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D Beach

Publications and source records attributed to D Beach.

At least 19 recordsLinked to original sources
PubMed1992

D type cyclins associate with multiple protein kinases and the DNA replication and repair factor PCNA.

Human cyclin D1 has been associated with a wide variety of proliferative diseases but its biochemical role is unknown. In diploid fibroblasts we find that cyclin D1 is complexed with many other cellular proteins. Among them are protein kinase catalytic subunits CDK2, CDK4 (previously called PSK-J3), and CDK5 (also called PSSALRE). In addition, polypeptides of 21 kd and 36 kd are identified in association with cyclin D1. We show that the 36 kd protein is the proliferating cell nuclear antigen, PCNA. Cyclin D3 also associates with multiple protein kinases, p21 and PCNA. It is proposed that there exists a quaternary complex of D cyclin, CDK, PCNA, and p21 and that many combinatorial variations (cyclin D1, D3, CDK2, 4, and 5) may assemble in vivo. These findings link a human putative G1 cyclin that is associated with oncogenesis with a well-characterized DNA replication and repair factor.

Amino Acid Sequence↗
PubMed1992

Growth-regulated expression of D-type cyclin genes in human diploid fibroblasts.

The human CCND1 cyclin D1/PRAD1 gene was previously identified by a genetic screen for G1 cyclin function in Saccharomyces cerevisiae and also was identified as the putative BCL1 oncogene. However, its role in human cell proliferation is not known. To determine if expression of human D-type cyclin genes correlates with the state of cell growth, we examined the level of mRNAs for CCND1 and a related gene, CCND3, in normal human diploid fibroblasts (HDF). The levels of both mRNAs decrease upon serum depletion or at high cell densities. Following stimulation of quiescent fibroblasts with serum, the mRNA levels increase gradually to a peak at about 12 hr, prior to the onset of S phase. Induction of cyclin gene expression by serum is reduced concomitantly with the decline in FOS induction in aging HDFs, suggesting a possible relationship to the decrease in the proliferative response to mitogens during cellular senescence. Cycloheximide partially blocks the induction of CCND1 and CCND3 gene expression by serum, suggesting that both de novo protein synthesis-dependent and -independent pathways contribute to induction. Treatment of HDFs with defined growth factors suggests a correlation between CCND mRNA induction and DNA synthesis. However, induction of these genes is not sufficient for the transition from quiescence through G1 into S phase.

Blood↗
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

Molecular cloning and chromosomal mapping of CCND genes encoding human D-type cyclins.

A human D-type cyclin gene (CCND1/cyclin D1/PRAD1) was previously isolated by virtue of its ability to complement a triple G1 cyclin (Cln) deficiency of Saccharomyces cerevisiae and was also identified as a candidate BCL1 oncogene. We now report the molecular cloning of two additional human D-type cyclin genes, CCND2 (cyclin D2) and CCND3 (cyclin D3). All three human D-type cyclin genes encode small (33-34 kDa) proteins that share an average of 57% identity over the entire coding region and 78% in the cyclin box. The D-type cyclins are most closely related to cyclin A (39% identity) and cyclin E (36%), followed by cyclin B (29%) and cyclin C (21%). Isolation and characterization of genomic clones revealed two pseudogenes corresponding to CCND2 and CCND3, respectively. All three cyclin D genes are interrupted by an intron at the same position. CCND2 has been mapped to chromosome 12p13, and CCND3 has been mapped to chromosome 6p21.

Amino Acid Sequence↗
PubMed1992

Human p53 inhibits growth in Schizosaccharomyces pombe.

Overexpression of wild-type p53 in mammalian cells blocks growth. We show here that the overexpression of wild-type human p53 in the fission yeast Schizosaccharomyces pombe also blocks growth, whereas the overexpression of mutant forms of p53 does not. The p53 polypeptide is located in the nucleus and is phosphorylated at both the cdc2 site and the casein kinase II site in S. pombe. A new dominant mutation of p53, resulting in the change of a cysteine to an arginine at amino acid residue 141, was identified. The results presented here demonstrate that S. pombe could provide a simple system for studying the mechanism of action of human p53.

Alleles↗
PubMed1991

Specific activation of cdc25 tyrosine phosphatases by B-type cyclins: evidence for multiple roles of mitotic cyclins.

Two previously unidentified human cdc25 genes have been isolated, cdc25A and cdc25B. Both genes rescue a cdc25ts mutant of fission yeast. Microinjection of anti-cdc25A antibodies into HeLa cells causes their arrest in mitosis. cdc25A and cdc25B display endogenous tyrosine phosphatase activity that is stimulated several-fold, in the absence of cdc2, by stoichiometric addition of either cyclin B1 or B2 but not A or D1. Association between cdc25A and cyclin B1/cdc2 was detected in the HeLa cells. These findings indicate that B-type cyclins are multifunctional proteins that not only act as M phase regulatory subunits of the cdc2 protein kinase, but also activate the cdc25 tyrosine phosphatase, of which cdc2 is the physiological substrate. A region of amino acid similarity between cyclins and tyrosine PTPases has been detected. This region is absent in cdc25 phosphatases. The motif may represent an activating domain that has to be provided to cdc25 by intermolecular interaction with cyclin B.

Amino Acid Sequence↗
PubMed1991

Cell cycle regulation of histone H1 kinase activity associated with the adenoviral protein E1A.

Several cellular proteins form stable complexes with the proteins encoded by the adenovirus early region 1A (E1A) gene in extracts derived from adenovirus infected or transformed cells. Two of the cellular proteins that bind to E1A have been identified; one, a 105-kilodalton protein (pRb), is the product of the retinoblastoma gene, and the other, a 60-kilodalton protein, is a human cyclin A. Two other proteins that bind E1A have now been shown to be related to p34cdc2. This E1A complex displayed histone H1-specific kinase activity; the kinase activity was modulated during the cell division cycle, and association of pRb with E1A apparently was not required for this activity.

Adenovirus Early Proteins↗
PubMed1991

Premature initiation of mitosis in yeast lacking RCC1 or an interacting GTPase.

A fission yeast mutant is described in which the onset of mitosis is uncoupled from the completion of DNA replication. pim1 (premature initiation of mitosis) cells can undergo mitotic chromosome condensation and mitotic spindle formation without completion of S phase and without the cdc25 mitotic inducer. The M phase kinase is required for pim1-induced mitosis and becomes activated. pim1 encodes a homolog of the human RCC1 nuclear protein. pim1 mutants are fully rescued by overexpression of spi1, a newly identified essential gene whose predicted product shares 81% identity with human TC4. spi1 and TC4 define a new subclass within the "ras-like" GTPase superfamily that is structurally distinct from the ras, rho, or sec4 families. Diploid yeast that carry one wild-type and one disrupted copy of spi1 have multiple satellite nuclei, and mitotic haploidization occurs at very high frequency. spi1 appears to interact with pim1 in the maintenance of a coordinated cell cycle.

Amino Acid Sequence↗
PubMed1991

Human D-type cyclin.

A cDNA library prepared from a human glioblastoma cell line has been introduced into a budding yeast strain that lacks CLN1 and CLN2 and is conditionally deficient for CLN3 function. We rescued a gene that we call cyclin D1. It is related to A-, B-, and CLN-type cyclins, but appears to define a new subclass within the cyclin gene family. Transcription of the cyclin D1 gene gives rise to two major transcripts through alternative polyadenylation. The cyclin D1 gene transcript and its 34 kd product are both abundant in the glioblastoma cell line of origin.

Amino Acid Sequence↗
PubMed1991

mik1 and wee1 cooperate in the inhibitory tyrosine phosphorylation of cdc2.

wee1 acts antagonistically to cdc25 in the tyrosine dephosphorylation and activation of cdc2, yet biochemical evidence suggests that wee1 is not required for tyrosine phosphorylation and its role is obscure. We show here that a related 66 kd kinase, called mik1, acts redundantly with wee1 in the negative regulation of cdc2 in S. pombe. A null allele of mik1 has no discernible phenotype, but a mik1 wee1 double mutant is hypermitotically lethal: all normal M phase checkpoints are bypassed, including the requirement for initiation of cell cycle "start," completion of S phase, and function of the cdc25+ mitotic activator. In the absence of mik1 and wee1 activity, cdc2 rapidly loses phosphate on tyrosine, both in strains undergoing mitotic lethality and in those that are viable owing to a compensating mutation within cdc2. The data suggest that mik1 and wee1 act cooperatively on cdc2, either directly as the inhibitory tyrosine kinase or as essential activators of that kinase.

Amino Acid Sequence↗
PubMed1991

Interaction between ran1+ protein kinase and cAMP dependent protein kinase as negative regulators of fission yeast meiosis.

In fission yeast, meiosis is initiated by transcriptional activation of the mei3+ gene under the combined influence of the four mating type genes. The mei3+ gene product acts as a meiotic inducer by binding to and inhibiting the ran1+ protein kinase. Inactivation of ran1+ kinase is both necessary and sufficient to allow meiotic differentiation. We describe a class of mutants which are unable to undergo both normal meiosis and meiosis induced by inactivation of ran1+. In addition to these defects, the cells are sterile and unable to enter stationary phase. We have determined that the mutants define two complementation groups, designated cgs1+ and cgs2+ (continues to grow in stationary). The wild type allele of each gene has been isolated and sequence analysis of cgs1+ shows that it encodes a protein homologous to the regulatory subunit of cyclic AMP dependent protein kinase (cAPK). Biochemical studies demonstrate that in cgs1-1 containing cells, cAPK activity is unregulated by cyclic AMP (cAMP). Sequence analysis of cgs2+ shows that the predicted protein it encodes shares homology with a phosphodiesterase from Dictyostelium discoideum and biochemical studies demonstrate that cells containing a mutant allele of cgs2+ have elevated levels of cAMP. Thus, both genes encode proteins that regulate the activity of cAPK. We have previously shown that cells overproducing ran1+ kinase are meiotically defective. Here, we provide direct evidence that the meiotic defect caused by either unregulated cAPK activity or unregulated ran1+ kinase activity is due to inability to induce transcription of the mei2+ gene, which is required for meiotic initiation. We propose that the switch from vegetative growth to meiosis in fission yeast requires inactivation of ran1+ kinase and is prevented by unregulated levels of cAPK.

Amino Acid Sequence↗
PubMed1991

A cdc2-like kinase phosphorylates histone H1 in the amitotic macronucleus of Tetrahymena.

Genetic and biochemical studies have shown that cdc2 protein kinase plays a pivotal role in a highly conserved mechanism controlling the entry of cells into mitosis. It is generally believed that one function of cdc2 kinase is to phosphorylate histone H1 which in turn promotes mitotic chromosome condensation. However, direct evidence linking H1 phosphorylation to mitotic chromatin condensation is limited and the exact cellular function(s) of H1 phosphorylation remains unclear. In this study, we show that mammalian cdc2 kinase phosphorylates H1 from the amitotic macronucleus of Tetrahymena with remarkable fidelity. Furthermore, we demonstrate that macronuclei from Tetrahymena contain a growth-associated H1 kinase activity which closely resembles cdc2 kinase from other eukaryotes. Using polyclonal antibodies raised against yeast p34cdc2, we have detected a 36 kd immunoactive polypeptide in macronuclei which binds to Suc1 (p13)-coated beads and closely follows H1 kinase activity. Since macronuclei divide without mitotic chromosome condensation, these data demonstrate that H1 phosphorylation by cdc2 kinase may be necessary, but is not sufficient to promote mitotic chromatin condensation. The fact that an activity which strongly resembles mammalian cdc2 kinase is active during cell growth in a nucleus which does not undergo mitosis and chromosome condensation suggests that other factors are needed for a true mitotic division to occur. These data also reinforce the notion that H1 phosphorylation has important functions outside mitosis both in Tetrahymena and in mammalian cells.

Animals↗
PubMed1991

A role for the p34cdc2 kinase and phosphatases in the regulation of phosphorylation and disassembly of lamin B2 during the cell cycle.

While the p34cdc2 kinase is considered to be a critical regulator of mitosis, its function has not yet been directly linked to one of the key events during the onset of mitosis: nuclear envelope breakdown. Here we show that a major structural protein of the nuclear envelope, lamin B2, is phosphorylated by p34cdc2. Results from two-dimensional phosphopeptide mapping experiments demonstrate that the p34cdc2-specific phosphopeptides represent both mitotic and interphase specific phosphorylations of lamin B2 and include the major interphase phosphorylation site. In mitotic cells we detected two distinct forms of lamin B2 which differ in electrophoretic mobility and in degree of phosphorylation. The phosphorylation pattern of lamin B2 generated in vitro by p34cdc2 was more closely related to the less phosphorylated mitotic lamin B2, suggesting that another kinase(s) in addition to p34cdc2 is involved in generating the mitotic phosphorylation pattern. In addition, we show that treatment of interphase cells with okadaic acid, a potent phosphatase inhibitor, leads to the acquisition of mitosis-specific phosphopeptides and can reversibly increase the detergent-solubility of lamin B2. However, the M-phase-like phosphorylation of lamin B2 in itself is not sufficient to induce its disassembly from the nuclear lamina suggesting that an additional event(s) besides phosphorylation is required.

Animals↗
PubMed1991

A complex containing p34cdc2 and cyclin B phosphorylates the nuclear lamin and disassembles nuclei of clam oocytes in vitro.

Cell-free extracts prepared from activated clam oocytes contain factors which induce phosphorylation of the single 67-kD lamin (L67), disassemble clam oocyte nuclei, and cause chromosome condensation in vitro (Dessev, G., R. Palazzo, L. Rebhun, and R. Goldman. 1989. Dev. Biol. 131:469-504). To identify these factors, we have fractionated the oocyte extracts. The nuclear lamina disassembly (NLD) activity, together with a protein kinase activity specific for L67, appear as a single peak throughout a number of purification steps. This peak also contains p34cdc2, cyclin B, and histone H1-kinase activity, which are components of the M-phase promoting factor (MPF). The NLD/L67-kinase activity is depleted by exposure of this purified material to Sepharose conjugated to p13suc1, and is restored upon addition of a p34cdc2/p62 complex from HeLa cells. The latter complex phosphorylates L67 and induces NLD in the absence of other clam oocyte proteins. Our results suggest that a single protein kinase activity (p34cdc2-H1 kinase, identical with MPF) phosphorylates the lamin and is involved in the meiotic breakdown of the nuclear envelope in clam oocytes.

Animals↗
PubMed1991

Isolation and culture of human decidual capillary endothelial cells in serum-free medium supplemented with human uterine angiogenic factor.

Human decidual capillary endothelial (HDCE) cells, obtained from decidual fragments of legally induced first-trimester terminations of pregnancies, were cultured in a serum-free medium supplemented with human uterine angiogenic factor (HUAF). The method of isolating the cells from the decidual tissue is described. Identification of the decidual endothelial cells was based on light- and electron-microscopic observations as well as on antifactor VIII immunoperoxidase-staining technique. The HDCE cell culture in the serum-free medium lasted for 25 weeks through eight subcultures. The cells frozen in glycerin yielded 80% viability after thawing. Electron-microscopic observations of the monolayer demonstrated Weibel-Palade bodies, desmosomes and tight junctions. HUAF is mitogenic to HDCE at 10-100 ng/ml. The dependency of HDCE cells grown in culture on HUAF may explain in part the mechanism involved in the dynamic vascular expansion occurring during gestation.

Angiogenesis Inducing Agents↗
PubMed1991

Spi1 GTPase interacts with RCC1 to maintain interdependency of cell cycle events.

A mutant which can enter mitosis at any cell cycle stage has been isolated and characterized in fission yeast. The pim1 (premature initiation of mitosis) mutant prearrested at G1/S can develop a mitotic spindle and has tightly condensed chromosomes upon shift to the restrictive temperature. pim1-induced mitosis requires maturation promoting factor (MPF) activity, but not the essential mitotic inducer, cdc25. The pim1+ gene encodes a homolog of regulator of chromosome condensation 1 (RCC1), a regulator of onset of mitosis in mammalian cells. A multicopy suppressor of pim1, spi1, was isolated, and found to encode a 25 kDa GTPase. The primary sequence of the spi1 GTPase shows extensive identity (80%) to human TC4, whose function is unknown. The spi1/TC4 GTPase defines a novel class in the "ras-like" GTPase family, which is distinct from ras, rho, or ypt. Disruption of the spi1+ gene causes genomic instability in a heterozygous diploid. These genetic data suggest that pim1+ and spi1+ interact to coordinate correct entry into mitosis. Immunological experiments demonstrate that the pim1+ and spi1+ products are physically associated. Mutation in the pim1 gene results in lowered affinity of the protein for the spi1 protein in vitro, which may explain why high dosages of the spi1 protein can rescue the pim1 mutant in vivo. The pim1/spi1 complex dissociates in the presence of Mg2+ and GTP. The current data suggests that pim1+ acts as a GTP exchanger for the spi1 GTPase.

Amino Acid Sequence↗
PubMed1990

Distinct nuclear and spindle pole body population of cyclin-cdc2 in fission yeast.

Cyclins, as subunits of the protein kinase encoded by the cdc2 gene are major controlling elements of the eukaryotic cell cycle. The fission yeast Schizosaccharomyces pombe has a B-type cyclin, which is a nuclear protein encoded by the cdc13 gene. Here we demonstrate the presence of two spatially distinct cdc13 cyclin populations in the nucleus of S. pombe, one of which is associated with the mitotic spindle poles. Both populations colocalize with the product of the cdc2 gene (p34cdc2). Treatment of cells with the antimicrotubule drug thiabendazole prevents cyclin degradation and blocks the tyrosine dephosphorylation and activation of cdc2. These results suggest a key regulatory role of the cdc2-cyclin complex in the initiation of mitotic spindle formation and also that mitotic microtubule function is required for cdc2 activation.

CDC2 Protein Kinase↗