels remained low during the G2 delay. After irradiation in G2 phase, cyclin B mRNA was readily detectable although at slightly lower levels than in the controls. However, cyclin B protein was markedly decreased in amount.

Cell Cycle↗

PubMed1992

Dephosphorylation of cdc2 on threonine 161 is required for cdc2 kinase inactivation and normal anaphase.

Exit from metaphase of the cell cycle requires inactivation of MPF, a stoichiometric complex between the cdc2 catalytic and the cyclin B regulatory subunits, as well as that of cyclin A-cdc2 kinase. Inactivation of both complexes depends on proteolytic degradation of the cyclin subunit, yet cyclin proteolysis is not sufficient to inactivate the H1 kinase activity of cdc2. Genetic evidence strongly suggests that type 1 phosphatase plays a key role in the metaphase-anaphase transition of the cell cycle. Here we report that inhibition of both type 1 and type 2A phosphatases by okadaic acid allows cyclin degradation to occur, but prevents cdc2 kinase inactivation. Complete inhibition of type 2A phosphatase alone is not sufficient to prevent cdc2 kinase inactivation following cyclin proteolysis. We show further that residue 161 of cdc2 is phosphorylated in active cyclin A or cyclin B complexes at metaphase, whilst unassociated cdc2 is not phosphorylated. Proteolysis of cyclin releases a free cdc2 subunit, which subsequently undergoes dephosphorylation and then migrates more slowly than its Thr161 phosphorylated counterpart in Laemmli gels. Removal of phosphothreonine 161 requires cyclin proteolysis. However, it does not occur even after cyclin proteolysis, when both type 1 and type 2A phosphatases are inhibited. We conclude that both cyclin degradation and dephosphorylation of Thr161 on cdc2, catalysed at least in part by type 1 phosphatase, are required to inactivate either cyclin B- or cyclin A-cdc2 kinases and thus for cells to exit from M phase.

Amino Acid Sequence↗

PubMed1992

Cyclins and cdc2 kinases in Drosophila: genetic analyses in a higher eukaryote.

Cyclin proteins and the kinases with which they associate are encoded by gene families in multicellular eukaryotes. A variety of cyclin/kinase complexes with different functions may exist. We have started a genetic dissection of this complexity in Drosophila. We have done experiments to investigate a potential functional overlap between two kinases (Dmcdc2 and Dmcdc2c) and two cyclins (cyclin A and cyclin B). No functional overlap was observed between the Dmcdc2 and the Dmcdc2c kinases. The phenotype resulting from mutations in Dmcdc2 was not affected by altering the level of Dmcdc2c. Our results concerning cyclin A and cyclin B strongly suggest that these two cyclins have largely overlapping functions. Cell proliferation was observed in the absence of either cyclin A or cyclin B, but not if both cyclins were absent. Cyclin A also has essential functions that cannot be taken over by cyclin B, but these functions appear to be required at defined developmental stages in specific tissues only.

Animals↗

PubMed1992

Characterization of four B-type cyclin genes of the budding yeast Saccharomyces cerevisiae.

The previously described CLB1 and CLB2 genes encode a closely related pair of B-type cyclins. Here we present the sequences of another related pair of B-type cyclin genes, which we term CLB3 and CLB4. Although CLB1 and CLB2 mRNAs rise in abundance at the time of nuclear division, CLB3 and CLB4 are turned on earlier, rising early in S phase and declining near the end of nuclear division. When all possible single and multiple deletion mutants were constructed, some multiple mutations were lethal, whereas all single mutants were viable. All lethal combinations included the clb2 deletion, whereas the clb1 clb3 clb4 triple mutant was viable, suggesting a key role for CLB2. The inviable multiple clb mutants appeared to have a defect in mitosis. Conditional clb mutants arrested as large budded cells with a G2 DNA content but without any mitotic spindle. Electron microscopy showed that the spindle pole bodies had duplicated but not separated, and no spindle had formed. This suggests that the Clb/Cdc28 kinase may have a relatively direct role in spindle formation. The two groups of Clbs may have distinct roles in spindle formation and elongation.

Amino Acid Sequence↗

PubMed1992

Identification of a mouse B-type cyclin which exhibits developmentally regulated expression in the germ line.

To begin to examine the function of cyclins in mammalian germ cells, we have screened an adult mouse testis cDNA library for the presence of B-type cyclins. We have isolated cDNAs that encode a murine B-type cyclin, which has been designated cycB1. cycB1 was shown to be expressed in several adult tissues and in the midgestation mouse embryo. In the adult tissues, the highest levels of cycB1 transcripts were seen in the testis and ovary, which contain germ cells at various stages of differentiation. The major transcripts corresponding to cycB1 are 1.7 and 2.5 kb, with the 1.7 kb species being the predominant testicular transcript and the 2.5 kb species more abundant in the ovary. Examination of cDNAs corresponding to the 2.5 kb and 1.7 kb mRNAs revealed that these transcripts encode identical proteins, differing only in the polyadenylation signal used and therefore in the length of their 3' untranslated regions. Northern blot and in situ hybridization analyses revealed that the predominant sites of cycB1 expression in the testis and ovary were in the germinal compartment, particularly in early round spermatids in the testis and growing oocytes in the ovary. Thus cycB1 is expressed in both meiotic and postmeiotic cells. This pattern of cycB1 expression further suggests that cycB1 may have different functions in the two cell types, only one of which correlates with progression of the cell cycle.

Amino Acid Sequence↗

PubMed1992

Relationships between cdc2 kinase, DNA cross-linking, and cell cycle perturbations induced by nitrogen mustard.

M phase-promoting factor (MPF) consists of a p34cdc2 (cdc2) kinase and cyclin B complex which in its active form promotes G2 to M transition. The role of MPF in G2 arrest following DNA damage, however, has remained largely uncharacterized. We have investigated whether nitrogen mustard (HN2) interfered with either the formation of MPF or its activation. For this purpose, we measured cdc2 kinase activity relative to cdc2 and cyclin B protein turnover and the phosphorylation status of cdc2. Studies were performed in two exceptional human lymphoma cell lines, which differed in HN2 sensitivity by 5-fold (CA46, 50% growth-inhibitory dose = 1.0 microM; JLP119, 50% growth-inhibitory dose = 0.2 microM) but exhibited virtually identical DNA interstrand and DNA-protein cross-link exposure. Following HN2 treatment, CA46 cells ceased to enter mitosis and exhibited a marked delay in G2 phase. Failure to enter mitosis paralleled inhibition of cdc2 kinase. Inhibition was not due to decreased levels of cdc2 or cyclin B protein; rather, G2 arrest correlated with the accumulation of both tyrosine-phosphorylated cdc2 and cyclin B. These findings implied that G2 arrest resulted from a down-regulation of the processes that activate MPF. We also found that JLP119 cells, within a few hours of mitosis at the time of drug treatment, evaded checkpoint control and continued cell division unabated by DNA damage. Furthermore, despite similar DNA cross-link exposure, JLP119 cells within the window of checkpoint control were more susceptible to S phase delay than CA46 cells. Altered cell cycle responses correlated with the greater susceptibility of JLP119 cells to the cytotoxic effects of HN2.

CDC2 Protein Kinase↗

PubMed1992

Cyclin A-Cys41 does not undergo cell cycle-dependent degradation in Xenopus extracts.

Truncated cyclin A and cyclin B lacking the N-terminal domain comprising the 'destruction box' escape from proteolysis and arrest cells at metaphase. Mutation of a conserved arginine residue of the destruction domain makes cyclin B resistant to proteolysis. Here we show that mutation of the same residue also makes cyclin A resistant to proteolysis, in either of two situations in which the cyclin degradation pathway is turned on: (i) in Xenopus extracts of activated eggs where the degradation pathway has been permanently turned on by adding a recombinant undegradable cyclin B in which the arginine residue of the destruction box has been substituted by alanine; (ii) in extracts of metaphase II-arrested oocytes after Ca(2+)-dependent inactivation of the cytostatic factor (CSF).

Adenosine Triphosphate↗