Genetic and biochemical studies in lower eukaryotes have identified several proteins that ensure accurate segregation of chromosomes. These include the Drosophila aurora and yeast Ipl1 kinases that are required for centrosome maturation and chromosome segregation. We have identified two human homologues of these genes, termed aurora1 and aurora2, that encode cell-cycle-regulated serine/threonine kinases. Here we demonstrate that the aurora2 gene maps to chromosome 20q13, a region amplified in a variety of human cancers, including a significant number of colorectal malignancies. We propose that aurora2 may be a target of this amplicon since its DNA is amplified and its RNA overexpressed, in more than 50% of primary colorectal cancers. Furthermore, overexpression of aurora2 transforms rodent fibroblasts. These observations implicate aurora2 as a potential oncogene in many colon, breast and other solid tumors, and identify centrosome-associated proteins as novel targets for cancer therapy.

The APC suggests that deregulation of cell adhesion may be involved. Calcium-dependent cell-cell adhesion is maintained by interactions between transmembrane cadherin molecules which require the association of catenins with their cytoplasmic domains (8-10). The stability of the (3-catenincadherin complex is in turn modulated by a posttranscriptional mechanism that affects the relative stability of ,B-catenin itself (11). This mechanism is engaged by the product of the WNT1 oncogene which promotes the accumulation of (3-and ly-catenins and strengthens calcium-dependent cell adhesion (11,12). In addition to simply supporting cell adhesion, a role for P-catenin in signal transduction has also been proposed. In Drosophila, the appearance of armadillo, the f3-catenin homolog, in the cytoplasm is dependent upon expression of wingless, the WNT1 homolog (13 Lipofectin (BRL) was added to the washed cells. Transfection medium was removed after 20-24 hr and 2 ml of growth medium (Leibovitz L-15 medium with penicillin, streptomycin, L-glutamine, and 10% fetal bovine serum; Irvine Scientific) was added to each well. Twenty-four hours later cells were harvested or were analyzed by immunofluorescence. Detection of 83-galactosidase expression was performed by the protocol published by Stratagene.Immunological Procedures. The general procedures for immunofluorescence analysis, microscopy, and photography have been described (15). Transfected cells were analyzed 48 hr after transfection. Cells were grown on coverslips, washed with phosphate-buffered saline, and then fixed in methanol at -20°C. After blocking in 10% powdered milk solution, detection of 3-catenin was performed with either a 1:200 dilution of rabbit polyclonal anti-,B-catenin serum (gift of B. Gumbiner, Sloan-Kettering) or, in the case of costaining experiments (see Fig. 2C), a 1:50 dilution of a mouse monoclonal antibody (Transduction Laboratories, Lexington, KY). APC protein was detected with affinity-purified rabbit polyclonal antibodies (6). For secondary antibodies, fluorescein-conjugated goatanti-rabbit serum (Sigma) or Texas Red-conjugated donkey anti-mouse antibodies (Cappel) were used at dilutions of 1:32 and 1:60, respectively. Except where noted, all SDS/PAGE was performed with 8% polyacrylamide gels. Protein blots were developed overnight with the following antibodies: a 1:2500 dilution of rabbit polyclonal anti-/3-catenin serum (B. Gumbiner); a 1:5000 dilution of anti-p120GAP (GTPaseactivating protein) serum (16), or a 1:500 dilution of anti-acatenin serum (J. Papkov, Sugen, Redwood City, CA), or a mixture of affinity-purified anti-APC2 and anti-APC3 antibodies (6), each at 0.2 ,ug/ml. After a 1-hr incubation in 25 mM Tris-buffered saline containing 0.05% Tween 20 and 125I1 labeled protein A (Amersham) at 1 ,uCi/ml (1 ,uCi = 37 kBq) blots were washed, exposed to x-ray film, and then quantitated by a 12-hr exposure on an Ambis model 4000 3 scanner. The ECL system (Amersham) was used for detection of the protein blots shown in Figs. 4 B and 5 A an...

Mutations in the human APC gene are linked to familial adenomatous polyposis and to the progression of sporadic colorectal and gastric tumors. To gain insight into APC function, APC-associated proteins were identified by immunoprecipitation experiments. Antibodies to APC precipitated a 95-kilodalton protein that was purified and identified by sequencing as beta-catenin, a protein that binds to the cell adhesion molecule E-cadherin. An antibody specific to beta-catenin also recognized the 95-kilodalton protein in the immunoprecipitates. These results suggest that APC is involved in cell adhesion.

No abstract

Proteins that associate with the GTP‐bound forms of the Ras superfamily of proteins are potential effector targets for these molecular switches. A 195 kDa protein was purified from cell lysates by affinity chromatography on immobilized cdc42Hs‐GTP and a corresponding cDNA was isolated. Sequence analysis revealed localized identities to calponin, the WW domain, unconventional myosins and to the rasGAP‐related domain (GRD) contained in IRA, NF‐1, SAR1 and rasGAP. p195 was found to be identical to IQGAP1, a protein previously reported to bind ras. Purified recombinant p195/IQGAP1 bound to and inhibited the GTPase activity of cdc42Hs and rac whereas no interaction with ras was detected. The C‐terminal half of IQGAP1 containing the GRD bound to cdc42 and rac in a GRD‐dependent fashion, but a smaller fragment containing only the GRD did not. Cdc42 was also co‐immunoprecipitated from cell lysates with antibody specific to p195/IQGAP1. Calmodulin also co‐immunoprecipitated with p195/IQGAP1 and was found to associate with fragments containing the IQ domain. Expression of a cDNA fragment encoding the GRD inhibited the CDC24/CDC42 pathway in yeast, but no effect on ras was observed. In mammalian cells, both endogenous and ectopically expressed p195/IQGAP1 were localized to lamellipodia and ruffling cell membranes, where co‐localization with actin was apparent. These results suggest that IQGAP1 is an effector target for cdc42Hs and may mediate the effects of this GTPase on cell morphology.

In the budding yeast Saccharomyces cerevisiae, the process of conjugation of haploid cells of genotype MATa and MAT alpha to form MATa/alpha diploids is triggered by pheromones produced by each mating type. These pheromones stimulate a cellular response by interaction with receptors linked to a heterotrimeric G protein. Although genetic analysis indicates that the pheromone signal is transmitted through the G beta gamma dimer, the initial target(s) of G protein activation remain to be determined. Temperature-sensitive cells with mutations of the CDC24 and CDC42 genes, which are incapable of budding and of generating cell polarity at the restrictive temperature, are also unable to mate. Cdc24 acts as a guanylyl-nucleotide-exchange factor for the Rho-type GTPase Cdc42, which has been shown to be a fundamental component of the molecular machinery controlling morphogenesis in eukaryotic cells. Therefore, the inability of cdc24 and cdc42 mutants to mate has been presumed to be due to a requirement for generation of cell polarity and related morphogenetic events during conjugation. But here we show that Cdc42 has a direct signalling role in the mating-pheromone response between the G protein and the downstream protein kinase cascade.

The tumor suppressor APC protein associates with the cadherin-binding proteins alpha- and beta-catenin. To examine the relationship between cadherin, catenins, and APC, we have tested combinatorial protein-protein interactions in vivo, using a yeast two-hybrid system, and in vitro, using purified proteins. beta-Catenin directly binds to APC at high and low affinity sites. alpha-Catenin cannot directly bind APC but associates with it by binding to beta-catenin. Plakoglobin, also known as gamma-catenin, directly binds to both APC and alpha-catenin and also to the APC-beta-catenin complex, but not directly to beta-catenin. beta-Catenin binds to multiple independent regions of APC, some of which include a previously identified consensus motif and others which contain the centrally located 20 amino acid repeat sequences. The APC binding site on beta-catenin may be discontinuous since neither the carboxyl- nor amino-terminal halves of beta-catenin will independently associate with APC, although the amino-terminal half independently binds alpha-catenin. The catenins bind to APC and E-cadherin in a similar fashion, but APC and E-cadherin do not associate with each other either in the presence or absence of catenins. Thus, APC forms distinct heteromeric complexes containing combinations of alpha-catenin, beta-catenin, and plakoglobin which are independent from the cadherin-catenin complexes.

MotivationCurrently there are no tools specifically designed for annotating genes in phages. Several tools are available that have been adapted to run on phage genomes, but due to their underlying design, they are unable to capture the full complexity of phage genomes. Phages have adapted their genomes to be extremely compact, having adjacent genes that overlap and genes completely inside of other longer genes. This non-delineated genome structure makes it difficult for gene prediction using the currently available gene annotators. Here we present PHANOTATE, a novel method for gene calling specifically designed for phage genomes. Although the compact nature of genes in phages is a problem for current gene annotators, we exploit this property by treating a phage genome as a network of paths: where open reading frames are favorable, and overlaps and gaps are less favorable, but still possible. We represent this network of connections as a weighted graph, and use dynamic programing to find the optimal path.ResultsWe compare PHANOTATE to other gene callers by annotating a set of 2133 complete phage genomes from GenBank, using PHANOTATE and the three most popular gene callers. We found that the four programs agree on 82% of the total predicted genes, with PHANOTATE predicting more genes than the other three. We searched for these extra genes in both GenBank’s non-redundant protein database and all of the metagenomes in the sequence read archive, and found that they are present at levels that suggest that these are functional protein-coding genes.Availability and implementation https://github.com/deprekate/PHANOTATE Supplementary information Supplementary data are available at Bioinformatics online.