Regulation of the EphA2 Kinase by the Low Molecular Weight Tyrosine Phosphatase Induces Transformation
2002; Elsevier BV; Volume: 277; Issue: 42 Linguagem: Inglês
10.1074/jbc.m207127200
ISSN1083-351X
AutoresKeith D. Kikawa, Derika R. Vidale, Robert L. Van Etten, Michael S. Kinch,
Tópico(s)Ubiquitin and proteasome pathways
ResumoIntracellular signaling by protein tyrosine phosphorylation is generally understood to govern many aspects of cellular behavior. The biological consequences of this signaling pathway are important because the levels of protein tyrosine phosphorylation are frequently elevated in cancer cells. In the classic paradigm, tyrosine kinases promote tumor cell growth, survival, and invasiveness, whereas tyrosine phosphatases negatively regulate these same behaviors. Here, we identify one particular tyrosine phosphatase, low molecular weight tyrosine phosphatase (LMW-PTP), which is frequently overexpressed in transformed cells. We also show that overexpression of LMW-PTP is sufficient to confer transformation upon non-transformed epithelial cells. Notably, we show that the EphA2 receptor tyrosine kinase is a prominent substrate for LMW-PTP and that the oncogenic activities of LMW-PTP result from altered EphA2 expression and function. These results suggest a role for LMW-PTP in transformation progression and link its oncogenic potential to EphA2. Intracellular signaling by protein tyrosine phosphorylation is generally understood to govern many aspects of cellular behavior. The biological consequences of this signaling pathway are important because the levels of protein tyrosine phosphorylation are frequently elevated in cancer cells. In the classic paradigm, tyrosine kinases promote tumor cell growth, survival, and invasiveness, whereas tyrosine phosphatases negatively regulate these same behaviors. Here, we identify one particular tyrosine phosphatase, low molecular weight tyrosine phosphatase (LMW-PTP), which is frequently overexpressed in transformed cells. We also show that overexpression of LMW-PTP is sufficient to confer transformation upon non-transformed epithelial cells. Notably, we show that the EphA2 receptor tyrosine kinase is a prominent substrate for LMW-PTP and that the oncogenic activities of LMW-PTP result from altered EphA2 expression and function. These results suggest a role for LMW-PTP in transformation progression and link its oncogenic potential to EphA2. low molecular weight tyrosine phosphatase Cancer arises when a population of cells gains the ability to inappropriately grow and survive. These biological behaviors often result from genetic and environmental abnormalities that work together to trigger specific signaling pathways that promote inappropriate cell growth and survival. In particular, increased levels of protein tyrosine phosphorylation are understood to initiate powerful signals that govern many different aspects of cell behavior (1Hunter T. Cell. 2000; 100: 113-127Abstract Full Text Full Text PDF PubMed Scopus (2253) Google Scholar). A popular paradigm suggests that a balance between tyrosine kinase and phosphatase activities determines the cellular levels of protein tyrosine phosphorylation and thereby governs cellular decisions regarding growth, survival, and invasiveness (2Pawson T. Scott J.D. Science. 1997; 278: 2075-2080Crossref PubMed Scopus (1891) Google Scholar). This model predicts that tyrosine kinases are oncogenic, whereas tyrosine phosphatases negatively regulate transformation. Although this paradigm has generally been supported by the identification of oncogenic tyrosine kinases, emerging evidence reveals a more complex interplay between tyrosine kinases and phosphatases. For example, the PTP(CAAX) tyrosine phosphatase has been recently implicated as an oncogene (3Cates C.A. Michael R.L. Stayrook K.R. Harvey K.A. Burke Y.D. Randall S.K. Crowell P.L. Crowell D.N. Cancer Lett. 1996; 110: 49-55Crossref PubMed Scopus (189) Google Scholar). Moreover, the enzymatic activity of Src family kinases is liberated by phosphatase-mediated dephosphorylation of critical tyrosine residues (4Cobb B.S. Parsons J.T. Oncogene. 1993; 8: 2897-2903PubMed Google Scholar, 5Liu X. Pawson T. Recent Prog. Horm. Res. 1994; 49: 149-160PubMed Google Scholar). In the latter situation, phosphatases can actually up-regulate protein tyrosine phosphorylation by increasing the enzymatic activity of kinases. The EphA2 receptor tyrosine kinase is overexpressed in a large number of human cancers. High levels of EphA2 apply to a large number of different cancers, including breast, prostate, colon, and lung carcinomas as well as metastatic melanomas (6Rosenberg I.M. Goke M. Kanai M. Reinecker H.C. Podolsky D.K. Am. J. Physiol. 1997; 273: G824-G832PubMed Google Scholar, 7Easty D.J. Guthrie B.A. Maung K. Farr C.J. Lindberg R.A. Toso R.J. Herlyn M. Bennett D.C. Cancer Res. 1995; 55: 2528-2532PubMed Google Scholar, 8Andres A.C. Zuercher G. Djonov V. Flueck M. Ziemiecki A. Int. J. Cancer. 1995; 63: 288-296Crossref PubMed Scopus (44) Google Scholar, 9Walker-Daniels J. Coffman K. Azimi M. Rhim J.S. Bostwick D.G. Snyder P. Kerns B.J. Waters D.J. Kinch M.S. Prostate. 1999; 41: 275-280Crossref PubMed Scopus (227) Google Scholar, 10Zelinski D.P. Zantek N.D. Stewart J.C. Irizarry A.R. Kinch M.S. Cancer Res. 2001; 61: 2301-2306PubMed Google Scholar). The highest levels of EphA2 are consistently found on the most aggressive cell models of human cancer (9Walker-Daniels J. Coffman K. Azimi M. Rhim J.S. Bostwick D.G. Snyder P. Kerns B.J. Waters D.J. Kinch M.S. Prostate. 1999; 41: 275-280Crossref PubMed Scopus (227) Google Scholar, 10Zelinski D.P. Zantek N.D. Stewart J.C. Irizarry A.R. Kinch M.S. Cancer Res. 2001; 61: 2301-2306PubMed Google Scholar, 11Zantek N.D. Walker-Daniels J. Stewart J.C. Hansen R.K. Robinson D. Miao H. Wang B. Kung H.J. Bissell M.J. Kinch M.S. Clin. Cancer Res. 2001; 7: 3640-3648PubMed Google Scholar). Moreover, EphA2 is not simply a marker of transformed disease as ectopic overexpression of EphA2 confers tumorigenic and metastatic potential upon non-transformed epithelial cells (10Zelinski D.P. Zantek N.D. Stewart J.C. Irizarry A.R. Kinch M.S. Cancer Res. 2001; 61: 2301-2306PubMed Google Scholar). In addition to its overexpression, EphA2 is functionally altered in transformed cells as compared with non-transformed epithelia (12Zantek N.D. Azimi M. Fedor-Chaiken M. Wang B. Brackenbury R. Kinch M.S. Cell Growth & Differ. 1999; 10: 629-638PubMed Google Scholar). In particular, EphA2 is prominently tyrosine phosphorylated in non-transformed epithelial cells. Yet, despite its overexpression, the EphA2 in transformed cells is not tyrosine phosphorylated (10Zelinski D.P. Zantek N.D. Stewart J.C. Irizarry A.R. Kinch M.S. Cancer Res. 2001; 61: 2301-2306PubMed Google Scholar, 11Zantek N.D. Walker-Daniels J. Stewart J.C. Hansen R.K. Robinson D. Miao H. Wang B. Kung H.J. Bissell M.J. Kinch M.S. Clin. Cancer Res. 2001; 7: 3640-3648PubMed Google Scholar, 12Zantek N.D. Azimi M. Fedor-Chaiken M. Wang B. Brackenbury R. Kinch M.S. Cell Growth & Differ. 1999; 10: 629-638PubMed Google Scholar). Recent studies indicate that these differences in EphA2 phosphotyrosine content are important because tyrosine phosphorylation of EphA2 causes it to interact with downstream signaling components that function to negatively regulate cell growth and invasiveness (10Zelinski D.P. Zantek N.D. Stewart J.C. Irizarry A.R. Kinch M.S. Cancer Res. 2001; 61: 2301-2306PubMed Google Scholar, 12Zantek N.D. Azimi M. Fedor-Chaiken M. Wang B. Brackenbury R. Kinch M.S. Cell Growth & Differ. 1999; 10: 629-638PubMed Google Scholar, 13Carles-Kinch K. Kilpatrick K.E. Stewart J.C. Kinch M.S. Cancer Res. 2001; 62: 2840-2847Google Scholar, 14Pandey A. Duan H. Dixit V.M. J. Biol. Chem. 1995; 270: 19201-19204Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 15Pandey A. Lazar D.F. Saltiel A.R. Dixit V.M. J. Biol. Chem. 1994; 269: 30154-30157Abstract Full Text PDF PubMed Google Scholar, 16Miao H. Burnett E. Kinch M.S. Simon E. Wang B. Nat. Cell. Bio. 2000; 2: 62-69Crossref PubMed Scopus (470) Google Scholar). In contrast, unphosphorylated EphA2 appears to adopt a different subcellular localization and interacts with different substrates (10Zelinski D.P. Zantek N.D. Stewart J.C. Irizarry A.R. Kinch M.S. Cancer Res. 2001; 61: 2301-2306PubMed Google Scholar, 11Zantek N.D. Walker-Daniels J. Stewart J.C. Hansen R.K. Robinson D. Miao H. Wang B. Kung H.J. Bissell M.J. Kinch M.S. Clin. Cancer Res. 2001; 7: 3640-3648PubMed Google Scholar, 12Zantek N.D. Azimi M. Fedor-Chaiken M. Wang B. Brackenbury R. Kinch M.S. Cell Growth & Differ. 1999; 10: 629-638PubMed Google Scholar). Most importantly, recent studies have shown that unphosphorylated EphA2 functions as a powerful oncoprotein, whereas restoration of EphA2 phosphotyrosine content is sufficient to reverse the oncogenic potential of EphA2 (10Zelinski D.P. Zantek N.D. Stewart J.C. Irizarry A.R. Kinch M.S. Cancer Res. 2001; 61: 2301-2306PubMed Google Scholar, 12Zantek N.D. Azimi M. Fedor-Chaiken M. Wang B. Brackenbury R. Kinch M.S. Cell Growth & Differ. 1999; 10: 629-638PubMed Google Scholar, 13Carles-Kinch K. Kilpatrick K.E. Stewart J.C. Kinch M.S. Cancer Res. 2001; 62: 2840-2847Google Scholar). Based on the differential behaviors of EphA2 in normal and transformed cells, our laboratory has been investigating the regulation of EphA2 phosphotyrosine content. Our recent studies have centered upon evidence that cancer cells often demonstrate decreased cell-cell contacts (17Birchmeier W. Bioessays. 1995; 17: 97-99Crossref PubMed Scopus (176) Google Scholar, 18Kinch M.S. Clark G.J. Der C.J. Burridge K. J. Cell Biol. 1995; 130: 461-471Crossref PubMed Scopus (280) Google Scholar, 19Volberg T. Geiger B. Dror R. Zick Y. Cell Regul. 1991; 2: 105-120Crossref PubMed Scopus (104) Google Scholar), which destabilize ligand binding (10Zelinski D.P. Zantek N.D. Stewart J.C. Irizarry A.R. Kinch M.S. Cancer Res. 2001; 61: 2301-2306PubMed Google Scholar, 12Zantek N.D. Azimi M. Fedor-Chaiken M. Wang B. Brackenbury R. Kinch M.S. Cell Growth & Differ. 1999; 10: 629-638PubMed Google Scholar). Our present studies investigate an additional possibility, which is that the phosphotyrosine content of EphA2 in transformed cells is negatively regulated by an associated phosphatase. We affirm this hypothesis and identify LMW-PTP1 as a phosphatase that regulates EphA2. Human LMW-PTP has been cloned, sequenced, expressed, and structurally characterized (20Wo Y.Y. Zhou M.M. Stevis P. Davis J.P. Zhang Z.Y. Van Etten R.L. Biochemistry. 1992; 31: 1712-1721Crossref PubMed Scopus (106) Google Scholar, 21Bryson G.L. Massa H. Trask B.J. Van E. RL. Genomics. 1995; 30: 133-140Crossref PubMed Scopus (36) Google Scholar, 22Zhang M. Stauffacher C.V. Lin D. Van Etten R.L. J. Biol. Chem. 1998; 273: 21714-21720Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). We also demonstrate that LMW-PTP is overexpressed in many transformed cell lines and that overexpression of LMW-PTP is sufficient to confer transformation upon non-transformed epithelial cells. Moreover, we demonstrate that the oncogenic activities of LMW-PTP require EphA2. Human breast (MCF-10Aneo, MCF-10AneoST, MCF-7, MDA-MB-231, MDA-MB-435, SK-BR-3) epithelial cells were cultured as described previously (23Paine T.M. Soule H.D. Pauley R.J. Dawson P.J. Int. J. Cancer. 1992; 50: 463-473Crossref PubMed Scopus (85) Google Scholar, 24Jacob A.N. Kalapurakal J. Davidson W.R. Kandpal G. Dunson N. Prashar Y. Kandpal R.P. Cancer Detect. Prev. 1999; 23: 325-332Crossref PubMed Scopus (43) Google Scholar, 25Shevrin D.H. Gorny K.I. Kukreja S.C. Prostate. 1989; 15: 187-194Crossref PubMed Scopus (48) Google Scholar). Monoclonal antibodies specific for phosphotyrosine (PY20) and β-catenin were purchased from Transduction Laboratories (Lexington, KY). Monoclonal antibodies specific for phosphotyrosine (4G10) and EphA2 (clone D7) were purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). Monoclonal antibodies against vinculin were purchased from NeoMarkers (Fremont, CA). Purified LMW-PTP was prepared as described (20Wo Y.Y. Zhou M.M. Stevis P. Davis J.P. Zhang Z.Y. Van Etten R.L. Biochemistry. 1992; 31: 1712-1721Crossref PubMed Scopus (106) Google Scholar). Cell lysates were harvested and normalized for equal loading as described previously (18Kinch M.S. Clark G.J. Der C.J. Burridge K. J. Cell Biol. 1995; 130: 461-471Crossref PubMed Scopus (280) Google Scholar). To confirm equal loading, blots were stripped as described previously and reprobed with antibodies specific to β-catenin or vinculin (18Kinch M.S. Clark G.J. Der C.J. Burridge K. J. Cell Biol. 1995; 130: 461-471Crossref PubMed Scopus (280) Google Scholar). Immunoprecipitation of EphA2 or LMW-PTP were performed using rabbit anti-mouse (Chemicon, Temecula, CA)-conjugated protein A-Sepharose (Sigma) as described previously (12Zantek N.D. Azimi M. Fedor-Chaiken M. Wang B. Brackenbury R. Kinch M.S. Cell Growth & Differ. 1999; 10: 629-638PubMed Google Scholar). To confirm equal loading, blots were stripped as described previously and reprobed with EphA2- or LMW-PTP-specific antibodies (18Kinch M.S. Clark G.J. Der C.J. Burridge K. J. Cell Biol. 1995; 130: 461-471Crossref PubMed Scopus (280) Google Scholar). Immunoblot analyses were performed on normalized cell lysates and immunoprecipitations as detailed (12Zantek N.D. Azimi M. Fedor-Chaiken M. Wang B. Brackenbury R. Kinch M.S. Cell Growth & Differ. 1999; 10: 629-638PubMed Google Scholar). Antibody binding was detected by enhanced chemiluminescence (ECL; Pierce) and visualized by autoradiography (Kodak X-OMAT; Eastman Kodak Co.). "Calcium switch" experiments were performed as described previously using MCF-10Aneo cells grown to 70% confluence in medium containing a final concentration of 4 mm EGTA (12Zantek N.D. Azimi M. Fedor-Chaiken M. Wang B. Brackenbury R. Kinch M.S. Cell Growth & Differ. 1999; 10: 629-638PubMed Google Scholar). Pervanadate was added to MDA-MB-231 in monolayer culture at a final concentration of 0, 1, 10, or 100 μm, and the treatment was allowed to incubate for 10 min at 37 °C, 5% CO2. For the combined EGTA-pervanadate treatment, MDA-MB-231 cells were first treated with 100 μm pervanadate and were then subjected to the EGTA treatment. To evaluate LMW-PTP activity against EphA2, EphA2 was immunoprecipitated from MCF-10Aneo cells and incubated with purified LMW-PTP protein at a concentration of 0.45, 7.8, or 62 μg/ml for 0, 5, 15, or 30 min. The assay was terminated through the addition of Laemmli sample buffer. The phosphotyrosine content of the EphA2 in the treatments was then observed using immunoblot analysis with antibodies specific to phosphotyrosine. To determine in vitro autophosphorylation activity, immunoprecipitated EphA2 was evaluated using in vitro kinase assays as detailed previously (12Zantek N.D. Azimi M. Fedor-Chaiken M. Wang B. Brackenbury R. Kinch M.S. Cell Growth & Differ. 1999; 10: 629-638PubMed Google Scholar). Monolayers of MCF-10Aneo cells were grown to 30–50% confluence and were transfected with pcDNA3.1-LMW-PTP or pcDNA3.1-LMW-PTPD129A using LipofectAMINE PLUS (Invitrogen). As a control for the transfection procedure, empty pcDNA3.1 vector was transfected into the same cell line in parallel. Transient transfections were allowed to grow for 48 h after transfection. For stable lines, neomycin-resistant cells were selected in growth medium containing 16 μg/ml neomycin (Mediatech, Inc., Herndon, VA). To confirm LMW-PTP overexpression, immunoblot analysis was performed using LMW-PTP-specific antibodies. Parental cells and cells transfected with empty pcDNA3.1 vector were used as negative controls. To evaluate cell growth using monolayer assays, 1 × 105 cells were seeded into tissue culture-treated multiwell dishes for 1, 2, 4, or 6 days in triplicate experiments. Cell numbers were evaluated by trypsin suspension of the samples followed by microscopic evaluation using a hemacytometer. Soft agar colony formation was performed and quantified as detailed (10Zelinski D.P. Zantek N.D. Stewart J.C. Irizarry A.R. Kinch M.S. Cancer Res. 2001; 61: 2301-2306PubMed Google Scholar,26Clark G.J. Kinch M.S. Gilmer T.M. Burridge K. Der C.J. Oncogene. 1996; 12: 169-176PubMed Google Scholar). For experiments with EphA2 antisense, cells were incubated with oligonucleotides prior to suspension in soft agar. The data shown are representative of at least three different experiments. Monolayers of MCF-10Aneo cells and MCF-10A cells stably overexpressing LMW-PTP were grown to 30% confluence and were transfected with EphA2 antisense oligonucleotides as detailed (13Carles-Kinch K. Kilpatrick K.E. Stewart J.C. Kinch M.S. Cancer Res. 2001; 62: 2840-2847Google Scholar). Samples that had been transfected with an inverted EphA2 antisense oligonucleotide or with the transfection reagent alone provided negative controls. Several independent lines of investigation suggested that EphA2 is regulated by an associated tyrosine phosphatase. EphA2 was rapidly dephosphorylated in non-transformed MCF-10Aneo epithelial cells upon disruption of ligand binding. Immunoblot analysis with phosphotyrosine antibodies (PY20 or 4G10) indicated lower levels of EphA2 phosphotyrosine content within 5 min following EGTA treatment, which destabilizes EphA2-ligand binding (12Zantek N.D. Azimi M. Fedor-Chaiken M. Wang B. Brackenbury R. Kinch M.S. Cell Growth & Differ. 1999; 10: 629-638PubMed Google Scholar) (Fig.1 A). Similarly, tyrosine phosphorylation of EphA2 decreased following incubation of non-transformed epithelial cells with dominant-negative inhibitors of EphA2-ligand binding (e.g. EphA2-Fc, not shown). Similar results were obtained using multiple non-transformed epithelial cell systems, including MCF-12A, MCF10-2, HEK293, Madin-Darby canine kidney, and Madin-Darby bovine kidney cells (not shown). Based on these findings, we asked whether tyrosine phosphatase inhibitors could prevent the loss of EphA2 phosphotyrosine content in response to EGTA treatment. Indeed, inhibitors such as sodium orthovanadate prevented the decrease in EphA2 phosphotyrosine following treatment of MCF-10Aneo cells with EGTA (Fig. 1 B). Previous studies by our laboratory have shown that the phosphotyrosine content of EphA2 is greatly reduced in transformed epithelial cells as compared with non-transformed epithelia (10Zelinski D.P. Zantek N.D. Stewart J.C. Irizarry A.R. Kinch M.S. Cancer Res. 2001; 61: 2301-2306PubMed Google Scholar, 12Zantek N.D. Azimi M. Fedor-Chaiken M. Wang B. Brackenbury R. Kinch M.S. Cell Growth & Differ. 1999; 10: 629-638PubMed Google Scholar). Thus, we asked whether tyrosine phosphatase activity could contribute to the reduced phosphotyrosine content of EphA2 in transformed cells. Although EphA2 was not tyrosine phosphorylated in transformed breast cancer cells (MDA-MB-231, MDA-435, MCFneoST, or PC-3 cells), incubation with increasing concentrations of sodium orthovanadate induced vigorous tyrosine phosphorylation of EphA2 (Fig. 1 C). As vanadate treatment of cells can often lead to exaggerated phosphorylation of physiologically irrelevant sites, we performed phosphopeptide mapping studies using EphA2 that had been labeled with [32P]ATP either in vitro or in vivo (not shown). These studies revealed identical patterns of tyrosine phosphorylation in non-transformed MCF-10Aneo cells and vanadate-treated MDA-MB-231 cells (not shown). Although the cytoplasmic domain contains multiple sites that could have been phosphorylated promiscuously, these were not phosphorylated under the conditions utilized here, suggesting that vanadate had not increased the phosphorylation of irrelevant sites. Altogether, these results indicate that EphA2 is regulated by an associated phosphatase that suppresses EphA2 phosphotyrosine content in transformed cells. To identify tyrosine phosphatases that might regulate EphA2 in transformed cells, we considered a recent report that LMW-PTP regulates a related molecule, EphB4 (27Stein E. Lane A.A. Cerretti D.P. Schoecklmann H.O. Schroff A.D. VanEtten R.L. Daniel T.O. Genes Dev. 1998; 12: 667-678Crossref PubMed Scopus (367) Google Scholar). Our initial experiments began by assessing the expression and function of LMW-PTP in non-transformed (MCF-10Aneo) and transformed (MCF-7, SK-BR-3, MDA-MB-435, MDA-MB-231) mammary epithelial cells (Fig. 2). Immunoblot analyses of whole cell lysates revealed relatively high levels of LMW-PTP in tumor-derived breast cancer cells as compared with non-transformed MCF-10Aneo mammary epithelial cells. The membranes were stripped and reprobed with antibodies against a control protein (vinculin), verifying that the high levels of LMW-PTP did not reflect a loading error or a generalized increase in protein levels in the transformed cells. A transformed variant of MCF-10Aneo, MCFneoST, also demonstrated elevated LMW-PTP expression, which was intriguing based on a recent report that EphA2 is not tyrosine phosphorylated in those cells (11Zantek N.D. Walker-Daniels J. Stewart J.C. Hansen R.K. Robinson D. Miao H. Wang B. Kung H.J. Bissell M.J. Kinch M.S. Clin. Cancer Res. 2001; 7: 3640-3648PubMed Google Scholar). The use of a genetically matched system also precluded potential differences due to cell origin or culture conditions. Thus, the highest levels of LMW-PTP were consistently found in transformed epithelial cells and were inversely related to EphA2 phosphotyrosine content. The results suggested that LMW-PTP negatively regulates the phosphotyrosine content of EphA2 in tumor cells. To explore this hypothesis further, we first asked whether the two molecules interactedin vivo. EphA2 was immunoprecipitated from MDA-MB-231 cells using specific antibodies (clone D7), and these complexes were resolved by SDS-PAGE. Subsequent immunoblot analyses revealed that LMW-PTP was prominently found within EphA2 immune complexes (Fig.3 A). The inverse experiment confirmed that EphA2 could similarly be detected in complexes of immunoprecipitated LMW-PTP (Fig. 3 B). Control immunoprecipitations with irrelevant antibodies confirmed the specificity of the interactions of the two molecules (not shown). The co-immunoprecipitation studies did not clarify whether EphA2 can serve as a substrate for LMW-PTP. To address this directly, EphA2 was immunoprecipitated from MCF-10Aneo cells, where it is normally tyrosine phosphorylated. The EphA2 immune complexes were then incubated with different concentrations of purified LMW-PTP before immunoblot analyses of EphA2 with phosphotyrosine-specific antibodies (PY20 and 4G10). These experiments demonstrated that purified LMW-PTP could dephosphorylate EphA2 in a dose- and time-dependent manner (Fig. 4). Although in vitro studies indicated that EphA2 could be dephosphorylated by LMW-PTP in vitro, we recognized thatin vitro studies are not always representative of the corresponding situation in vivo. Thus, LMW-PTP was ectopically overexpressed in MCF-10A cells. This particular cell system was selected because non-transformed MCF-10A cells have low levels of endogenous LMW-PTP and because the EphA2 in these non-transformed epithelial cells is normally tyrosine phosphorylated. Ectopic overexpression of LMW-PTP was achieved by stable transfection, as determined by immunoblot analyses with specific antibodies (Fig.5 A). Importantly, overexpression of LMW-PTP was sufficient to reduce the phosphotyrosine content of EphA2 as compared with vector-transfected negative controls (Fig. 5 B). Identical results were obtained using different experiments with different transfectants and in both stably and transiently transfected samples (not shown), thus eliminating potential concerns about clonal variation. Moreover, the decreased phosphotyrosine content was specific for EphA2 as the overall phosphotyrosine content of LMW-PTP-overexpressing cells was not decreased (Fig. 5 C). Tyrosine phosphorylated EphA2 negatively regulates tumor cell growth, whereas unphosphorylated EphA2 acts as a powerful oncoprotein (10Zelinski D.P. Zantek N.D. Stewart J.C. Irizarry A.R. Kinch M.S. Cancer Res. 2001; 61: 2301-2306PubMed Google Scholar, 12Zantek N.D. Azimi M. Fedor-Chaiken M. Wang B. Brackenbury R. Kinch M.S. Cell Growth & Differ. 1999; 10: 629-638PubMed Google Scholar, 13Carles-Kinch K. Kilpatrick K.E. Stewart J.C. Kinch M.S. Cancer Res. 2001; 62: 2840-2847Google Scholar). Thus, we asked whether overexpression of LMW-PTP would be sufficient to induce transformation. To address this question, we utilized the MCF-10A cells, described above, which had been transfected with either wild-type LMW-PTP or a vector control. Our initial studies evaluated the growth rates of control and LMW-PTP-overexpressing cells in monolayer culture. When evaluated using standard, two-dimensional culture conditions, the growth rates of LMW-PTP-overexpressing MCF-10A cells were significantly lower than the growth rates of matched controls (p < 0.05) (Fig.6 A). Two-dimensional assessments of growth often do not reflect the transformed character of tumor cells. Instead, three-dimensional analyses of cell behavior using soft agar and reconstituted basement membranes can provide a more relevant way of assessing transformed behavior. Whereas vector-transfected MCF-10Aneo cells were largely incapable of colonizing soft agar, LMW-PTP-overexpressing cells formed an average of 4.9 colonies per high powered microscope field (p < 0.01; Fig. 6 B). Based on recent findings with other three-dimensional assay systems, we also evaluated cell behavior using three-dimensional, reconstituted basement membranes. Consistent with a more aggressive phenotype, microscopic assessment of cell behavior in Matrigel confirmed the transformed character of LMW-PTP-overexpressing cells (not shown). When plated atop or within Matrigel, LMW-PTP-overexpressing cells formed larger colonies than vector-transfected cells (not shown). Altogether, consistent results with multiple and different systems suggest that overexpression of LMW-PTP is sufficient to induce transformation. Tyrosine phosphorylation of EphA2 induces its internalization and degradation (13Carles-Kinch K. Kilpatrick K.E. Stewart J.C. Kinch M.S. Cancer Res. 2001; 62: 2840-2847Google Scholar). Thus, we postulated that overexpression of LMW-PTP might increase the protein levels of EphA2. Indeed, immunoblot analyses of whole cell lysates revealed higher levels of EphA2 in MCF-10A cells that overexpress LMW-PTP as compared with vector-transfected controls (Fig. 5 A). Moreover, this EphA2 was not tyrosine phosphorylated (Fig. 5 B). However, immunoblot analyses revealed that the reduced phosphotyrosine content was selective for EphA2 as the general levels of phosphotyrosine were not altered in LMW-PTP-transformed cells (Fig. 5 C). The finding that overexpression of LMW-PTP increased EphA2 protein levels and decreased its phosphotyrosine content was intriguing since this phenotype was reminiscent of highly aggressive tumor cells (10Zelinski D.P. Zantek N.D. Stewart J.C. Irizarry A.R. Kinch M.S. Cancer Res. 2001; 61: 2301-2306PubMed Google Scholar,12Zantek N.D. Azimi M. Fedor-Chaiken M. Wang B. Brackenbury R. Kinch M.S. Cell Growth & Differ. 1999; 10: 629-638PubMed Google Scholar). Thus, we asked whether selective targeting of LMW-PTP in transformed cells would impact EphA2. To accomplish this, the catalytically inactive (28Zhang Z. Harms E. Van Etten R.L. J. Biol. Chem. 1994; 269: 25947-25950Abstract Full Text PDF PubMed Google Scholar) mutant LMW-PTPD129A was overexpressed in MDA-MB-231 cells, which have high levels of wild-type LMW-PTP (Fig. 2) and which also have elevated levels of EphA2 that is hypo-phosphorylated. Ectopic overexpression of LMW-PTPD129Awas found to decrease the levels of EphA2. Moreover, immunoblot analyses of immunoprecipitated material demonstrated that EphA2 was tyrosine phosphorylated (Fig. 5 D). These results indicate that overexpression of wild-type LMW-PTP is necessary and sufficient to confer the overexpression and functional alterations of EphA2 that have been observed in tumor-derived cells. Although the EphA2 in the LMW-PTP-overexpressing MCF-10A cells was not tyrosine phosphorylated, it retained enzymatic activity. In vitro kinase assays verified that the EphA2 from LMW-PTP-transformed MCF-10A cells had levels of enzymatic activity that were comparable with vector-transfected controls (Fig.7 A). To verify equal sample loading, two controls were performed. Equal amounts of input lysate were verified by immunoblot analyses with β-catenin antibodies (not shown). In addition, the immunoprecipitated EphA2 was divided, and half of the material was resolved by SDS-PAGE and analyzed by immunoblot analyses with EphA2 and phosphotyrosine-specific antibodies (Fig. 7, B and C). Thus phosphorylated and unphosphorylated EphA2 were both capable of enzymatic activity. Having determined that the levels of EphA2 were elevated in LMW-PTP-transformed cells, we asked to what extent the oncogenic activity of EphA2 contributed to this phenotype. To address this, we utilized our experience with antisense strategies to selectively decrease EphA2 expression in LMW-PTP-transformed cells (13Carles-Kinch K. Kilpatrick K.E. Stewart J.C. Kinch M.S. Cancer Res. 2001; 62: 2840-2847Google Scholar, 29Hess A.R. Seftor E.A. Gardner L.M. Carles-Kinch K. Schneider G.B. Seftor R.E. Kinch M.S. Hendrix M.J.C. Cancer Res. 2001; 61: 3250-3255PubMed Google Scholar). We verified the success of these strategies by immunoblot analyses (Fig.8 A) and then asked whether decreased EphA2 expression would alter soft agar colonization. Indeed, transfection with EphA2 antisense oligonucleotides decreased the soft agar colonization of LMW-PTP-transformed MCF-10A cells by at least 87% (p < 0.01; Fig. 8 B). As a control, transfection of these cells with an inverted antisense nucleotide did not significantly alter soft agar colonization. Thus, we were able to exclude that the results with the antisense oligonucleotides had resulted from nonspecific toxicities caused by the transfection procedure. Altogether, our results indicate that the oncogenic actions of overexpressed LMW-PTP require high levels of EphA2. The major finding of our present study is that EphA2 is regulated by an associated tyrosine phosphatase, and we identify LMW-PTP as a critical regulator of EphA2 tyrosine phosphorylation. We also demonstrate that LMW-PTP is overexpressed in metastatic cancer cells and that LMW-PTP overexpression is sufficient to confer transformation upon non-transformed epithelial cells. Finally, we demonstrate that LMW-PTP up-regulates the expression of EphA2 and that the oncogenic activities of LMW-PTP require this overexpression of EphA2. Recent reports from our laboratory and others have shown that many transformed epithelial cells express high levels of EphA2 that is not tyrosine phosphorylated (10Zelinski D.P. Zantek N.D. Stewart J.C. Irizarry A.R. Kinch M.S. Cancer Res. 2001; 61: 2301-2306PubMed Google Scholar, 12Zantek N.D. Azimi M. Fedor-Chaiken M. Wang B. Brackenbury R. Kinch M.S. Cell Growth & Differ. 1999; 10: 629-638PubMed Google Scholar). Previously, we had related these depressed levels of EphA2 tyrosine phosphorylation with decreased ligand binding. Transformed cells often have unstable cell-cell contacts, and we postulated that this decreases the ability of EphA2 to stably interact with its ligands, which are anchored to the membrane of adjacent cells (12Zantek N.D. Azimi M. Fedor-Chaiken M. Wang B. Brackenbury R. Kinch M.S. Cell Growth & Differ. 1999; 10: 629-638PubMed Google Scholar). In part, our present data suggest a new paradigm in which the phosphotyrosine content of EphA2 is also negatively regulated by an associated tyrosine phosphatase that is overexpressed in transformed cells. Given the relationship between EphA2 phosphorylation and cell-cell adhesion, we cannot exclude that cell-cell contacts could also regulate the expression or function of LMW-PTP, and future investigation should address this possibility. The fact that high levels of LMW-PTP were observed in several different cell models of metastatic cancer is notable given that LMW-PTP overexpression is sufficient to confer transformation. LMW-PTP-overexpressing cells gain the ability to colonize soft agar and acquire a transformed phenotype when cultured in three-dimensional basement membranes, such as Matrigel. Notably, however, LMW-PTP-overexpressing MCF-10A epithelial cells displayed reduced rates of cell growth as measured using two-dimensional assays of cell growth. This latter observation is consistent with recent reports that high levels of LMW-PTP similarly decrease the monolayer growth rates of other cell types (30Shimizu H. Shiota M. Yamada N. Miyazaki K. Ishida N. Kim S. Miyazaki H. Biochem. Biophys. Res. Commun. 2001; 289: 602-607Crossref PubMed Scopus (28) Google Scholar, 31Fiaschi T. Chiarugi P. Buricchi F. Giannoni E. Taddei M.L. Talini D. Cozzi G. Zecchi-Orlandini S. Raugei G. Ramponi G. J. Biol. Chem. 2001; 276: 49156-49163Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). Although such a finding had been interpreted to suggest that LMW-PTP might negatively regulate transformation, our findings support a very different conclusion. Consistent with this, recent studies by our laboratory and others have shown that transformation of MCF-10A cells is often accompanied by decreased monolayer growth rates and that the most aggressive variants of MCF-10A in vivo demonstrate the slowest growth in monolayer culture (10Zelinski D.P. Zantek N.D. Stewart J.C. Irizarry A.R. Kinch M.S. Cancer Res. 2001; 61: 2301-2306PubMed Google Scholar, 11Zantek N.D. Walker-Daniels J. Stewart J.C. Hansen R.K. Robinson D. Miao H. Wang B. Kung H.J. Bissell M.J. Kinch M.S. Clin. Cancer Res. 2001; 7: 3640-3648PubMed Google Scholar, 32Sun X.G. Rotenberg S.A. Cell Growth & Differ. 1999; 10: 343-352PubMed Google Scholar). These findings have implications for the design and interpretation of tests of oncogene function when using non-transformed epithelial cell systems. The biochemical consequences of EphA2 tyrosine phosphorylation remain largely unclear. Unlike other receptor tyrosine kinases, for which autophosphorylation is necessary for enzymatic activity, tyrosine phosphorylation of EphA2 is not required for its enzymatic activity. Consistent with our present results, EphA2 retains comparable levels of enzymatic activity in non-transformed and tumor-derived cells, despite dramatic differences in its phosphotyrosine content (12Zantek N.D. Azimi M. Fedor-Chaiken M. Wang B. Brackenbury R. Kinch M.S. Cell Growth & Differ. 1999; 10: 629-638PubMed Google Scholar). Similarly, antibody-mediated stimulation of EphA2 autophosphorylation does not change the levels of EphA2 enzymatic activity (13Carles-Kinch K. Kilpatrick K.E. Stewart J.C. Kinch M.S. Cancer Res. 2001; 62: 2840-2847Google Scholar). Phosphopeptide analyses of the EphA2 cytoplasmic domain provide one potential explanation. Although EphA2 has a predicted activation loop tyrosine at residue 772 (33Lindberg R.A. Hunter T. Mol. Cell. Biol. 1990; 10: 6316-6324Crossref PubMed Scopus (221) Google Scholar), neither in vitro nor in vivophosphopeptide analyses have identified phosphorylation of this site in normal or malignant cells. 2M. S. Kinch, unpublished information. Thus, the lack of a consensus activation loop tyrosine may account for the retention of EphA2 enzymatic activity in cells where it is not tyrosine phosphorylated. Tyrosine phosphorylation of EphA2 does not appear to be necessary for its intrinsic enzymatic activity. Instead, ligand-mediated tyrosine phosphorylation regulates EphA2 protein stability (13Carles-Kinch K. Kilpatrick K.E. Stewart J.C. Kinch M.S. Cancer Res. 2001; 62: 2840-2847Google Scholar). Specifically, tyrosine phosphorylation fates EphA2 to interact with the c-Cbl adapter protein and to subsequently be internalized and degraded within proteosomes. 3Walker-Daniels, J., Riese, D. J., and Kinch, M. S. (2002) Cell Growth & Differ., in press. Consequently, the phosphatase activity of LMW-PTP would be predicted to increase EphA2 protein stability. Indeed, the highest levels of EphA2 are consistently found in cells with high levels of LMW-PTP. One interesting implication of this finding is that it provides a mechanism, independent of genetic regulation of the EphA2 gene, to explain why high levels of EphA2 are found in many different tumors. An alternative possibility is that LMW-PTP up-regulates EphA2 gene expression, and our present findings do not formally eliminate this possibility. The fact that EphA2 antisense oligonucleotides reversed the transformed character of LMW-PTP-overexpressing cells suggests that the up-regulation of EphA2 is relevant to the cellular behaviors of LMW-PTP-mediated transformation. In summary, our present studies identify LMW-PTP as a new oncogene that is overexpressed in transformed cells. We also link the biochemical and biological actions of overexpressed LMW-PTP with EphA2. These findings have important implications for understanding the biochemical and biological mechanisms that contribute to the metastatic progression of epithelial cells. Moreover, our present studies identify an important signaling system that could ultimately provide an opportunity to target the large number of cancer cells that overexpress EphA2 or LMW-PTP. We thank Dr. Al Schroff and the Purdue Cancer Center for producing the monoclonal antibody against LMW-PTP. We also thank the current members of the Kinch and Van Etten laboratories for critical reading of the manuscript.
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