The EphA8 Receptor Regulates Integrin Activity through p110γ Phosphatidylinositol-3 Kinase in a Tyrosine Kinase Activity-Independent Manner

Abstract

Recent genetic studies suggest that ephrins may function in a kinase-independent Eph receptor pathway. Here we report that expression of EphA8 in either NIH 3T3 or HEK293 cells enhanced cell adhesion to fibronectin via ␣ 5 ␤ 1 -or ␤ 3 integrins. Interestingly, a kinase-inactive EphA8 mutant also markedly promoted cell attachment to fibronectin in these cell lines. Using a panel of EphA8 point mutants, we have demonstrated that EphA8 kinase activity does not correlate with its ability to promote cell attachment to fibronectin. Analysis using EphA8 extracellular and intracellular domain mutants has revealed that enhanced cell adhesion is dependent on ephrin A binding to the extracellular domain and the juxtamembrane segment of the cytoplasmic domain of the receptor. EphA8-promoted adhesion was efficiently inhibited by wortmannin, a phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor. Additionally, we found that EphA8 had associated PI 3-kinase activity and that the p110␥ isoform of PI 3-kinase is associated with EphA8. In vitro binding experiments revealed that the EphA8 juxtamembrane segment was sufficient for the formation of a stable complex with p110␥. Similar results were obtained in assay using cells stripped of endogenous ephrin A ligands by treatment with preclustered ephrin A5-Fc proteins. In addition, a membrane-targeted lipid kinase-inactive p110␥ mutant was demonstrated to stably associate with EphA8 and suppress EphA8-promoted cell adhesion to fibronectin. Taken together, these results suggest the presence of a novel mechanism by which the EphA8 receptor localizes p110␥ PI 3-kinase to the plasma membrane in a tyrosine kinase-independent fashion, thereby allowing access to lipid substrates to enable the signals required for integrin-mediated cell adhesion.The Eph receptor tyrosine kinases (RTKs), together with their ephrin ligands, regulate diverse developmental patterning processes including axon guidance, cell migration, and cell segregation (13). However, in contrast to other families of receptor tyrosine kinases, the Eph RTKs do not appear to regulate cell proliferation and survival. It was recently reported that activation of the Eph RTKs by their cognate ligands leads to changes in cell adhesion to various extracellular matrix proteins. For example, EphB1 promoted cell attachment to fibronectin or fibrinogen, whereas neither a kinase-inactive EphB1 mutant nor EphB1 point mutants defective for binding to either Nck or low-molecular-weight protein tyrosine phosphatase (LMW-PTP) showed this effect (21, 35). EphB2 was also shown to indirectly control integrin activity by inducing tyrosine phosphorylation of R-Ras, possibly through a novel signaling intermediate, Src homology 2 (SH2) domain-containing Eph receptor binding protein 1 (SHEP1) (9, 43). More recently, EphA2 kinase was reported to regulate integrin function by causing focal adhesion kinase dephosphorylation (26). These results are consistent with the concept that the kinase activity of the Eph RTKs plays a pivotal role in regulation of ce...

The EphA8 receptor induces sustained MAP kinase activation to promote neurite outgrowth in neuronal cells

“…However, differences with regard to increase in the number of neurites per cell and branched neurites in EphA8-transfected cells were statistically not significant, compared with dbcAMPtreated cells with no EphA8 expression (data not shown). EphA8 expression in NG108-15 cells induced neurite outgrowth in fibronectin-coated, but not BSA-coated substratum ( Figure 1a, lanes 3 and 5), possibly consistent with previous reports that the EphA8 receptor crosstalks with the fibronectin receptor (Gu and Park, 2001). …”

“…Significantly, ligand stimulation and EphA8 autokinase activity are not required for EphA8-mediated MAPK activation. In contrast to our previous finding that the juxtamembrane region is critical for EphA8-promoted cell adhesion (Gu and Park, 2001), data from this study indicate that the conserved tyrosine kinase domain is important for EphA8-induced MAPK activation and neurite outgrowth. Our findings suggest that the EphA8 receptor induces axonal projections through regulation of the MAPK signaling pathway.…”

“…It is possible that the EphA8 receptor is maximally active due to overexpression and thus not responsive to ephrinA5-Fc in both NIH3T3 and HEK293 cells. To rule out this possibility, we used a doxycycline-inducible expression system for the wildtype or kinase-inactive EphA8 receptor in HEK293 cells as described previously (Gu and Park, 2001). In this system, no EphA8 was detected in the absence of doxycycline, and the level of EphA8 expression correlated with the time of incubation with doxycycline (Figure 6a, first panel).…”

“…The EphA8 K666M mutant is defective in tyrosine kinase activity due to the presence of Met in place of Lys 666, the putative ATP-binding residue (Gu and Park, 2001). The deleted portions of the murine EphA8 receptor correspond to a highly conserved juxtamembrane (JM) domain, a tyrosine kinase domain (KD), a sterile alpha motif (SAM), and an immunoglubuline-like (Ig) domain, respectively (Gu and Park, 2001). EphA8 mutants were transiently expressed, together with EGFP in NG108-15 cells.…”

“…from three independent experiments. (c) HEK293 cells were transiently transfected with pcDNA3-neo vector as a control or wild-type EphA8 expression construct, and treated ( þ ) with preclustered ephrin A5-Fc for 30 min or left untreated (À), as described previously (Gu and Park, 2001). At 48 h posttransfection, equal amounts of proteins (500 mg) from cell lysates were immunoprecipitated with anti-EphA8 antibodies, and analysed by immunoblotting with anti-phosphotyrosine antibodies as a probe (first panel).…”