A novel bacterial tyrosine kinase essential for cell division and differentiation

Protein kinases play central roles in the regulation of eukaryotic and prokaryotic cell growth, division, and differentiation. The Caulobacter crescentus divL gene encodes a novel bacterial tyrosine kinase essential for cell viability and division. Although the DivL protein is homologous to the ubiquitous bacterial histidine protein kinases (HPKs), it differs from previously studied members of this protein kinase family in that it contains a tyrosine residue (Tyr-550) in the conserved H-box instead of a histidine residue, which is the expected site of autophosphorylation. DivL is autophosphorylated on Tyr-550 in vitro, and this tyrosine residue is essential for cell viability and regulation of the cell division cycle. Purified DivL also catalyzes phosphorylation of CtrA and activates transcription in vitro of the cell cycle-regulated fliF promoter. Suppressor mutations in ctrA bypass the conditional cell division phenotype of cold-sensitive divL mutants, providing genetic evidence that DivL function in cell cycle and developmental regulation is mediated, at least in part, by the global response regulator CtrA. DivL is the only reported HPK homologue whose function has been shown to require autophosphorylation on a tyrosine, and, thus, it represents a new class of kinases within this superfamily of protein kinases.

c-Myc enhances protein synthesis and cell size during B lymphocyte development

Members of the myc family of nuclear protooncogenes play roles in cell proliferation, differentiation, and apoptosis. Moreover, inappropriate expression of c-myc genes contributes to the development of many types of cancers, including B cell lymphomas in humans. Although Myc proteins have been shown to function as transcription factors, their immediate effects on the cell have not been well defined. Here we have utilized a murine model of lymphomagenesis (Eμ-myc mice) to show that constitutive expression of a c-myc transgene under control of the Ig heavy-chain enhancer (Eμ) results in an increase in cell size of normal pretransformed B lymphocytes at all stages of B cell development. Furthermore, we show that c-Myc-induced growth occurs independently of cell cycle phase and correlates with an increase in protein synthesis. These results suggest that Myc may normally function by coordinating expression of growth-related genes in response to mitogenic signals. Deregulated c-myc expression may predispose to cancer by enhancing cell growth to levels required for unrestrained cell division.

Increased sensitivity to apoptotic stimuli in c-abl-deficient progenitor B-cell lines

The protooncogene c-abl encodes a nonreceptor tyrosine kinase whose cellular function is unknown. To study the possible involvement of c-Abl in proliferation, differentiation, and cell cycle regulation of early B cells, long-term lymphoid bone marrow cultures were established from c-abl-deficient mice and their wild-type littermates. Interleukin 7-dependent progenitor B-cell clones and lines expressing B220 and CD43 could be generated from both mutant and wild-type mice. The mutant and wild-type lines displayed no difference in their proliferative capacity as measured by thymidine incorporation in response to various concentrations of interleukin 7. Similarly, c-abl deficiency did not interfere with the ability of mutant clones to differentiate into surface IgM-positive cells in vitro. Analysis of cultures after growth factor deprivation, however, revealed a strikingly accelerated rate of cell death in c-abl mutant cells, due to apoptosis as confirmed by terminal deoxynucleotidyltransferase-mediated UTP nick end labeling analysis. Furthermore, a greater susceptibility to apoptotic cell death in c-abl mutant cells was also observed after glucocorticoid treatment. These results suggest that mutant c-Abl renders the B-cell progenitors more sensitive to apoptosis, and may account for some of the phenotypes observed in c-abl-deficient animals.

An interaction between DNA ligase I and proliferating cell nuclear antigen: Implications for Okazaki fragment synthesis and joining

Although three human genes encoding DNA ligases have been isolated, the molecular mechanisms by which these gene products specifically participate in different DNA transactions are not well understood. In this study, fractionation of a HeLa nuclear extract by DNA ligase I affinity chromatography resulted in the specific retention of a replication protein, proliferating cell nuclear antigen (PCNA), by the affinity resin. Subsequent experiments demonstrated that DNA ligase I and PCNA interact directly via the amino-terminal 118 aa of DNA ligase I, the same region of DNA ligase I that is required for localization of this enzyme at replication foci during S phase. PCNA, which forms a sliding clamp around duplex DNA, interacts with DNA pol δ and enables this enzyme to synthesize DNA processively. An interaction between DNA ligase I and PCNA that is topologically linked to DNA was detected. However, DNA ligase I inhibited PCNA-dependent DNA synthesis by DNA pol δ. These observations suggest that a ternary complex of DNA ligase I, PCNA and DNA pol δ does not form on a gapped DNA template. Consistent with this idea, the cell cycle inhibitor p21, which also interacts with PCNA and inhibits processive DNA synthesis by DNA pol δ, disrupts the DNA ligase I–PCNA complex. Thus, we propose that after Okazaki fragment DNA synthesis is completed by a PCNA–DNA pol δ complex, DNA pol δ is released, allowing DNA ligase I to bind to PCNA at the nick between adjacent Okazaki fragments and catalyze phosphodiester bond formation.

Constitutive Bcl-2 expression throughout the hematopoietic compartment affects multiple lineages and enhances progenitor cell survival

Bcl-2, which can both reduce apoptosis and retard cell cycle entry, is thought to have important roles in hematopoiesis. To evaluate the impact of its ubiquitous overexpression within this system, we targeted expression of the human bcl-2 gene in mice by using the promoter of the vav gene, which is active throughout this compartment but rarely outside it. The vav-bcl-2 transgene was expressed in essentially all nucleated cells of hematopoietic tissues but not notably in nonhematopoietic tissues. Presumably because of enhanced cell survival, the mice displayed increases in myeloid cells as well as a marked elevation in B and T lymphocytes. The spleen was enlarged and the lymphoid follicles expanded. Although total thymic cellularity was normal, T cell development was altered: cells at the very immature and most mature stages were increased, whereas those at the intermediate stage were decreased. Unexpectedly, blood platelets were reduced by half, suggesting that their production from megakaryocytes is regulated by the Bcl-2 family. Colony formation by myeloid progenitor cells in vitro remained cytokine dependent, and the frequency of most progenitor and preprogenitor cells was normal. Macrophage progenitors were less frequent and yielded smaller colonies, however, perhaps reflecting inhibitory effects of Bcl-2 on cell cycling in specific lineages. After irradiation or factor deprivation, Bcl-2 markedly enhanced clonogenic survival of all tested progenitor and preprogenitor cells. Thus, Bcl-2 has multiple effects on the hematopoietic system. These mice should help to further clarify the role of apoptosis in the development and homeostasis of this compartment.

MAD2 associates with the cyclosome/anaphase-promoting complex and inhibits its activity

Cell cycle progression is monitored by checkpoint mechanisms that ensure faithful duplication and accurate segregation of the genome. Defects in spindle assembly or spindle-kinetochore attachment activate the mitotic checkpoint. Once activated, this checkpoint arrests cells prior to the metaphase-anaphase transition with unsegregated chromosomes, stable cyclin B, and elevated M phase promoting factor activity. However, the mechanisms underlying this process remain obscure. Here we report that upon activation of the mitotic checkpoint, MAD2, an essential component of the mitotic checkpoint, associates with the cyclin B-ubiquitin ligase, known as the cyclosome or anaphase-promoting complex. Moreover, purified MAD2 causes a metaphase arrest in cycling Xenopus laevis egg extracts and prevents cyclin B proteolysis by blocking its ubiquitination, indicating that MAD2 functions as an inhibitor of the cyclosome. Thus, MAD2 links the mitotic checkpoint pathway to the cyclin B destruction machinery which is critical in controlling the metaphase-anaphase transition.

Synergistic inhibition of human cancer cell growth by cytotoxic drugs and mixed backbone antisense oligonucleotide targeting protein kinase A

Protein kinase A type I plays a key role in neoplastic transformation, conveying mitogenic signals of different growth factors and oncogenes. Inhibition of protein kinase A type I by antisense oligonucleotides targeting its RIα regulatory subunit results in cancer cell growth inhibition in vitro and in vivo. A novel mixed backbone oligonucleotide HYB 190 and its mismatched control HYB 239 were tested on soft agar growth of several human cancer cell types. HYB 190 demonstrated a dose-dependent inhibition of colony formation in all cell lines whereas the HYB 239 at the same doses caused a modest or no growth inhibition. A noninhibitory dose of each mixed backbone oligonucleotide was used in OVCAR-3 ovarian and GEO colon cancer cells to study whether any cooperative effect may occur between the antisense and a series of cytotoxic drugs acting by different mechanisms. Treatment with HYB 190 resulted in an additive growth inhibitory effect with several cytotoxic drugs when measured by soft agar colony formation. A synergistic growth inhibition, which correlated with increased apoptosis, was observed when HYB 190 was added to cancer cells treated with taxanes, platinum-based compounds, and topoisomerase II selective drugs. This synergistic effect was also observed in breast cancer cells and was obtained with other related drugs such as docetaxel and carboplatin. Combination of HYB 190 and paclitaxel resulted in an accumulation of cells in late S-G2 phases of cell cycle and marked induction of apoptosis. A cooperative effect of HYB 190 and paclitaxel was also obtained in vivo in nude mice bearing human GEO colon cancer xenografts. These results are the first report of a cooperative growth inhibitory effect obtained in a variety of human cancer cell lines by antisense mixed backbone oligonucleotide targeting protein kinase A type I-mediated mitogenic signals and specific cytotoxic drugs.

Unusual Centrosome Cycle in Dictyostelium: Correlation of Dynamic Behavior and Structural Changes

Centrosome duplication and separation are of central importance for cell division. Here we provide a detailed account of this dynamic process in Dictyostelium. Centrosome behavior was monitored in living cells using a γ-tubulin–green fluorescent protein construct and correlated with morphological changes at the ultrastructural level. All aspects of the duplication and separation process of this centrosome are unusual when compared with, e.g., vertebrate cells. In interphase the Dictyostelium centrosome is a box-shaped structure comprised of three major layers, surrounded by an amorphous corona from which microtubules emerge. Structural duplication takes place during prophase, as opposed to G1/S in vertebrate cells. The three layers of the box-shaped core structure increase in size. The surrounding corona is lost, an event accompanied by a decrease in signal intensity of γ-tubulin–green fluorescent protein at the centrosome and the breakdown of the interphase microtubule system. At the prophase/prometaphase transition the separation into two mitotic centrosomes takes place via an intriguing lengthwise splitting process where the two outer layers of the prophase centrosome peel away from each other and become the mitotic centrosomes. Spindle microtubules are now nucleated from surfaces that previously were buried inside the interphase centrosome. Finally, at the end of telophase, the mitotic centrosomes fold in such a way that the microtubule-nucleating surface remains on the outside of the organelle. Thus in each cell cycle the centrosome undergoes an apparent inside-out/outside-in reversal of its layered structure.

Liz1p, a Novel Fission Yeast Membrane Protein, Is Required for Normal Cell Division When Ribonucleotide Reductase Is Inhibited

Ribonucleotide reductase activity is required for generating deoxyribonucleotides for DNA replication. Schizosaccharomyces pombe cells lacking ribonucleotide reductase activity arrest during S phase of the cell cycle. In a screen for hydroxyurea-sensitive mutants in S. pombe, we have identified a gene, liz1+, which when mutated reveals an additional, previously undescribed role for ribonucleotide reductase activity during mitosis. Inactivation of ribonucleotide reductase, by either hydroxyurea or a cdc22-M45 mutation, causes liz1− cells in G2 to undergo an aberrant mitosis, resulting in chromosome missegregation and late mitotic arrest. liz1+ encodes a 514-amino acid protein with strong similarity to a family of transmembrane transporters, and localizes to the plasma membrane of the cell. These results reveal an unexpected G2/M function of ribonucleotide reductase and establish that defects in a transmembrane protein can affect cell cycle progression.

Modulation of Cell Proliferation and Differentiation through Substrate-dependent Changes in Fibronectin Conformation

Integrin-mediated cell adhesion to extracellular matrices provides signals essential for cell cycle progression and differentiation. We demonstrate that substrate-dependent changes in the conformation of adsorbed fibronectin (Fn) modulated integrin binding and controlled switching between proliferation and differentiation. Adsorption of Fn onto bacterial polystyrene (B), tissue culture polystyrene (T), and collagen (C) resulted in differences in Fn conformation as indicated by antibody binding. Using a biochemical method to quantify bound integrins in cultured cells, we found that differences in Fn conformation altered the quantity of bound α5 and β1 integrin subunits but not αv or β3. C2C12 myoblasts grown on these Fn-coated substrates proliferated to different levels (B > T > C). Immunostaining for muscle-specific myosin revealed minimal differentiation on B, significant levels on T, and extensive differentiation on C. Differentiation required binding to the RGD cell binding site in Fn and was blocked by antibodies specific for this site. Switching between proliferation and differentiation was controlled by the levels of α5β1 integrin bound to Fn, and differentiation was inhibited by anti-α5, but not anti-αv, antibodies, suggesting distinct integrin-mediated signaling pathways. Control of cell proliferation and differentiation through conformational changes in extracellular matrix proteins represents a versatile mechanism to elicit specific cellular responses for biological and biotechnological applications.

MOB1, an Essential Yeast Gene Required for Completion of Mitosis and Maintenance of Ploidy

Mob1p is an essential Saccharomyces cerevisiae protein, identified from a two-hybrid screen, that binds Mps1p, a protein kinase essential for spindle pole body duplication and mitotic checkpoint regulation. Mob1p contains no known structural motifs; however MOB1 is a member of a conserved gene family and shares sequence similarity with a nonessential yeast gene, MOB2. Mob1p is a phosphoprotein in vivo and a substrate for the Mps1p kinase in vitro. Conditional alleles of MOB1 cause a late nuclear division arrest at restrictive temperature. MOB1 exhibits genetic interaction with three other yeast genes required for the completion of mitosis, LTE1, CDC5, and CDC15 (the latter two encode essential protein kinases). Most haploid mutant mob1 strains also display a complete increase in ploidy at permissive temperature. The mechanism for the increase in ploidy may occur through MPS1 function. One mob1 strain, which maintains stable haploidy at both permissive and restrictive temperature, diploidizes at permissive temperature when combined with the mps1–1 mutation. Strains containing mob2Δ also display a complete increase in ploidy when combined with the mps1-1 mutation. Perhaps in addition to, or as part of, its essential function in late mitosis, MOB1 is required for a cell cycle reset function necessary for the initiation of the spindle pole body duplication.

Taxol Suppresses Dynamics of Individual Microtubules in Living Human Tumor Cells

Microtubules are intrinsically dynamic polymers, and their dynamics play a crucial role in mitotic spindle assembly, the mitotic checkpoint, and chromosome movement. We hypothesized that, in living cells, suppression of microtubule dynamics is responsible for the ability of taxol to inhibit mitotic progression and cell proliferation. Using quantitative fluorescence video microscopy, we examined the effects of taxol (30–100 nM) on the dynamics of individual microtubules in two living human tumor cell lines: Caov-3 ovarian adenocarcinoma cells and A-498 kidney carcinoma cells. Taxol accumulated more in Caov-3 cells than in A-498 cells. At equivalent intracellular taxol concentrations, dynamic instability was inhibited similarly in the two cell lines. Microtubule shortening rates were inhibited in Caov-3 cells and in A-498 cells by 32 and 26%, growing rates were inhibited by 24 and 18%, and dynamicity was inhibited by 31 and 63%, respectively. All mitotic spindles were abnormal, and many interphase cells became multinucleate (Caov-3, 30%; A-498, 58%). Taxol blocked cell cycle progress at the metaphase/anaphase transition and inhibited cell proliferation. The results indicate that suppression of microtubule dynamics by taxol deleteriously affects the ability of cancer cells to properly assemble a mitotic spindle, pass the metaphase/anaphase checkpoint, and produce progeny.

14-3-3 Proteins Act as Negative Regulators of the Mitotic Inducer Cdc25 in Xenopus Egg Extracts

Cdc25, the dual-specificity phosphatase that dephosphorylates the Cdc2–cyclin B complex at mitosis, is highly regulated during the cell cycle. In Xenopus egg extracts, Cdc25 is associated with two isoforms of the 14-3-3 protein. Cdc25 is complexed primarily with 14-3-3ε and to a lesser extent with 14-3-3ζ. The association of these 14-3-3 proteins with Cdc25 varies dramatically during the cell cycle: binding is high during interphase but virtually absent at mitosis. Interaction with 14-3-3 is mediated by phosphorylation of Xenopus Cdc25 at Ser-287, which resides in a consensus 14-3-3 binding site. Recombinant Cdc25 with a point mutation at this residue (Cdc25-S287A) is incapable of binding to 14-3-3. Addition of the Cdc25-S287A mutant to Xenopus egg extracts accelerates mitosis and overrides checkpoint-mediated arrests of mitotic entry due to the presence of unreplicated and damaged DNA. These findings indicate that 14-3-3 proteins act as negative regulators of Cdc25 in controlling the G2–M transition.

Identification of Novel Temperature-sensitive Lethal Alleles in Essential β-Tubulin and Nonessential α2-Tubulin Genes as Fission Yeast Polarity Mutants

We have screened for temperature-sensitive (ts) fission yeast mutants with altered polarity (alp1–15). Genetic analysis indicates that alp2 is allelic to atb2 (one of two α-tubulin genes) and alp12 to nda3 (the single β-tubulin gene). atb2+ is nonessential, and the ts atb2 mutations we have isolated are dominant as expected. We sequenced two alleles of ts atb2 and one allele of ts nda3. In the ts atb2 mutants, the mutated residues (G246D and C356Y) are found at the longitudinal interface between α/β-heterodimers, whereas in ts nda3 the mutated residue (Y422H) is situated in the domain located on the outer surface of the microtubule. The ts nda3 mutant is highly sensitive to altered gene dosage of atb2+; overexpression of atb2+ lowers the restrictive temperature, and, conversely, deletion rescues ts. Phenotypic analysis shows that contrary to undergoing mitotic arrest with high viability via the spindle assembly checkpoint as expected, ts nda3 mutants execute cytokinesis and septation and lose viability. Therefore, it appears that the ts nda3 mutant becomes temperature lethal because of irreversible progression through the cell cycle in the absence of activating the spindle assembly checkpoint pathway.

Comprehensive Identification of Cell Cycle–regulated Genes of the Yeast Saccharomyces cerevisiae by Microarray HybridizationD⃞

We sought to create a comprehensive catalog of yeast genes whose transcript levels vary periodically within the cell cycle. To this end, we used DNA microarrays and samples from yeast cultures synchronized by three independent methods: α factor arrest, elutriation, and arrest of a cdc15 temperature-sensitive mutant. Using periodicity and correlation algorithms, we identified 800 genes that meet an objective minimum criterion for cell cycle regulation. In separate experiments, designed to examine the effects of inducing either the G1 cyclin Cln3p or the B-type cyclin Clb2p, we found that the mRNA levels of more than half of these 800 genes respond to one or both of these cyclins. Furthermore, we analyzed our set of cell cycle–regulated genes for known and new promoter elements and show that several known elements (or variations thereof) contain information predictive of cell cycle regulation. A full description and complete data sets are available at http://cellcycle-www.stanford.edu