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PubMed2003

The EphB6 receptor inhibits JNK activation in T lymphocytes and modulates T cell receptor-mediated responses.

EphB6 is the most recently identified member of the Eph receptor tyrosine kinase family. EphB6 is primarily expressed in thymocytes and a subpopulation of T cells, suggesting that it may be involved in regulation of T lymphocyte differentiation and functions. We show here that overexpression of EphB6 in Jurkat T cells and stimulation with the EphB6 ligand, ephrin-B1, results in the selective inhibition of TCR-mediated activation of JNK but not the MAPK pathway. EphB6 appears to suppress the JNK pathway by preventing T cell receptor (TCR)-induced activation of the small GTPase Rac1, a critical event in initiating the JNK cascade. Furthermore, EphB6 blocked anti-CD3-induced secretion of IL-2 and CD25 expression in a ligand-dependent manner. Dominant negative EphB6 suppressed the inhibitory activity of the endogenous receptor and enhanced anti-CD3-induced JNK activation, CD25 expression, and IL-2 secretion, confirming the requirement for EphB6-specific signaling. Activation of the JNK pathway and the establishment of an IL-2/IL-2R autocrine loop have been shown to play a role in the negative selection of CD4(+)CD8(+) self-reacting thymocytes. In agreement, stimulation of murine thymocytes with ephrin-B1 not only blocked anti-CD3-induced CD25 up-regulation and IL-2 production, but also inhibited TCR-mediated apoptosis. Thus, EphB6 may play an important role in regulating thymocyte differentiation and modulating responses of mature T cells.

Enzyme Activation↗

PubMed2001

The kinase-null EphB6 receptor undergoes transphosphorylation in a complex with EphB1.

Uniquely for the Eph family of receptor tyrosine kinases, the EphB6 receptor is catalytically inactive due to the alteration of several critical residues in its kinase domain. This has cast doubt upon its ability to participate in cytoplasmic signaling events. We show here that despite its lack of kinase activity, EphB6 undergoes inducible tyrosine phosphorylation upon stimulation with the Eph-B receptor subfamily ligand ephrin-B1. We also demonstrate, for the first time, evidence of cross-talk between Eph receptors. Overexpression of a catalytically active member of the Eph-B subfamily, EphB1, resulted in increased EphB6 phosphorylation. EphB1-induced EphB6 phosphorylation was ligand-dependent and required the functional catalytic activity of EphB1. EphB1 not only transphosphorylated EphB6, but together they also formed a stable hetero-complex. In addition, we identify the proto-oncogene c-Cbl as an EphB6-binding protein. Although EphB6-Cbl association appeared to be constitutive, Cbl required a functional phosphotyrosine binding domain in order to bind the receptor, whereas its RING finger motif ubiquitin-transfer domain was not necessary. Our findings demonstrate that EphB6 is an actively signaling receptor that undergoes transphosphorylation upon ligand binding and that can initiate specific cytoplasmic signaling events.

Animals↗

PubMed2005

Transcriptional silencing of EphB6 receptor tyrosine kinase in invasive breast carcinoma cells and detection of methylated promoter by methylation specific PCR.

The receptor tyrosine kinase EphB6 is expressed at reasonable levels in normal breast cells. It shows decreased abundance in non-invasive breast carcinoma cells and is transcriptionally silenced in invasive breast carcinoma cells. We have characterized EphB6 promoter and correlated the expression of EphB6 transcript to differential methylation of the promoter region. The demethylation of promoter sequence in vivo by growth in media containing 5-aza-2'-deoxycytidine restores the expression of EphB6 to normal levels in breast carcinoma cells, and the ability of the promoter to initiate transcription of a reporter gene is lost after methylation of the promoter sequence. The promoter region has binding sites for various factors such as SP1 and p300. The specific methylation of CpG dinucleotides has allowed us to design primers that can selectively amplify the methylated promoter and thus facilitate identification of normal, non-invasive, and invasive breast cells. The potential significance of EphB6 to serve as a diagnostic and prognostic indicator is discussed.

Biomarkers, Tumor↗

PubMed2005

Biphasic functions of the kinase-defective Ephb6 receptor in cell adhesion and migration.

EphB6 is a unique member in the Eph family of receptor tyrosine kinases in that its kinase domain contains several alterations in conserved amino acids and is catalytically inactive. Although EphB6 is expressed both in a variety of embryonic and adult tissues, biological functions of this receptor are largely unknown. In the present study, we examined the function of EphB6 in cell adhesion and migration. We demonstrated that EphB6 exerted biphasic effects in response to different concentrations of the ephrin-B2 ligand; EphB6 promoted cell adhesion and migration when stimulated with low concentrations of ephrin-B2, whereas it induced repulsion and inhibited migration upon stimulation with high concentrations of ephrin-B2. A truncated EphB6 receptor lacking the cytoplasmic domain showed monophasic-positive effects on cell adhesion and migration, indicating that the cytoplasmic domain is essential for the negative effects. EphB6 is constitutively associated with the Src family kinase Fyn. High concentrations of ephrin-B2 induced tyrosine phosphorylation of EphB6 through an Src family kinase activity. These results indicate that EphB6 can both positively and negatively regulate cell adhesion and migration, and suggest that tyrosine phosphorylation of the receptor by an Src family kinase acts as the molecular switch for the functional transition.

Animals↗

PubMed2000

Ephrin-B2 is a candidate ligand for the Eph receptor, EphB6.

No ligand has hitherto been designated for the Eph receptor tyrosine kinase family member, EphB6. Here, expression of an EphB6 ligand in the pro-B leukemic cell line, Reh, is demonstrated by binding of soluble EphB6-Fc fusion protein to the Reh cells. The ligand belongs to the subgroup of membrane spanning ligands, as suggested by the fact that phosphatidylinositol-specific phospholipase C treatment did not abrogate binding of EphB6-Fc. Two transmembrane Eph receptor ligands, ephrin-B1 and ephrin-B2, were identified in Reh cells. Analysis of EphB6-Fc fusion protein binding to ephrin-B1 or ephrin-B2 transfected COS cells revealed a high-affinity saturable binding between EphB6-Fc and ephrin-B2, but not with ephrin-B1. In mice, EphB6 has previously been shown to be expressed in thymus. Here, we show expression of EphB6 in human thymus, as well as the expression of ephrin-B2 in both human and mouse thymus. We conclude that ephrin-B2 may be a physiological ligand for the EphB6 receptor.

Animals↗

PubMed2000

T-cell-specific expression of kinase-defective Eph-family receptor protein, EphB6 in normal as well as transformed hematopoietic cells.

Although most kinase-defective growth factor receptor proteins are associated with pathogenic conditions, a kinase-defective Eph-family receptor protein, EphB6, is expressed in normal human tissues. We generated monoclonal antibodies specific for human EphB6 to characterize its expression on human hematopoietic cells. A very small population of normal human peripheral white blood cells (0.57 +/- 0.07%, n = 12) expressed EphB6. The EphB6-positive cells were CD2+, CD7+, CD3+ and CD4+ or CD8+ lymphocytes, but they did not express CD19 or CD11b. In human bone marrow, only 1.5 +/- 0.19% of lymphocytes expressed EphB6. Compared with the expression in peripheral lymphocytes, prominent expression of EphB6 protein was demonstrated in CD4+CD8+ double-positive mouse thymocytes. The T-cell lineage-specific expression was strictly conserved in human leukemia/lymphoma cells. Among T-cell-derived leukemia cells, the expression level of EphB6 seemed to decrease with maturation of the cells. These results suggest that EphB6 expression is regulated in T-cell development.

Animals↗

PubMed2006

EphB2 and ephrin-B1 expressed in the adult kidney regulate the cytoarchitecture of medullary tubule cells through Rho family GTPases.

Eph receptors and ephrin ligands are membrane-bound cell-cell communication molecules with well-defined functions in development, but their expression patterns and functions in many adult tissues are still largely unknown. We have detected substantial levels of the EphB2 and EphB6 receptors and the ephrin-B1 ligand in the adult mouse kidney by RT-PCR amplification. Immunolocalization experiments revealed that EphB2 is localized in the tubules of the inner and outer medulla and EphB6 is in the tubules of the outer medulla and cortex. By contrast, ephrin-B1 was detected in tubules throughout the whole nephron. Consistent with the overlapping expression of the EphB2 receptor and the ephrin-B1 ligand in the medulla, EphB2 is tyrosine-phosphorylated, and therefore activated, in the kidney. In the outer medulla, however, EphB2 signaling may be attenuated by the co-expressed kinase-inactive EphB6 receptor. Interestingly, we found that EphB signaling induces RhoA activation and Rac1 inactivation as well as cell retraction, enlargement of focal adhesions and prominent stress fibers in primary cultures of medullary tubule cells. These results suggest that EphB receptor signaling through Rho family GTPases regulates the cytoarchitecture and spatial organization of the tubule cells in the adult kidney medulla and, therefore, may affect the reabsorption ability of the kidney.

Animals↗

PubMed2004

High-throughput analysis of genome-wide receptor tyrosine kinase expression in human cancers identifies potential novel drug targets.

Novel high-throughput analyses in molecular biology allow sensitive and rapid identification of disease-related genes and drug targets. We have used quantitative real-time reverse transcription-PCR reactions (n = 23000) to analyze expression of all human receptor tyrosine kinases (n = 56) in malignant tumors (n = 313) of different origins and normal control samples (n = 58). The different tumor types expressed very different numbers of receptor tyrosine kinases: whereas brain tumors and testicular cancer expressed 50 receptor tyrosine kinases, acute myeloid leukemia (AML) samples expressed only 20 different ones. Specimens of similar tumor origin exhibited characteristic receptor tyrosine kinase expression patterns and were grouped together in hierarchical cluster analyses. When we focused on specific tumor entities, receptor tyrosine kinases were identified that were disease and/or stage specific. Leukemic blasts from AML bone marrow samples differed significantly in receptor tyrosine kinase expression compared with normal bone marrow and purified CD34+ cells. Among the differentially expressed receptor tyrosine kinases, we found FLT3, c-kit, CSF1 receptor, EPHB6, leukocyte tyrosine kinase, and ptk7 to be highly overexpressed in AML samples. Whereas expression changes of some of these were associated with altered differentiation patterns (e.g., CSF1 receptor), others, such as FLT3, were genuinely overexpressed in leukemic blasts. These data and the associated database (http://medweb.uni-muenster.de/institute/meda/research/) provide a comprehensive view of receptor tyrosine kinase expression in human cancer. This information can assist in the definition of novel drug targets.

Antigens, CD34↗

PubMed2001

Semiquantitative expression analysis of ephrine-receptor tyrosine kinase mRNA's in a rat model of traumatic brain injury.

The molecular mechanisms involved in recovery of function of the central nervous system (CNS) after injury to the brain are incompletely understood. Here the expression of ephrine (Eph) kinases following traumatic brain injury (subdural haematoma) was analysed in order to find out whether these developmentally regulated genes may be involved in tissue remodelling after brain damage. mRNA was isolated from ipsilateral cortices 7, 18, and 28 days after surgery and semiquantitative reverse transcription-polymerase chain reaction was performed. Most Eph kinases did not show significant regulation at gene expression level during the time course of recovery from acute brain injury but there is some evidence that mRNA of EphB1 might be slightly upregulated.

Animals↗

PubMed2000

Prognostic significance of EPHB6, EFNB2, and EFNB3 expressions in neuroblastoma.

BACKGROUND: EPH family receptor tyrosine kinases and their ligand ephrins play pivotal roles in development. High-level expression of transcripts encoding EPHB6 receptors (EPHB6), its ligands ephrin-B2 and ephrin-B3 (EFNB2, EFNB3) is predictive of favorable disease outcome of neuroblastoma (NB). When combined with TrkA expression, the expression of EPHB6, EFNB2, or EFNB3 predicts more accurately the disease outcome than each of the four variables alone. PROCEDURE: Cox regression and Kaplan-Meier analyses were used to assess the prognostic significance of EPHB6, EFNB2, EFNB3, and TrkA expressions in NB without MYCN amplification. RESULTS: High-level expression of EFNB3 or TrkA predicted favorable NB outcome of NB without MYCN amplification (p < 0.03). As found in the general NB population, EPHB6, EFNB2, or EFNB3 expression in combination with TrkA expression was significantly predictive of the disease outcome of normal MYCN NB (p < 0.01). CONCLUSIONS: EPHB6, EFNB2, and EFNB3 expressions may permit further refinement of the prognostic stratification of NB into favorable and unfavorable groups.

Child↗

PubMed2002

Developmental expression of EphB6 in the thymus: lessons from EphB6 knockout mice.

A member of the largest family of receptor protein kinases, EphB6, lacks its intrinsic kinase activity, but it is expressed in normal human tissues. To investigate the physiological function of EphB6, we generated EphB6 deficient mice. EphB6(-/-) mice developed normally, revealed no abnormality in general appearance, and were fertile. Although a developmental increase of EphB6 in the fetal thymus was confirmed, T-cell development in various lymphoid organs of EphB6(-/-) mice was comparable to those of EphB6(+/+). Even in fetal thymus organ cultures, any developmental differences of EphB6(-/-) and EphB6(+/+) thymocytes were undetectable. The different binding characteristics to ephrin-Fc proteins between EphB6(-/-) and EphB6(+/+) thymocytes demonstrated that ephrin-B2 is the unique ligand for EphB6 among eight known ephrins. While EphB6 was a dominant receptor that binds to ephrin-B2 in adult thymocytes, fetal ones also expressed another EphB that binds to ephrin-B2. Overlapping expression of the EphB subfamily in the fetal thymus might compensate for the loss of EphB6 during the thymic development.

Animals↗

PubMed2000

Implications of EPHB6, EFNB2, and EFNB3 expressions in human neuroblastoma.

Neuroblastoma (NB) is a common pediatric tumor that exhibits a wide range of biological and clinical heterogeneity. EPH (erythropoietin-producing hepatoma amplified sequence) family receptor tyrosine kinases and ligand ephrins play pivotal roles in neural and cardiovascular development. High-level expression of transcripts encoding EPHB6 receptors (EPHB6) and its ligands ephrin-B2 and ephrin-B3 (EFNB2, EFNB3) is associated with low-stage NB (stages 1, 2, and 4S) and high TrkA expression. In this study, we showed that EFNB2 and TrkA expressions were associated with both tumor stage and age, whereas EPHB6 and EFNB3 expressions were solely associated with tumor stage, suggesting that these genes were expressed in distinct subsets of NB. Kaplan-Meier and Cox regression analyses revealed that high-level expression of EPHB6, EFNB2, and EFNB3 predicted favorable NB outcome (P<0.005), and their expression combined with TrkA expression predicted the disease outcome more accurately than each variable alone (P<0.00005). Interestingly, if any one of the four genes (EPHB6, EFNB2, EFNB3, or TrkA) was expressed at high levels in NB, the patient survival was excellent (>90%). To address whether a good disease outcome of NB was a consequence of high-level expression of a "favorable NB gene," we examined the effect of EPHB6 on NB cell lines. Transfection of EPHB6 cDNA into IMR5 and SY5Y expressing little endogenous EPHB6 resulted in inhibition of their clonogenicity in culture. Furthermore, transfection of EPHB6 suppressed the tumorigenicity of SY5Y in a mouse xenograft model, demonstrating that high-level expressions of favorable NB genes, such as EPHB6, can in fact suppress malignant phenotype of unfavorable NB.

Animals↗

PubMed2004

[Analysis of gene expression profiles among 3 epithelial ovarian tumor subtypes using cDNA and tissue microarrays].

BACKGROUND & OBJECTIVE: Ovarian cancer is the leading cause of death among the gynecological malignancy mainly due to lacking of effective early diagnostic methods. To identify novel candidate genes and further explore their clinical significance of epithelial ovarian tumors, we developed a new method in our laboratory by combining cDNA microarray with RNA in situ hybridization in frozen tissue microarray. METHODS: cDNA microarrays were used to seek differentially expressed genes among 3 subtypes of ovarian tumors (serous borderline ovarian tumors, serous ovarian cancers, and endometrioid ovarian carcinomas). RNA in situ hybridization in frozen tissue microarray was used to further confirm the findings from cDNA microarrays. RESULTS: In the study of cDNA microarray, 40 genes and ESTs showed significant differential expression between low and high-malignancy, as well as serous and endometrioid carcinomas. EPHB6, PTPRF, GFER, ERG25, PLRP1, FLJ22060, and WISP2 were further validated by RNA in situ hybridization in tissue microarray. CONCLUSIONS: cDNA microarray combined with RNA in situ hybridization in frozen tissue microarray is an ideal choice for identifying novel oncogenes. EPHB6, PTPRF, GFER, ERG25, PLRP1, FLJ22060, and WISP2 might become the new candidate oncogenes for epithelial ovarian cancer.

CCN Intercellular Signaling Proteins↗

PubMed1996

Distinct and overlapping expression patterns of ligands for Eph-related receptor tyrosine kinases during mouse embryogenesis.

Recent studies have implicated Eph-related receptor tyrosine kinases and their membrane-bound ligands in restricting or stimulating the movement of cells and axons. Members of these large families of receptors and ligands fall into two major binding specificity classes, in which the GPI-anchored subgroup of ligands can each bind to all members of a subgroup of receptors, whereas the transmembrane ligands interact with a distinct subgroup of receptors. Analysis of expression patterns is therefore important in order to understand which receptor-ligand interactions occur in vivo. We have cloned mouse orthologues of five members of the ligand family and analysed in detail their developmental expression, in comparison with each other, and with the receptor specificity class they can interact with. We find that B61, AL-1/RAGS, LERK4, and ELF-1, members of the GPI-anchored subgroup of ligands, have both distinct and overlapping aspects to their expression in early mesoderm, somites, and branchial arches; in complex, dynamic patterns in the limb; and in spatial domains and specific neurons in the CNS. Similarly, Elk-L is expressed in hindbrain segments, the roof plate, and floor plate, which overlaps with that of other transmembrane ligands, but has distinct expression in somites. The expression domains of ligands are complementary to those of the corresponding receptors in a number of tissues, including the midbrain, hindbrain, and differentiating limbs, consistent with potential roles in restricting cell movement. In addition, we find that there are some overlaps in expression of receptors and ligands, for example in somites and the early limb. Taken together with previous studies showing that Eph-related receptors also have distinct but overlapping expression patterns, these data indicate that each ligand may have stage- and tissue-specific interactions with an individual member or multiple members of the receptor family.

Amino Acid Sequence↗

PubMed1998

Ammonium/methylammonium permeases of a Cyanobacterium. Identification and analysis of three nitrogen-regulated amt genes in synechocystis sp. PCC 6803.

Ammonium is an important nitrogen source for many microorganisms and plants. Ammonium transporters whose activity can be probed with [14C]methylammonium have been described in several organisms including some cyanobacteria, and amt genes encoding ammonium/methylammonium permeases have been recently identified in yeast, Arabidopsis thaliana, and some bacteria. The unicellular cyanobacterium Synechocystis sp. PCC 6803 exhibited a [14C]methylammonium uptake activity that was inhibited by externally added ammonium. Three putative amt genes that are found in the recently published complete sequence of the chromosome of strain PCC 6803 were inactivated by insertion of antibiotic resistance-encoding gene-cassettes. The corresponding mutant strains were impaired in uptake of [14C]methylammonium. Open reading frame sll0108 (amt1) was responsible for a high affinity uptake activity (Ks for methylammonium, 2.7 microM), whereas open reading frames sll1017 (amt2) and sll0537 (amt3) made minor contributions to uptake at low substrate concentrations. Expression of the three amt genes was higher in nitrogen-starved cells than in cells incubated in the presence of a source of nitrogen (either ammonium or nitrate), but amt1 was expressed at higher levels than the other two amt genes. Transcription of amt1 was found to take place from a promoter bearing the structure of the cyanobacterial promoters activated by the nitrogen control transcription factor, NtcA.

Bacterial Proteins↗

PubMed2003

[Establishment and identification of recombinant NS-1 cell strain that stably expresses chimeric HBc containing HBV multiepitope short peptides].

OBJECTIVE: To establish recombinant NS-1 cell strain that is capable of stable expression of chimeric HBc particle containing HBV multi epitope short peptides. METHODS: The recombinant plasmid, pHBc-Mep, was transfected into NS-1 cells via Lipofectamine, and the recombinant cell strain was screened with G418 and subclone screening. The expression products of the cells were examined by RT-PCR, ELISA, indirect immunofluorescence assay (IFA) and Western blotting. RESULTS: The results of RT-PCR, ELISA, IFA and Western blotting demonstrated that the recombinant protein HBc-Mep was expressed in the screened cells after continuous cloning for 3 times, but not in cells transfected with pcDNA3.1 or nontransfected cells. CONCLUSION: The recombinant cell strain stably expressing chimeric HBc particle containing multi epitope short peptides of HBV, designated as NS/HBc-Mep, has been established successfully.

Animals↗

PubMed1996

A new member of the Eph family of receptors that lacks protein tyrosine kinase activity.

Using a PCR-based screen to identify tyrosine kinases involved in T cell development, we have cloned a new member of the Eph-family of receptor tyrosine kinases (Mep, for murine eph-family protein). At the amino acid level Mep is 60% identical to the chicken embryonic kinase Cek9. Sequence analysis indicates that the predicted extracellular portion of Mep bears an Ig-like domain, a cysteine-rich region, and sequences homologous to fibronectin type III. The transmembrane region of Mep is followed by a kinase domain. Surprisingly, this kinase domain carries amino acid substitutions in the highly conserved consensus motifs found in all protein tyrosine kinases and known to be crucial for kinase activity. We demonstrate that a bacterial fusion protein of the Mep kinase domain does not have protein tyrosine kinase activity. Analysis of Mep mRNA levels in a variety of mouse tissues shows that Mep is highly expressed in thymus and brain. We have also isolated two additional Mep cDNA clones from thymocytes which are predicted to encode secreted forms of the Mep extracellular domain; mRNAs encoding these secreted isoforms are also expressed in mouse brain.

Alternative Splicing↗

PubMed2004

EphB6-null mutation results in compromised T cell function.

So far, there is very limited knowledge about the role of Eph kinases, the largest family of receptor tyrosine kinases, in the immune system. Here, using EphB6(-/-) mice, we demonstrated that in vitro and in vivo T cell responses such as lymphokine secretion, proliferation, and the development of delayed-type skin hypersensitivity and experimental autoimmune encephalitis in EphB6(-/-) mice were compromised. On the other hand, humoral immune responses, such as serum levels of different Ig isotypes and IgG response to tetanus toxoid, were normal in these mice. Mechanistically, we showed that EphB6 migrated to the aggregated TCRs and rafts after TCR activation. Further downstream, in the absence of EphB6, ZAP-70 activation, LAT phosphorylation, the association of PLCgamma1 with SLP-76, and p44/42 MAPK activation were diminished. Thus, we have shown that EphB6 is pivotal in T cell function.

Animals↗