Biomedical subjects

B Ducommun

Publications and source records attributed to B Ducommun.

At least 37 records · Page 2Linked to original sources

PubMed1998

Identification of a fission yeast dynamin-related protein involved in mitochondrial DNA maintenance.

Members of the dynamin-related proteins family have been identified in a wide range of organisms, however their precise functions remain elusive. We have identified a new member of that GTPase family in the fission yeast Schizosaccharomyces pombe. We show that Msp1+ is an essential nuclear gene encoding a 101 kDa protein whose closest homologue is the S. cerevisiae MGM1 gene product. We also report that msp1 conditional loss of function affects the maintenance of mitochondrial DNA and leads to growth arrest associated with respiratory deficiency.

Adenosine Triphosphatases↗

PubMed1998

p21 binding to PCNA causes G1 and G2 cell cycle arrest in p53-deficient cells.

A unique feature of p21 that distinguishes it from the other cyclin-dependent kinase (CDK) inhibitors is its ability to associate with the proliferating cell nuclear antigen (PCNA), an auxiliary factor for DNA polymerases delta and epsilon. While it is now well established that inhibition of cyclin/CDK complexes by p21 can result in G1 cell cycle arrest, the consequences of p21/PCNA interaction on cell cycle progression have not yet been determined. Here, we show, using a tetracycline-regulated system, that expression of wild-type p21 in p53-deficient DLD1 human colon cancer cells inhibits DNA synthesis and causes G1 and G2 cell cycle arrest. Similar effects are observed in cells expressing p21CDK-, a mutant impaired in the interaction with CDKs, but not in cells expressing p21PCNA-, a mutant deficient for the interaction with PCNA. Analysis of cells treated with a p21-derived PCNA-binding peptide provides additional evidence that the growth inhibitory effects of p21 and p21CDK result from their ability to bind to PCNA. Our results suggest that p21 might inhibit cell cycle progression by two independent mechanisms, inhibition of cyclin/CDK complexes, and inhibition of PCNA function resulting in both G1 and G2 arrest.

Amino Acid Sequence↗

PubMed1997

Phosphorylation of human CDC25B phosphatase by CDK1-cyclin A triggers its proteasome-dependent degradation.

In eukaryotes the activity of CDK1 (CDC2), a cyclin-dependent kinase that initiates the structural changes that culminate in the segregation of chromosomes at mitosis, is regulated by the synergistic and opposing activities of a cascade of kinases and phosphatases. Dephosphorylation of threonine 14 and tyrosine 15 of CDK1 by the CDC25 phosphatases is a key step in the activation of the CDK1-cyclin B protein kinase. Little is currently known about the role and the regulation of CDC25B. Here we report in vitro and in vivo data that indicate that CDC25B is degraded by the proteasome. This degradation is dependent upon phosphorylation by the CDK1-cyclin A complex but not by CDK1-cyclin B. These results indicate that CDK1-cyclin A phosphorylation targets CDC25B for degradation and that this might be an important component of cell cycle regulation at the G2/M transition.

CDC2 Protein Kinase↗

PubMed1997

Use of the two-hybrid system to identify protein-protein interaction temperature-sensitive mutants: application to the CDK2/p21Cip1 interaction.

We describe the application of the two-hybrid system to the identification of protein-protein interaction temperature-sensitive mutants. We applied this strategy to the interaction between the human CDK2 cell cycle regulator and the p21Cip1 regulatory subunit. A library of randomly generated CDK2 mutant proteins was screened for interaction with p21Cip1 at different temperatures. This approach resulted in the isolation of single point mutations in CDK2 causing temperature-sensitive interaction with p21Cip1. Our results demonstrate that the two-temperature two-hybrid screen is an efficient approach for the rational design and screening of protein-protein interaction conditional mutations.

CDC2-CDC28 Kinases↗

PubMed1997

Evidence for a mammalian Nim1-like kinase pathway acting at the G0-1/S transition.

In fission yeast the Nim1 kinase phosphorylates and inactivates the cdc2-inhibitory Weel tyrosine kinase. In addition, Nim1 is necessary for an efficient cellular response to nutritional starvation leading to cell cycle arrest in G1. Given the remarkable evolutionary conservation of the cell cycle regulatory mechanism we have investigated the effect of Nim1 expression on the control of the mammalian cell cycle using a plasmid microinjection approach. In synchronised IMR90 human fibroblasts, expression of Nim1 strongly inhibited entry into S-phase. This effect was dependent on the catalytic activity of Nim1 and did not require its regulatory domain. Furthermore we show that co-expression of human Wee1 kinase reverted the inhibitory effect, indicating that Nim1 was acting in a Wee1-dependent manner. These results provide evidence for the existence of a Nim1-like kinase pathway acting at the G0-1/S transition in human cells.

Fibroblasts↗

PubMed1997

Alternative splicing of the human CDC25B tyrosine phosphatase. Possible implications for growth control?

CDC25B2, a protein tyrosine phosphatase closely related to the putative CDC25B oncogene, was identified in a Burkitt lymphoma cDNA library. CDC25B2 differs from CDC25B by a 14 residue insertion and a 41 residue deletion, which are both located in the amino-terminal region of the protein, upstream of the catalytic domain. Examination of the genomic sequence revealed that CDC25B1 (formerly B) and CDC25B2 are splice variants of the same gene. A third variant, CDC25B3, that carries both the 14 and the 41 residue sequences was also identified in the same cDNA library. All three variants were detected in a panel of human primary culture and cell lines, although at different levels. In primary fibroblasts and in HeLa cells the CDC25B expression is cell cycle regulated, reaching a maximum in G2-phase. In vitro, CDC25B1 phosphatase is slightly more active than CDC25B2 and B3. However, episomal overexpression of the three CDC25B variants in fission yeast reveals that in vivo, CDC25B2 is largely more active than either B1 or B3 (B2>B3>B1) both to complement a thermosensitive S pombe CDC25 activity and to act as a mitotic inducer. Alternative splicing of CDC25B may therefore contribute to the control of cell proliferation.

Alternative Splicing↗

PubMed1997

Role of the fission yeast nim 1 protein kinase in the cell cycle response to nutritional signals.

The fission yeast cdr1/nim1 protein kinase phosphorylates and inactivates the weel cdc2-inhibitory kinase. We have investigated the role played by cdr1/nim1 in the connection between nutritional signals and the cell cycle machinery. We show that loss of nim1 activity impairs the appropriate cellular adaptation to nutritional changes. However, the reduction in cell size at division in response to nitrogen starvation is independent of nim1. Moreover, we report that nim1 is an unstable protein that is rapidly degraded upon starvation, through a mechanism that is dependent upon protein synthesis. We propose that nim1, as a constitutive indirect activator of cdc2 at mitosis, favors the cellular response to starvation but does not actively participate in it. On the contrary, upon nitrogen starvation nim1 must be actively destroyed to protect the cells from a commitment into the cell cycle under unfavourable growth conditions.

Cell Cycle↗

PubMed1997

Escherichia coli cytolethal distending toxin blocks the HeLa cell cycle at the G2/M transition by preventing cdc2 protein kinase dephosphorylation and activation.

Cytolethal distending toxins (CDT) constitute an emerging heterogeneous family of bacterial toxins whose common biological property is to inhibit the proliferation of cells in culture by blocking their cycle at G2/M phase. In this study, we investigated the molecular mechanisms underlying the block caused by CDT from Escherichia coli on synchronized HeLa cell cultures. To this end, we studied specifically the behavior of the two subunits of the complex that determines entry into mitosis, i.e., cyclin B1, the regulatory unit, and cdc2 protein kinase, the catalytic unit. We thus demonstrate that CDT causes cell accumulation in G2 and not in M, that it does not slow the progression of cells through S phase, and that it does not affect the normal increase of cyclin B1 from late S to G2. On the other hand, we show that CDT inhibits the kinase activity of cdc2 by preventing its dephosphorylation, an event which, in normal cells, triggers mitosis. This inhibitory activity was demonstrated for the three partially related CDTs so far described for E. coli. Moreover, we provide evidence that cells exposed to CDT during G2 and M phases are blocked only at the subsequent G2 phase. This observation means that the toxin triggers a mechanism of cell arrest that is initiated in S phase and therefore possibly related to the DNA damage checkpoint system.

Bacterial Toxins↗

PubMed1996

Interaction studies between the p21Cip1/Waf1 cyclin-dependent kinase inhibitor and proliferating cell nuclear antigen (PCNA) by surface plasmon resonance.

The cyclin-dependent kinase (CDK) inhibitor p21Cip1 consists of two domains that interact with CDKs and proliferating cell nuclear antigen (PCNA), respectively. We have investigated the interaction between p21Cip1 and PCNA using surface plasmon resonance (SPR) technology and compared the results with those obtained from other sources such as the yeast two-hybrid system. Whilst other methods are only semi-quantitative, the SPR technique allowed us to determine the kinetic parameters of the interaction. The apparent equilibrium constant KD calculated for these kinetic parameters was 3.2 x 10(-7) M. We further demonstrate the use of SPR to study the interaction between mutant proteins and to determine their actual KD. The interaction between p21Cip1/PCNA is shown to be dependent upon the trimeric conformation of PCNA since a point mutant that abolishes PCNA-PCNA interaction also abolishes PCNA's interaction with p21Cip1. Finally, we demonstrate that SPR can be used to characterise the interaction of p21Cip1 and PCNA in the presence of short competitive peptides.

Amino Acid Sequence↗

PubMed1996

Characterisation of human cdc2 lysine 33 mutations expressed in the fission yeast Schizosaccharomyces pombe.

The mammalian p34cdc2 protein kinase, a universal cell cycle regulator, complements cdc2/CDC28 temperature-sensitive mutations in yeasts. We report the biochemical characterisation of two substitutions of human cdc2 at lysine 33, a residue involved in nucleotide binding, that differently alter the fission yeast cell cycle. K33A-hscdc2 and K33R-hscdc2 mutants are both catalytically inactive, but overexpression of K33R-cdc2 is lethal while K33A-cdc2 is not. We show that human K33R-cdc2 acts as a dominant negative allele that associates yeast cdc13/cyclinB and therefore renders endogeneous Schizosaccharomyces pombe cdc2 unactivatable. These results are discussed on the light of the molecular modeling of the mutants in the cdc2 model structure.

Betaine↗

PubMed1996

Heterologous expression of the human cyclin-dependent kinase inhibitor p21Cip1 in the fission yeast, Schizosaccharomyces pombe reveals a role for PCNA in the chk1+ cell cycle checkpoint pathway.

Fission yeast cells expressing the human gene encoding the cyclin-dependent kinase inhibitor protein p21Cip1 were severely compromised for cell cycle progress. The degree of cell cycle inhibition was related to the level of p21Cip1 expression. Inhibited cells had a 2C DNA content and were judged by cytology and pulsed field gel electrophoresis to be in the G2 phase of the cell cycle. p21Cip1 accumulated in the nucleus and was associated with p34cdc2 and PCNA. Thus, p21Cip1 interacts with the same targets in fission yeast as in mammalian cells. Elimination of p34cdc2 binding by mutation within the cyclin-dependent kinase binding domain of p21Cip1 exaggerated the cell cycle delay phenotype. By contrast, elimination of PCNA binding by mutation within the PCNA-binding domain completely abolished the cell cycle inhibitory effects. Yeast cells expressing wild-type p21Cip1 and the mutant form that is unable to bind p34cdc2 showed enhanced sensitivity to UV. Cell cycle inhibition by p21Cip1 was largely abolished by deletion of the chk1+ gene that monitors radiation damage and was considerably enhanced in cells deleted for the rad3+ gene that monitors both DNA damage and the completion of DNA synthesis. Overexpression of PCNA also resulted in cell cycle arrest in G2 and this phenotype was also abolished by deletion of chk1+ and enhanced in cells deleted for rad3+. These results formally establish a link between PCNA and the products of the rad3+ and chk1+ checkpoint genes.

Amino Acid Sequence↗

PubMed1995

Mechanism of inhibition of proliferating cell nuclear antigen-dependent DNA synthesis by the cyclin-dependent kinase inhibitor p21.

It is known that the direct binding of the cyclin-dependent kinase (Cdk) inhibitor p21, also called Cdk-interacting protein 1 (p21), to proliferating cell nuclear antigen (PCNA) results in the inhibition of PCNA-dependent DNA synthesis. We provide evidence that p21 first inhibits the replication factor C-catalyzed loading of PCNA onto DNA and second prevents the binding of DNA polymerase delta core to the PCNA clamp assembled on DNA. The second effect contributes most to the inhibition of pol delta holoenzyme activity. p21 primarily inhibited the DNA synthesis resulting from multiple reassembly of DNA polymerase delta holoenzyme. On the other hand, an ability of the PCNA clamp to translocate along double-stranded DNA was not affected by p21. These data were confirmed with a mutant of p21 that is unable to bind PCNA and therefore neither inhibited clamp assembly nor prevented the loading of DNA polymerase delta core onto DNA. Our data suggest that p21 does not discriminate in vitro "repair" and "replication" DNA synthesis based on template length but does act preferentially on polymerization which encounters obstacles to progress.

Binding, Competitive↗

PubMed1995

Identification of binding domains on the p21Cip1 cyclin-dependent kinase inhibitor.

Members of the recently discovered family of cyclin-dependent kinases inhibitors (CKIs) appear to play an essential regulatory role in the control of cell proliferation. To investigate the molecular basis of the interaction between these proteins and the cyclin-dependent kinases (CDKs), we performed a systematic mutagenesis of the CKI family member p21Cip1 using the alanine-scanning strategy. We have examined the interaction between in vitro translated human cdk2, cyclins A and D1, purified proliferating cell nuclear antigen (PCNA) and a set of human p21Cip1 mutants fused to glutathione S-transferase. Independent domains that are required for the interaction with cdk2 and with PCNA have been identified. The cdk2 binding domain is located in the N-terminal part of the protein, between residues 45 and 60, a region that is fully conserved in the p27Kip1 inhibitor. A PCNA binding region was localised to the C-terminus of the protein, between residues 142 and 163. These findings define protein motifs that are highly conserved between members of the CKI family and that are likely to play an essential function in the regulation of the G1/S transition.

Amino Acid Sequence↗

PubMed1995

Effects of TGF-beta 1 (transforming growth factor-beta 1) on the cell cycle regulation of human breast adenocarcinoma (MCF-7) cells.

The antiproliferative effects of TGF-beta 1 were investigated in a human breast adenocarcinoma cell line (MCF-7). We report that TGF-beta 1 inhibits proliferation through cell cycle arrest in G1. A MCF-7 cell subline (MCF-7(-)), in which the type II TGF-beta receptor is not detected, was shown to be resistant to TGF-beta 1 growth inhibitory effect. Cdk2 kinase activity was inhibited in the MCF-7 sensitive cell subline in parallel with the inhibition of cell cycle progression. In both sensitive and resistant cell lines, TGF-beta 1 treatment did not affect cdk2, cdk4, cyclin E and cyclin D1 mRNA and protein levels. However, in the MCF-7 sensitive cell subline, a time-dependent increase in cells positive for p21WAF1/CIP1 nuclear localization was observed after TGF-beta 1 treatment. These findings suggest that TGF-beta 1 inhibition of MCF-7 cell proliferation is achieved through a type II receptor-dependent down-regulation of Cdk2 kinase activity without modification of Cdk and cyclin expression, but correlated with an increase in p21WAF1/CIP1 nuclear accumulation.

Adenocarcinoma↗

PubMed1995

Effects of phleomycin-induced DNA damage on the fission yeast Schizosaccharomyces pombe cell cycle.

The effect of phleomycin, a bleomycin-like antibiotic, has been investigated in the fission yeast, Schizosaccharomyces pombe. We report that in response to phleomycin-induced DNA damage, growth was inhibited and S. pombe cells arrested in the G2-phase of the cell cycle. DNA repair mutants rad9 and rad17 did not arrest and were hypersensitive to phleomycin. Cell cycle mutants that entered mitosis without monitoring the completion of DNA replication also displayed an increased sensitivity to this DNA-damaging agent. Thus, phleomycin could be used as a tool in the fission yeast S. pombe model system for the study of DNA damage and cell cycle checkpoints, or as a new selective agent.

Cell Cycle↗

PubMed1995

The fission yeast Nim1/Cdr1 kinase: a link between nutritional state and cell cycle control.

Close connections appear to exist between extra-cellular signals that regulate cell proliferation and the protein kinases that control the cell cycle machinery. The fission yeast nim1 kinase is an inducer of cdc2 kinase activity acting through the inhibition of wee1 kinase. Nim1 function is required for a correct cellular response to nutritional starvation. In the absence of nim1, starved cells are unable to decrease their size at mitosis, to arrest their cycle in G1 and to enter G0. Here, we review our current knowledge on the role and the regulation of nim1 in connecting cell cycle and nutritional pathways.

CDC2 Protein Kinase↗

PubMed1995

Effect of phenylarsine oxide on the fission yeast Schizosaccharomyces pombe cell cycle.

Phosphotyrosyl turnover is an essential regulatory mechanism for many biological processes, and the balance between tyrosine kinases and phosphatases plays a major role in the control of cell proliferation. Phenylarsine oxide (PAO), a potent inhibitor of tyrosine phosphatases (PTPase), was used to investigate the involvement of PTPase in the growth and control of the cell cycle of the fission yeast Schizosaccharomyces pombe. Cell proliferation was arrested by treatment with PAO, which was found to inhibit cdc25 PTPase in vitro but appeared not to act in vivo on this mitosis inducer. The PAO-treated cells displayed a mono- or binucleated phenotype and a DNA content that was either 2C or 4C, indicating a cell cycle arrest with a failure to complete cytokinesis. Entry into the cell division cycle from the G0 quiescent stage was also delayed by treatment with PAO. These results suggest that a number of key events in the mitotic cell cycle are regulated by as yet unidentified PTPases.

Arsenicals↗

PubMed1995

Subcellular localisation of human wee1 kinase is regulated during the cell cycle.

Wee1 kinase-dependent phosphorylation of cdc2 maintains the cdc2/cyclin B complex in an inert form until it is activated by the cdc25 tyrosine phosphatase at the end of G2. As described for cdc25, cell cycle-linked changes in the intracellular localisation of wee1 may constitute an important aspect of the temporal regulation of cdc2 activity. Here we report that the subcellular distribution of human wee1 changes during the cell cycle in HeLa and IMR90 cells. During interphase, wee1 is found almost exclusively in the nucleus. When the cell enters mitosis, wee1 is relocalised into the cytoplasm. During cell division, wee1 becomes restricted to the mitotic equator and by the end of mitosis it is found exclusively in association with midbody bridges, a phenomenon that is dependent on microtubule assembly. The relocalisations of wee1 and its association with subcellular structures may play key regulatory roles at different stages of the cell cycle and during mitosis.

Biological Transport↗