Growth suppression of glioma cells by PTEN requires a functional phosphatase catalytic domain

Deletions of all or part of chromosome 10 are the most common genetic alterations in high-grade gliomas. The PTEN gene (also called MMAC1 and TEP1) maps to chromosome region 10q23 and has been implicated as a target of alteration in gliomas and also in other cancers such as those of the breast, prostate, and kidney. Here we sought to provide a functional test of its candidacy as a growth suppressor in glioma cells. We used a combination of Northern blot analysis, protein truncation assays, and sequence analysis to determine the types and frequency of PTEN mutations in glioma cell lines so that we could define appropriate recipients to assess the growth suppressive function of PTEN by gene transfer. Introduction of wild-type PTEN into glioma cells containing endogenous mutant alleles caused growth suppression, but was without effect in cells containing endogenous wild-type PTEN. The ectopic expression of PTEN alleles, which carried mutations found in primary tumors and have been shown or are expected to inactivate its phosphatase activity, caused little growth suppression. These data strongly suggest that PTEN is a protein phosphatase that exhibits functional and specific growth-suppressing activity.

Cleavage Furrows Formed between Centrosomes Lacking an Intervening Spindle and Chromosomes Contain Microtubule Bundles, INCENP, and CHO1 but Not CENP-E

PtK1 cells containing two independent mitotic spindles can cleave between neighboring centrosomes, in the absence of an intervening spindle, as well as at the spindle equators. We used same-cell video, immunofluorescence, and electron microscopy to compare the structure and composition of normal equatorial furrows with that of ectopic furrows formed between spindles. As in controls, ectopic furrows contained midbodies composed of microtubule bundles and an electron-opaque matrix. Despite the absence of an intervening spindle and chromosomes, the midbodies associated with ectopic furrows also contained the microtubule-bundling protein CHO1 and the chromosomal passenger protein INCENP. However, CENP-E, another passenger protein, was not found in ectopic furrows but was always present in controls. We also examined cells in which the ectopic furrow initiated but relaxed. Although relaxing furrows contained overlapping microtubules from opposing centrosomes, they lacked microtubule bundles as well as INCENP and CHO1. Together these data suggest that the mechanism defining the site of furrow formation during mitosis in vertebrates does not depend on the presence of underlying microtubule bundles and chromosomes or on the stable association of INCENP or CHO1. The data also suggest that the completion of cytokinesis requires the presence of microtubule bundles and specific proteins (e.g., INCENP, CHO1, etc.) that do not include CENP-E.

Short DNA Fragments without Sequence Similarity Are Initiation Sites for Replication in the Chromosome of the Yeast Yarrowia lipolytica

We have previously shown that both a centromere (CEN) and a replication origin are necessary for plasmid maintenance in the yeast Yarrowia lipolytica (Vernis et al., 1997). Because of this requirement, only a small number of centromere-proximal replication origins have been isolated from Yarrowia. We used a CEN-based plasmid to obtain noncentromeric origins, and several new fragments, some unique and some repetitive sequences, were isolated. Some of them were analyzed by two-dimensional gel electrophoresis and correspond to actual sites of initiation (ORI) on the chromosome. We observed that a 125-bp fragment is sufficient for a functional ORI on plasmid, and that chromosomal origins moved to ectopic sites on the chromosome continue to act as initiation sites. These Yarrowia origins share an 8-bp motif, which is not essential for origin function on plasmids. The Yarrowia origins do not display any obvious common structural features, like bent DNA or DNA unwinding elements, generally present at or near eukaryotic replication origins. Y. lipolytica origins thus share features of those in the unicellular Saccharomyces cerevisiae and in multicellular eukaryotes: they are discrete and short genetic elements without sequence similarity.

WRS-85D: A Tryptophanyl-tRNA Synthetase Expressed to High Levels in the Developing Drosophila Salivary Gland

In a screen for genes expressed in the Drosophila embryonic salivary gland, we identified a tryptophanyl-tRNA synthetase gene that maps to cytological position 85D (WRS-85D). WRS-85D expression is dependent on the homeotic gene Sex combs reduced (Scr). In the absence of Scr function, WRS-85D expression is lost in the salivary gland primordia; conversely, ectopic expression of Scr results in expression of WRS-85D in new locations. Despite the fact that WRS-85D is a housekeeping gene essential for protein synthesis, we detected both WRS-85D mRNA and protein at elevated levels in the developing salivary gland. WRS-85D is required for embryonic survival; embryos lacking the maternal contribution were unrecoverable, whereas larvae lacking the zygotic component died during the third instar larval stage. We showed that recombinant WRS-85D protein specifically charges tRNATrp, and WRS-85D is likely to be the only tryptophanyl-tRNA synthetase gene in Drosophila. We characterized the expression patterns of all 20 aminoacyl-tRNA synthetases and found that of the four aminoacyl-tRNA synthetase genes expressed at elevated levels in the salivary gland primordia, WRS-85D is expressed at the highest level throughout embryogenesis. We also discuss the potential noncanonical activities of tryptophanyl-tRNA synthetase in immune response and regulation of cell growth.

Membrane-anchored Plakoglobins Have Multiple Mechanisms of Action in Wnt Signaling

In Wnt signaling, β-catenin and plakoglobin transduce signals to the nucleus through interactions with TCF-type transcription factors. However, when plakoglobin is artificially engineered to restrict it to the cytoplasm by fusion with the transmembrane domain of connexin (cnxPg), it efficiently induces a Wnt-like axis duplication phenotype in Xenopus. In Xenopus embryos, maternal XTCF3 normally represses ventral expression of the dorsalizing gene Siamois. Two models have been proposed to explain the Wnt-like activity of cnxPg: 1) that cnxPg inhibits the machinery involved in the turnover of cytosolic β-catenin, which then accumulates and inhibits maternal XTCF3, and 2) that cnxPg directly acts to inhibit XTCF3 activity. To distinguish between these models, we created a series of N-terminal deletion mutations of cnxPg and examined their ability to induce an ectopic axis in Xenopus, activate a TCF-responsive reporter (OT), stabilize β-catenin, and colocalize with components of the Wnt signaling pathway. cnxPg does not colocalize with the Wnt pathway component Dishevelled, but it does lead to the redistribution of APC and Axin, two proteins involved in the regulation of β-catenin turnover. Expression of cnxPg increases levels of cytosolic β-catenin; however, this effect does not completely explain its signaling activity. Although cnxPg and Wnt-1 stabilize β-catenin to similar extents, cnxPg activates OT to 10- to 20-fold higher levels than Wnt-1. Moreover, although LEF1 and TCF4 synergize with β-catenin and plakoglobin to activate OT, both suppress the signaling activity of cnxPg. In contrast, XTCF3 suppresses the signaling activity of both β-catenin and cnxPg. Both exogenous XLEF1 and XTCF3 are sequestered in the cytoplasm of Xenopus cells by cnxPg. Based on these data, we conclude that, in addition to its effects on β-catenin, cnxPg interacts with other components of the Wnt pathway, perhaps TCFs, and that these interactions contribute to its signaling activity.

Regulation of G2/M Progression by the STE Mitogen-activated Protein Kinase Pathway in Budding Yeast Filamentous Growth

Inoculation of diploid budding yeast onto nitrogen-poor agar media stimulates a MAPK pathway to promote filamentous growth. Characteristics of filamentous cells include a specific pattern of gene expression, elongated cell shape, polar budding pattern, persistent attachment to the mother cell, and a distinct cell cycle characterized by cell size control at G2/M. Although a requirement for MAPK signaling in filamentous gene expression is well established, the role of this pathway in the regulation of morphogenesis and the cell cycle remains obscure. We find that ectopic activation of the MAPK signal pathway induces a cell cycle shift to G2/M coordinately with other changes characteristic of filamentous growth. These effects are abrogated by overexpression of the yeast mitotic cyclins Clb1 and Clb2. In turn, yeast deficient for Clb2 or carrying cdc28-1N, an allele of CDK defective for mitotic functions, display enhanced filamentous differentiation and supersensitivity to the MAPK signal. Importantly, activation of Swe1-mediated inhibitory phosphorylation of Thr-18 and/or Tyr-19 of Cdc28 is not required for the MAPK pathway to affect the G2/M delay. Mutants expressing a nonphosphorylatable mutant Cdc28 or deficient for Swe1 exhibit low-nitrogen-dependent filamentous growth and are further induced by an ectopic MAPK signal. We infer that the MAPK pathway promotes filamentous growth by a novel mechanism that inhibits mitotic cyclin/CDK complexes and thereby modulates cell shape, budding pattern, and cell-cell connections.

MAL, an Integral Element of the Apical Sorting Machinery, Is an Itinerant Protein That Cycles between the Trans-Golgi Network and the Plasma Membrane

The MAL proteolipid is a nonglycosylated integral membrane protein found in glycolipid-enriched membrane microdomains. In polarized epithelial Madin-Darby canine kidney cells, MAL is necessary for normal apical transport and accurate sorting of the influenza virus hemagglutinin. MAL is thus part of the integral machinery for glycolipid-enriched membrane–mediated apical transport. At steady state, MAL is predominantly located in perinuclear vesicles that probably arise from the trans-Golgi network (TGN). To act on membrane traffic and to prevent their accumulation in the target compartment, integral membrane elements of the protein-sorting machinery should be itinerant proteins that cycle between the donor and target compartments. To establish whether MAL is an itinerant protein, we engineered the last extracellular loop of MAL by insertion of sequences containing the FLAG epitope or with sequences containing residues that became O-glycosylated within the cells or that displayed biotinylatable groups. The ectopic expression of these modified MAL proteins allowed us to investigate the surface expression of MAL and its movement through different compartments after internalization with the use of a combination of assays, including surface biotinylation, surface binding of anti-FLAG antibodies, neuraminidase sensitivity, and drug treatments. Immunofluorescence and flow cytometric analyses indicated that, in addition to its Golgi localization, MAL was also expressed on the cell surface, from which it was rapidly internalized. This retrieval implies transport through the endosomal pathway and requires endosomal acidification, because it can be inhibited by drugs such as chloroquine, monensin, and NH4Cl. Resialylation experiments of surface MAL treated with neuraminidase indicated that ∼30% of the internalized MAL molecules were delivered to the TGN, probably to start a new cycle of cargo transport. Together, these observations suggest that, as predicted for integral membrane members of the late protein transport machinery, MAL is an itinerant protein cycling between the TGN and the plasma membrane.

Checkpoint Defects Leading to Premature Mitosis Also Cause Endoreplication of DNA in Aspergillus nidulans

The G2 DNA damage and slowing of S-phase checkpoints over mitosis function through tyrosine phosphorylation of NIMXcdc2 in Aspergillus nidulans. We demonstrate that breaking these checkpoints leads to a defective premature mitosis followed by dramatic rereplication of genomic DNA. Two additional checkpoint functions, uvsB and uvsD, also cause the rereplication phenotype after their mutation allows premature mitosis in the presence of low concentrations of hydroxyurea. uvsB is shown to encode a rad3/ATR homologue, whereas uvsD displays homology to rad26, which has only previously been identified in Schizosaccharomyces pombe. uvsBrad3 and uvsDrad26 have G2 checkpoint functions over mitosis and another function essential for surviving DNA damage. The rereplication phenotype is accompanied by lack of NIMEcyclinB, but ectopic expression of active nondegradable NIMEcyclinB does not arrest DNA rereplication. DNA rereplication can also be induced in cells that enter mitosis prematurely because of lack of tyrosine phosphorylation of NIMXcdc2 and impaired anaphase-promoting complex function. The data demonstrate that lack of checkpoint control over mitosis can secondarily cause defects in the checkpoint system that prevents DNA rereplication in the absence of mitosis. This defines a new mechanism by which endoreplication of DNA can be triggered and maintained in eukaryotic cells.

Phosphorylation of protein kinase N by phosphoinositide-dependent protein kinase-1 mediates insulin signals to the actin cytoskeleton

Growth factors such as insulin regulate phosphatidylinositol 3-kinase-dependent actin cytoskeleton rearrangement in many types of cells. However, the mechanism by which the insulin signal is transmitted to the actin cytoskeleton remains largely unknown. Yeast two-hybrid screening revealed that the phosphatidylinositol 3-kinase downstream effector phosphoinositide-dependent protein kinase-1 (PDK1) interacted with protein kinase N (PKN), a Rho-binding Ser/Thr protein kinase potentially implicated in a variety of cellular events, including phosphorylation of cytoskeletal components. PDK1 and PKN interacted in vitro and in intact cells, and this interaction was mediated by the kinase domain of PDK1 and the carboxyl terminus of PKN. In addition to a direct interaction, PDK1 also phosphorylated Thr774 in the activation loop and activated PKN. Insulin treatment or ectopic expression of the wild-type PDK1 or PKN, but not protein kinase Cζ, induced actin cytoskeleton reorganization and membrane ruffling in 3T3-L1 fibroblasts and Rat1 cells that stably express the insulin receptor (Rat1-IR). However, the insulin-stimulated actin cytoskeleton reorganization in Rat1-IR cells was prevented by expression of kinase-defective PDK1 or PDK1-phosphorylation site-mutated PKN. Thus, phosphorylation by PDK1 appears to be necessary for PKN to transduce signals from the insulin receptor to the actin cytoskeleton.

Novel gene conversion between X-Y homologues located in the nonrecombining region of the Y chromosome in Felidae (Mammalia)

Genes located on the mammalian Y chromosome outside of the pseudoautosomal region do not recombine with those on the X and are predicted to either undergo selection for male function or gradually degenerate because of an accumulation of deleterious mutations. Here, phylogenetic analyses of X-Y homologues, Zfx and Zfy, among 26 felid species indicate two ancestral episodes of directed genetic exchange (ectopic gene conversion) from X to Y: once during the evolution of pallas cat and once in a common predecessor of ocelot lineage species. Replacement of the more rapidly evolving Y homologue with the evolutionarily constrained X copy may represent a mechanism for adaptive editing of functional genes on the nonrecombining region of the mammalian Y chromosome.

Computation, prediction, and experimental tests of fitness for bacteriophage T7 mutants with permuted genomes

We created a simulation based on experimental data from bacteriophage T7 that computes the developmental cycle of the wild-type phage and also of mutants that have an altered genome order. We used the simulation to compute the fitness of more than 105 mutants. We tested these computations by constructing and experimentally characterizing T7 mutants in which we repositioned gene 1, coding for T7 RNA polymerase. Computed protein synthesis rates for ectopic gene 1 strains were in moderate agreement with observed rates. Computed phage-doubling rates were close to observations for two of four strains, but significantly overestimated those of the other two. Computations indicate that the genome organization of wild-type T7 is nearly optimal for growth: only 2.8% of random genome permutations were computed to grow faster, the highest 31% faster, than wild type. Specific discrepancies between computations and observations suggest that a better understanding of the translation efficiency of individual mRNAs and the functions of qualitatively “nonessential” genes will be needed to improve the T7 simulation. In silico representations of biological systems can serve to assess and advance our understanding of the underlying biology. Iteration between computation, prediction, and observation should increase the rate at which biological hypotheses are formulated and tested.

Leptin can induce proliferation, differentiation, and functional activation of hemopoietic cells

Many cytokines exert their biological effect through members of the hemopoietin receptor family. Using degenerate oligonucleotides to the common WSXWS motif, we have cloned from human hemopoietic cell cDNA libraries various forms of the receptor that was recently shown to bind the obesity hormone, leptin. mRNAs encoding long and short forms of the human leptin receptor were found to be coexpressed in a range of human and murine hemopoietic organs, and a subset of cells from these tissues bound leptin at the cell surface. Ectopic expression in murine Ba/F3 and M1 cell lines revealed that the long, but not the short, form of the leptin receptor can signal proliferation and differentiation, respectively. In cultures of murine or human marrow cells, human leptin exhibited no capacity to stimulate cell survival or proliferation, but it enhanced cytokine production and phagocytosis of Leishmania parasites by murine peritoneal macrophages. Our data provide evidence that, in addition to its role in fat regulation, leptin may also be able to regulate aspects of hemopoiesis and macrophage function.

Purification of the β-cell glucose-sensitive factor that transactivates the insulin gene differentially in normal and transformed islet cells

The β cell-specific glucose-sensitive factor (GSF), which binds the A3 motif of the rat I and human insulin promoters, is modulated by extracellular glucose. A single mutation in the GSF binding site of the human insulin promoter abolishes the stimulation by high glucose only in normal islets, supporting the suggested physiological role of GSF in the glucose-regulated expression of the insulin gene. GSF binding activity was observed in all insulin-producing cells. We have therefore purified this activity from the rat insulinoma RIN and found that a single polypeptide of 45 kDa was responsible for DNA binding. Its amino acid sequence, determined by microsequencing, provided direct evidence that GSF corresponds to insulin promoter factor 1 (IPF-1; also known as PDX-1) and that, in addition to its essential roles in development and differentiation of pancreatic islets and in β cell-specific gene expression, it functions as mediator of the glucose effect on insulin gene transcription in differentiated β cells. The human cDNA coding for GSF/IPF-1 has been cloned, its cell and tissue distribution is described. Its expression in the glucagon-producing cell line αTC1 transactivates the wild-type human insulin promoter more efficiently than the mutated construct. It is demonstrated that high levels of ectopic GSF/IPF-1 inhibit the expression of the human insulin gene in normal islets, but not in transformed βTC1 cells. These results suggest the existence of a control mechanism, such as requirement for a coactivator of GSF/IPF-1, which may be present in limiting amounts in normal as opposed to transformed β cells.

A functional genetic screen identifies regions at the C-terminal tail and death-domain of death-associated protein kinase that are critical for its proapoptotic activity

Death-associated protein kinase (DAP-kinase) is a Ca+2/calmodulin-regulated serine/threonine kinase with a multidomain structure that participates in apoptosis induced by a variety of signals. To identify regions in this protein that are critical for its proapoptotic activity, we performed a genetic screen on the basis of functional selection of short DAP-kinase-derived fragments that could protect cells from apoptosis by acting in a dominant-negative manner. We expressed a library of randomly fragmented DAP-kinase cDNA in HeLa cells and treated these cells with IFN-γ to induce apoptosis. Functional cDNA fragments were recovered from cells that survived the selection, and those in the sense orientation were examined further in a secondary screen for their ability to protect cells from DAP-kinase-dependent tumor necrosis factor-α-induced apoptosis. We isolated four biologically active peptides that mapped to the ankyrin repeats, the “linker” region, the death domain, and the C-terminal tail of DAP-kinase. Molecular modeling of the complete death domain provided a structural basis for the function of the death-domain-derived fragment by suggesting that the protective fragment constitutes a distinct substructure. The last fragment, spanning the C-terminal serine-rich tail, defined a new regulatory region. Ectopic expression of the tail peptide (17 amino acids) inhibited the function of DAP-kinase, whereas removal of this region from the complete protein caused enhancement of the killing activity, indicating that the C-terminal tail normally plays a negative regulatory role. Altogether, this unbiased screen highlighted functionally important regions in the protein and revealed an additional level of regulation of DAP-kinase apoptotic function that does not affect the catalytic activity.

Regulation of fatty acid homeostasis in cells: Novel role of leptin

It is proposed that an important function of leptin is to confine the storage of triglycerides (TG) to the adipocytes, while limiting TG storage in nonadipocytes, thus protecting them from lipotoxicity. The fact that TG content in nonadipocytes normally remains within a narrow range, while that of adipocytes varies enormously with food intake, is consistent with a system of TG homeostasis in normal nonadipocytes. The facts that when leptin receptors are dysfunctional, TG content in nonadipocytes such as islets can increase 100-fold, and that constitutively expressed ectopic hyperleptinemia depletes TG, suggest that leptin controls the homeostatic system for intracellular TG. The fact that the function and viability of nonadipocytes is compromised when their TG content rises above or falls below the normal range suggests that normal homeostasis of their intracellular TG is critical for optimal function and to prevent lipoapoptosis. Thus far, lipotoxic diabetes of fa/fa Zucker diabetic fatty rats is the only proven lipodegenerative disease, but the possibility of lipotoxic disease of skeletal and/or cardiac muscle may require investigation, as does the possible influence of the intracellular TG content on autoimmune and neoplastic processes.