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L Meijer

Publications and source records attributed to L Meijer.

At least 73 records · Page 4Linked to original sources
PubMed1997

Roscovitine, a novel cyclin-dependent kinase inhibitor, characterizes restriction point and G2/M transition in tobacco BY-2 cell suspension.

Although the developmental programs of plants and animals differ, key regulatory components of their cell cycle have been conserved. Particular attention has been paid to the role of the complexes between highly conserved cyclin and cyclin-dependent kinases in regulating progression through the cell cycle. The recent demonstration that roscovitine is a potent and selective inhibitor of the animal cyclin-dependent kinases cdc2 (CDK1), CDK2 and CDK5 prompted an investigation into its effects on progression through the plant cell cycle. Roscovitine induced arrests both in late G1 and late G2 phase in BY-2 tobacco cell suspensions. Both block were fully reversible when roscovitine was used at concentrations similar to those used in the animal system. Stationary-phase cells subcultured in the presence of roscovitine were arrested at a 2C DNA content. This arrest was more efficient without exogenous addition of plant growth regulator. Roscovitine induced a block in G1 earlier than that induced by aphidicolin. S-phase synchronized cells treated with roscovitine were arrested at a 4C DNA content at the G2/ M transition. The expression analysis of a mitotic cyclin (NTCYC1) indicated that the roscovitine-induced G2 block probably occurs in late G2. Finally, cells in metaphase were insensitive to roscovitine. The purified CDK/cyclin kinase activities of late G1 and early M arrested cells were inhibited in vitro by roscovitine. The implications of these experimental observations for the requirement for CDK activity during progression through the plant cell cycle are discussed.

2,4-Dichlorophenoxyacetic Acid↗
PubMed1996

Sequential dephosphorylation of p34(cdc2) on Thr-14 and Tyr-15 at the prophase/metaphase transition.

The G2-M transition of the cell cycle is triggered by the p34(cdc2)/cyclin B kinase. During the prophase/metaphase transition, the inactive, Thr-14/Tyr-15 phosphorylated form of p34(cdc2) (TP-YP) is modified to an active, Thr-14/Tyr-15 dephosphorylated form (T-Y) by the cdc25 dual-specificity phosphatase. Using highly synchronized starfish oocytes as a cellular model, we show that dephosphorylation in vivo and in vitro occurs in two steps: Thr-14 dephosphorylation precedes Tyr-15 dephosphorylation. The transient intermediate form (T-YP), which can be obtained in vitro by treatment of TP-YP by protein phosphatase 2A, displays low but significant kinase activity. These results raise the possibility that the intermediate form T-YP may be involved in the autocatalytic amplification of the p34(cdc2)/cyclin B complex through phosphorylation/activation of the cdc25 phosphatase and phosphorylation/inactivation of the wee1 kinase.

Amino Acid Sequence↗
PubMed1996

Chemical inhibitors of cyclin-dependent kinases.

Transient activation o f cyclin-dependent kinases (CDKs) is responsible for transition through the successive phases of the cell-division cycle. Major changes in the expression and regulation of CDKs have been described in human tumours. Enzymatic screening is starting to uncover chemical inhibitors o f CDKs that arrest the cell cycle at various steps. This review summarizes our knowledge of the first generation inhibitors, their molecular mechanisms of action and their effects on the cell cycle and apoptosis, and discusses their potential as synchronizing agents, as ligands for affinity chromatography and as therapeutic agents.

Journal Article↗
PubMed1995

Multiple modes of ligand recognition: crystal structures of cyclin-dependent protein kinase 2 in complex with ATP and two inhibitors, olomoucine and isopentenyladenine.

Cyclin-dependent kinases (CDKs) are conserved regulators of the eukaryotic cell cycle with different isoforms controlling specific phases of the cell cycle. Mitogenic or growth inhibitory signals are mediated, respectively, by activation or inhibition of CDKs which phosphorylate proteins associated with the cell cycle. The central role of CDKs in cell cycle regulation makes them a potential new target for inhibitory molecules with anti-proliferative and/or anti-neoplastic effects. We describe the crystal structures of the complexes of CDK2 with a weakly specific CDK inhibitor, N6-(delta 2-isopentenyl)adenine, and a strongly specific inhibitor, olomoucine. Both inhibitors are adenine derivatives and bind in the adenine binding pocket of CDK2, but in an unexpected and different orientation from the adenine of the authentic ligand ATP. The N6-benzyl substituent in olomoucine binds outside the conserved binding pocket and is most likely responsible for its specificity. The structural information from the CDK2-olomoucine complex will be useful in directing the search for the next generation inhibitors with improved properties.

Adenine↗
PubMed1995

Chemical inhibitors of cyclin-dependent kinases.

The eukaryotic cell division cycle is regulated by a family of protein kinases, the cyclin-dependent kinases (cdk's), constituted of at least two subunits, a catalytic subunit (cdk1-7) associated with a regulatory subunit (cyclin A-H). Transient activation of cdk's is responsible for transition through the different phases of the cell cycle. Major abnormalities of cdk's expression and regulation have been described in human tumours. Enzymatic screening is starting to uncover chemical inhibitors of cdk's with anti-mitotic activities. This review summarizes our knowledge of these first inhibitors, their mechanism of action, their effects on the cell cycle, and discusses the potential of such type of inhibitors as anti-tumour agents.

4-Butyrolactone↗
PubMed1994

Olomoucine, an inhibitor of the cdc2/cdk2 kinases activity, blocks plant cells at the G1 to S and G2 to M cell cycle transitions.

The cdc2/cdk2 protein kinases play key roles in the cell cycle at two control points: the G1/S transition and the entry into mitosis. Olomoucine, a specific inhibitor of these kinases, was tested in two plant cell systems: Petunia mesophyll protoplasts induced to divide and Arabidopsis thaliana cell suspension cultures. The cell cycle status was analysed from DNA histograms or through continuous labelling of cells with 5-bromodeoxyuridine (BrdUrd) followed by double staining with bis-benzimide (Hoechst 33258) and propidium iodide (PI). Such analyses resolve cells from several generations according to the extent of their DNA replication. Olomoucine was shown to reversibly arrest differentiated Petunia cells induced to divide at G1 phase and cycling Arabidopsis cells in late G1 and G2. A comparison of the effects of aphidicolin, oryzalin and olomoucine suggests that in the Arabidopsis cell suspension culture, a cdc2/cdk2-like kinase is activated at a restriction point in late G1.

Arabidopsis↗
PubMed1994

Purification of a 15-kDa cdk4- and cdk5-binding protein.

Yeasts p13suc1/p18CKS and their human homologues, p9CKShs1/p9CKShs2, strongly interact with p34cdc2 and p34cdk2. While attempting to purify the starfish oocyte p13suc1 homologue, we discovered a 15-kDa protein cross-reactive with anti-p9CKShs2/anti-p13suc1 antibodies. p15cdk-BP-Sepharose binds an anti-PSTAIRE cross-reactive protein of 33 kDa when loaded with starfish oocyte extracts. The p15cdk-BP-bound "PSTAIRE signal" is part of a 250-kDa complex distinct from p34cdc2/cyclin B. p15cdk-BP-Sepharose beads retain a kinase phosphorylating HMG I/Y, P1, and myelin basic protein (among 24 substrates tested). Major cdc2 kinase substrates are not phosphorylated by the p15cdk-BP-bound kinase. Phosphopeptide maps of P1 phosphorylated by the p15cdk-BP-bound kinase, p34cdc2/cyclin B, p 33cdk5/p25, and casein kinase 2 showed that these kinases phosphorylate P1 on different sites. Phosphopeptide maps of P1 phosphorylated by the p15cdk-BP-bound starfish kinase and p15cdk-BP-bound human p34cdk4/cyclin D are largely coincident. To investigate the nature of the p15cdk-BP-bound kinase, extracts of mammalian tissues and cells were loaded on p9CKShs1- and p15cdk-BP-Sepharose and the bound proteins were analyzed using specific anti-cdk antibodies. cdc2 and cdk2 bind to p9CKShs1-Sepharose, but not to p15cdk-BP. cdk4 and cdk5 bind to p15cdk-BP-Sepharose, but not to p9CKShs1-Sepharose. We conclude that p15cdk-BP specifically binds the cdk4/cyclin D and cdk5 kinases and, along with p13suc1 and p9CKShs, may be part of a larger family of cdk-binding proteins.

Amino Acid Sequence↗
PubMed1994

Expression and activation of cdks (1 and 2) and cyclins in the cell cycle progression during liver regeneration.

In normal adult liver, hepatocytes are arrested in G0, and they rapidly respond to a mass loss by a definite number of divisions. Thus, taking advantage of the in vivo regenerative capacity of the liver following partial hepatectomy, we have analyzed both expression and activation of p34cdc2 (= cdk1) and p33cdk2 through the cell cycle, particularly during the long lasting G1 phase and in the G1/S transition. While p33cdk2 is constantly expressed during the cell cycle, p34cdc2 is completely absent in resting hepatocytes and remains unexpressed for up to 20 h after partial hepatectomy, a time period corresponding to the G1 phase and G1/S transition, and then accumulates in the S, G2, and M phases. No histone H1 kinase activity is detected during the G1 phase, while two peaks of p34cdc2 kinase activity are observed during the S and M phases and only one peak of p33cdk2 kinase activity in the S phase. p34cdc2 forms complexes with both cyclins A and B while p33cdk2 is associated with cyclin A only. Surprisingly, cyclins E and D1 are present in resting liver and with modest variation throughout the cell cycle. Taken together, our data provide evidence that the pattern of G1-associated proteins in hepatocytes during liver regeneration is distinct from that described in other cell types.

Animals↗
PubMed1993

8-Dimethylamino-1-methyladenine, a novel potent antagonist of the 1-methyladenine receptor in starfish oocytes.

Starfish oocytes are naturally arrested in the late G2 phase of the first meiotic division. The follicle-cell-derived hormone, 1-methyladenine, is responsible for highly synchroneous induction of oocyte maturation. A series of systematically modified 1-methyladenine analogues was used to map the essential molecular interactions between 1-methyladenine and its stereospecific receptors, which are localized on the oocyte plasma membrane. The earlier hypothesis about structure/activity relationships has been confirmed. Quantum-chemical calculations indicated additional dipole-dipole interactions and presumably a charge-transfer interaction, with the nucleobase as pi-electron donor. Among the 49 compounds tested, a series of novel inhibitors of 1-methyladenine-induced maturation was found. With the synthesis of 8-dimethylamino-1-methyladenine (concentration for 50% inhibition 1 microM), a very potent antagonist of the natural hormone was obtained which may become an important tool for investigating the mechanism of 1-methyladenine-induced maturation of the starfish oocytes, an important model for cell-cycle-control studies. The results are discussed in the context of preexisting biological data. In conclusion, we propose a modified model of the molecular interactions between 1-methyladenine and its receptor.

Adenine↗
PubMed1993

The labour supply of married and cohabiting women in the Netherlands, 1981-1989.

"In this article we investigate the differences in labour supply of married women and cohabiting women in the Netherlands; we try to answer the question how these differences can be explained. From this study, it can be concluded that differences between both categories of women in participation and in weekly hours worked can predominantly be explained by differences in characteristics (e.g. age, net wage rate, and age of children), than by differences in behaviour. The empirical results indicate that cohabiting women are more economically independent than married women. However for married women we found evidence that there was increased economic independence during the eighties; i.e. their weekly hours work has become less affected by the income of their partners." (SUMMARY IN FRE)

Demography↗
PubMed1992

Interaction between the cell-cycle-control proteins p34cdc2 and p9CKShs2. Evidence for two cooperative binding domains in p9CKShs2.

A universal intracellular factor, the 'M-phase-promoting factor' (MPF), displaying histone H1 kinase activity and constituted of at least two subunits, p34cdc2 and cyclin Bcdc13, triggers the G2----M transition of the cell cycle in all organisms. The yeast p13suc1 and p18CKS1 subunits and their functionally interchangeable human homologues, p9CKShs1 and p9CKShs2, directly interact with p34cdc2 and may actually be part of the MPF complex. We have chemically synthesized p9CKShs2 and several of its peptide domains in order to investigate the binding of p9CKShs2 and p34cdc2. Several arguments support the hypothesis that the N-terminal half (peptide B) and the C-terminal half (peptide E) each contain a p34cdc2-binding site and that these two binding domains cooperate in establishing a stable p9CKShs2-p34cdc2 complex: (a) only the combination of peptides B + E, and not B or E alone, is able to elute the cdc2 kinase from p9CKShs1-Sepharose beads; (b) only immobilized peptides B + E, and not immobilized B or E, bind the cdc2 kinase; (c) only the peptides B + E combination, and not B or E alone, can compete with p9CKShs1 for cdc2 kinase binding; (d) only when supplemented with E or B free peptide does the cdc2 kinase bind to B- or E-Sepharose beads, respectively. No binding occurs in the absence of free peptide. This additivity cannot be attributed to the formation of a B-E complex mimicking the full-length p9CKShs2. The cyclin B subunit is not required for the formation of the p9CKShs2-p34cdc2 complex through these two binding domains. The implications of the existence of two cooperative p34cdc2-binding domains in p9CKShs2 on the structure of the active M-phase-specific kinase is discussed.

Amino Acid Sequence↗
PubMed1991

Allene oxide and aldehyde biosynthesis in starfish oocytes.

Allene oxides are a very unusual type of epoxide that, in biological systems, are formed by the enzymic dehydration of fatty acid hydroperoxides (lipoxygenase products). This reaction occurs widely in plants, in which allene oxide synthesis is a key step in the conversion of linolenic acid to jasmonic acid, the plant growth regulator. We report biosynthesis of the allene oxide (8R)-8,9-epoxyeicosa-(5Z,9,11Z,14Z)-tetraenoic acid via the (8R)-lipoxygenase metabolism of arachidonic acid in starfish oocytes. Formation of the allene oxide was deduced from high pressure liquid chromatography, UV, gas chromatography-mass spectrometry and 1H-NMR analyses of the precise structure and mechanism of biosynthesis of its major hydrolysis product, the alpha-ketol 8-hydroxy-9-ketoeicosa-(5Z,11Z,14Z)-trienoic acid. A second enzymic activity detected in the oocytes (hydroperoxide lyase) cleaves specifically the (8R)-hydroperoxy substrate into C7 and C13 fragments, identified as the hydroxyacid, (5Z)-7-hydroxyheptenoic acid, and two aldehydes, (2E,4Z,7Z)-tridecenal and its 4E isomer. Discovery of the allene oxide synthase and hydroperoxide lyase marks the first definitive localization of these enzymic activities to an animal cell. It was established previously that the (8R)-lipoxygenase metabolite (8R)-HETE will activate the maturation (re-initiation of meiosis) of starfish oocytes. The individual 8-lipoxygenase products may be involved at distinct stages of cell development.

Aldehydes↗
PubMed1991

High-mobility-group proteins P1, I and Y as substrates of the M-phase-specific p34cdc2/cyclincdc13 kinase.

All dividing cells entering the M phase of the cell cycle undergo the transient activation of an M-phase-specific histone H1 kinase which was recently shown to be constituted of at least two subunits, p34cdc2 and cyclincdc13. The DNA-binding high-mobility-group (HMG) proteins 1, 2, 14, 17, I, Y and an HMG-like protein, P1, were investigated as potential substrates of H1 kinase. Among these HMG proteins, P1 and HMG I and Y are excellent substrates of the M-phase-specific kinase obtained from both meiotic starfish oocytes and mitotic sea urchin eggs. Anticyclin immunoprecipitates, extracts purified on specific p34cdc2-binding p13suc1-Sepharose and affinity-purified H1 kinase display strong HMG I, Y and P1 phosphorylating activities, demonstrating that the p34cdc2/cyclincdc13 complex is the active kinase phosphorylating these HMG proteins. HMG I and P1 phosphorylation is competitively inhibited by a peptide mimicking the consensus phosphorylation sequence of H1 kinase. HMG I, Y and P1 all possess the consensus sequence for phosphorylation by the p34cdc2/cyclincdc13 kinase (Ser/Thr-Pro-Xaa-Lys/Arg). HMG I is phosphorylated in vivo at M phase on the same sites phosphorylated in vitro by H1 kinase. P1 is phosphorylated by H1 kinase on sites different from the sites of phosphorylation by casein kinase II. The three thermolytic phosphopeptides of P1 phosphorylated in vitro by purified H1 kinase are all present in thermolytic peptide maps of P1 phosphorylated in vivo in proliferating HeLa cells. These phosphopeptides are absent in nonproliferating cells. These results demonstrate that the DNA-binding proteins HMG I, Y and P1 are natural substrates for the M-phase-specific protein kinase. The phosphorylation of these proteins by p34cdc2/cyclincdc13 may represent a crucial event in the intense chromatin condensation occurring as cells transit from the G2 to the M phase of the cell cycle.

Animals↗
PubMed1991

Cyclin B targets p34cdc2 for tyrosine phosphorylation.

A universal intracellular factor, the 'M phase-promoting factor' (MPF), triggers the G2/M transition of the cell cycle in all organisms. In late G2, it is present as an inactive complex of tyrosine-phosphorylated p34cdc2 and unphosphorylated cyclin Bcdc13. In M phase, its activation as an active MPF displaying histone H1 kinase (H1K) originates from the concomitant tyrosine dephosphorylation of the p34cdc2 subunit and the phosphorylation of the cylin Bcdc13 subunit. We have investigated the role of cyclin in the formation of this complex and the tyrosine phosphorylation of p34cdc2, using highly synchronous mitotic sea urchin eggs as a model. As cells leave the S phase and enter the G2 phase, a massive tyrosine phosphorylation of p34cdc2 occurs. This large p34cdc2 tyrosine phosphorylation burst does not arise from a massive increase in p34cdc2 concentration. It even appears to affect only a fraction (non-immunoprecipitable by anti-PSTAIR antibodies) of the total p34cdc2 present in the cell. Several observations point to an extremely close association between accumulation of unphosphorylated cyclin and p34cdc2 tyrosine phosphorylation: (i) both events coincide perfectly during the G2 phase; (ii) both tyrosine-phosphorylated p34cdc2 and cyclin are not immunoprecipitated by anti-PSTAIR antibodies; (iii) accumulation of unphosphorylated cyclin by aphidicolin treatment of the cells, triggers a dramatic accumulation of tyrosine-phosphorylated p34cdc2; and (iv) inhibition of cyclin synthesis by emetine inhibits p34cdc2 tyrosine phosphorylation without affecting the p34cdc2 concentration. These results show that, as it is synthesized, cyclin B binds and recruits p34cdc2 for tyrosine phosphorylation; this inactive complex then requires the completion of DNA replication before it can be turned into fully active MPF. These results fully confirm recent data obtained in vitro with exogenous cyclin added to cycloheximide-treated Xenopus egg extracts.

Animals↗
PubMed1990

Activation of M-phase-specific histone H1 kinase by modification of the phosphorylation of its p34cdc2 and cyclin components.

An M-phase-specific histone H1 kinase (H1K) has been described in a wide variety of eukaryotic cell types undergoing the G2/M transition in the cell division cycle. We have used p13suc1-Sepharose affinity chromatography to purify H1K to near homogeneity from matured starfish oocytes. A yield of 67% was obtained. Active H1K behaves as a 90- to 100-kD protein and appears to be constituted of equimolar amounts of cyclin and p34cdc2. The p34cdc2 subunit becomes tyrosine-dephosphorylated as the H1K is activated during entry of the oocytes into M phase, whereas the cyclin subunit is reciprocally phosphorylated. Acid phosphatase treatment of inactive p34cdc2/cyclin complex induces p34cdc2 dephosphorylation and three- to eightfold stimulation of the enzyme activity. These results suggest that active M-phase-specific H1K is constituted of both dephosphorylated p34cdc2 and phosphorylated cyclin.

Acid Phosphatase↗
PubMed1989

Cyclin is a component of the sea urchin egg M-phase specific histone H1 kinase.

A so-called 'growth-associated' or 'M-phase specific' histone H1 kinase (H1K) has been described in a wide variety of eukaryotic cell types; p34cdc2 has previously been shown to be a catalytic subunit of this protein kinase. In fertilized sea urchin eggs the activity of H1K oscillates during the cell division cycle and there is a striking temporal correlation between H1K activation and the accumulation of a phosphorylated form of cyclin. H1K activity declines in parallel with proteolytic cyclin destruction of the end of the first cell cycle. By virtue of the high affinity of the fission yeast p13suc1 for the p34cdc2 protein, H1K strongly binds to p13-Sepharose beads. Cyclin, p34cdc2 and H1K co-purify on this affinity reagent as well as through several conventional chromatographic procedures. Anticyclin antibodies immunoprecipitate the M-phase specific H1K in crude extracts or in purified fractions. Sea urchin eggs appear to contain much less cyclin than p34cdc2, suggesting that p34cdc2 may interact with other proteins. These results demonstrate that cyclin and p34cdc2 are major components of the M-phase specific H1K.

Animals↗
PubMed1989

Starfish oocyte maturation: evidence for a cyclic AMP-dependent inhibitory pathway.

Oocyte maturation (meiosis reinitiation) in starfish is induced by the natural hormone 1-methyladenine (1-MeAde). Cyclic AMP seems to play a negative role in maturation since 1-MeAde triggers a decrease of the oocyte cAMP concentration and since intracellular microinjections of cAMP delay or inhibit maturation. Cyclic GMP is also inhibitory but other nucleotides such as cCMP, cIMP, and cUMP are inactive. The involvement of cAMP and cGMP in the control of oocyte maturation has been further investigated by the use of the stereoisomers of the phosphodiesterase-stable adenosine and guanosine 3',5'-phosphorothioates (cAMPS and cGMPS). The Sp isomers of cAMPS and cGMPS respectively activate cAMP-dependent protein kinase and cGMP-dependent kinase, while the Rp isomers inhibit the kinases. Extracellular addition of these cAMPS and cGMPS isomers has no effect on the oocytes. Intracellular microinjection of the kinase-activating (Sp)-cAMPS and (Sp)-cGMPS delays or inhibits 1-MeAde-induced maturation in a concentration-dependent manner (I50, 30 and 300 microM, respectively). Microinjections of (Rp)-cAMPS and (Rp)-cGMPS have no inhibitory effects and neither trigger nor facilitate maturation. Using various analogs, we found that the delaying or inhibiting effect is restricted to the compounds activating cAMP-dependent kinase, while the compounds inactive on or inhibiting the kinase have no effects on maturation. The inhibitory effect of (Sp)-cAMPS can be reversed by comicroinjection of the heat-stable inhibitor of cAMP-dependent protein kinase, by comicroinjection of the antagonist (Rp)-cAMPS, or by an increase in the 1-MeAde concentration. The negative effects of (Sp)-cAMPS or (Sp)-cGMPS are observed only when these isomers are microinjected during the hormone-dependent period. These results suggest that a cAMP-dependent inhibitory pathway participates in the maintenance of the prophase arrest of oocytes and that 1-MeAde acts both by inhibiting this negative pathway (dis-inhibitory pathway) and by stimulating a parallel activatory pathway leading to oocyte maturation. The generality of this mechanism is discussed.

Adenine↗