INTRODUCTION

Top Abstract Introduction Materials and Methods Results Discussion References

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 cell adhesion through integrins. In contrast, some studies indicate that the Eph RTKs might function in a kinase activity-independent mechanism. Evidence supporting this possibility comes from genetic studies using the nematode Caenorhabditis elegans. For example, mutations in the Eph receptor VAB-1 kinase domain caused a less penetrant embryonic arrest phenotype than a null mutation in the same gene, suggesting that VAB-1 may function partly in a kinase-independent signaling pathway (15). Moreover, C. elegans ephrin vab-2 mutations enhanced vab-1 kinase mutations, resembling vab-1 null mutations in the resulting phenotype (6). Two different mechanisms of kinase activity-independent signaling by Eph RTKs are possible. First, the ephrin ligands could transmit signals via the Eph receptor cytoplasmic region in a way that does not involve the tyrosine kinase activity. Interestingly, the native EphB6/Mep protein lacks tyrosine kinase activity due to many amino acid substitutions in conserved kinase domain sequence motifs which are important for catalysis; this may reflect an intrinsic signaling function of the kinase-inactive receptor (17). Second, the Eph receptors could function in reverse signaling via ephrin ligands. Vertebrate ephrins either are membrane anchored by glycosylphosphatidylinositol (GPI) anchors (ephrin A subgroup) or are transmembrane proteins (ephrin B subgroup). (12). The observation that ephrin B protein become phosphorylated on cytoplasmic tyrosine residues in response to Eph receptor binding provides evidence for the reverse signaling hypothesis (4, 20). More recently, consistent with a guidance role for the EphB2 extracellular domain (19), it was shown that retinal ganglion cell axon pathfinding within the retina was partially mediated by EphB receptors acting in a kinase-independent manner (3). It was also demonstrated that upon receptor binding, the ephrin A5 ligand could induce a signaling event through the Fyn protein tyrosine kinase, concomitant with alterations in the adhesive properties of the ligand-expressing cells (8). However, there have been no indications to date that the known intracellular signals transmitted by mammalian Eph receptors elicit biological responses without the tyrosine kinase activity.

We have previously reported that the EphA8 receptor can regulate cellular cytoskeletal modification through a kinase-dependent signaling mechanism (7). For example, EphA8 defective in binding to Fyn kinase fails to attenuate cell attachment responses to uncoated glass surfaces. However, unlike the case for EphB receptors, we found no strong correlation between tyrosine phosphorylation of EphA8 and alterations in cell attachment to a specific extracellular matrix protein such as fibronectin. On the basis of this observation, we asked whether the EphA8-mediated cell attachment to fibronectin could be regulated in a kinase activity-independent manner. In this study, we used cell lines that express endogenous ephrin A ligand to evaluate whether expression of diverse EphA8 kinase mutants resulted in different responses to fibronectin. In these systems, we observed that expression of the EphA8 receptor significantly promoted cell attachment to fibronectin without altering cellular integrin levels. Surprisingly, no significant differences in the ability to promote cell attachment to fibronectin were observed between wild-type EphA8 and kinase-inactive mutants. Moreover, our results identify p110 phosphotidylinositol (PI) 3-kinase as a key bridging molecule between EphA8 and integrins. Our results provide the first example of kinase-independent communication between the mammalian EphA receptors and integrins.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results Discussion References

Cell culture and transfection. NIH 3T3 cells were maintained in Dulbecco's modified Eagle's medium (DMEM; Sigma) supplemented with 5% heat-inactivated fetal calf serum (Life Technologies. Inc) as described previously (30). HEK293 cells were routinely cultured in alpha-MEM (Sigma) containing 10% heat-inactivated fetal bovine serum (BioWhittaker). Tetracycline-free serum (Clontech) was used in experiments involving doxycycline-induced EphA8 expression to prevent leakage expression. The calcium phosphate precipitation method was used as described previously (16) to transfect cells with various expression plasmids. Stable G418-resistant clones were selected by supplementing the culture medium with G418 (400 µg/ml). The clones were periodically cultured in the same selection medium to maintain stable expression. Transient transfections were carried out using LipofectAMINE (Life Technologies) according to the manufacturer's instructions. Doxycycline-inducible EphA8 expression cell lines were constructed by cotransfection with the pTRE-EphA8 and pTet-On (Clontech) constructs, followed by selection with G418. Positive clones were directly selected by Western analysis on the basis of their ability to express EphA8 in the presence of doxycycline (2 µg/ml; Sigma). At least three independent clones of each of the wild-type and kinase-inactive EphA8 receptors were obtained. PI-specific phospholipase C (PI-PLC) was treated to eliminate GPI-linked ephrin A subgroup ligands from NIH 3T3 or HEK293 cells as described previously (30). For treatment of preclustered ephrin A5-Fc proteins, purified ephrin A5-Fc (30) was aggregated using anti-human Fc (Jackson Immunoresearch) for 1 h at 4°C, and stimulations were for 20 min at 37°C.

Construction of expression vectors. The murine EphA8 cDNA (pSP38), EphA8-TrkB chimeric cDNA, and EphA8 cDNA tagged with the nine-amino-acid hemagglutinin (HA) epitope (YPYDVPDYA) at its COOH terminus have been described elsewhere (7, 30). The EphA8-III deletion mutant was generated as follows. First, a 689-bp EcoRI/NcoI fragment (nucleotides [nt] 2 to 690 of the insert in pSP38) was ligated to a 224-bp NcoI/XhoI-digested PCR product amplified using primers matching nt 899 to 918 and 1101 to 1120 of the insert in pSP38. The resulting EcoRI/XhoI fragment was subcloned into the corresponding region of full-length EphA8 cDNA. To construct the EphA8-V-VII deletion mutant, a 1,095-bp PstI fragment (nt 814 to 1908 of the insert in pSP38) was subcloned into pGEM5z (Promega) and then subjected to two separate PCRs. A 248-bp PCR product was amplified using the 5' T7 universal primer and a 3' primer matching nt 1037 to 1054 of the pSP38 insert, and a 301-bp PCR product was amplified using a 5' primer matching nt 1679 to 1696 of the pSP38 insert and the 3' SP6 universal primer. The two partially complementary PCR fragments thus generated were annealed and used as the template in another PCR with the 5' T7 and 3' SP6 universal primers. The resulting 385-bp product was digested with PstI and subcloned into the corresponding region of full-length EphA8 cDNA. The EphA8 juxtamembrane region (JM) deletion mutant (EphA8-JM) was also generated by PCR as follows: a 324-bp PCR product was amplified using primers matching nt 1524 to 1543 and 1822 to 1840 of the insert in pSP38, and a 255-bp PCR product was generated using primers nt 2008 to 2022 and 2230 to 2249 of the insert in pSP38. The partially complementary fragments generated by these PCRs were annealed and reamplified with the primers used for the 5' end of the first PCR product and the 3' end of the second PCR product. The resulting 481-bp product was digested with StuI and XbaI and subcloned into the corresponding region of full-length EphA8 cDNA. To construct the EphA8 sterile alpha motif (SAM) deletion mutant (EphA8-SAM), a 1,010-bp XbaI/EcoRI fragment (nt 2235 to 3244 of the pSP38 insert) was subcloned into pGEM11z (Promega) and used as the template in two separate PCRs: a 634-bp PCR product was amplified using the 5' SP6 universal primer and a 3' primer matching nt 2839 to 2853 of the pSP38 insert, and a 239-bp PCR product was amplified using a 5' primer matching nt 3049 to 3068 of the insert in pSP38 and the 3' T7 universal primer. The resulting PCR fragments were annealed and used as the template in another PCR with the 5' SP6 and 3' T7 universal primers. The resulting 865-bp fragment was digested with NcoI and ApaI and subcloned into the corresponding region of the XbaI/EcoRI EphA8 fragment in pGEM11z. Regions amplified by PCR were sequenced to exclude the possibility of errors introduced by the polymerase.

RT-PCR analysis. For RT-PCR analysis, total RNA was isolated from transfected HEK293 cells using an RNeasy mini kit (Qiagen) and then used to synthesize first-strand cDNA using oligo (dT) primers and Omniscript reverse transcriptase (Qiagen). The resulting cDNA was then used in PCR to amplify murine p110, EphA8, and human p110 DNA fragments. A 546-bp DNA fragment of EphA8 was amplified using a 5' primer matching nt 2569 to 2588 of the insert in pSP38 and a 3' primer matching the HA epitope sequence appended at the carboxy terminus of EphA8. A 330-bp DNA fragment of murine p110 was amplified using a 5' primer matching nt 3322 to 3341 of the murine p110 cDNA and a 3' primer matching the FLAG epitope sequence appended at the carboxy terminus of p110. A 229-bp DNA fragment of the human p110 was amplified using a 5' primer matching nt 3401 to 3420 and a 3' primer matching nt 3609 to 3628 of the human p110 cDNA sequence (GenBank accession no. X83368). PCR was performed using HotStar Taq DNA polymerase and 18 cycles of amplification with a 1-min denaturation step at 94°C, a 1-min annealing step at 56°C, and a 1.5-min extension step at 72°C.

Immunoprecipitation, Western blotting, cell surface biotinylation, and pulse-chase experiments. Immunoprecipitation and Western blotting were performed as previously described (7). For cell surface biotinylation, confluent cells were detached by trypsin treatment, washed once with complete medium (DMEM or alpha-MEM containing 10% heat-inactivated fetal bovine serum) and counted prior to cell surface biotinylation. The cells were placed on ice, washed five times with phosphate-buffered saline (PBS) and biotinylated by incubation with sulfo-N-hydroxysuccinimide-biotin (0.5 mg/ml; Pierce) for 30 min. The reaction was stopped by washing three times with PBS. Labeled cells were lysed with PLC lysis buffer as described above. Extracted cell proteins were immunoprecipitated with the relevant antibodies, and the immunocomplexes were bound to protein A-Sepharose (integrin ₅ polyclonal antibody) or rabbit anti-mouse immunoglobulin G (IgG)-Sepharose (integrin ₅₁ monoclonal antibody) beads. Bound material was eluted by boiling the beads in 5% sodium dodecyl sulfate (SDS) and resolved by electrophoresis on SDS-7.5 polyacrylamide gels (7.5% SDS-PAGE) under nonreducing conditions. Biotinylated integrins were detected by incubating blots with streptavidin-horseradish peroxidase (HRP) (Pierce). For pulse-chase experiments, cells were transiently transfected with FLAG-tagged p110 and then treated with doxycycline to induce the wild-type EphA8 protein. Cells were washed once with PBS and starved for 2 h in methionine- and cysteine-free medium containing 10% dialyzed fetal calf serum. The cells were then pulse-labeled for 15 min with 200 µCi of [³⁵S]methionine and [³⁵S]cysteine (NEN), per ml, washed once to remove unincorporated radioactivity, and chased for 30 min in unlabeled normal medium with or without preclustered EphA5-Fc. At the indicated times, cells were detergent extracted and subjected to immunoprecipitation with anti-FLAG antibody.

Binding assay, GST-mixing experiment, far-Western analysis, and PI 3-kinase assay. Binding assays using EphA5-Fc proteins and glutathione S-transferase (GST)-mixing experiments were performed as described previously (7, 30). Far-Western blot assays were performed as described previously (11). GST-JM fusion protein was radiolabeled using [-³²P]ATP and anti-HA immunoprecipitates containing wild-type EphA8 as described previously (7). The labeled GST-JM proteins were purified using a PD-10 column (Amersham-Pharmacia) and then used to probe membranes containing proteins transferred from 7.5% SDS-polyacrylamide gels. After incubation at room temperature for 3 h, the membranes were washed five times with Tris-buffered saline (10 mM Tris-HCl [pH 7.5], 0.9% NaCl) before exposure to film. PI 3-kinase activity was measured as previously described (14), with some modifications. Briefly, proteins were immunoprecipitated by incubating cell extracts with an anti-HA antibody or a p85 subunit-specific PI 3-kinase antibody and protein A-Sepharose as described above. The Sepharose beads were washed three times with HNTG buffer (7), once with 1% Nonidet P-40 in PBS, once with 100 mM Tris-HCl (pH 7.5) containing 500 mM LiCl, and once with 50 mM Tris-HCl buffer (pH 7.2) containing 150 mM NaCl. After removal of the last wash, the beads were resuspended in kinase buffer (20 mM HEPES [pH 7.2], 50 mM NaCl, 1 mM EGTA) containing 4 µg of PI (Sigma), 10 µM ATP, 5 mM MnCl₂, and 10 µCi of [-³²P]ATP and incubated for 20 min at 30°C. The reaction was stopped by the addition of 100 µl of 1 N HCl and 200 µl of a 1:1 mixture of chloroform and methanol. The lipids were extracted and resolved on potassium oxalate-pretreated thin-layer chromatography (TLC) plates (EM Sciences) with 35 ml of 2 N acetic acid and 65 ml of 1-propanol as the mobile phase. Dried plates were exposed to either Fuji-Imaging plates for quantitation or Kodak X-ray film for autoradiography. Analysis of ephrin A5-stimulated accumulation of 3'-phosphorylated inositol phospholipids was performed as described elsewhere (18, 32). Briefly, HEK293 cells transiently transfected with p110 were treated with doxycyline for 12 h to induce the expression of the EphA8 receptors. These cells were serum starved in phosphate-deficient DMEM for 2 h prior to labeling with ³²P_i (2 mCi/ml for 2 h in a 60-mm-diameter dish) and then treated with preclustered ephrin A5-Fc for the indicated times. Lipid extraction, deacylation, and analysis of the deacylated lipids by anion-exchange high-pressure liquid chromatography (HPLC) were performed as previously described (32).

Cell attachment assays. Attachment assays were performed on 22-mm² coverslips coated with different matrix proteins. Coverslips were placed in 35-mm-diameter dishes, coated by overnight incubation at 4°C, and postcoated for 1 h with 1% bovine serum albumin in PBS. Fibronectin (Promega) and laminin (Upstate Biotechnology, Inc.) were used at 100 and 125 µg/ml, respectively, in PBS. in cell attachment assays using PI 3-kinase inhibitors, wortmannin (stored in dimethyl sulfoxide [DMSO] in the dark at 20°C) was added directly to the culture medium for 30 min. Cells were collected by brief trypsinization, washed twice with complete medium, and then replated in triplicate onto coverslips (5 × 10⁵ cells/coverslip). After 15 (NIH 3T3) or 30 (HEK293) min at 37°C, unattached cells were removed by washing twice with complete medium, and adherent cells were incubated until 2 h after replating. Adherent cells on coverslips were counted directly by a hemocytometer. Alternatively, cells were fixed in 4% paraformaldehyde, stained with 0.5% crystal violet in 20% methanol, and quantified by measurement of the optical density at 570 nm. Control coverslips were incubated for 2 h without washing and used to measure total cell numbers. The ratio of attached cells to total cell numbers was calculated for each of three coverslips. Data are expressed as mean ± standard error (SE) and are representative of three independent experiments. In experiments using integrin blocking antibodies, cells were preincubated with the relevant antibodies (5 µg/ml) for 5 min at room temperature before replating.

Antibodies. Polyclonal rabbit antibody specific for the EphA8 JM was described previously (7). Monoclonal antiphosphotyrosine antibody (4G10) and polyclonal anti-p85 subunit PI 3-kinase antibody were purchased from Upstate Biotechnology. Polyclonal rabbit anti-p110, -p110, and -p110 antibodies were from Santa Cruz Biotechnology. Polyclonal rabbit anti-HA antibody was obtained from Zymed. Monoclonal mouse anti-FLAG antibody was from Sigma. Monoclonal anti-human integrin ₅₁ (JBS5) and murine ₅₁ (BMA5) and polyclonal anti-murine integrin ₅ antibodies were from Chemicon. Monoclonal anti-murine integrin ₃ antibody was from Transduction Laboratories. The HRP-conjugated secondary antibodies were from Amersham Pharmacia Biotech, and streptavidin-HRP was from Pierce.

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

Both wild-type and kinase-inactive EphA8 proteins promote cell attachment to fibronectin. To determine whether EphA8 signaling can promote cell adhesion through integrins, wild-type and kinase-inactive EphA8 receptors and a chimeric EphA8-TrkB receptor consisting of the extracellular domain of the EphA8 receptor plus the transmembrane and cytoplasmic domains of TrkB were stably expressed in NIH 3T3 fibroblasts. Lysates prepared from representative cell lines were analyzed by immunoprecipitation with either anti-TrkB or anti-EphA8 antibodies and then assayed by immunoblotting using a mixture of anti-EphA8 and anti-TrkB antibodies as a probe (Fig. 1A, top). High levels of chimeric EphA8-TrkB receptor, wild-type EphA8, and kinase-inactive EphA8 were detected in the correspondingly transfected fibroblasts. When the same blot was stripped and reprobed with antiphosphotyrosine antibody (Fig. 1A, bottom), it was evident that both EphA8-TrkB and EphA8 proteins were highly tyrosine phosphorylated due to the endogenous expression of EphA subgroup ligands as described previously (Fig. 1A, bottom, lanes 3 to 5) (30). In contrast, the EphA8 mutant containing Met in place of Lys-666, the putative ATP binding residue, was not tyrosine phosphorylated (Fig. 1A, bottom, lane 6). The kinase-inactive EphA8 mutant appeared to be weakly tyrosine phosphorylated because it was very weakly detected after long exposure (data not shown). This phosphorylation is possibly due to cross-phosphorylation induced by heterodimerization of EphA8 with other EphA family members present in low levels in these cells, as assessed by a binding assay using ephrin A5-Fc (see Fig. 4B). Cell attachment assays were performed by detaching the cells from culture dishes and replating them onto coverslips coated with fibronectin or laminin. As shown in Fig. 1B, adhesion of two independent NIH 3T3 clones onto a fibronectin matrix was significantly enhanced by expression of the wild-type EphA8 receptor, whereas the EphA8-TrkB chimeric receptor did not promote cell attachment to fibronectin. Unexpectedly, expression of a kinase-inactive EphA8 receptor also markedly promoted cell adhesion to fibronectin, producing an effect similar to that exerted by the wild-type EphA8 receptor. No consistent differences in adhesion to laminin-coated surfaces were observed in response to EphA8. In our cell adhesion assays where cells were seeded onto coverslips, most attached to the surface as well-separated cells that did not aggregate. These results strongly suggest that the intracytoplasmic region of the EphA8 receptor plays a critical role in promoting cell attachment to fibronectin and that the kinase activity of the receptor is not necessary for this action.

View larger version (38K): [in this window] [in a new window]   FIG. 1.   (A) Stable expression and in vivo tyrosine phosphorylation of EphA8, EphA8-TrkB, and the EphA8 mutant containing Met in place of Lys-666. NIH 3T3 cells were stably transfected with pMEXneo-derived expression plasmids containing, the indicated cDNAs, and individual G418-resistant clones were isolated. Cells were lysed in PLC lysis buffer, and proteins from each cell lysate were immmuprecipitated with either anti-TrkB or anti-EphA8 antibody. Immunoprecipitates were separated by 7.5% SDS-PAGE and immunoblotted with a mixture of anti-TrkB and anti-EphA8 antibodies (top); the same blot was stripped and reprobed with antiphosphotyrosine antibody (bottom). (B) Cell attachment responses to two different extracellular matrix proteins. Control NIH 3T3 fibroblasts (either parental or vector transfected) or stably transfected cells expressing the indicated proteins were plated on coverslips coated with fibronectin or laminin. Cells were allowed to adhere for 15 min. then nonadherent cells were removed by washing, and incubation was continued for 105 min. The percentage of cells attached after 15 min is shown. Data from three separate experiments are presented as means ± SE.

EphA8-stimulated cell attachment is mediated through ₅₁ and ₃ integrins in NIH 3T3 fibroblasts. We next investigated whether the observed promotion of cell attachment by EphA8 was mediated by integrins by testing the effects of monoclonal anti-₅₁ and anti-₃ integrin blocking antibodies on EphA8-stimulated cell attachment to fibronectin. Neither anti-₅₁ nor anti-₃ integrin antibodies had any significant effect on the attachment of a parental NIH 3T3 fibroblasts to fibronectin, which stayed at a consistent level of 45 to 55% (Fig. 2A). This level of adhesion seems to be the basal level, and it is possible that it is not integrin mediated, since it could not be reduced by the use of the integrin blocking antibodies, including those specific for ₄ and _v integrins (data not shown). In contrast, EphA8-stimulated cell adhesion in NIH 3T3 fibroblasts expressing the wild-type or kinase-inactive EphA8 receptor was partially inhibited by anti-₅₁ integrin blocking antibodies. The simultaneous addition of anti-₅₁ and anti-₃ integrin blocking antibodies markedly inhibited adhesion of these transfected fibroblasts onto a fibronectin matrix. The ₃ integrin subunit is known to heterodimerize with the _v or _IIb integrin subunit (1). Since _IIb3 integrin is expressed only in platelets, it is likely that _v3 integrin is involved in EphA8 modulation of cell adhesion in NIH 3T3 fibroblasts. This possibility could not be confirmed in the present study, as no specific blocking antibody against murine _v3 integrin was available. Nonetheless, our data strongly suggest that EphA8 action modulates cell adhesion through ₅₁ and possibly _v3 integrin proteins in NIH 3T3 fibroblasts.

View larger version (53K): [in this window] [in a new window]   FIG. 2.   (A) Effects of anti-51 and anti-3 integrin blocking antibodies (Ab) on NIH 3T3 cell adhesion to fibronectin. NIH 3T3 fibroblasts stably expressing wild-type or kinase-inactive EphA8 were detached and incubated for 5 min with either anti-51 or anti-3 integrin blocking antibody (5 µg/ml). The cells were then replated on fibronectin-coated coverslips and allowed to adhere for 15 min. Data from three independent experiments are presented as means ± SE. (B) Induction of wild-type EphA8 protein expression in NIH 3T3 fibroblasts. Cells were incubated with 2 µg of doxycycline (Dox) per ml for the indicated periods of time and then lysed, and proteins from the lysates were immunoprecipitated with anti-EphA8 antibody. Immunoprecipitates were separated by 7.5% SDS-PAGE and immunoblotted with anti-EphA8 antibody (top); the same blot was stripped of antibodies and reprobed with antiphosphotyrosine antibody (bottom). (C) Effects of anti-51 and anti-3 integrin blocking antibodies on NIH 3T3 cell attachment stimulated by the induced expression of EphA8. Cells were treated with doxycycline for the indicated periods of time, and cell attachment assays were performed after incubation with either anti-51 or anti-3 blocking antibodies as described above. (D) Analysis of 51 and 3 integrins expressed in NIH 3T3 cells, concomitant with the induced expression of EphA8. EphA8 expression was induced for the indicated times, then the cells were harvested and cell surface proteins were biotinylated; integrins were immunoprecipitated with anti-5 polyclonal antibody, and biotinylated integrins were detected using streptavidin-HRP (top). Cells were lysed in PLC lysis buffer, and protein concentrations were equalized (bottom). Cell lysates were fractionated by SDS-PAGE and then analyzed by immunobloting using anti-3 antibody as a probe.

EphA8-stimulated cell attachment is mediated mainly through ₅₁ integrin in HEK293 epithelial cells. To investigate whether regulation of integrin activity by the EphA8 receptor is dependent on cell type, we also expressed wild-type or kinase-inactive EphA8 receptors in HEK293 epithelial cells, once again using a powerful doxycycline-inducible expression system. In response to doxycycline, the time-dependent EphA8 induction (Fig. 3A, top) and tyrosine phosphorylation (Fig. 3A, middle) profiles in HEK293 cells were very similar to the patterns observed for the EphA8 receptor inducibly expressed in NIH 3T3 fibroblasts. Likewise, the kinase-inactive EphA8 mutant was also induced by doxycycline (Fig. 3C, top), and as we expected, no tyrosine phosphorylation of the mutant EphA8 protein was detectable (Fig. 3C, bottom). The adhesive properties of these cells in response to doxycycline were tested in cell attachment assays. Induction of the EphA8 proteins in these cells dramatically promoted cell attachment to a fibronectin matrix within 12 h, regardless of the EphA8 kinase activity (Fig. 3B and D). To test whether the cells adhered to fibronectin through ₅₁ integrin receptors, we examined the effects of anti-₅₁ blocking antibodies on cell adhesion to fibronectin at each induction time point (Fig. 3B and D). It was evident that anti-₅₁ blocking antibodies alone greatly inhibited cell adhesion to fibronectin. In contrast, no inhibition of cell adhesion onto fibronectin could be detected by adding anti-_v3 blocking antibodies (data not shown). These results indicate that in HEK293 cells, ₅₁ integrin is a major fibronectin receptor which interacts with EphA8 in a kinase activity-independent fashion. In addition, as observed in NIH 3T3 fibroblasts, the expression of EphA8 did not alter the surface expression of ₅₁ integrin in HEK293 epithelial cells (Fig. 3A, bottom). Taken together, our data indicate that EphA8 modulation of cell adhesion through alteration of integrin activity is a global mechanism and not restricted to a small subset of cell types.

View larger version (54K): [in this window] [in a new window]   FIG. 3.   (A) Induction of wild-type EphA8 protein expression in HEK293 epithelial cells. Cells were incubated with 2 µg of doxycycline (Dox) per ml for the indicated times. Proteins from cell lysates were immmunoprecipitated with anti-EphA8 antibody, then separated by 7.5% SDS-PAGE, and immunoblotted with the same antibody (top); the same blot was stripped and then reprobed with antiphosphotyrosine antibody (middle); Bottom, analysis of 51 integrin expressed in HEK293 cells, concomitant with the induced expression of EphA8. EphA8 expression was induced for the indicated times, then the cells were harvested, and cell surface proteins were biotinylated. Labeled integrins were immunoprecipitated with anti-51 monoclonal antibody and then detected with streptavidin-HRP. (B) Effect of anti-51 integrin blocking antibodies (Ab) on the cell attachment stimulated by the induced expression of EphA8 in HEK293 cells. The cells were treated with doxycycline for the indicated times, and then cell attachment assays were performed after incubation with anti-51 blocking antibodies as described in the legend to Fig. 2A. Note that HEK293 cells were allowed to adhere for 30 min before being washed to remove nonadherent cells, and then incubation was continued for 90 min. The percentage of cells attached after 30 min is shown. Data from three independent experiments are presented as means ± SE. (C) Induction of kinase-inactive EphA8 protein expression in HEK293 epithelial cells. As a positive control, HEK293 cells inducibly expressing the wild-type EphA8 protein were treated with doxycycline for 12 h. Proteins from cell lysates were immmunoprecipitated with anti-EphA8 antibody, then separated by 7.5% SDS-PAGE, and immunoblotted with anti-EphA8 antibody (top); the same blot was stripped and then reprobed with antiphosphotyrosine antibody (bottom). (D) Effects of anti-51 integrin blocking antibodies on cell attachment stimulated by the induced expression of kinase-inactive EphA8 receptor in HEK293 cells. Cell attachment assays were performed as described for panel B.

Ligand binding of EphA8 is critical for the increased cell attachment to fibronectin. To investigate whether regulation of integrin activity by EphA8 is a ligand-dependent process, we constructed two different deletion mutants lacking structural motifs of the EphA8 ectodomain. According to the recently published genomic organization of the murine EphA8 gene, the globular domain and a stretch of cysteine-rich sequences are encoded, possibly as a functional unit, by exon III, whereas two fibronectin type III (FNIII) domains are encoded by exons V to VII (22). It is well known that the N-terminal globular domain of Eph receptors is critical for ligand binding (23). It has also been suggested that FNIII domains play a role in receptor dimerization (24). Thus, exon boundaries were used to generate EphA8 deletion mutants lacking these two important structural domains. The deletion mutants were named EphA8III and EphA8V-VII, according to their deleted exons. These deletion mutants were stably expressed in NIH 3T3 fibroblasts, and two independent clones of each deletion mutant were studied in order to eliminate the possibility of clonal selection for the results.

View larger version (24K): [in this window] [in a new window]   FIG. 4.   (A) Expression and tyrosine phosphorylation of the exogenous EphA8III and EphA8V-VII deletion mutants in NIH 3T3 fibroblasts. NIH 3T3 cells were transfected with pMEXneo-derived expression plasmids containing recombinant cDNAs encoding the indicated deletion proteins and selected in G418-containing medium. Two independent cell lines per constructs were analyzed by immunoprecipitation with anti-EphA8 antibody. Western blot analysis was performed with anti-EphA8 antibody (top); the same blot was stripped and then reprobed with antiphosphotyrosine antibody (bottom). C, control. (B) The domain encoded by exon III of EphA8 is critical for efficient binding of the ephrin A5 ligand. NIH 3T3 cells stably expressing wild-type EphA8 or the indicated deletion mutants were treated with 40 nM chimeric ephrin A5-Fc proteins followed by radioiodinated goat anti-human lgG, washed, and solubilized for determination of specific binding. Nonspecific binding was assessed as binding in the absence of chimeric Fc proteins. Specific binding, shown on the y axis, was calculated by subtracting nonspecific binding from total counts bound. (C) Deletion of exon III abrogates the EphA8-stimulated cell attachment to fibronectin. Cell attachment assays were performed as described in previous figure legends.

Tyrosine phosphorylations in EphA8 do not correlate with EphA8-stimulated cell attachment to fibronectin To test whether the EphA8-stimulated integrin activity was independent of tyrosine phosphorylation of EphA8, the codons for Tyr-615 and Tyr-792 were replaced with phenylalanine codons in the EphA8 cDNA to construct EphA8^Y615F and EphA8^Y792F point mutants. We have previously demonstrated that Tyr-615 constitutes a major autophosphorylation site and mediates preferential binding to the Fyn SH2 domain (7). Tyr-792 is located in the activation loop of the EphA8 tyrosine kinase domain, and corresponding tyrosine residues are found in all known members of the Eph receptor family. HEK293 cells were transiently transfected with pcDNA3 constructs containing wild-type or mutated EphA8 cDNAs. In contrast to the wild-type protein, the EphA8 mutant proteins immunoprecipitated from lysates of EphA8^Y615F- or EphA8^Y792F-transfected cells contained markedly reduced levels of phosphotyrosine (Fig. 5A, bottom left) compared with the overall expression levels of these proteins (Fig. 5A, top left). Nonetheless, the attachment responses of HEK293 cells expressing EphA8^Y615F or EphA8^Y792F were stimulated to a degree similar to that observed in cells expressing the wild-type EphA8 protein (Fig. 5B).

View larger version (59K): [in this window] [in a new window]   FIG. 5.   (A) Transient expression and tyrosine phosphorylation of EphA8 point mutants and kinase-inactive proteins. HEK293 cells were transiently transfected with pcDNA3-derived expression plasmids containing the recombinant cDNAs encoding the indicated EphA8 mutant proteins and were lysed for immunoprecipitation with anti-EphA8 antibody at 24 h posttransfection. Western blot analysis was performed with anti-EphA8 antibody to verify the level of EphA8 expression (top); the same blot was stripped and then reprobed with antiphosphotyrosine antibody to evaluate the level of autophosphorylation of the transfected EphA8 mutant proteins (bottom). (B) Mutations of two major tyrosine phosphorylation sites and the ATP binding site lysine of the EphA8 receptor do not affect EphA8-stimulated cell attachment to fibronectin. Cell attachment assays were performed as described in previous figure legends. (C) Top, schematic representation of the EphA8 cytoplasmic domain deletion mutants. Middle, HEK293 cells were transiently transfected with the indicated constructs, and cells were lysed for immunoprecipitation with anti-HA polyclonal antibody at 24 h posttransfection. Western blot analysis was performed with same antibody to verify the expression level of the deletion mutant. Note that the anti-EphA8 antibody does not recognize the EphA8 mutant lacking the JM encoded by exon X. Bottom, the same blot was stripped and then reprobed with antiphosphotyrosine antibody. (D) Cell attachment assays performed as described in previous figure legends.

The JM encoded by exon X is crucial for interaction with integrin To investigate the structural determinant of EphA8 that controls integrin activity, we used exon-intron boundaries of the murine EphA8 gene to design deletion mutants. The exon X- and XVI-encoded portions of the murine EphA8 receptor correspond to a highly conserved JM and a sterile alpha motif (SAM), respectively (22). The EphA8 cytoplasmic domain deletion mutants were transiently expressed as HA epitope-tagged proteins in HEK293 cells. The HA epitope-tagged deletion mutants were expressed at the expected molecular masses and at similar levels, indicating the stability of these constructs (Fig. 5C, middle). Although the JM deletion mutant contained reduced levels of phosphotyrosine (Fig. 5C, bottom, lane 3) due to the lack of a major autophosphorylation site, Tyr-615, it had retained efficient tyrosine kinase activity, as demonstrated by its action on an exogenous substrate (enolase) in an in vitro kinase assay (data not shown). We then monitored the effects of each deletion mutant on the adhesive properties of the cells. HEK293 cells expressing wild-type EphA8 or the SAM deletion mutant showed increased adhesion to fibronectin relative to cells transiently transfected with empty vector (Fig. 5D). In contrast, cells expressing the JM deletion mutant showed only a slight increase in adhesion to fibronectin, much less than the increase observed in cells expressing the SAM deletion mutant or wild-type EphA8. This result indicates that the EphA8 JM encoded by exon X is required for interaction with integrin.

The p85 subunit-associated PI 3-kinase does not contribute to EphA8-stimulated integrin activation. In a preliminary approach to identifying the signaling mechanism by which EphA8 regulates integrin function, we assessed the effects of the PI 3-kinase inhibitor wortmannin on HEK293 cells that had been induced to express wild-type or kinase-inactive EphA8 receptors. Treatment of HEK293 cells with up to 100 nM wortmannin had no effect on the basal adhesion to fibronectin (Fig. 6A). Regardless of the kinase activity, doxycycline-induced expression of EphA8 proteins in HEK293 cells stimulated cell attachment to fibronectin in the absence of wortmannin. However, EphA8-stimulated cell attachment was partially inhibited by 10 nM wortmannin and almost completely abolished by wortmannin concentrations of 50 to 100 nM (Fig. 6A). We also observed that the PI 3-kinase inhibitor LY294002 had a similar inhibitory effect on EphA8-stimulated cell attachment (data not shown). These results imply that PI 3-kinase may couple the EphA8 receptor to integrin activation. To further explore the possibility of an association between the EphA8 receptor and PI 3-kinase activity, we performed in vitro kinase assays using HEK293 cells induced to express EphA8 proteins. After induction of EphA8 proteins with doxycycline, extracts were immunoprecipitated with anti-HA antibody to capture the activated EphA8 proteins, and the immunoprecipitates were then assayed for the ability to phosphorylate PI in vitro. An increase in PI 3-kinase activity, as indicated by the appearance of PI(3)P, was observed upon expression of the wild-type or kinase-inactive EphA8 protein (Fig. 6B, first and second panels). Interestingly, an in vitro PI 3-kinase assay using antiPhosphotyrosine immunoprecipitates has revealed that PI 3-kinase activity was significantly increased by the induction of expression of the wild-type EphA8 but not when kinase-inactive EphA8 was induced (Fig. 6B, third panel). More importantly, wild-type or SAM deletion EphA8 proteins stimulated PI 3-kinase activity to a greater extent than did the JM deletion EphA8 mutant in HEK293 transfectants (Fig. 6C and D). However, we observed that transient expression of the PI 3-kinase p85 subunit lacking the p110 binding site (p85) did not inhibit EphA8-promoted cell adhesion to fibronectin (data not shown). Consistent with this observation, the EphA8-expressing cells did not show any significant alteration in PI 3-kinase activity in anti-p85 subunit immunoprecipitates, whereas p85-associated PI 3-kinase activity in the same cells was elevated in response to serum stimulation (Fig. 6E). We also generated stable HEK293 cell lines expressing 10-fold-excess amounts of p85. The anti-p85 subunit immunoprecipitates from these cells showed no changes in PI 3-kinase activity in response to serum stimulation (Fig. 7A, left, lanes 1 and 2). In contrast, transient expression of the wild-type or kinase-inactive EphA8 proteins in the same cells reproducibly induced a marked increase in PI 3-kinase activity (Fig. 7A, left, lanes 4 and 5) and also promoted cell attachment to a fibronectin matrix (Fig. 7A, right). In addition, immunoprecipitates obtained with antiserum against the p110 or p110 PI 3-kinase catalytic subunit showed similar levels of PI 3-kinase activity, regardless of the auto-kinase activity of the expressed EphA8 protein (Fig. 7B, first and second panels). These results strongly indicate that the EphA8 receptor can regulate integrin activity independently of the p85 regulatory subunit by interacting with PI 3-kinase.

View larger version (55K): [in this window] [in a new window]   FIG. 6.    (A) Inhibition of EphA8-stimulated cell attachment by wortmannin. Expression of wild-type or kinase-inactive EphA8 proteins in HEK293 cells was induced with doxycycline (Dox) for 24 h, and cells were incubated with wortmannin at concentrations ranging from 10 to 100 nM for 30 min prior to cell attachment assays. Cells that had not been treated with doxycycline were included as controls. Wortmannin was dissolved in DMSO. Note that cells were treated with DMSO for 30 min prior to the cell attachment assay in the absence of wortmannin (0 nM on the x axis). Treatment of cells with DMSO (5 to 10 µl) had no effect on the basal level of adhesion or EphA8-stimulated cell adhesion. (B) The EphA8 proteins were induced with doxycycline for the indicated times and then immunoprecipitated with either anti-HA antibody (top, left and middle) or antiphosphotyrosine antibody (top, right). Immunoprecipitates were incubated with 4 µg of PI and 10 µCi of [-32P]ATP for 10 min, and the reaction was analyzed by TLC followed by autoradiography. Western blot analysis of anti-HA immunoprecipitates was performed with the same antibody to verify the expression levels of wild-type and kinase-inactive EphA8 proteins at the indicated induction times (bottom). (C and D) The EphA8 JM is necessary for association with PI 3-kinase activity. HEK293 cells were transiently transfected with the indicated constructs, and cells were lysed for immunoprecipitation of the EphA8 deletion mutants with anti-HA polyclonal antibody at 24 h posttransfection. Western blot analysis was performed with same antibody to verify the expression level of the deletion mutant (C, bottom) PI 3-kinase activity was determined by TLC and autoradiography (C, top) and also by measuring formation of radiolabeled PIP from PI, using a phosphorimaging system (D). Shown are mean values (±SE) of three independent experiments. (E) p85-dependent heterodimeric PI 3-kinase is not stimulated to response to EphA8 expression. The kinase-inactive EphA8 protein was induced with doxycycline for the indicated times, and then p85-p110 heterodimeric PI 3-kinase was immunoprecipitated with anti-p85 polyclonal antibody. As a control, the same cells were starved of serum for 24 h and then stimulated with (+) or without () serum for 15 min in the absence of doxycycline. Immunoprecipitates were analyzed by in vitro PI 3-kinase assay, and lipids were extracted and analyzed by TLC and autoradiography as described above.

View larger version (53K): [in this window] [in a new window]   FIG. 7.   Evidence that p110 PI 3-kinase is tightly associated with the EphA8 receptor. (A) Left, HEK293 cells were transfected with the p85 cDNA, and individual G418-resistant clones were isolated. A representative cell line was treated with (+) or without () serum for 15 min, cells were lysed in PLC lysis buffer, and p110-p85 heterodimers from the cell lysates were immmuprecipitated with anti-p85 polyclonal antibody (lanes 1 and 2). The stable p85 transfectants were transiently transfected with the indicated constructs and lysed for immunoprecipitation of the EphA8 proteins with anti-HA polyclonal antibody at 24 h posttransfection (lanes 3 to 5). PI-3 kinase activity was measured as described in previous figure legends. Right, cell attachment assays performed as in previous figure legends. (B) EphA8 protein expression in HEK293 cells was induced with doxycycline treatment for the indicated times, and then PI 3-kinases were immunoprecipitated (IP) with the indicated anti-p110 isotype antibodies. PI 3-kinase activity was measured as described in previous figure legends. (C) Expression of wild-type or kinase-inactive EphA8 proteins in HEK293 cells was induced with doxycycline for the indicated times. Top, proteins from cell lysates were immunoprecipitated with anti-p110 antibody and then analyzed by immunoblot using anti-EphA8 antibody as a probe (top); the same blot was stripped and reprobed with anti-p110 antibody (bottom).

The tight association between p110 PI 3-kinase and the EphA8 receptor. To identify the EphA8-specific PI 3-kinase isotype, we performed immunoprecipitations using a p110-specific antibody (Fig. 7B, third panel). Anti-p110 immunoprecipitates contained low levels of PI-3 kinase activity in the absence of EphA8 protein (lanes 1 and 3). Expression of the wild-type or kinase-inactive EphA8 proteins resulted in a substantial increase in p110 PI 3-kinase activity (lanes 2 and 4). Moreover, stringently washed anti-p110 immunoprecipitates contained high levels of wild-type or kinase-inactive EphA8 protein (Fig. 7C, top, lanes 2 and 4). Interestingly, we reproducibly observed that the p110 protein level was substantially increased in EphA8-expressing cells (Fig. 7C, bottom), suggesting that the p110 PI 3-kinase may be stabilized by the EphA8 receptor. In addition, these results suggest that the increase in PI 3-kinase activity from either EphA8 or p110 immunoprecipitates was due to the presence of more p110 protein that had been stabilized by the activated EphA8 receptor. Taken together, our results demonstrate that p110 PI 3-kinase mediates EphA8-dependent regulation of integrins.

View larger version (50K): [in this window] [in a new window]   FIG. 8.   The JM deletion EphA8 mutant suppresses EphA8-promoted p110 PI 3-kinase activity and integrin activation by inhibiting the association of EphA8 with p110. HEK293 cells expressing EphA8 in response to doxycycline (Dox) were transiently transfected with the indicated constructs; 12 h after transfection, EphA8 protein expression was induced for 12 h. (A) Cell attachment assays performed as described in previous figure legends. (B) Proteins from cell lysates were immunoprecipitated with anti-p110 antibody, and then PI-3 kinase activity was measured as described in previous figure legends. (C) Proteins from cell lysates were immunoprecipitated with anti-p110 antibody and then analyzed by immunoblotting with anti-EphA8 antibody as a probe (top); the same blot was stripped and reprobed with anti-HA and anti-p110 antibodies (middle and bottom, respectively). (D) The EphA8 JM is sufficient for association with p110. A FLAG-tagged murine p110 construct was transiently transfected into parental cells (lane 5) or HEK293 cells expressing EphA8 in response to doxycycline treatment (lanes 1 to 4). As a control, the FLAG-tagged p110 protein transiently expressed in parental HEK293 cells was directly immunoprecipitated with anti-FLAG antibody (lane 5). The induction of EphA8 began 12 h after transfection and continued for 24 h (lanes 2 and 4). Proteins from each cell lysate were mixed with approximately equal amounts of purified GST or with GST-JM fusion protein bound to glutathione-Sepharose beads (lanes 1 to 4). The washed beads were separated by 7.5% SDS-PAGE and Western blotted using anti-FLAG antibody as a probe. (E) The EphA8 JM is sufficient for direct association with p110. HEK293 cells expressing both exogenous p110 and wild-type EphA8 were prepared as described for panel D. FLAG-tagged p110 was directly immunoprecipitated with anti-FLAG antibody, and then the washed beads were directly separated by 7.5% SDS-PAGE and transferred to membranes. The blots were probed with 32P-labeled GST-JM protein to detect p110. The labeling of GST-JM protein was performed in an in vitro kinase assay with [-32P]ATP and anti-HA immunoprecipitates containing wild-type EphA8 as previously described. Note that the GST-JM fusion protein contained Tyr-615, which is a major phosphorylation site.

Association of p110 with EphA8 is rapidly induced by oligomeric ephrin A5-Fc added to cells stripped of ephrin A ligands. To further exclude the possibility that EphA8 promotes integrin activity by activating ligand signaling pathways, NIH 3T3 cells were treated with PI-PLC, which eliminates GPI-linked ephrin A subgroup ligands from the cell surface as previously reported (30). We then compared the ability of the ephrin A-stripped cells to adhere to surfaces coated with fibronectin with or without preclustered ephrin A5-Fc soluble ligand stimulation (see Fig. 11A). As we expected, a prominent increase in cell attachment was induced in cells stimulated by preclustered ephrin A5-Fc. Furthermore, the ability of wild-type or kinase-inactive EphA8 receptor to associate with the p110 protein was markedly enhanced by stimulation with preclustered ephrin A5-Fc (Fig. 9A, top, lanes 2 and 4). The p110 PI 3-kinase activity was also significantly increased in the cells stimulated by preclustered ephrin A5-Fc (Fig. 9A. bottom, lanes 2 and 4). We also transiently expressed wild-type or lipid kinase-inactive p110 in HEK293 cells expressing induced wild-type EphA8. These cells were also treated with PI-PLC and preclustered ephrin A5-Fc. Interestingly, we reproducibly observed that transient expression of a murine p110 mutant lacking lipid kinase activity, K833R, did not significantly alter cell attachment promoted by EphA8 activation (See Fig. 11B). Consistent with these results, association of the lipid kinase-inactive p110 protein with EphA8 was not detected in cells stimulated with preclustered ephrin A5-Fc (Fig. 9B, lane 5). These results suggest that the lipid kinase-active form of p110 may be important for stable association with EphA8 at the plasma membrane. In contrast, exogenous wild-type p110 expression markedly strengthened cell attachment promoted by EphA8 activation until 4 h after EphA8 induction (see Fig. 11B). In addition, the rapid and stable association of the FLAG-tagged wild-type p110 PI-3 kinase with EphA8 occurred upon stimulation with preclustered ephrin A5-Fc (Fig. 9B, lane 3). We also investigated whether EphA8 increases the level of 3' phosphoinositides in vivo upon stimulation with preclustered ephrin A5-Fc (Fig. 10A). For these experiments, we transiently expressed wild-type p110 in HEK293 cells expressing induced wild-type or kinase-inactive EphA8. These cells were treated with PI-PLC during the period of serum starvation for 2 h, then labeled with ³²P_i, and exposed to preclustered ephrin A5-Fc for the indicated times. The ³²P-labeled phospholipids were then analyzed as described previously (18, 32). As expected, the ephrin A5-Fc treatment markedly increased the amount of PI(3, 4, 5)P₃ in HEK293 cells expressing either wild-type or kinase-inactive EphA8. The EphA5-stimulated accumulation of PI(3, 4)P₂ in these cell lines was similar to that of PI(3, 4, 5)P₃, but the levels of PI(3)P PI(4)P and PI(4, 5)P₂ did not significantly increase with ephrin A5 treatment (data not shown). From results obtained from multiple experiments, it became evident that endogenously expressed p110 protein levels were significantly elevated upon stimulation with preclustered ephrin A5-Fc (Fig. 9A, middle). In addition, the exogenously expressed wild-type p110 protein level was consistently increased by EphA8 activation (Fig. 9B, bottom, lane 3). These results suggest that ephrin A ligands enhance the stability of p110 bound to EphA8 rather than increasing p110 mRNA because the p110 cDNA used for exogenous expression did not contain its own basal promoter and transcriptionally regulated sequences. To further investigate whether p110 mRNA levels can be upregulated by activated EphA8 expression in HEK293 cells, we used a semiquantitative RT-PCR approach to measure p110 mRNA levels. Primer sets that discriminate between exogenous murine p110 and endogenous human p110 were used (Fig. 9C). In all PCR experiments, the quality and amount of cDNA were initially assessed by PCR with primers specific for -actin (data not shown). Similar levels of human p110 mRNA were detected regardless of activated EphA8 induction (Fig. 9C, bottom). In addition, a similar level of murine p110 mRNA was observed in cells transfected with wild-type murine p110 or the lipid kinase-inactive p110 mutant (Fig. 9C, top). As expected, in the absence of doxycycline treatment, the murine EphA8 mRNA level was barely detectable. In contrast, it was abundant in the same cells treated with doxycycline. To further analyze the rate of degradation of the exogenously expressed p110 protein, we transiently expressed wild-type EphA8 in HEK293 cells expressing induced wild-type EphA8. These cells were serum starved and treated with PI-PLC simultaneously and then pulsed labeled with [³⁵S]cysteine and [³⁵S]methionine for 15 min without preclustered ephrin A5-Fc treatment. The amount of p110 was then measured in the 30 min following the chase with or without preclustered ephrin A5-Fc treatment. As shown in Fig. 10B, the rate of degradation of p110 was much higher in untreated cells (lane 3). The half-life of p110 in untreated cells was estimated to be less than 10 min (data not shown). In contrast, degradation of p110 in the ligand-treated cells was almost undetectable for at least 30 min (lane 2). These results strongly suggest that the level of p110 in cells is increased by the activated EphA8 receptor through protein stabilization. It should be noted that in these experiments, the Eph receptor was not efficiently labeled, probably because of the absence of doxycycline treatment during the period of serum starvation and pulse-chase. Taken together, these results strongly indicate that induced EphA8 expression does not affect p110 mRNA levels; rather, it probably stabilizes the p110 protein that is associated with EphA8 at the plasma membrane. We also estimated the fraction of EphA8 associated with the exogenously expressed p110 protein in HEK293 cells that had been transiently transfected with p110 and then treated with doxycycline for 12 h. These cells were also treated with PI-PLC and preclustered ephrin A5-Fc. The relative amount of EphA8 was calculated by comparing the amount of EphA8 recovered using anti-FLAG antibody to the total cellular EphA8 in the same lysate. These amounts were estimated using immunoblot analysis and anti-EphA8 antibody as a probe (Fig. 10C). To achieve maximum recovery of p110, three consecutive immunoprecipitations of the same lysate were combined and analyzed. The results indicate that about 10% of total cellular EphA8 was recovered by anti-FLAG antibody through association with p110 (Fig.