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Department of Basic Medical Sciences and Purdue Cancer Center, Purdue University, West Lafayette, Indiana 47907 [N. D. Z., M. A., M. S. K.]; Department of Cell Biology, Neurobiology and Anatomy, University of Cincinnati, Cincinnati, Ohio 45267 [M-F. C.]; and Department of Medicine, Rammelkamp Center for Research, MetroHealth Campus, Case Western Reserve University, Cleveland, Ohio 44109 [B. W.]
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Introduction |
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There is much interest in how tyrosine kinases like EphA2 regulate cell growth and differentiation. One often unappreciated mechanistic hint is the observation that substrates of tyrosine kinases are found almost exclusively within sites of cellular adhesion (14) . In epithelial cells, for example, tyrosine-phosphorylated proteins are predominantly located in E-cadherin-associated adherens junctions (14 , 15) . E-cadherin mediates calcium-dependent cell-cell adhesions through homophillic interactions with E-cadherin on apposing cells (16 , 17) . In cancer cells, E-cadherin function is frequently destabilized, either by loss of E-cadherin expression (18) or by disruption of linkages between E-cadherin and the actin cytoskeleton (19, 20, 21, 22, 23) . Restoration of E-cadherin function, either by E-cadherin transfection (24 , 25) or treatment with pharmacological reagents (21) , is sufficient to block cancer cell growth and induce epithelial differentiation. However, the mechanisms by which E-cadherin imparts these tumor suppressor functions are largely unknown. Whereas E-cadherin-mediated stabilization of cell-cell contacts undoubtedly is involved, there is recent evidence that E-cadherin also generates intracellular signals that could contribute to tumor suppression (15 , 26 , 27) .
Previous studies by our laboratory have linked E-cadherin with signaling by tyrosine phosphorylation. E-cadherin aggregation into assembling adherens junctions initiates a signaling cascade involving tyrosine phosphorylation that may contribute to E-cadherin’s tumor suppressor function (28) . In addition, we have demonstrated that transformed epithelial cells have elevated levels of tyrosine phosphorylation that destabilize E-cadherin function (21) . To identify tyrosine kinases and their substrates in breast cancer, we recently generated monoclonal antibodies that are specific for tyrosine-phosphorylated proteins in Ras-transformed breast epithelial cells (15) . Using these antibodies, we identified the EphA2 tyrosine kinase as a protein that is tyrosine-phosphorylated upon E-cadherin-mediated adhesion. We also show that E-cadherin regulates the functioning of EphA2.
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Results |
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TOPAbstractIntroductionResultsDiscussionMaterials and MethodsReferences |
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and data not shown), with 2–5-fold more EphA2 in neoplastic cells, as determined by Western blot analysis using multiple EphA2 antibodies and by Northern blot analysis (data not shown).
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. The decreased phosphotyrosine content was confirmed using different EphA2 antibodies (D7, B2D6, and rabbit polyclonal antibodies) for immunoprecipitation and with multiple phosphotyrosine antibodies (PY20, 4G10, and rabbit polyclonal antibodies) for Western blot analysis (data not shown). Decreased EphA2 phosphorylation was also observed in other metastatic breast cancer cell lines as well as invasive tumor cell lines derived from colon, pancreatic, ovarian, and lung cancers (data not shown).
Further comparison of EphA2 in nonneoplastic and metastatic cells revealed other changes in EphA2 distribution and function. Immunofluorescence staining with EphA2-specific antibodies revealed that EphA2 in nonneoplastic cells was mostly found within sites of cell-cell contact (Fig. 2)
, with little staining of membrane that was not in contact with neighboring cells. In contrast, EphA2 in metastatic cells was absent from sites of cell-cell contacts. Instead, the EphA2 in these cells was either diffusely distributed or enriched within membrane ruffles at the leading edge of migrating cells. The enrichment within membrane ruffles was confirmed by colocalization of EphA2 with f-actin (data not shown). This localization within membrane ruffles was not observed in nontransformed epithelia, even at low cell density. These differences in subcellular distribution were confirmed using three different EphA2-specific antibodies (D7, B2D6, and rabbit polyclonal antibodies). The correlation between EphA2 localization and phosphotyrosine content forms the basis for much of the remainder of this study.
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. Despite the low phosphotyrosine content of EphA2 in metastatic cells, this EphA2 demonstrated enzymatic activity that was comparable with or higher than the activity of EphA2 isolated from nonneoplastic cells. This activity was unaffected by the basal phosphotyrosine content of EphA2 because unlabeled phosphate was rapidly exchanged with labeled phosphate during the autophosphorylation assays as described previously (33
, 34) . KOH treatment of the membranes prior to autoradiography did not significantly reduce the level of phosphorylation, indicating that the observed enzymatic activity represented mostly phosphorylation on tyrosine residues. It is also notable that the phosphotyrosine content of EphA2 (Fig. 1B)
was not predictive of its enzymatic activity (Fig. 3)
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). In contrast, a control chimera (Ctrl-IgG) did not alter EphA2 phosphorylation. Clustering EphA2 at the cell surface with specific antibodies (EK166B or B2D6) also induced levels of EphA2 activation that were comparable with that nonneoplastic cells (Fig. 4A)
. Receptor aggregation, not simply antibody binding, was necessary for EphA2 phosphorylation as incubation with anti-EphA2 (Fig. 4
, 1°) alone did not increase EphA2 phosphorylation relative to matched controls. This effect was specific for EphA2 as neither secondary (Fig. 4
, 2°) antibodies alone or clustering of isotype-matched control antibodies (which recognize an inaccessible cytoplasmic epitope on EphA2) did not induce tyrosine phosphorylation of EphA2 (data not shown). Analysis of the timing of EphA2 phosphorylation revealed EphA2 phosphorylation within 2 min after cross-linking, with optimal phosphorylation detected after 5 min (Fig. 4B)
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Because both EphA2 and E-cadherin are found at sites of cell-cell contact, we first examined whether the two proteins colocalize using two-color immunofluorescence microscopy. This revealed an overlapping distribution of EphA2 and E-cadherin along the lateral membranes of epithelial cells and, specifically, within sites of cell-cell contact (Fig. 5)
. Vertical sectioning by confocal microscopy confirmed colocalization of E-cadherin and EphA2 within sites of cell-cell contact (data not shown).
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and induced either a diffuse or membrane ruffle pattern of staining (Fig. 6C)
, which was reminiscent of EphA2 in metastatic cells. Subsequent restoration of normal levels of extracellular calcium restored normal levels of EphA2 phosphorylation and cell-cell localization within 5 min (Fig. 6, A and C)
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and remained diffuse or present within membrane ruffles (Fig. 7C)
. In contrast, isotype-matched control antibodies and scrambled peptides did not prevent EphA2 phosphorylation or localization within intercellular junctions. Specific inhibition of E-cadherin with these inhibitors also blocked EphA2 phosphorylation and cell-cell localization upon treatment of confluent cell monolayers (data not shown), thus confirming that EphA2 phosphorylation and localization are sensitive to the functioning of E-cadherin.
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. In contrast, the EphA2 in 231-E-cad redistributed into sites of cell-cell contacts and had levels of phosphotyrosine that were comparable with that of MCF-10A cells (Fig. 9A)
. These changes in EphA2 phosphorylation and localization increased with cell density (data not shown), consistent with an idea that E-cadherin function regulates EphA2 phosphorylation and localization.
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. Whereas parental and 231-neo cells were mesenchymal in appearance and readily grew atop one another, the E-cadherin-transfected cells had more prominent cell-cell adhesions and grew as single-cell monolayers. Analysis of cell-ECM3
attachments by staining with paxillin-specific antibodies revealed numerous focal adhesions in control MDA-MB-231 cells, whereas 231-E-cad cells had fewer focal adhesions. The decrease in focal adhesions was most prominent in 231-E-cad cells within colonies (Fig. 8
, bottom right), whereas individual cells had focal adhesions that were comparable with controls (data not shown).
EphA2 activation contributes to the decreased cell-ECM adhesion. To activate EphA2 in MDA-MB-231 cells, we aggregated EphA2 at the cell surface with specific antibodies (as described above) and found that this caused a rapid loss of focal adhesions within 5 min. This was confirmed by paxillin staining (Fig. 10)
and by interference reflection microscopy (data not shown). Similar results were obtained in other neoplastic cell lines (data not shown). In contrast, treatment with either primary or secondary antibodies alone did not alter focal adhesions.
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, EphA2 activation decreased the proliferation in MDA-MB-231 cells (31% reduction; P < 0.001), whereas control conditions (primary or secondary antibodies alone and isotype controls) did not change cell growth. The short duration of EphA2 signaling that is induced by antibody aggregation (Fig. 4B)
likely underestimates EphA2’s growth-inhibitory potential. A similar decrease in cell growth was obtained following EphA2 activation in other cell types, including MDA-MB-435 cells (22% reduction; P < 0.0005) and MCF-10A cells (16% reduction; P < 0.01). For experiments with MCF-10A, we plated cells at low cell density and scored individual cells (to preclude cell-cell contacts that might otherwise activate EphA2).
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Discussion |
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Decreased EphA2 Phosphorylation in Metastatic Cells.
We originally identified EphA2 using antibodies that recognize tyrosine-phosphorylated proteins in Ras-transformed MCF-10A-neoT cells (15)
. MCF-10A-neoT cells express E-cadherin (21)
and, consequently, EphA2 is tyrosine-phosphorylated (data not shown). Notably, EphA2 was tyrosine-phosphorylated in nonneoplastic mammary epithelial cell lines but not in metastatic cell lines. In this respect, EphA2 differs from many other tyrosine kinases (e.g., cErbB2, epidermal growth factor receptor, platelet-derived growth factor receptor, and Src), the phosphorylation of which increases in cancer cells (2
, 41
, 42)
. For these kinases, phosphorylation elevates tyrosine kinase activity, triggering signal transduction cascades that promote cell proliferation.
The phosphotyrosine content of EphA2 does not relate to its intrinsic enzymatic activity in mammary epithelial cells. In vitro assays revealed that, despite its low phosphotyrosine content, the enzymatic activity of EphA2 in metastatic cells is comparable with or increased over the activity of phosphorylated EphA2 in nonneoplastic epithelial cells. This is consistent with evidence that the phosphorylation of EphB2 also has little effect on its kinase activity (43) . Our results suggest that, rather than controlling enzymatic activity, the phosphotyrosine content of EphA2 might influence the choice or availability of substrates and interacting proteins. In addition, changes in the phosphotyrosine content of EphA2 might provide signals that are independent of EphA2 enzymatic activity, which is supported by recent reports that other Eph kinases (VAB-1 and EphB2) have kinase-independent functions (44 , 45) . This suggests that protein interactions, localization, phosphotyrosine content, and enzymatic activity all contribute to Eph receptor function.
There are several possible explanations for the loss of EphA2 phosphorylation in metastatic cells. The primary sites of receptor autophosphorylation are not mutated because the sites that become autophosphorylated in vitro are the same in nontransformed and neoplastic cells.4 Consistent with this, EphA2 tyrosine phosphorylation was restored by cross-linking EphA2 with antibodies or by transfection with E-cadherin. Another possible cause for decreased EphA2 phosphorylation could be loss of EphA2 ligands (ephrin-A class molecules). However, our ability to restore EphA2 phosphorylation in E-cadherin-transfected cells appears to exclude this possibility. A third possibility is that the phosphotyrosine content of EphA2 is repressed by an associated tyrosine-phosphatase. Consistent with this, treatment of neoplastic cells with tyrosine-phosphatase inhibitors restores normal levels of EphA2 tyrosine phosphorylation.5 However, the identities of the phosphatases responsible for this are presently unknown.
Regulation of EphA2 Activation by E-Cadherin.
We focused on the possibility that decreased stability of cell-cell contacts inhibits tyrosine phosphorylation of EphA2 in metastatic cells. Both Eph family receptor tyrosine kinases and their ephrin ligands are bound to the cell surface (1
, 6
, 7)
, so cells must be in close contact to facilitate Eph-ephrin interactions. Little is known, however, about the nature of these contacts and their precise effects on Eph-ephrin interactions.
Because many breast tumors lack E-cadherin and have unstable cell-cell junctions (18 , 46) , we investigated how expression of E-cadherin affects EphA2 phosphorylation in mammary epithelial cells. We found inhibition of E-cadherin function either by removal of Ca2+ or with function-blocking antibodies or peptides reduced EphA2 phosphorylation and caused EphA2 to redistribute into membrane ruffles. Conversely, expression of E-cadherin in MDA-MB-231 cells restored EphA2 phosphorylation and localization to sites of cell-cell contact. The simplest explanation for these results is that E-cadherin stabilizes cell-cell contacts and, thereby, facilitates interactions between EphA2 and its ligands.
At present, there is no evidence for or against a direct interaction between E-cadherin and EphA2. The two proteins are expressed in overlapping patterns. but we have not been able to coimmunoprecipitate EphA2 and E-cadherin.5 EphA2 also does not cocluster with E-cadherin at the cell surface in response to antibody-mediated aggregation of either molecule,6 which is consistent with our biochemical evidence. We cannot exclude that experimental conditions used for protein extraction dissociate such interactions or that a small fraction of activated EphA2 coclusters with E-cadherin. Direct interaction between the two molecules may not be necessary if E-cadherin primarily serves to stabilize cell-cell contacts and thereby promote interactions between EphA2 and its ligands. Other aspects of E-cadherin function, such as signaling (28) , cytoskeletal association (47) , and junction formation (16) might also target EphA2 to sites of cell-cell contact.
EphA2 Regulates Cell-ECM Adhesion and Growth.
An immediate consequence of EphA2 activation is decreased cell-ECM contact at focal adhesions. Focal adhesions are sites of membrane-cytoskeletal interaction that provide anchorage for cell migration and invasion (48)
. Focal adhesions also play critical roles in signal transduction, where they organize intracellular signals that control cell growth and survival (39
, 40)
. We propose that E-cadherin-mediated stabilization of ligand binding induces EphA2 to block focal adhesions. Consistent with this, it is understood that epithelial cells balance their cell-cell and cell-ECM adhesions and that this is linked with the proper functioning of E-cadherin (49
, 50) . Individual epithelial cells have more focal adhesions than cells within colonies, whereas cells with decreased E-cadherin function have increased cell-matrix adhesion, regardless of cell density (21)
. Although the molecular mechanisms responsible for this are unknown, many proteins that interact with Eph kinases regulate cell adhesion or cytoskeletal organization, including the p85 subunit of phosphatidylinositol 3'-kinase, Src, Fyn, and Ras-GAP (35
, 51, 52, 53)
.
Focal adhesions initiate signals that promote cell growth, and it follows that loss of these structures may contribute to decreased cell growth following EphA2 activation. By inference, loss of EphA2 activation might contribute to deregulated growth of neoplastic cells by increasing signals from focal adhesions. This would be consistent with evidence that neoplastic cells have increased signaling by focal adhesion proteins (e.g., FAK; Ref. 54 ). Although EphA2 activation decreases cell growth, the expression pattern of EphA2 does not fit the classic pattern of a tumor suppressor. Most tumor suppressors are inactivated either because of decreased expression or loss of enzymatic activity. In contrast, neoplastic cells express high levels of EphA2, which, although nonphosphorylated, retains comparable levels of enzymatic activity. An alternative explanation is that EphA2 positively regulates cell growth but that this signaling is reduced in nontransformed epithelia. Support for this includes evidence that EphA2 is overexpressed in neoplastic cells and is supported by the fact that other Eph kinases (e.g., EphA1) are oncogenic (55) . In this scenario, EphA2 "activation" by E-cadherin or receptor aggregation might decrease EphA2 function, perhaps by reducing EphA2 expression levels. It is intriguing that the lowest levels of EphA2 are found in cells where it is phosphorylated and that ligand-mediated aggregation decreases EphA2 expression levels. A third possibility is that EphA2 functions very differently in normal and neoplastic epithelia. The phosphotyrosine content and subcellular localization of EphA2 differ in normal and neoplastic cells, and either property could alter substrate specificity or availability. Indeed, tyrosine-phosphorylated EphA2 (but not unphosphorylated EphA2) interacts with the phosphatidylinositol 3'-kinase and the SLAP adapter protein (56) . SLAP was recently shown to negatively regulate cell growth (57) , which is supportive of our evidence that EphA2 also regulates cell proliferation. Future studies will be necessary to define EphA2’s role as a positive and/or negative regulator of cell growth and to determine whether these properties differ between normal and neoplastic epithelia.
Conclusions.
Loss of E-cadherin in carcinomas promotes invasion (18
, 58)
, cell motility (27)
, and cell proliferation (26)
. In this study, we have identified the receptor tyrosine kinase EphA2 as one protein that is phosphorylated after cell-cell contact and demonstrated that both the phosphorylation and localization of EphA2 are sensitive to changes in E-cadherin function and expression. We also find that EphA2 activation negatively regulates cell-ECM adhesion and cell growth. These findings raise the possibility that important effects of E-cadherin on tumor cell behavior may occur via effects on EphA2.
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Materials and Methods |
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TOPAbstractIntroductionResultsDiscussionMaterials and MethodsReferences |
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Western Blot Analysis.
Unless noted otherwise, all experiments used confluent cell monolayers that were extracted in a buffer containing 1% Triton X-100 or in RIPA buffer containing 1% Triton X-100, 0.5% sodium deoxycholate, and 0.1% SDS for 6 min on ice, as described previously (21)
. After protein concentrations were measured by Coomassie Blue staining (Pierce, Rockford, IL) or Bio-Rad DC Protein Assay (Hercules, CA), equal amounts of protein were resolved by SDS-PAGE and transferred to nitrocellulose (Protran, Schleicher & Schuell, Keene, NH), and Western blot analysis was performed as described previously (21)
. Antibody binding was detected by enhanced chemiluminescence as recommended by the manufacturer (Pierce). To reprobe, we stripped blots as described previously (21)
.
Immunofluorescence and Confocal Microscopy.
Immunostaining was performed as described previously (21)
. In brief, cells were grown on glass coverslips to visualize individual cells. Cells were observed at both high cell density (
70% confluence) and low cell density (20% confluence) by seeding 1 x 106 cells onto either a 3.5- or 10-cm tissue culture plate overnight at 37°C. At high cell density, extensive overlapping of neoplastic cells precludes accurate subcellular visualization. The samples were fixed in 3.7% formaldehyde solution, extracted in 0.5% Triton X-100, and stained. Immunostaining was visualized using rhodamine-conjugated donkey antimouse antibodies (Chemicon, Temecula, CA) and FITC-conjugated donkey antirabbit (Chemicon) and epifluorescence microscopy (model BX60, x600, Olympus Lake Success, NY) and recorded onto T-Max 400 film (Eastman-Kodak, Rochester, NY). For confocal microscopy, samples were viewed on a Nikon Diaphot 300 outfitted with a Bio-Rad MRC 1024 UV/Vis System and Coherent Innova Enterprise model 622 60-mW output water-cooled lasers.
Immunoprecipitation.
Immunoprecipitation experiments were performed as described (21)
for 1.5 h at 4°C with the appropriate EphA2-specific monoclonal antibodies (D7 or B2D6) and rabbit antimouse (Chemicon) conjugated protein A-Sepharose (Sigma). Immunoprecipitates were washed three times in lysis buffer, resuspended in SDS sample buffer (Tris buffer containing 5% SDS, 3.8% DTT, 25% glycerol, and 0.1% bromphenol blue), and resolved by 10% SDS-PAGE.
In Vitro Kinase Assays.
For in vitro autophosphorylation assays, immunoprecipitated EphA2 was washed in lysis buffer and incubated in 10 mM PIPES, 3 mM MnCl2, 5 mM PNPP (Sigma 104 phosphatase substrate; Sigma), 1 mM NaVO4, 1 µM ATP, and 10 µCi of [
-32P]ATP (New England Nuclear, Boston, MA) at 25°C for the times shown. The reactions were terminated by the addition of 5x Laemmli sample buffer at multiple time points before saturation. After resolving samples by 10% SDS-PAGE, the gel was transferred to nitrocellulose (Schleicher & Schuell) or Immobilon P (Pierce), and incorporated material was detected by autoradiography. To hydrolyze phosphoserine/threonine, we treated the membranes with 1 N KOH at 65°C for 1 h and reassessed them by autoradiography. After several half-lives, Western blot analysis was performed to determine EphA2 loading.
Cross-Linking of EphA2 Receptors.
For antibody cross-linking experiments, cells grown as a monolayer were incubated at 4°C for 20 min with 4 µg/ml EphA2 antibody (either clone EK166B or B2D6) or purified fusion protein of ephrin-A1 fused to IgG (B61-IgG; Ref. 10
). Primary antibody alone, rabbit antimouse IgG alone and control fusion proteins were used as controls. The samples were washed with medium, incubated with 20 µg/ml rabbit antimouse IgG in conditioned medium at 4°C for 10 min, and warmed to 37°C for 10 min before extraction and immunoprecipitation. To determine the optimal time for activation, we incubated the plates in the presence of cross-linking antibody at 37°C for 0–120 min.
EGTA and Antibody Treatments.
"Calcium switch" experiments were performed as described previously (28)
. Monolayers of MCF-10A cells were grown to 80% confluence. EGTA was added to growth medium to a final concentration of 4 mM, and the cells were incubated at 37°C for 30 min. The medium was removed, and calcium concentrations restored with normal growth medium. To block E-cadherin function, we supplemented the medium with E-cadherin antibodies (1:100 dilution; DECMA-1; Sigma) or 10 µg/ml peptide corresponding to the E-cadherin HAV sequence (YTLFSHAVSSNGN). Controls include isotype control antibodies (rat anti-HA antibody; Boehringer Mannheim, Indianapolis, IN) and matched, scrambled peptides (SGATNSLHNFSVY). The Purdue Laboratory for Macromolecular Structure synthesized peptides. Cells were then incubated for the indicated times at 37°C and extracted for Western blot analysis and immunoprecipitation. Cell monolayers grown on glass coverslips were treated in the same manner and immunostained for EphA2.
E-Cadherin Expression and Function.
MDA-MB-231 cells were cotransfected with pBATEM2, a mouse E-cadherin expression vector (59)
and pSV2neo (60)
using FuGENE 6 Transfection Reagent (Boehringer Mannheim), following the manufacturer’s instructions. Transfected cells were selected in growth media supplemented with 400 µg/ml G418. Immunostaining and Western blot analysis with specific antibodies confirmed E-cadherin expression.
Proliferation Assay.
Cells were plated onto glass coverslips and cultured overnight in growth medium. EphA2 antibodies (EK166B or B2D6, extracellular or D7, intracellular) or ligand fusion protein (B61-IgG) were added to the media at 1 µg/ml and incubated at 4°C for 20 min, washed with medium, and incubated with 20 µg/ml rabbit antimouse plus 3 µg/ml BrdUrd at 37°C for 4 h. Cells were fixed in cold methanol for 8 min, extracted with 2 N HCl at 37°C for 30 min and stained with a BrdUrd antibody to indicate proliferating cells and Hoechst dye to label the nuclei of all cells on the coverslip. A minimum of six random fields were selected in a double-blind study, and at least 150 cells were assessed in each sample. Each experiment was repeated at least three times.
Statistical Methods.
All statistical analyses were performed using the SAS System for Windows, Version 6.12. An ANOVA model was used to compare the percentage of cells that grew in each field, within each specimen, in the control group to the percentage of cells that grew in each field, within each specimen, in the experimental group. Group (control versus experimental) was treated as a fixed effect and specimen within each group was treated as a random effect. A normal probability plot of the residuals was used to assess the homogeneity of the variances of the mean percentage cell growth for the control and experimental groups. P < 0.05 was considered statistically significant.
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Acknowledgments |
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Footnotes |
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1 Supported by American Cancer Society Grant RPG CSM-86522 (to M. S. K.), NIH Grant AR44713 (to R. B. and M. S. K.), and U. S. Army Medical Research and Material Command Grants 17-98-1-8146 (to M. S. K.) and 17-98-1-8292 (to R. B.). N. D. Z. is a Howard Hughes Medical Institute Predoctoral Fellow. 
2 To whom requests for reprints should be addressed, at Department of Basic Medical Sciences, Purdue University, West Lafayette, IN 47907-1246. E-mail: msk{at}vet.purdue.edu
3 The abbreviations used are: ECM, extracellular matrix; BrdUrd, bromodeoxyuridine.
4 M. S. Kinch, unpublished results.
5 N. D. Zantek, unpublished results.
6 M. Fedor-Chaiken and M. S. Kinch, unpublished results.
Received for publication 4/13/99. Revision received 7/ 2/99. Accepted for publication 7/28/99.
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A. B. Larsen, M. W. Pedersen, M.-T. Stockhausen, M. V. Grandal, B. v. Deurs, and H. S. Poulsen Activation of the EGFR Gene Target EphA2 Inhibits Epidermal Growth Factor-Induced Cancer Cell Motility Mol. Cancer Res., March 1, 2007; 5(3): 283 - 293. [Abstract] [Full Text] [PDF]
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S. Abraham, D. W. Knapp, L. Cheng, P. W. Snyder, S. K. Mittal, D. S. Bangari, M. Kinch, L. Wu, J. Dhariwal, and S. I. Mohammed Expression of EphA2 and Ephrin A-1 in Carcinoma of the Urinary Bladder Clin. Cancer Res., January 15, 2006; 12(2): 353 - 360. [Abstract] [Full Text] [PDF]
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 M. Tanaka, R. Kamata, and R. Sakai EphA2 Phosphorylates the Cytoplasmic Tail of Claudin-4 and Mediates Paracellular Permeability J. Biol. Chem., December 23, 2005; 280(51): 42375 - 42382. [Abstract] [Full Text] [PDF]
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 H. Andersen, J. Mejlvang, S. Mahmood, I. Gromova, P. Gromov, E. Lukanidin, M. Kriajevska, J. K. Mellon, and E. Tulchinsky Immediate and Delayed Effects of E-Cadherin Inhibition on Gene Regulation and Cell Motility in Human Epidermoid Carcinoma Cells Mol. Cell. Biol., October 15, 2005; 25(20): 9138 - 9150. [Abstract] [Full Text] [PDF]
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J. Wykosky, D. M. Gibo, C. Stanton, and W. Debinski EphA2 as a Novel Molecular Marker and Target in Glioblastoma Multiforme Mol. Cancer Res., October 1, 2005; 3(10): 541 - 551. [Abstract] [Full Text] [PDF]
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 A. P. Putzke, S. T. Hikita, D. O. Clegg, and J. H. Rothman Essential kinase-independent role of a Fer-like non-receptor tyrosine kinase in Caenorhabditis elegans morphogenesis Development, July 15, 2005; 132(14): 3185 - 3195. [Abstract] [Full Text] [PDF]
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 A. I. Ivanov, A. A. Steiner, A. C. Scheck, and A. A. Romanovsky Expression of Eph receptors and their ligands, ephrins, during lipopolysaccharide fever in rats Physiol Genomics, April 14, 2005; 21(2): 152 - 160. [Abstract] [Full Text] [PDF]
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 H. Xu, W. Tian, J. N. Lindsley, T. T. Oyama, J. M. Capasso, C. J. Rivard, H. T. Cohen, S. M. Bagnasco, S. Anderson, and D. M. Cohen EphA2: expression in the renal medulla and regulation by hypertonicity and urea stress in vitro and in vivo Am J Physiol Renal Physiol, April 1, 2005; 288(4): F855 - F866. [Abstract] [Full Text] [PDF]
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H. Miao, K. Strebhardt, E. B. Pasquale, T.-L. Shen, J.-L. Guan, and B. Wang Inhibition of Integrin-mediated Cell Adhesion but Not Directional Cell Migration Requires Catalytic Activity of EphB3 Receptor Tyrosine Kinase: ROLE OF RHO FAMILY SMALL GTPases J. Biol. Chem., January 14, 2005; 280(2): 923 - 932. [Abstract] [Full Text] [PDF]
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C. J. Herrem, T. Tatsumi, K. S. Olson, K. Shirai, J. H. Finke, R. M. Bukowski, M. Zhou, A. L. Richmond, I. Derweesh, M. S. Kinch, et al. Expression of EphA2 is Prognostic of Disease-Free Interval and Overall Survival in Surgically Treated Patients with Renal Cell Carcinoma Clin. Cancer Res., January 1, 2005; 11(1): 226 - 231. [Abstract] [Full Text] [PDF]
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M. Hu, K. L. Carles-Kinch, D. P. Zelinski, and M. S. Kinch EphA2 Induction of Fibronectin Creates a Permissive Microenvironment for Malignant Cells Mol. Cancer Res., October 1, 2004; 2(10): 533 - 540. [Abstract] [Full Text] [PDF]
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P. H. Thaker, M. Deavers, J. Celestino, A. Thornton, M. S. Fletcher, C. N. Landen, M. S. Kinch, P. A. Kiener, and A. K. Sood EphA2 Expression Is Associated with Aggressive Features in Ovarian Carcinoma Clin. Cancer Res., August 1, 2004; 10(15): 5145 - 5150. [Abstract] [Full Text] [PDF]
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 M. D. Westfall and J. A. Pietenpol p63: molecular complexity in development and cancer Carcinogenesis, June 1, 2004; 25(6): 857 - 864. [Abstract] [Full Text] [PDF]
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P. Dobrzanski, K. Hunter, S. Jones-Bolin, H. Chang, C. Robinson, S. Pritchard, H. Zhao, and B. Ruggeri Antiangiogenic and Antitumor Efficacy of EphA2 Receptor Antagonist Cancer Res., February 1, 2004; 64(3): 910 - 919. [Abstract] [Full Text] [PDF]
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 B. de Saint-Vis, C. Bouchet, G. Gautier, J. Valladeau, C. Caux, and P. Garrone Human dendritic cells express neuronal Eph receptor tyrosine kinases: role of EphA2 in regulating adhesion to fibronectin Blood, December 15, 2003; 102(13): 4431 - 4440. [Abstract] [Full Text] [PDF]
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R. L. Pratt and M. S. Kinch Ligand Binding Up-Regulates EphA2 Messenger RNA Through the Mitogen-Activated Protein/Extracellular Signal-Regulated Kinase Pathway Mol. Cancer Res., December 1, 2003; 1(14): 1070 - 1076. [Abstract] [Full Text] [PDF]
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G. Zeng, Z. Hu, M. S. Kinch, C.-X. Pan, D. A. Flockhart, C. Kao, T. A. Gardner, S. Zhang, L. Li, L. A. Baldridge, et al. High-Level Expression of EphA2 Receptor Tyrosine Kinase in Prostatic Intraepithelial Neoplasia Am. J. Pathol., December 1, 2003; 163(6): 2271 - 2276. [Abstract] [Full Text]
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K. T. Coffman, M. Hu, K. Carles-Kinch, D. Tice, N. Donacki, K. Munyon, G. Kifle, R. Woods, S. Langermann, P. A. Kiener, et al. Differential EphA2 Epitope Display on Normal versus Malignant Cells Cancer Res., November 15, 2003; 63(22): 7907 - 7912. [Abstract] [Full Text] [PDF]
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M. Lu, K. D. Miller, Y. Gokmen-Polar, M.-H. Jeng, and M. S. Kinch EphA2 Overexpression Decreases Estrogen Dependence and Tamoxifen Sensitivity Cancer Res., June 15, 2003; 63(12): 3425 - 3429. [Abstract] [Full Text] [PDF]
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A. Palmer and R. Klein Multiple roles of ephrins in morphogenesis, neuronal networking, and brain function Genes & Dev., June 15, 2003; 17(12): 1429 - 1450. [Full Text] [PDF]
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J. Walker-Daniels, A. R. Hess, M. J.C. Hendrix, and M. S. Kinch Differential Regulation of EphA2 in Normal and Malignant Cells Am. J. Pathol., April 1, 2003; 162(4): 1037 - 1042. [Full Text] [PDF]
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M. S. Kinch, M.-B. Moore, and D. H. Harpole Jr. Predictive Value of the EphA2 Receptor Tyrosine Kinase in Lung Cancer Recurrence and Survival Clin. Cancer Res., February 1, 2003; 9(2): 613 - 618. [Abstract] [Full Text] [PDF]
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M. Koolpe, M. Dail, and E. B. Pasquale An Ephrin Mimetic Peptide That Selectively Targets the EphA2 Receptor J. Biol. Chem., November 27, 2002; 277(49): 46974 - 46979. [Abstract] [Full Text] [PDF]
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J. Walker-Daniels, D. J. Riese II, and M. S. Kinch c-Cbl-Dependent EphA2 Protein Degradation Is Induced by Ligand Binding Mol. Cancer Res., November 1, 2002; 1(1): 79 - 87. [Abstract] [Full Text] [PDF]
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K. D. Kikawa, D. R. Vidale, R. L. Van Etten, and M. S. Kinch Regulation of the EphA2 Kinase by the Low Molecular Weight Tyrosine Phosphatase Induces Transformation J. Biol. Chem., October 11, 2002; 277(42): 39274 - 39279. [Abstract] [Full Text] [PDF]
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F. Wang, R. K. Hansen, D. Radisky, T. Yoneda, M. H. Barcellos-Hoff, O. W. Petersen, E. A. Turley, and M. J. Bissell Phenotypic Reversion or Death of Cancer Cells by Altering Signaling Pathways in Three-Dimensional Contexts J Natl Cancer Inst, October 2, 2002; 94(19): 1494 - 1503. [Abstract] [Full Text] [PDF]
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K. Carles-Kinch, K. E. Kilpatrick, J. C. Stewart, and M. S. Kinch Antibody Targeting of the EphA2 Tyrosine Kinase Inhibits Malignant Cell Behavior Cancer Res., May 1, 2002; 62(10): 2840 - 2847. [Abstract] [Full Text] [PDF]
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G. Williams, E.-J. Williams, and P. Doherty Dimeric Versions of Two Short N-cadherin Binding Motifs (HAVDI and INPISG) Function as N-cadherin Agonists J. Biol. Chem., February 1, 2002; 277(6): 4361 - 4367. [Abstract] [Full Text] [PDF]
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N. Munarini, R. Jager, S. Abderhalden, G. Zuercher, V. Rohrbach, S. Loercher, B. Pfanner-Meyer, A.-C. Andres, and A. Ziemiecki Altered mammary epithelial development, pattern formation and involution in transgenic mice expressing the EphB4 receptor tyrosine kinase J. Cell Sci., January 1, 2002; 115(1): 25 - 37. [Abstract] [Full Text] [PDF]
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A. W. Boyd and M. Lackmann Signals from Eph and Ephrin Proteins: A Developmental Tool Kit Sci. Signal., December 11, 2001; 2001(112): re20 - re20. [Abstract] [Full Text] [PDF]
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N. D. Zantek, J. Walker-Daniels, J. Stewart, R. K. Hansen, D. Robinson, H. Miao, B. Wang, H.-J. Kung, M. J. Bissell, and M. S. Kinch MCF-10A-NeoST: A New Cell System for Studying Cell-ECM and Cell-Cell Interactions in Breast Cancer Clin. Cancer Res., November 1, 2001; 7(11): 3640 - 3648. [Abstract] [Full Text] [PDF]
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A. R. Hess, E. A. Seftor, L. M. G. Gardner, K. Carles-Kinch, G. B. Schneider, R. E. B. Seftor, M. S. Kinch, and M. J. C. Hendrix Molecular Regulation of Tumor Cell Vasculogenic Mimicry by Tyrosine Phosphorylation: Role of Epithelial Cell Kinase (Eck/EphA2) Cancer Res., April 1, 2001; 61(8): 3250 - 3255. [Abstract] [Full Text]
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D. P. Zelinski, N. D. Zantek, J. C. Stewart, A. R. Irizarry, and M. S. Kinch EphA2 Overexpression Causes Tumorigenesis of Mammary Epithelial Cells Cancer Res., March 1, 2001; 61(5): 2301 - 2306. [Abstract] [Full Text]
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C. Y. Sasaki, H. Lin, P. J. Morin, and D. L. Longo Truncation of the Extracellular Region Abrogrates Cell Contact but Retains the Growth-suppressive Activity of E-cadherin Cancer Res., December 1, 2000; 60(24): 7057 - 7065. [Abstract] [Full Text]
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S Orsulic and R Kemler Expression of Eph receptors and ephrins is differentially regulated by E-cadherin J. Cell Sci., January 5, 2000; 113(10): 1793 - 1802. [Abstract] [PDF]
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