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PubMed2000

Endogenous protein kinase CK2 participates in Wnt signaling in mammary epithelial cells.

Protein kinase CK2 (formerly casein kinase II) is a serine/threonine kinase overexpressed in many human tumors, transformed cell lines, and rapidly proliferating tissues. Recent data have shown that many cancers involve inappropriate reactivation of Wnt signaling through ectopic expression of Wnts themselves, as has been seen in a number of human breast cancers, or through mutation of intermediates in the Wnt pathway, such as adenomatous polyposis coli or beta-catenin, as described in colon and other cancers. Wnts are secreted factors that are important in embryonic development, but overexpression of certain Wnts, such as Wnt-1, leads to proliferation and transformation of cells. We report that upon stable transfection of Wnt-1 into the mouse mammary epithelial cell line C57MG, morphological changes and increased proliferation are accompanied by increased levels of CK2, as well as of beta-catenin. CK2 and beta-catenin co-precipitate with the Dvl proteins, which are Wnt signaling intermediates. A major phosphoprotein of the size of beta-catenin appears in in vitro kinase reactions performed on the Dvl immunoprecipitates. In vitro translated beta-catenin, Dvl-2, and Dvl-3 are phosphorylated by CK2. The selective CK2 inhibitor apigenin blocks proliferation of Wnt-1-transfected cells, abrogates phosphorylation of beta-catenin, and reduces beta-catenin and Dvl protein levels. These results demonstrate that endogenous CK2 is a positive regulator of Wnt signaling and growth of mammary epithelial cells.

Adaptor Proteins, Signal Transducing↗
PubMed1999

A leucine-rich nuclear export signal in the p53 tetramerization domain: regulation of subcellular localization and p53 activity by NES masking.

Appropriate subcellular localization is crucial for regulating p53 function. We show that p53 export is mediated by a highly conserved leucine-rich nuclear export signal (NES) located in its tetramerization domain. Mutation of NES residues prevented p53 export and hampered tetramer formation. Although the p53-binding protein MDM2 has an NES and has been proposed to mediate p53 export, we show that the intrinsic p53 NES is both necessary and sufficient for export. This report also demonstrates that the cytoplasmic localization of p53 in neuroblastoma cells is due to its hyperactive nuclear export: p53 in these cells can be trapped in the nucleus by the export-inhibiting drug leptomycin B or by binding a p53-tetramerization domain peptide that masks the NES. We propose a model in which regulated p53 tetramerization occludes its NES, thereby ensuring nuclear retention of the DNA-binding form. We suggest that attenuation of p53 function involves the conversion of tetramers into monomers or dimers, in which the NES is exposed to the proteins which mediate their export to the cytoplasm.

3T3 Cells↗
PubMed1999

An HMG1-like protein facilitates Wnt signaling in Caenorhabditis elegans.

We show that during Caenorhabditis elegans male spicule development, the specification of a glial versus neuronal cell fate in a canonical neurogenic sublineage is dependent on Wnt signaling. Inactivation of a Wnt signaling pathway mediated by the Wnt receptor LIN-17 transforms the SPD sheath cell into its sister, the SPD neuron. We discovered a new mutant, son-1, that displays this same cell fate transformation. The son-1 mutation enhances the phenotypes of reduction-of-function lin-17 mutants in several developmental processes, including vulva development, somatic gonad development, and male tail patterning. son-1 encodes an HMG1/2-like DNA-binding protein and is localized in all cell nuclei through development as revealed by a GFP reporter construct. Disruption of son-1 function by RNA-mediated interference results in the same spicule defect as caused by overexpression of POP-1, a TCF/LEF class HMG protein known to act downstream of the Wnt signaling pathway. Our results provide in vivo evidence for the functional involvement of an HMG1/2-like protein, SON-1, in Wnt signaling. The sequence nonspecific HMG protein SON-1 and the sequence specific HMG protein POP-1 might both act in the Wnt responding cells to regulate gene transcription in opposite directions.

Amino Acid Sequence↗
PubMed2000

A C. elegans mediator protein confers regulatory selectivity on lineage-specific expression of a transcription factor gene.

The Caenorhabditis elegans caudal homolog, pal-1, is required for neurogenesis in the male tail. We show that expression of pal-1 in the postembryonic neuroblast cell V6 can be initiated by two alternate pathways. One pathway, acting in wild type, requires a regulatory element in the fifth pal-1 intron. The other pathway, independent of this element, is normally repressed by the newly identified gene sop-1, which encodes a homolog of the mammalian Mediator complex protein TRAP230. In sop-1 mutants, pal-1 is activated by a pathway that is stimulated by bar-1/beta-catenin, a component of the Wnt signal transduction pathway. The results support a physiological role of the Mediator complex in conveying regulatory signals to the transcriptional apparatus.

Amino Acid Sequence↗
PubMed2000

New thoughts on the role of the beta-gamma subunit in G-protein signal transduction.

Heterotrimeric G proteins are involved in numerous biological processes, where they mediate signal transduction from agonist-bound G-protein-coupled receptors to a variety of intracellular effector molecules and ion channels. G proteins consist of two signaling moieties: a GTP-bound alpha subunit and a beta-gamma heterodimer. The beta-gamma dimer, recently credited as a significant modulator of G-protein-mediated cellular responses, is postulated to be a major determinant of signaling fidelity between G-protein-coupled receptors and downstream effectors. In this review we have focused on the role of beta-gamma signaling and have included examples to demonstrate the heterogeneity in the heterodimer composition and its implications in signaling fidelity. We also present an overview of some of the effectors regulated by beta-gamma and draw attention to the fact that, although G proteins and their associated receptors play an instrumental role in development, there is rather limited information on beta-gamma signaling in embryogenesis.

Animals↗
PubMed2001

The role of the homeodomain protein Bozozok in zebrafish axis formation.

The zebrafish bozozok (boz) gene encoding a homeodomain protein (also named Dharma/Nieuwkoid) is required during blastula stages for the formation of a complete Spemann-Mangold gastrula organizer and subsequent development of axial mesoderm and anterior neural structures. Expression of bozin the dorsal yolk syncytial layer (YSL) and overlying marginal blastomeres is activated by beta-catenin. Bozozok itself acts as a transcriptional repressor, and promotes organizer formation by directly inhibiting expression of the bmp2b (swirl) gene and by negatively regulating Wnt signaling by an unknown mechanism. boz cooperates with the Nodal-related secreted factors, Cyclops and Squint, in organizer formation. The incomplete organizer in boz mutants is deficient in expression of a number of factors such as Chordin that antagonize Bone morphogenetic proteins (Bmps), and Dickkopf 1, a Wnt antagonist. Conversely, the dorsal blastoderm of boz mutants exhibits ectopic expression of genes normally excluded from the dorsal midline such as wnt8 or tbx6. boz specifies the formation of anterior neuroectoderm by regulating Bmp and Wnt pathways in a fashion consistent with Nieuwkoop's two-step neural patterning model. boz promotes neural induction by limiting the anti-neuralizing activity of Bmp morphogens. In addition, by negative regulation of Wnt signaling, boz limits posteriorization of neuroectoderm. bozozok chordino double mutants exhibit a synergistic loss of head and trunk. This synthetic phenotype is due to dramatically increased Bmp signaling and consequent massive accumulation of cells in the tailbud at the expense of dorso-anterior structures. Therefore, boz and din act in overlapping pathways that provide the main mechanism to limit Bmp signaling in the zebrafish gastrula and allow for head and trunk development. Notably, Bozozok appears to function by repressing transcription of target genes such as swr (bmp2b) gene, and as such is the earliest acting repressor that the nascent dorsal axis is using to antagonize ventral influences.

Animals↗
PubMed2005

Retinoic acid-metabolizing enzyme Cyp26a1 is essential for determining territories of hindbrain and spinal cord in zebrafish.

Retinoic acid (RA) plays a critical role in neural patterning and organogenesis in the vertebrate embryo. Here we characterize a mutant of the zebrafish named giraffe (gir) in which the gene for the RA-degrading enzyme Cyp26a1 is mutated. The gir mutant displayed patterning defects in multiple organs including the common cardinal vein, pectoral fin, tail, hindbrain, and spinal cord. Analyses of molecular markers suggested that the lateral plate mesoderm is posteriorized in the gir mutant, which is likely to cause the defects of the common cardinal vein and pectoral fin. The cyp26a1 expression in the rostral spinal cord was strongly upregulated in the gir mutant, suggesting a strong feedback control of its expression by RA signaling. We also found that the rostral spinal cord territory was expanded at the expense of the hindbrain territory in the gir mutant. Such a phenotype is the opposite of that of the mutant for Raldh2, an enzyme that synthesizes RA. We propose a model in which Cyp26a1 attenuates RA signaling in the prospective rostral spinal cord to limit the expression of hox genes and to determine the hindbrain-spinal cord boundary.

Animals↗
PubMed1996

Cell fate specification in the inner ear.

From its origin as a single ectodermal patch, the inner ear becomes a labyrinth of chambers housing six to eight sensory organs. Along the way, specific cell fates are realized. The secrets underlying these cell fate specifications are beginning to be revealed through the application of several molecular-genetic approaches. Recent papers describing such approaches have included gene expression studies in the early otic epithelium and inner ear sensory epithelia. large-scale screens of zebrafish mutants to identify ear defects, and targeted gene perturbations of neurotrophins. of their receptors or of the Brn-3.1 transcription factor in mice.

Animals↗
PubMed1996

Mutations disrupting the ordering and topographic mapping of axons in the retinotectal projection of the zebrafish, Danio rerio.

Retinal ganglion cells connect to their target organ, the rectum, in a highly ordered fashion. We performed a large-scale screen for mutations affecting the retinotectal projection of the zebrafish, which resulted in the identification of 114 mutations. 44 of these mutations disturb either the order of RGC axons in the optic nerve and tract, the establishment of a topographic map on the tectum, or the formation of proper termination fields. Mutations in three genes, boxer, dackel and pinscher, disrupt the sorting of axons in the optic tract but do not affect mapping on the tectum. In these mutants, axons from the dorsal retina grow along both the ventral and the dorsal branch of the optic tract. Mutations in two genes, nevermind and who-cares, affect the dorsoventral patterning of the projection. In embryos homozygous for either of these mutations, axons from dorsal retinal ganglion cells terminate ventrally and dorsally in the tectum. In nevermind, the retinotopic order of axons along the optic nerve and tract is changed in a characteristic way as well, while it appears to be unaffected in who-cares. Two mutations in two complementation groups, gnarled and macho, affect the anteroposterior patterning of the projection. In these mutants, nasodorsal axons branch and terminate too soon in the anterior tectum. In 27 mutants belonging to six complementation groups, retinal axons do not form normal termination fields. Some implications for models concerning the formation of topographic projections are discussed.

Animals↗
PubMed2001

RefSeq and LocusLink: NCBI gene-centered resources.

Thousands of genes have been painstakingly identified and characterized a few genes at a time. Many thousands more are being predicted by large scale cDNA and genomic sequencing projects, with levels of evidence ranging from supporting mRNA sequence and comparative genomics to computing ab initio models. This, coupled with the burgeoning scientific literature, makes it critical to have a comprehensive directory for genes and reference sequences for key genomes. The NCBI provides two resources, LocusLink and RefSeq, to meet these needs. LocusLink organizes information around genes to generate a central hub for accessing gene-specific information for fruit fly, human, mouse, rat and zebrafish. RefSeq provides reference sequence standards for genomes, transcripts and proteins; human, mouse and rat mRNA RefSeqs, and their corresponding proteins, are discussed here. Together, RefSeq and LocusLink provide a non-redundant view of genes and other loci to support research on genes and gene families, variation, gene expression and genome annotation. Additional information about LocusLink and RefSeq is available at http://www.ncbi.nlm.nih.gov/LocusLink/.

Animals↗
PubMed2003

Lazy eyes zebrafish mutation affects Müller glial cells, compromising photoreceptor function and causing partial blindness.

A behavioral assay based on the optokinetic reflex was used to screen chemically mutagenized zebrafish larvae for deficits in visual function. A homozygous recessive mutation, lazy eyes (lze), was isolated based on the observation that 5-day postfertilization (dpf) mutants displayed weaker and less frequent eye movements than wild-type fish in response to moving stripes. Electroretinographic (ERG) recordings revealed that mutants had severely reduced a- and b-wave amplitudes relative to wild-type fish, indicating outer retinal dysfunction. Retinal lamination and cellular differentiation were normal in the lze retina; however, mutant photoreceptor cells had small outer segments and pyknotic nuclei were occasionally observed in the outer retina and the marginal zone of lze. Cone, rod, amacrine, bipolar, and Müller cell marker analyses indicated that the typical lze retina contained fewer rod photoreceptors and fewer Müller cells than wild-type fish at 5 dpf. At 3 dpf, however, mutant retinas had normal numbers of rod photoreceptors and Müller cells, suggesting that the initial differentiation of these cell types occurred normally. Rod photoreceptor histology was normal at this early stage, but Müller cells were often hypertrophied, suggesting that they were unhealthy. Constant light rearing of mutant animals accelerated the Müller cell degeneration, severely worsened the visual deficit, but had no obvious affect on the photoreceptors. When ERG responses and Müller cell degeneration from the same mutant animals were analyzed, the extent of the Müller cell loss matched closely the degree to which ERG responses were reduced. In summary, the lze gene appears to be required for Müller cell viability and normal visual function. The lze mutant may be a model for the study of the involvement of Müller cells in photoreceptor development and function.

Adaptation, Ocular↗
PubMed2002

Modulation of BMP activity in dorsal-ventral pattern formation by the chordin and ogon antagonists.

We analyzed the interactions between mutations in antagonistic BMP pathway signaling components to examine the roles that the antagonists play in regulating BMP signaling activity. The dorsalized mutants swirl/bmp2b, snailhouse/bmp7, lost-a-fin/alk8, and mini fin/tolloid were each analyzed in double mutant combinations with the ventralized mutants chordino/chordin and ogon, whose molecular nature is not known. Similar to the BMP antagonist chordino, we found that the BMP ligand mutants swirl/bmp2b and snailhouse/bmp7 are also epistatic to the putative BMP pathway antagonist, ogon, excluding a class of intracellular antagonists as candidates for ogon. In ogon;mini fin double mutants, we observed a mutual suppression of the ogon and mini fin mutant phenotypes, frequently to a wild type phenotype. Thus, the Tolloid/Mini fin metalloprotease that normally cleaves and inhibits Chordin activity is dispensable, when Ogon antagonism is reduced. These results suggest that Ogon encodes a Tolloid and Chordin-independent antagonistic function. By analyzing genes whose expression is very sensitive to BMP signaling levels, we found that the absence of Ogon or Chordin antagonism did not increase the BMP activity remaining in swirl/bmp2b or hypomorphic snailhouse/bmp7 mutants. These results, together with other studies, suggest that additional molecules or mechanisms are essential in generating the presumptive gastrula BMP activity gradient that patterns the dorsal-ventral axis. Lastly we observed a striking increased penetrance of the swirl/bmp2b dominant dorsalized phenotype, when Chordin function is also absent. Loss of the BMP antagonist Chordin is expected to increase BMP signaling levels in a swirl heterozygote, but instead we observed an apparent decrease in BMP signaling levels and a loss of ventral tail tissue. As has been proposed for the fly orthologue of chordin, short gastrulation, our paradoxical results can be explained by a model whereby Chordin both antagonizes and promotes BMP activity.

Animals↗
PubMed2004

Differential cooperation between dHAND and three different E-proteins.

dHAND is a transcription factor belonging to the class B basic helix-loop-helix protein family and is expressed during embryogenesis in the heart, branchial arches, limb buds, and neural crest derivatives. Despite much study, the molecular mechanisms involved in the regulation of dHAND activity are not well understood. We therefore carried out yeast two-hybrid screening using full-length dHAND as bait, which led to identification of several dHAND-binding proteins, including three E-proteins: E2A, ME2, and ALF1. Subsequent analysis revealed that although their heterodimerization and transcriptional activities were similar, dHAND/E-protein heterodimers bind to an E-box element with differing affinities, suggesting they have distinct DNA binding specificities. Moreover, in situ hybridization showed that E-protein genes are expressed fairly ubiquitously among embryonic tissues, including the branchial arches and limb buds. By contrast, little signal was detected in the heart, suggesting that dHAND complexes with partners other than E-proteins in cardiac tissue.

Animals↗
PubMed2002

Presenilin couples the paired phosphorylation of beta-catenin independent of axin: implications for beta-catenin activation in tumorigenesis.

The Alzheimer's disease-linked gene presenilin 1 (PS1) is required for intramembrane proteolysis of APP and Notch. In addition, recent observations strongly implicate PS1 as a negative regulator of the Wnt/beta-catenin signaling pathway, although the mechanism underlying this activity is unknown. Here, we show that presenilin functions as a scaffold that rapidly couples beta-catenin phosphorylation through two sequential kinase activities independent of the Wnt-regulated Axin/CK1alpha complex. Thus, presenilin deficiency results in increased beta-catenin stability in vitro and in vivo by disconnecting the stepwise phosphorylation of beta-catenin, both in the presence and absence of Wnt stimulation. These findings highlight an aspect of beta-catenin regulation outside of the canonical Wnt-regulated pathway and a function of presenilin separate from intramembrane proteolysis.

Alzheimer Disease↗
PubMed2003

Mutagenesis study on the zebra fish SOX9 high-mobility group: comparison of sequence and non-sequence specific HMG domains.

A unique class of proteins, containing high-mobility group (HMG) domain(s), recognizes unusual DNA structures and/or bends specific to AT-rich linear double-stranded DNA. The DNA binding feature of these proteins is exhibited in the HMG domain(s). Although the sequence specific and non-sequence specific HMG domains exhibit very high degrees of sequence similarity, the reasons for the difference between their DNA recognition mechanisms are unclear. A series of zebra fish SOX9 HMG domain mutants was prepared in an effort to elucidate the importance of various residues on protein stability and DNA binding. This study is the first of a comprehensive mutagenesis study on a sequence specific HMG domain. Comparing how various residues influence sequence specific and non-sequence specific HMG domains helps us to rationalize their mode of action. Positively charged amino acids concentrated at the surface of sequence specific HMG domains recognize specific, linear AT-rich DNA segments. After the negative charges at the surface of the DNA are neutralized, the hydrophobic residues of the protein may intercalate DNA. Phenylalanine at position 12 plays a crucial role in the sequence specific HMG domain. The differences in pI values, the instability index, and DNA contact regions between sequence and non-sequence specific HMG domains are associated with their functional modes.

Amino Acid Sequence↗
PubMed2003

Mammary gland development requires syndecan-1 to create a beta-catenin/TCF-responsive mammary epithelial subpopulation.

Mice with a null mutation in the cell surface heparan sulfate (HS) proteoglycan, syndecan-1 (Sdc1), develop almost normally, but resist mammary tumor development in response to Wnt-1. Here, we test the hypothesis that Sdc1 promotes Wnt-1-induced tumor development by interacting with the Wnt cell surface signaling complex. Thus, the response of Sdc1-/- mammary epithelial cells (mecs) to the intracellular, activated Wnt signal transducer, DeltaNbeta-catenin, was assayed both in vitro and in vivo, to test whether beta-catenin/TCF transactivation was Sdc1-independent. Surprisingly, we found that the expression of a canonical Wnt pathway reporter, TOP-FLASH, was reduced by 50% in both unstimulated Sdc1-/- mecs and in stimulated cells responding to Wnt1 or DeltaNbeta-catenin. Tumor development in response to DeltaNbeta-catenin was also significantly delayed on a Sdc1-/- background. Furthermore, the average beta-catenin/TCF transactivation per cell was normal in Sdc1-/- mec cultures, but the number of responsive cells was reduced by 50%. Sdc1-/- mecs show compensatory changes that maintain the number of HS chains, hence these experiments cannot test the coreceptor activity of HS for Wnt signaling. We propose that TCF-dependent transactivational activity is suppressed in 50% of cells in Sdc1-/- glands, and conclude that the major effect of Sdc1 does not map to the activity of the Wnt signaling complex, but to another pathway to create or stabilize the beta-catenin/TCF-responsive tumor precursor cells in mouse mammary gland.

Animals↗