EphrinB1 promotes cancer cell migration and invasion through the interaction with RhoGDI1.

Eph receptors and their corresponding ephrin ligands have been associated with regulating cell-cell adhesion and motility, and thus have a critical role in various biological processes including tissue morphogenesis and homeostasis, as well as pathogenesis of several diseases. Aberrant regulation of Eph/ephrin signaling pathways is implicated in tumor progression of various human cancers. Here, we show that a Rho family GTPase regulator, Rho guanine nucleotide dissociation inhibitor 1 (RhoGDI1), can interact with ephrinB1, and this interaction is enhanced upon binding the extracellular domain of the cognate EphB2 receptor. Deletion mutagenesis revealed that amino acids 327-334 of the ephrinB1 intracellular domain are critical for the interaction with RhoGDI1. Stimulation with an EphB2 extracellular domain-Fc fusion protein (EphB2-Fc) induces RhoA activation and enhances the motility as well as invasiveness of wild-type ephrinB1-expressing cells. These Eph-Fc-induced effects were markedly diminished in cells expressing the mutant ephrinB1 construct (Δ327-334) that is ineffective at interacting with RhoGDI1. Furthermore, ephrinB1 depletion by siRNA suppresses EphB2-Fc-induced RhoA activation, and reduces motility and invasiveness of the SW480 and Hs578T human cancer cell lines. Our study connects the interaction between RhoGDI1 and ephrinB1 to the promotion of cancer cell behavior associated with tumor progression. This interaction may represent a therapeutic target in cancers that express ephrinB1.

Oncogenic Met receptor induces ectopic structures in Xenopus embryos.

When aberrantly expressed or activated, the Met receptor tyrosine kinase is involved in tumor invasiveness and metastasis. In this study, we have used the Xenopus embryonic system to define the role of various Met proximal-binding partners and downstream signaling pathways in regulating an induced morphogenetic event. We show that expression of an oncogenic derivative of the Met receptor (Tpr-Met) induces ectopic morphogenetic structures during Xenopus embryogenesis. Using variant forms of Tpr-Met that are engineered to recruit a specific signaling molecule of choice, we demonstrate that the sole recruitment of either the Grb2 or the Shc adaptor protein is sufficient to induce ectopic structures and anterior reduction, while the recruitment of PI-3Kinase (PI-3K) is necessary but not sufficient for this effect. In contrast, the recruitment of PLCgamma can initiate the induction, but fails to maintain or elongate supernumerary structures. Finally, evidence indicates that the Ras/Raf/MAPK pathway is necessary, but not sufficient to induce these structures. This study also emphasizes the importance of examining signaling molecules in the regulatory context that is provided by receptor/effector interactions when assessing a role in cell growth and differentiation.

Xmeis1, a protooncogene involved in specifying neural crest cell fate in Xenopus embryos.

Meis1 (Myeloid Ecotropic viral Integration Site 1) is a homeobox gene that was originally isolated as a common site of viral integration in myeloid tumors of the BXH-2 recombinant inbred mice strain. We previously isolated a Xenopus homolog of Meis1 (Xmeis1). Here we show that Xmeis1 may play a significant role in neural crest development. In developing Xenopus embryos, Xmeis1 displays a broad expression pattern, but strong expression is observed in tissue of neural cell fate, such as midbrain, hindbrain, the dorsal portion of the neural tube, and neural crest derived branchial arches. In animal cap explants, overexpression of Xmeis1b, an alternatively spliced form of Xmeis1, induces expression of neural crest marker genes in the absence of mesoderm. Moreover, Xmeis1b induces XGli-3 and XZic3, pre-pattern genes involved at the earliest stages of neural crest development, and like these two genes, can induce ectopic pigmented cell masses when overexpressed in developing embryos. Misexpression of Xmeis1b also induces ectopic expression of neural crest markers along the antero-posterior axis of the neural tube in developing Xenopus embryos. In contrast, Xmeis1a, another splice variant, is much less effective at inducing these effects. These data suggest that Xmeis1b is involved in neural crest cell fate specification during embryogenesis, and can functionally intersect with the Gli/Zic signal transduction pathway.

Involvement of BMP-4/msx-1 and FGF pathways in neural induction in the Xenopus embryo.

The msx homeodomain protein is a downstream transcription factor of the bone morphogenetic protein (BMP)-4 signal and a key regulator for neural tissue differentiation. Xmsx-1 antagonizes the dorsal expression of noggin and cerberus, as revealed by in situ hybridization and reverse transcription-polymerase chain reaction assays. In animal cap explants, Xmsx-1 and BMP-4 inhibit the neural tissue differentiation induced by noggin or cerberus. A loss-of-function study using the Xmsx-1/VP-16 fusion construct indicated that neural tissue formation was directly induced by the injection of fusion ribonucleic acid, although the expression of neural cell adhesion molecule (N-CAM) in the cap was less than that in the cap injected with tBR or noggin. In contrast to the single cap assay, unexpectedly, both BMP-4 and Xmsx-1 failed to inhibit neurulation in the ectodermal explants to which the organizer mesoderm was attached. The results of cell-lineage tracing experiments indicated that the neural cells were differentiated from the animal pole tissue where the excess RNA of either BMP-4 or Xmsx-1 was injected, whereas notochord was differentiated from the organizer mesoderm. Neural tissue differentiated from BMP-4-injected ectodermal cells strongly expressed posterior neural markers, such as hoxB9 and krox20, suggesting that the posterior neural cells differentiated regardless of the existence of the BMP signal. The introduction of a dominant-negative form of the fibroblast growth factor (FGF) receptor (XFD) into the ectodermal cells drastically reduced the expression of pan and posterior neural markers (N-CAM and hoxB-9) if co-injected with BMP-4 RNA, although XFD alone at the same dose did not shut down the expression of N-CAM in the combination explants. Therefore, it is proposed that an FGF-related molecule was involved in the direct induction of posterior neural tissue in the inducing signals from the organizer mesoderm in vivo.

Fibroblast growth factor receptor-mediated rescue of x-ephrin B1-induced cell dissociation in Xenopus embryos.

The Eph family of receptor tyrosine kinases and their membrane-bound ligands, the ephrins, have been implicated in regulating cell adhesion and migration during development by mediating cell-to-cell signaling events. Genetic evidence suggests that ephrins may transduce signals and become tyrosine phosphorylated during embryogenesis. However, the induction and functional significance of ephrin phosphorylation is not yet clear. Here, we report that when we used ectopically expressed proteins, we found that an activated fibroblast growth factor (FGF) receptor associated with and induced the phosphorylation of ephrin B1 on tyrosine. Moreover, this phosphorylation reduced the ability of overexpressed ephrin B1 to reduce cell adhesion. In addition, we identified a region in the cytoplasmic tail of ephrin B1 that is critical for interaction with the FGF receptor; we also report FGF-induced phosphorylation of ephrins in a neural tissue. This is the first demonstration of communication between the FGF receptor family and the Eph ligand family and implicates cross talk between these two cell surface molecules in regulating cell adhesion.

Xenopus CRMP-2 is an early response gene to neural induction.

A neural specific protein, CRMP-2 (for Collapsin Response Mediator Protein-2), is considered to mediate collapsin-induced growth cone collapse during neural development. We have isolated the Xenopus homologue of the CRMP-2 (XCRMP-2) cDNA and studied the expression of XCRMP-2 mRNA and protein during neural induction. Induction of XCRMP-2 mRNA and protein expression, like N-CAM, occurred at the midgastrula stage and increased through early neural developmental stages. Whole mount in situ hybridization demonstrated that expression of XCRMP-2 mRNA was localized in neural tissues such as the neural plate and tube at early stages, while its expression in the brain, spinal cord, and eyes was observed at later stages. Immunostaining of Xenopus embryos with the antibody against CRMP-2 also showed that the protein was specifically expressed in the neural tissues at early stages. XCRMP-2 expression was induced by neural inducers such as noggin and chordin which antagonize a neural inhibitor, BMP4. A dominant negative BMP receptor also induced XCRMP-2 expression, suggesting that transcription of XCRMP-2 gene was negatively regulated by the BMP4 signaling. These results indicate that expression of XCRMP-2 is an early response marking neural commitment, and that transcriptional control of XCRMP-2 gene, is one of the targets of BMP4 signaling.

Loss of cell adhesion in Xenopus laevis embryos mediated by the cytoplasmic domain of XLerk, an erythropoietin-producing hepatocellular ligand.

The erythropoietin-producing hepatocellular (Eph) family of ligands and receptors has been implicated in the control of axon guidance and the segmental restriction of cells during embryonic development. In this report, we show that ectopic expression of XLerk, a Xenopus homologue of the murine Lerk-2 (ephrin-B1) transmembrane ligand, causes dissociation of Xenopus embryonic blastomeres by the mid-blastula transition. Moreover, a mutant that lacks the extracellular receptor binding domain can induce this phenotype. The carboxyl-terminal 19 amino acids of the cytoplasmic domain of XLerk are necessary but not sufficient to induce cellular dissociation. Basic fibroblast growth factor, but not activin, can rescue both the loss of cell adhesion and mesoderm induction in ectodermal explants expressing XLerk. Collectively, these results show that the cytoplasmic domain of XLerk has a signaling function that is important for cell adhesion, and fibroblast growth factor signaling modulates this function.

Identification of XLerk, an Eph family ligand regulated during mesoderm induction and neurogenesis in Xenopus laevis.

We have isolated and characterized the first Xenopus transmembrane Eph ligand, XLerk (Xenopus Ligand for Eph Receptor Tyrosine Kinases). While this ligand has 72% identity with the closest mammalian family member, Lerk-2, it is the cytoplasmic domain of this molecule that is the most conserved domain with 95% identity. XLerk exists as a maternally expressed mRNA, however, expression of transcripts and protein increase during gastrulation and again in the late swimming tadpole stage. In the adult, XLerk is expressed at low levels in most adult tissues with increased levels observed in the kidney, oocytes, ovary and testis. While low levels of XLerk expression are observed in the adult brain, in situ hybridization analysis demonstrates prominent expression in the developing olfactory system, retina, hindbrain, cranial ganglia, and somites. Furthermore, we have shown that XLerk transcripts are significantly elevated during mesoderm induction caused by activin and FGF, but not during noggin-induced neuralization. These results suggest a role for XLerk in the developing mesenchymal and nervous tissue.

Identification of a conserved family of Meis1-related homeobox genes.

Meis1 locus was isolated as a common site of viral integration involved in myeloid leukemia in BXH-2 mice. Meis1 encodes a novel homeobox protein belonging to the TALE (three amino acid loop extension) family of homeodomain-containing proteins. The homeodomain of Meis1 is the only known motif within the entire 390-amino-acid protein. Southern blot analyses using the Meis1 homeodomain as a probe revealed the existence family of Meis1-related genes (Mrgs) in several diverged species. In addition, the 3' untranslated region (UTR) Meis1 was remarkably conserved in evolution. To gain a further understanding of the role Meis1 plays in leukemia and development, as well as to identify conserved regions of the protein that might reveal function, we cloned and characterized Mrgs from the mouse and human genomes. We report the sequence of Mrg1 and MRG2 as well as their chromosomal locations in murine and human genomes. Both Mrgs share a high degree of sequence identity with the protein coding region of Meis1. We have also cloned the Xenopus laevis ortholog of (XMeis1). Sequence comparison of the murine and Xenopus clones reveals that Meis1 is highly conserved throughout its coding sequence as well as the 3' UTR. Finally, comparison of Meis1 and the closely related Mrgs to known homeoproteins suggests that Meis1 represents a new subfamily of TALE homeobox genes.

Meis1, a PBX1-related homeobox gene involved in myeloid leukemia in BXH-2 mice.

Leukemia results from the accumulation of multiple genetic alterations that disrupt the control mechanisms of normal growth and differentiation. The use of inbred mouse strains that develop leukemia has greatly facilitated the identification of genes that contribute to the neoplastic transformation of hematopoietic cells. BXH-2 mice develop myeloid leukemia as a result of the expression of an ecotropic murine leukemia virus that acts as an insertional mutagen to alter the expression of cellular proto-oncogenes. We report the isolation of a new locus, Meis1, that serves as a site of viral integration in 15% of the tumors arising in BXH-2 mice. Meis1 was mapped to a distinct location on proximal mouse chromosome 11, suggesting that it represents a novel locus. Analysis of somatic cell hybrids segregating human chromosomes allowed localization of MEIS1 to human chromosome 2p23-p12, in a region known to contain translocations found in human leukemias. Northern (RNA) blot analysis demonstrated that a Meis1 probe detected a 3.8-kb mRNA present in all BXH-2 tumors, whereas tumors containing integrations at the Meis1 locus expressed an additional truncated transcript. A Meis1 cDNA clone that encoded a novel member of the homeobox gene family was identified. The homeodomain of Meis1 is most closely related to those of the PBX/exd family of homeobox protein-encoding genes, suggesting that Meis1 functions in a similar fashion by cooperative binding to a distinct subset of HOX proteins. Collectively, these results indicate that altered expression of the homeobox gene Meis1 may be one of the events that lead to tumor formation in BXH-2 mice.

Expression of an amphibian homolog of the Eph family of receptor tyrosine kinases is developmentally regulated.

In order to study the function of tyrosine kinase receptors during Xenopus development, we have isolated Xek (Xenopus Elk-like kinase), a tyrosine kinase receptor, which shows significant homology to rat Elk and chicken cek5, members of the Eph family. Xek exists as a maternally expressed mRNA which decreases in expression at the mid blastula transition and reappears at late neurulation in Xenopus. Xek mRNA is expressed at higher levels in the anterior and dorsal regions of embryonic stages 16, 24 and 37. In adult Xenopus tissues, Xek appears to be ubiquitously expressed with higher expression observed in brain and ovary. In situ hybridization analysis demonstrates localized mRNA expression in the brain, brachial arches, trigeminal facial ganglion, and the retina of the swimming tadpole stage of development. The similarities in sequence and expression pattern suggest that Xek is an amphibian member of the Eph family and may play a role in the development or function of the central nervous system.

Critical tyrosine residues regulate the enzymatic and biological activity of Raf-1 kinase.

The serine/threonine kinase activity of the Raf-1 proto-oncogene product is stimulated by the activation of many tyrosine kinases, including growth factor receptors and pp60v-src. Recent studies of growth factor signal transduction pathways demonstrate that Raf-1 functions downstream of activated tyrosine kinases and p21ras and upstream of mitogen-activated protein kinase. However, coexpression of both activated tyrosine kinases and p21ras is required for maximal activation of Raf-1 in the baculovirus-Sf9 expression system. In this study, we investigated the role of tyrosine kinases and tyrosine phosphorylation in the regulation of Raf-1 activity. Using the baculovirus-Sf9 expression system, we identified Tyr-340 and Tyr-341 as the major tyrosine phosphorylation sites of Raf-1 when coexpressed with activated tyrosine kinases. Introduction of a negatively charged residue that may mimic the effect of phosphorylation at these sites activated the catalytic activity of Raf-1 and generated proteins that could transform BALB/3T3 cells and induce the meiotic maturation of Xenopus oocytes. In contrast, substitution of noncharged residues that were unable to be phosphorylated produced a protein that could not be enzymatically activated by tyrosine kinases and that could block the meiotic maturation of oocytes induced by components of the receptor tyrosine kinase pathway. These findings demonstrate that maturation of the tyrosine phosphorylation sites can dramatically alter the function of Raf-1. In addition, this is the first report that a transforming Raf-1 protein can be generated by a single amino acid substitution.

Requirement for Raf and MAP kinase function during the meiotic maturation of Xenopus oocytes.

The role of Raf and MAPK (mitogen-activated protein kinase) during the maturation of Xenopus oocytes was investigated. Treatment of oocytes with progesterone resulted in a shift in the electrophoretic mobility of Raf at the onset of germinal vesicle breakdown (GVBD), which was coincident with the activation of MAPK. Expression of a kinase-defective mutant of the human Raf-1 protein (KD-RAF) inhibited progesterone-mediated MAPK activation. MAPK activation was also inhibited by KD-Raf in oocytes expressing signal transducers of the receptor tyrosine kinase (RTK) pathway, including an activated tyrosine kinase (Tpr-Met), a receptor tyrosine kinase (EGFr), and Ha-RasV12. KD-RAF completely inhibited GVBD induced by the RTK pathway. In contrast, KD-RAF did not inhibit GVBD and the progression to Meiosis II in progesterone-treated oocytes. Injection of Mos-specific antisense oligodeoxyribonucleotides inhibited MAPK activation in response to progesterone and Tpr-Met, but failed to inhibit these events in oocytes expressing an oncogenic deletion mutant of Raf-1 (delta N'Raf). Injection of antisense oligodeoxyribonucleotides to Mos also reduced the progesterone- and Tpr-Met-induced electrophoretic mobility shift of Xenopus Raf. These results demonstrate that RTKs and progesterone participate in distinct yet overlapping signaling pathways resulting in the activation of maturation or M-phase promoting factor (MPF). Maturation induced by the RTK pathway requires activation of Raf and MAPK, while progesterone-induced maturation does not. Furthermore, the activation of MAPK in oocytes appears to require the expression of Mos.

tpr-met oncogene product induces maturation-producing factor activation in Xenopus oocytes.

tpr-met, a tyrosine kinase oncogene, is the activated form of the met proto-oncogene that encodes the receptor for hepatocyte growth factor/scatter factor. The tpr-met product (p65tpr-met) was tested for its ability to induce meiotic maturation in Xenopus oocytes. While src and abl tyrosine kinase oncogene products have previously been shown to be inactive in this assay, p65tpr-met efficiently induced maturation-promoting factor (MPF) activation and germinal vesicle breakdown (GVBD) together with the associated increase in ribosomal S6 subunit phosphorylation. tpr-met-mediated MPF activation and GVBD was dependent on the endogenous c-mosxe, while the increase in S6 protein phosphorylation was not significantly affected by the loss of mos function. The phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine inhibits tpr-met-mediated GVBD at concentrations that prevent insulin- but not progesterone-induced oocyte maturation. Moreover, maturation triggered by tpr-met is also inhibited by cyclic AMP-dependent protein kinase. This is the first demonstration that a tyrosine kinase oncogene product, p65tpr-met, can induce meiotic maturation in Xenopus oocytes and activate MPF through a mos-dependent pathway, possibly the insulin or insulinlike growth factor 1 pathway.

Premature translation termination mediates triosephosphate isomerase mRNA degradation.

We characterized an anemia-inducing mutation in the human gene for triosephosphate isomerase (TPI) that resulted in the production of prematurely terminated protein and mRNA with a reduced cytoplasmic half-life. The mutation converted a CGA arginine codon to a TGA nonsense codon and generated a protein of 188 amino acids, instead of the usual 248 amino acids. To determine how mRNA primary structure and translation influence mRNA stability, in vitro-mutagenized TPI alleles were introduced into cultured L cells and analyzed for their effect on TPI RNA metabolism. Results indicated that mRNA stability is decreased by all nonsense and frameshift mutations. To determine the relative contribution of the changes in mRNA structure and translation to the altered half-life, the effects of individual mutations were compared with the effects of second-site reversions that restored translation termination to normal. All mutations that resulted in premature translation termination reduced the mRNA half-life solely or mainly by altering the length of the mRNA that was translated. The only mutation that altered translation termination and that reduced the mRNA half-life mainly by affecting the mRNA structure was an insertion that shifted termination to a position downstream of the normal stop codon.

Human triose-phosphate isomerase deficiency: a single amino acid substitution results in a thermolabile enzyme.

Triose-phosphate isomerase (TPI; D-glyceraldehyde-3-phosphate ketol-isomerase, EC 5.3.1.1) deficiency is a recessive disorder that results in hemolytic anemia and neuromuscular dysfunction. To determine the molecular basis of this disorder, a TPI allele from two unrelated patients homozygous for TPI deficiency was compared with an allele from a normal individual. Each disease-associated sequence harbors a G X C----C X G transversion in the codon for amino acid-104 and specifies a structurally altered protein in which a glutamate residue is replaced by an aspartate residue. The importance of glutamate-104 to enzyme structure and function is implicated by its conservation in the TPI protein of all species that have been characterized to date. The glutamate-to-aspartate substitution results in a thermolabile enzyme as demonstrated by assays of TPI activity in cultured fibroblasts of each patient and cultured Chinese hamster ovary (CHO) cells that were stably transformed with the mutant alleles. Although this substitution conserves the overall charge of amino acid-104, the x-ray crystal structure of chicken TPI indicates that the loss of a side-chain methylene group (-CH2CH2COO- ---- -CH2COO-) is sufficient to disrupt the counterbalancing of charges that normally exists within a hydrophobic pocket of the native enzyme.

Characterization of the functional gene and several processed pseudogenes in the human triosephosphate isomerase gene family.

The functional gene and three intronless pseudogenes for human triosephosphate isomerase were isolated from a recombinant DNA library and characterized in detail. The functional gene spans 3.5 kilobase pairs and is split into seven exons. Its promoter contains putative TATA and CCAAT boxes and is extremely rich in G and C residues (76%). The pseudogenes share a high degree of homology with the functional gene but contain mutations that preclude the synthesis of an active triosephosphate isomerase enzyme. Sequence divergence calculations indicate that these pseudogenes arose approximately 18 million years ago. We present evidence that there is a single functional gene in the human triosephosphate isomerase gene family.