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.

EphB6 promotes anoikis by modulating EphA2 signaling.

Anoikis is a specific type of apoptosis induced by detachment of epithelial cells from extracellular matrix, and acquiring resistance to anoikis is an important step that enables cancer cells to metastasize. EphA2, which is overexpressed in a variety of human cancers, is phosphorylated by Akt on serine 897 and mediates ligand ephrin-independent promotion of anoikis resistance through the RhoG activator Ephexin4. EphB6 is frequently silenced in invasive and metastatic cancers; however, its role in cancer progression is poorly understood. Here we show that EphB6 interacts with EphA2 and suppresses EphA2-mediated promotion of anoikis resistance in MCF7 breast cancer cells. On the other hand, knockdown of EphB6 promotes anoikis resistance. We further show that expression of EphB6 decreases serine 897 phosphorylation of EphA2 and suppresses EphA2-Ephexin4 interaction and the RhoG activation. These findings implicate EphB6 as a negative regulator of EphA2 oncogenic signaling.

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.

EphB6 crosslinking results in costimulation of T cells.

Erythropoietin-producing hepatocyte (Eph) kinases represent the largest receptor tyrosine kinase family. Some of them are expressed in the T cell compartment, but their function in T cells is unknown. In peripheral blood, EphB6 was predominantly expressed on T cells, and was upregulated after culture. EphB6 crosslinking by anti-EphB6 mAb or ephrinB2 in the presence of suboptimal T cell receptor (TCR) stimulation led to drastic T cell proliferation, suggesting that EphB6 can co-stimulate T cells. The proliferation was accompanied by enhanced production of several lymphokines, such as IFN-gamma, IL-6, IL-10, TGF-beta, TNF-alpha, and GM-CSF, but not IL-2 and IL-4. Sorted EphB6(+) T cells had significantly stronger response to anti-CD3 and anti-CD28 stimulation than EphB6(-) T cells had. Taken together, these data suggest an important role of EphB6 in normal T cell activation. Within two minutes of anti-CD3 and anti-CD28 stimulation, EphB6 aggregated and colocalized with TCR, and this provides a morphological basis for EphB6 to enhance TCR signaling. The capping was followed by p38 MAPK activation, showing that EphB6 is capable of signaling, in spite of its lack of intrinsic kinase activity. This study demonstrates that interaction between EphB6 and its ligands facilitates T cell responses to antigen.