The EphB6 Receptor: Kinase-Dead but Very Much Alive.

The Eph receptor tyrosine kinase member EphB6 is a pseudokinase, and similar to other pseudoenzymes has not attracted an equivalent amount of interest as its enzymatically-active counterparts. However, a greater appreciation for the role pseudoenzymes perform in expanding the repertoire of signals generated by signal transduction systems has fostered more interest in the field. EphB6 acts as a molecular switch that is capable of modulating the signal transduction output of Eph receptor clusters. Although the biological effects of EphB6 activity are well defined, the molecular mechanisms of EphB6 function remain enigmatic. In this review, we use a comparative approach to postulate how EphB6 acts as a scaffold to recruit adaptor proteins to an Eph receptor cluster and how this function is regulated. We suggest that the evolutionary repurposing of EphB6 into a kinase-independent molecular switch in mammals has involved repurposing the kinase activation loop into an SH3 domain-binding site. In addition, we suggest that EphB6 employs the same SAM domain linker and juxtamembrane domain allosteric regulatory mechanisms that are used in kinase-positive Eph receptors to regulate its scaffold function. As a result, although kinase-dead, EphB6 remains a strategically active component of Eph receptor signaling.

The receptor tyrosine kinase EPHB6 regulates catecholamine exocytosis in adrenal gland chromaffin cells.

The erythropoietin-producing human hepatocellular receptor EPH receptor B6 (EPHB6) is a receptor tyrosine kinase that has been shown previously to control catecholamine synthesis in the adrenal gland chromaffin cells (AGCCs) in a testosterone-dependent fashion. EPHB6 also has a role in regulating blood pressure, but several facets of this regulation remain unclear. Using amperometry recordings, we now found that catecholamine secretion by AGCCs is compromised in the absence of EPHB6. AGCCs from male knockout (KO) mice displayed reduced cortical F-actin disassembly, accompanied by decreased catecholamine secretion through exocytosis. This phenotype was not observed in AGCCs from female KO mice, suggesting that testosterone, but not estrogen, contributes to this phenotype. Of note, reverse signaling from EPHB6 to ephrin B1 (EFNB1) and a 7-amino acid-long segment in the EFNB1 intracellular tail were essential for the regulation of catecholamine secretion. Further downstream, the Ras homolog family member A (RHOA) and FYN proto-oncogene Src family tyrosine kinase (FYN)-proto-oncogene c-ABL-microtubule-associated monooxygenase calponin and LIM domain containing 1 (MICAL-1) pathways mediated the signaling from EFNB1 to the defective F-actin disassembly. We discuss the implications of EPHB6's effect on catecholamine exocytosis and secretion for blood pressure regulation.

EPHB6 controls catecholamine biosynthesis by up-regulating tyrosine hydroxylase transcription in adrenal gland chromaffin cells.

EPHB6 is a member of the erythropoietin-producing hepatocellular kinase (EPH) family and a receptor tyrosine kinase with a dead kinase domain. It is involved in blood pressure regulation and adrenal gland catecholamine (CAT) secretion, but several facets of EPHB6-mediated CAT regulation are unclear. In this study, using biochemical, quantitative RT-PCR, immunoblotting, and gene microarray assays, we found that EPHB6 up-regulates CAT biosynthesis in adrenal gland chromaffin cells (AGCCs). We observed that epinephrine content is reduced in the AGCCs from male Ephb6-KO mice, caused by decreased expression of tyrosine hydroxylase, the rate-limiting enzyme in CAT biosynthesis. We demonstrate that the signaling pathway from EPHB6 to tyrosine hydroxylase expression in AGCCs involves Rac family small GTPase 1 (RAC1), MAP kinase kinase 7 (MKK7), c-Jun N-terminal kinase (JNK), proto-oncogene c-Jun, activator protein 1 (AP1), and early growth response 1 (EGR1). On the other hand, signaling via extracellular signal-regulated kinase (ERK1/2), p38 mitogen-activated protein kinase, and ELK1, ETS transcription factor (ELK1) was not affected by EPHB6 deletion. We further report that EPHB6's effect on AGCCs was via reverse signaling through ephrin B1 and that EPHB6 acted in concert with the nongenomic effect of testosterone to control CAT biosynthesis. Our findings elucidate the mechanisms by which EPHB6 modulates CAT biosynthesis and identify potential therapeutic targets for diseases, such as hypertension, caused by dysfunctional CAT biosynthesis.

EPHB6 and testosterone in concert regulate epinephrine release by adrenal gland chromaffin cells.

Erythropoietin-producing human hepatocellular receptor (EPH) B6 (EPHB6) is a member of the receptor tyrosine kinase family. We previously demonstrated that EPHB6 knockout reduces catecholamine secretion in male but not female mice, and castration reverses this phenotype. We showed here that male EPHB6 knockout adrenal gland chromaffin cells presented reduced acetylcholine-triggered Ca2+ influx. Such reduction depended on the non-genomic effect of testosterone. Increased large conductance calcium-activated potassium channel current densities were recorded in adrenal gland chromaffin cells from male EPHB6 knockout mice but not from castrated knockout or female knockout mice. Blocking of the large conductance calcium-activated potassium channel in adrenal gland chromaffin cells from male knockout mice corrected their reduced Ca2+ influx. We conclude that the absence of EPHB6 and the presence of testosterone would lead to augmented large conductance calcium-activated potassium channel currents, which limit voltage-gated calcium channel opening in adrenal gland chromaffin cells. Consequently, acetylcholine-triggered Ca2+ influx is reduced, leading to lower catecholamine release in adrenal gland chromaffin cells from male knockout mice. This explains the reduced resting-state blood catecholamine levels, and hence the blood pressure, in male but not female EPHB6 knock mice. These findings have certain clinical implications.

EPHA7 and EPHA10 Physically Interact and Differentially Co-localize in Normal Breast and Breast Carcinoma Cell Lines, and the Co-localization Pattern Is Altered in EPHB6-expressing MDA-MB-231 Cells.

Erythropoietin-producing hepatocellular carcinoma cell (EPH) receptors comprise the most abundant receptor tyrosine kinase family characterized to date in mammals including humans. These proteins are involved in axon guidance, tissue organization, vascular development and the intricate process of various diseases including cancer. These diverse functions of EPH receptors are attributed, in part, to their abilities for heterodimerization. While the interacting partners of kinase-deficient EPHB6 receptor have been characterized, the interaction of the kinase-dead EPHA10 with any other receptor has not been identified. By using co-immunoprecipitation, we demonstrated physical interaction between kinase-deficient EPHA10 with kinase-sufficient EPHA7 receptor. Immunocytochemical analyses have revealed that these two receptors co-localize on the cell surface, and soluble portions of the receptors exist as a complex in the cytoplasm as well as the nuclei. While EPHA7 and EPHA10 co-localize similarly on the membrane in MCF10A and MCF7 cells, they were differentially co-localized in MDA-MB-231 cells stably transfected with empty pcDNA vector (MDA-MB-231-PC) or an expression construct of EPHB6 (MDA-MB-231-B6). The full-length isoforms of these receptors were co-localized on the cell surface, and the soluble forms were present as a complex in the cytoplasm as well as the nucleus in MDA-MB-231-PC cells. MDA-MB-231-B6 cells, on the other hand, were distinguished by the absence of any signal in the nuclei. Our results represent the first demonstration of physical interaction between EPHA10 and EPHA7 and their cellular co-localization. Furthermore, these observations also suggest gene-regulatory functions of the complex of the soluble forms of these receptors in breast carcinoma cells of differential invasiveness.

Mutations of the EPHB6 receptor tyrosine kinase induce a pro-metastatic phenotype in non-small cell lung cancer.

Alterations of Eph receptor tyrosine kinases are frequent events in human cancers. Genetic variations of EPHB6 have been described but the functional outcome of these alterations is unknown. The current study was conducted to screen for the occurrence and to identify functional consequences of EPHB6 mutations in non-small cell lung cancer. Here, we sequenced the entire coding region of EPHB6 in 80 non-small cell lung cancer patients and 3 tumor cell lines. Three potentially relevant mutations were identified in primary patient samples of NSCLC patients (3.8%). Two point mutations led to instable proteins. An in frame deletion mutation (del915-917) showed enhanced migration and accelerated wound healing in vitro. Furthermore, the del915-917 mutation increased the metastatic capability of NSCLC cells in an in vivo mouse model. Our results suggest that EPHB6 mutations promote metastasis in a subset of patients with non-small cell lung cancer.

Receptor tyrosine kinase Ephb6 regulates vascular smooth muscle contractility and modulates blood pressure in concert with sex hormones.

Eph kinases constitute the largest receptor tyrosine kinase family, and their ligands, ephrins (Efns), are also cell surface molecules. Our study is the first to assess the role of Ephb6 in blood pressure (BP) regulation. We observed that EphB6 and all three of its Efnb ligands were expressed on vascular smooth muscle cells (VSMC) in mice. We discovered that small arteries from castrated Ephb6 gene KO males showed increased contractility, RhoA activation, and constitutive myosin light chain phosphorylation ex vivo compared with their WT counterparts. Consistent with this finding, castrated Ephb6 KO mice presented heightened BP compared with castrated WT controls. In vitro experiments in VSMC revealed that cross-linking Efnbs but not Ephb6 resulted in reduced VSMC contractions, suggesting that reverse signaling through Efnbs was responsible for the observed BP phenotype. The reverse signaling was mediated by an adaptor protein Grip1. Additional experiments demonstrated decreased 24-h urine catecholamines in male Ephb6 KO mice, probably as a compensatory feedback mechanism to keep their BP in the normal range. After castration, however, such compensation was abolished in Ephb6 KO mice and was likely the reason why BP increased overtly in these animals. It suggests that Ephb6 has a target in the nervous/endocrine system in addition to VSMC, regulating a testosterone-dependent catecholamine compensatory mechanism. Our study discloses that Ephs and Efns, in concert with testosterone, play a critical role in regulating small artery contractility and BP.

Eph/ephrin profiling in human breast cancer reveals significant associations between expression level and clinical outcome.

Pre-clinical studies provide compelling evidence that Eph family receptor tyrosine kinases (RTKs) and ligands promote cancer growth, neovascularization, invasion, and metastasis. Tumor suppressive roles have also been reported for the receptors, however, creating a potential barrier for clinical application. Determining how these observations relate to clinical outcome is a crucial step for translating the biological and mechanistic data into new molecularly targeted therapies. We investigated eph and ephrin expression in human breast cancer relative to endpoints of overall and/or recurrence-free survival in large microarray datasets. We also investigated protein expression in commercial human breast tissue microarrays (TMA) and Stage I prognostic TMAs linked to recurrence outcome data. We found significant correlations between ephA2, ephA4, ephA7, ephB4, and ephB6 and overall and/or recurrence-free survival in large microarray datasets. Protein expression in TMAs supported these trends. While observed no correlation between ephrin ligand expression and clinical outcome in microarray datasets, ephrin-A1 and EphA2 protein co-expression was significantly associated with recurrence in Stage I prognostic breast cancer TMAs. Our data suggest that several Eph family members are clinically relevant and tractable targets for intervention in human breast cancer. Moreover, profiling Eph receptor expression patterns in the context of relevant ligands and in the context of stage may be valuable in terms of diagnostics and treatment.

Dancing with the dead: Eph receptors and their kinase-null partners.

Eph receptor tyrosine kinases and their ligands, ephrins, are membrane proteins coordinating a wide range of biological functions both in developing embryos and in adult multicellular organisms. Numerous studies have implicated Eph receptors in the induction of opposing responses, including cell adhesion or repulsion, support or inhibition of cell proliferation and cell migration, and progression or suppression of multiple malignancies. Similar to other receptor tyrosine kinases, Eph receptors rely on their ability to catalyze tyrosine phosphorylation for signal transduction. Interestingly, however, Eph receptors also actively utilize three kinase-deficient receptor tyrosine kinases, EphB6, EphA10, and Ryk, in their signaling network. The accumulating evidence suggests that the unusual flexibility of the Eph family, allowing it to initiate antagonistic responses, might be partially explained by the influence of the kinase-dead participants and that the exact outcome of an Eph-mediated action is likely to be defined by the balance between the signaling of catalytically potent and catalytically null receptors. We discuss in this minireview the emerging functions of the kinase-dead EphB6, EphA10, and Ryk receptors both in normal biological responses and in malignancy, and analyze currently available information related to the molecular mechanisms of their action in the context of the Eph family.

DNA-based assay for EPHB6 expression in breast carcinoma cells as a potential diagnostic test for detecting tumor cells in circulation.

The early detection of breast cancer is critical for improved treatment and better management of the disease. The dissemination of tumor cells into the blood stream is known to occur early in tumor progression and these circulating tumor cells (CTCs) may be detectable before the occurrence of tumor metastasis. Methylation-specific polymerase chain reaction (MSP) can be exploited for detecting CTCs on the basis of differential methylation of numerous gene promoters in normal and carcinoma cells. In this study, we describe the relationship between loss of Ephrin receptor B6 (EPHB6) expression and the aggressiveness of breast carcinoma cell lines (BCCLs). The loss of EPHB6 expression in more aggressive BCCLs is regulated in a methylation-dependent manner. We demonstrate the ability of an EPHB6 MSP to distinguish between methylated and unmethylated EPHB6 promoters, and to predict expression of the EPHB6 transcript and protein. The sensitivity of MSP was related to the volume of blood processed for DNA isolation. As few as 50 tumor cells in 5 ml blood were detectable with a high efficiency. However, the detection of 10 tumor cells/5 ml was not as efficient. On the other hand, 5 tumor cells or 100 pg of free DNA in 200 microl of blood was also easily detectable. Our results suggest that MSP could be applied to detect even a single cell in 1 ml of blood by employing appropriate modifications. The EPHB6 MSP has clinical implications for the prognosis and/or diagnosis of breast and other cancer types including neuroblastoma, melanoma, and non-small cell lung carcinoma wherein EPHB6 expression is lost in more aggressive forms of the disease.

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.

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.

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.