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D J Lew

Publications and source records attributed to D J Lew.

At least 37 records · Page 2Linked to original sources
PubMed1996

Regulatory roles of cyclin dependent kinase phosphorylation in cell cycle control.

Cyclins and cyclin-dependent kinases (Cdks) are universal regulators of cell cycle progression in eukaryotic cells. Cdk activity is controlled by phosphorylation at three conserved sites, and many of the enzymes that act on these sites have now been identified. Although the biochemistry of Cdk phosphorylation is relatively well understood, the regulatory roles of such phosphorylation are, in many cases, obscure. Recent studies have uncovered new and unexpected potential roles, and prompted re-examination of previously assumed roles, of Cdk phosphorylation.

Cell Cycle↗
PubMed1996

Cdc28 tyrosine phosphorylation and the morphogenesis checkpoint in budding yeast.

A morphogenesis checkpoint in budding yeast delays nuclear division (and subsequent cell cycle progression) in cells that have failed to make a bud. We show that the ability of this checkpoint to delay nuclear division requires the SWE1 gene, encoding a protein kinase that inhibits the master cell cycle regulatory kinase Cdc28. The timing of nuclear division in cells that cannot make a bud is exquisitely sensitive to the dosage of SWE1 and MIH1 genes, which control phosphorylation of Cdc28 at tyrosine 19. In contrast, the timing of nuclear division in budded cells does not rely on Cdc28 phosphorylation, suggesting that the morphogenesis checkpoint somehow turns on this regulatory pathway. We show that SWE1 mRNA levels fluctuate during the cell cycle and are elevated in cells that cannot make a bud. However, regulation of SWE1 mRNA levels by the checkpoint is indirect, acting through a feedback loop requiring Swe1 activity. Further, the checkpoint is capable of delaying nuclear division even when SWE1 transcription is deregulated. We propose that the checkpoint delays nuclear division through post-translational regulation of Swe1 and that transcriptional feedback loops enhance the efficacy of the checkpoint.

CDC28 Protein Kinase, S cerevisiae↗
PubMed1995

Cell cycle control of morphogenesis in budding yeast.

A detailed description of the cytoskeletal rearrangements that orchestrate bud formation is beginning to emerge from studies on yeast morphogenesis. In this review, we focus on recent advances in our understanding of how the timing of these rearrangements is controlled. Dramatic changes in cell polarity that occur in G1 (polarization to the bud site), G2 (depolarization within the bud), and mitosis (repolarization to the mother/bud neck) are triggered by changes in the kinase activity of Cdc28, the universal regulator of cell cycle progression. The hunt for Cdc28 morphogenesis substrates is on.

CDC28 Protein Kinase, S cerevisiae↗
PubMed1995

A cell cycle checkpoint monitors cell morphogenesis in budding yeast.

Checkpoint controls are regulatory pathways that inhibit cell cycle progression in cells that have not faithfully completed a prior step in the cell cycle. In the budding yeast Saccharomyces cerevisiae, DNA replication and spindle assembly are monitored by checkpoint controls that prevent nuclear division in cells that have failed to complete these processes. During the normal cell cycle, bud formation is temporally coincident with DNA replication and spindle assembly, and the nucleus divides along the mother-bud axis in mitosis. In this report, we show that inhibition of bud formation also causes a dramatic delay in nuclear division. This allows cells to recover from a transient disruption of cell polarity without becoming binucleate. The delay occurs after DNA replication and spindle assembly, and results from delayed activation of the master cell cycle regulatory kinase, Cdc28. Cdc28 activation is inhibited by phosphorylation of Cdc28 on tyrosine 19, and by delayed accumulation of the B-type cyclins Clb1 and Clb2. These results suggest the existence of a novel checkpoint that monitors cell morphogenesis in budding yeast.

CDC28 Protein Kinase, S cerevisiae↗
PubMed1993

Morphogenesis in the yeast cell cycle: regulation by Cdc28 and cyclins.

Analysis of cell cycle regulation in the budding yeast Saccharomyces cerevisiae has shown that a central regulatory protein kinase, Cdc28, undergoes changes in activity through the cell cycle by associating with distinct groups of cyclins that accumulate at different times. The various cyclin/Cdc28 complexes control different aspects of cell cycle progression, including the commitment step known as START and mitosis. We found that altering the activity of Cdc28 had profound effects on morphogenesis during the yeast cell cycle. Our results suggest that activation of Cdc28 by G1 cyclins (Cln1, Cln2, or Cln3) in unbudded G1 cells triggers polarization of the cortical actin cytoskeleton to a specialized pre-bud site at one end of the cell, while activation of Cdc28 by mitotic cyclins (Clb1 or Clb2) in budded G2 cells causes depolarization of the cortical actin cytoskeleton and secretory apparatus. Inactivation of Cdc28 following cyclin destruction in mitosis triggers redistribution of cortical actin structures to the neck region for cytokinesis. In the case of pre-bud site assembly following START, we found that the actin rearrangement could be triggered by Cln/Cdc28 activation in the absence of de novo protein synthesis, suggesting that the kinase may directly phosphorylate substrates (such as actin-binding proteins) that regulate actin distribution in cells.

Actins↗
PubMed1993

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

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.

CDC28 Protein Kinase, S cerevisiae↗
PubMed1992

Different G1 cyclins control the timing of cell cycle commitment in mother and daughter cells of the budding yeast S. cerevisiae.

Growth of S. cerevisiae cells by budding gives rise to asymmetric progeny cells: a larger "mother" cell and a smaller "daughter" cell. The mother cell transits a brief G1 phase before forming a new bud and beginning DNA replication. The daughter cell stays in G1 for a longer period, growing in size before initiating a new cell cycle. We show that the timing of cell cycle initiation in mother and daughter cells is governed by different G1 cyclins. In daughter cells, transcription of CLN1 and CLN2 is induced in a size-dependent manner, and these cyclins are necessary for the normal timing of cell cycle initiation. CLN3 is not required in daughter cells, but is crucial for mother cells, in which the G1 phase is much longer in the absence of this cyclin.

CDC28 Protein Kinase, S cerevisiae↗
PubMed1992

Induction of cyclin mRNA and cyclin-associated histone H1 kinase during liver regeneration.

Cyclins and cyclin-associated cdc kinases are key regulators of oocyte maturation (Maller, J. L. (1990) in The Biology and Medicine of Signal Transduction (Nishizuka, Y., Endo, M., and Tanaka, C., eds) pp. 323-328, Raven Press, New York), yeast cell cycles (Nurse, P. (1990) Nature 344, 503-508), DNA replication in cell-free systems (D'Urso, F., Marraccino, R. L., Marshak, R. R., and Roberts, J. M. (1990) Science 250, 786-791), and amphibian cell proliferative transitions (Hunt, T. (1991) Nature 350, 462-463). The extent to which these regulatory molecules participate in the growth control of differentiated epithelial cells like hepatocytes is unknown. Therefore, we investigated the expression of "G1" (E, C, and D) and "G2/M" (A, B1, and B2) cyclin mRNAs, the relative levels of cyclin A- and B1-associated histone H1-kinase activity, and the appearance of cyclin-associated kinases (p32/p33cdk2 and p33/p34cdc2) in regenerating rat liver and in control tissues from sham hepatectomized rats. To do this, we exploited a battery of human cyclin cDNAs and cyclin antisera that recognize rat molecules. The results suggest an apparent sequence of regeneration-specific changes: 1) elevated and induced expression of cyclins E (2.1 kilobases (kb)) and C (4 kb), and D mRNAs (4 kb), within 12 h, respectively; 2) induction of cyclins A (3.4 and 1.8 kb), B1 (2.5 and 1.8 kb), and B2 (1.9 kb) mRNAs at 24 h; 3) induction of cyclin A- and B1-associated nuclear histone H1 kinase at 24 h; and 4) enhanced levels of PSTAIRE-containing proteins of Mr approximately 32-33 and 33-34 kDa in nuclear extracts from 24-h regenerating liver that co-immunoprecipitate with cyclin A and B1 antisera, respectively. These observations provide an intellectual framework that unifies the biology of hepatocyte mitogenesis, proto-oncogene expression, and the machinery of the cell cycle.

Animals↗
PubMed1992

G1 control in yeast and animal cells.

In budding yeast, Saccharomyces cerevisiae, the cell cycle is controlled at the G1/S phase transition by regulating the activity of the CDC28 protein kinase. This is the budding yeast homologue of the cdc2 protein kinase associated in most organisms with control of mitosis. In budding yeast CDC28 controls both the G1/S phase transition and the G2/M phase transition by being differentially activated by two distinct classes of positive regulatory subunits known as G1 cyclins or CLNs and B-type cyclins or CLBs, respectively. To establish whether a similar dual role for Cdc2-related kinases exists in animal cells, we and others have sought human homologues of yeast G1 cyclins. Of several candidates, cyclin E is the most promising in that it accumulates prior to S phase and is associated with a pre-S phase protein kinase activity. The kinetics of accumulation of cyclin E-associated protein kinase activity is consistent with a role at the mammalian cell cycle restriction point.

Animals↗
PubMed1992

A proliferation of cyclins.

Cyclins are regulatory subunits of the serine/threonine protein kinases that play key roles in cell cycle control. The roster of known cyclins has expanded significantly in the past year, revealing a large and very diverse family of proteins. Although cyclins were originally characterized by their periodic accumulation during interphase and destruction in mitosis (these were the 'mitotic' cyclins that control entry into mitosis), the newly identified cyclins do not conform to this pattern. Here we review what is known about the functions of the nonmitotic cyclins in yeast and in mammalian cells.

Journal Article↗
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↗
PubMed1991

Isolation of three novel human cyclins by rescue of G1 cyclin (Cln) function in yeast.

We have isolated a number of cDNAs derived from human mRNAs that are able to substitute for G1 cyclin genes in S. cerevisiae. Several of these encode human cyclins A, B1, and B2. Three novel genes have been identified, which we call cyclins C, D, and E. The novel proteins are sufficiently distantly related to the other members of the cyclin family and to each other as to constitute three new classes of cyclins. Cyclin C and E mRNAs accumulate periodically through the cell cycle, peaking at different times in G1.

Amino Acid Sequence↗
PubMed1991

A cyclin B homolog in S. cerevisiae: chronic activation of the Cdc28 protein kinase by cyclin prevents exit from mitosis.

A cyclin B homolog was identified in Saccharomyces cerevisiae using degenerate oligonucleotides and the polymerase chain reaction. The protein, designated Scb1, has a high degree of similarity with B-type cyclins from organisms ranging from fission yeast to human. Levels of SCB1 mRNA and protein were found to be periodic through the cell cycle, with maximum accumulation late, most likely in the G2 interval. Deletion of the gene was found not to be lethal, and subsequently other B-type cyclins have been found in yeast functionally redundant with Scb1. A mutant allele of SCB1 that removes an amino-terminal fragment of the encoded protein thought to be required for efficient degradation during mitosis confers a mitotic arrest phenotype. This arrest can be reversed by inactivation of the Cdc28 protein kinase, suggesting that cyclin-mediated arrest results from persistent protein kinase activation.

Alleles↗
PubMed1991

Cytoplasmic activation of GAF, an IFN-gamma-regulated DNA-binding factor.

We have investigated events following treatment of cells with interferon-gamma (IFN-gamma) that lead to the immediate transcriptional activation of an inducible gene. A gamma-interferon activation factor (GAF) was activated in the cytoplasm of human fibroblasts immediately after IFN-gamma treatment and bound to a newly identified target DNA sequence, the gamma-interferon activation site (GAS). The time course of activation of GAF was different in fibroblasts and HeLa cells and correlated well with IFN-gamma-induced transcriptional activation in both cell types. IFN-gamma-dependent activation of GAF also occurred in enucleated cells (cytoplasts), showing that an inactive cytoplasmic precursor is converted to the active factor. These findings support the concept that ligand-specific signals originating at the cell surface are transmitted through latent cytoplasmic proteins which are activated to bind specific DNA sites and then move to the nucleus to activate the transcription of specific sets of genes.

Base Sequence↗
PubMed1991

Characterization of constitutive exocytosis in the yeast Saccharomyces cerevisiae.

Constitutive exocytosis was investigated in the yeast Saccharomyces cerevisiae using temperature-sensitive mutant (sec) strains which do not allow vesicle fusion to the plasma membrane at the restrictive temperature. Secretory vesicles were accumulated in the cell at the restrictive temperature and then protein synthesis was blocked with cycloheximide. Upon returning the cells to the permissive temperature the contents of the accumulated vesicles were secreted. This allowed the study of constitutive exocytosis independent of the processes responsible for vesicular biosynthesis. Neither the kinetics nor magnitude of exocytosis were affected by removal of external Ca2+ or perturbations of cytosolic Ca2+. This suggests that in those systems where calcium is required for exocytosis it is a regulatory molecule and not part of the mechanism of membrane fusion. Release occurred over a very broad range of pH and in media with different ionic compositions, suggesting that ionic and potential gradients across the plasma membrane play no role in exocytosis in yeast. High osmolarity inhibited the rate, but not the extent, of release. A novel inhibitory effect of azide was detected which occurred only at low pH. Vanadate also inhibited release in a pH-independent manner. Secretion occurred at the same rate in cells with or without accumulated vesicles. This infers a rate-limiting step following vesicle accumulation, perhaps a limiting number of release sites on the plasma membrane.

Calcium↗
PubMed1991

G1 control in yeast and animal cells.

In yeast G1, cyclins control the Cdc28 protein kinase in order to regulate the primary cell cycle gating event known as START. Environmental and internal signals that control the cell cycle do so, apparently, by controlling the synthesis and/or stability of G1 cyclins, hence controlling the activity of the Cdc28 kinase. The substrates of the Cdc28 kinase that are critical for passage through START are not known. One simple hypothesis is that the G1 kinase phosphorylates and thus activates a transcription factor required for the initiation of S phase. The synthesis of an origin of replication-binding factor might be regulated in this fashion. Recent evidence suggests that Cdc28 protein kinase activity directly regulates the transcription of a family of genes whose products are required for DNA replication (N. Marini and S. Reed, in prep.). However, it is not yet known whether this transcriptional activation constitutes the execution of START. The situation in animal cells is more complex. A number of new cyclins and p34s have been identified. It is not clear yet which of these, if any, have functions in G1 and if they do, what functions these might be. If G1 cyclins and p34 kinases do have critical G1 roles, by analogy with yeast, they may couple signals mediated by both positive and negative growth factors to cell cycle progression. Candidates for the critical G1 substrates of these putative G1 protein kinases are the tumor suppressors such as the RB (retinoblastoma) gene product (p105RB).(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1991

Overlapping elements in the guanylate-binding protein gene promoter mediate transcriptional induction by alpha and gamma interferons.

The gene encoding a 67-kDa cytoplasmic guanylate-binding protein (GBP) is transcriptionally induced in cells exposed to interferon of either type I (alpha interferon [IFN-alpha] or type II (IFN-gamma). The promoter of the GBP gene was cloned and found to contain an IFN-alpha-stimulated response element, which mediated the response of the GBP gene to IFN-alpha. On the basis of transfection experiments with recombinant plasmids, two different elements were delineated. Both were required to obtain the maximal response of the GBP gene to IFN-gamma: the IFN-alpha-stimulated response element and an overlapping element termed the IFN-gamma activation site. Different proteins that act on each element were investigated, and their possible involvement in IFN-gamma-induced transcriptional regulation is discussed.

Base Sequence↗