CD98hc (SLC3A2) Interaction with β1 Integrins Is Required for Transformation
2004; Elsevier BV; Volume: 279; Issue: 52 Linguagem: Inglês
10.1074/jbc.m408700200
ISSN1083-351X
AutoresNeil C. Henderson, Elizabeth A. Collis, Alison C. MacKinnon, Kenneth J. Simpson, Christopher Haslett, Roy Zent, Mark H. Ginsberg, Tariq Sethi,
Tópico(s)Cellular transport and secretion
ResumoCD98hc (SLC3A2) constitutively and specifically associates with β1 integrins and is highly expressed on the surface of human tumor cells irrespective of the tissue of origin. We have found here that expression of CD98hc promotes both anchorage- and serum-independent growth. This oncogenic activity is dependent on β1 integrin-mediated phosphoinositol 3-hydroxykinase stimulation and the level of surface expression of CD98hc. Using chimeras of CD98hc and the type II membrane protein CD69, we show that the transmembrane domain of CD98hc is necessary and sufficient for integrin association in cells. Furthermore, CD98hc/β1 integrin association is required for focal adhesion kinase-dependent phosphoinositol 3-hydroxykinase activation and cellular transformation. Amino acids 82–87 in the putative cytoplasmic/transmembrane region appear to be critical for the oncogenic potential of CD98hc and provide a novel mechanism for tumor promotion by integrins. These results explain how high expression of CD98hc in human cancers contributes to transformation; furthermore, the transmembrane association of CD98hc and β1 integrins may provide a new target for cancer therapy. CD98hc (SLC3A2) constitutively and specifically associates with β1 integrins and is highly expressed on the surface of human tumor cells irrespective of the tissue of origin. We have found here that expression of CD98hc promotes both anchorage- and serum-independent growth. This oncogenic activity is dependent on β1 integrin-mediated phosphoinositol 3-hydroxykinase stimulation and the level of surface expression of CD98hc. Using chimeras of CD98hc and the type II membrane protein CD69, we show that the transmembrane domain of CD98hc is necessary and sufficient for integrin association in cells. Furthermore, CD98hc/β1 integrin association is required for focal adhesion kinase-dependent phosphoinositol 3-hydroxykinase activation and cellular transformation. Amino acids 82–87 in the putative cytoplasmic/transmembrane region appear to be critical for the oncogenic potential of CD98hc and provide a novel mechanism for tumor promotion by integrins. These results explain how high expression of CD98hc in human cancers contributes to transformation; furthermore, the transmembrane association of CD98hc and β1 integrins may provide a new target for cancer therapy. The CD98 family is composed of widely expressed cell-surface disulfide-linked 125-kDa heterodimeric membrane glycoproteins containing a common glycosylated 80-kDa heavy chain (CD98hc, 4F2hc, SLC3A2) and a group of ∼45-kDa light chains. Early studies of peripheral blood T lymphocytes implicated CD98hc in the regulation of cellular activation (1Haynes B.F. Hemler M.E. Mann D. Eisenbarth G. Shelhamer J. Mostowski H.S. Thomas C.A. Strominger J.L. Fauci A. J. Immunol. 1981; 126: 1409-1414PubMed Google Scholar). Although expressed at low levels on the surface of quiescent cells, CD98hc is rapidly up-regulated early in transition from G0 to G1 phase following cellular activation and remains at elevated levels until the cell cycle is complete (2Azzarone B. Malpiece Y. Zaech P. Moretta L. Fauci A. Suarez H. Exp. Cell Res. 1985; 159: 451-462Crossref PubMed Scopus (19) Google Scholar, 3Suomalainen H.A. J. Immunol. 1986; 137: 422-427PubMed Google Scholar, 4Parmacek M.S. Karpinski B.A. Gottesdiener K.M. Thompson C.B. Leiden J.M. Nucleic Acids Res. 1989; 17: 1915-1931Crossref PubMed Scopus (86) Google Scholar). All embryonic fibroblasts express CD98hc, and expression gradually diminishes on cells with maturity. CD98hc is highly expressed on the surface of tumor cells, irrespective of the tissue of origin (5Bellone G. Alloatti G. Levi R. Geuna M. Tetta C. Peruzzi L. Letarte M. Malavasi F. Eur. J. Immunol. 1989; 19: 1-8Crossref PubMed Scopus (26) Google Scholar, 6Dixon W.T. Sikora L.K. Demetrick D.J. Jerry L. M Int. J. Cancer. 1990; 45: 59-68Crossref PubMed Scopus (27) Google Scholar). Deletion of CD98hc in embryonic stem cells blocks their ability to form teratomas in mice, 1C. Feral and M. H. Ginsberg, submitted for publication. and overexpression of CD98hc in murine fibroblasts results in anchorage-independent growth (7Hara K. Kudoh H. Enomoto T. Hashimoto Y. Masuko T. Biochem. Biophys. Res. Commun. 1999; 262: 720-725Crossref PubMed Scopus (65) Google Scholar). In addition, increased CD98hc expression correlates with the development, progression, and metastatic potential of tumors (8Esteban F. Ruiz-Cabello F. Concha A. Perez Ayala M. Delgado M. Garrido F. Cancer. 1990; 66: 1493-1498Crossref PubMed Scopus (32) Google Scholar, 9Garber M.E. Troyanskaya O.G. Schluens K. Petersen S. Thaesler Z. Pacyna-Gengelbach M. van de Rijn M. Rosen G.D. Perou C.M. Whyte R.I. Altman R.B. Brown P.O. Botstein D. Petersen I. Proc. Natl. Acad. Sci. U. S. A. 2001; 98 (Correction (2002) Proc. Natl. Acad. Sci. U. S. A.99, 1098): 13784-13789Crossref PubMed Scopus (1103) Google Scholar, 10Yoon J.H. Kim Y.B. Kanai Y. Endou H. Kim K. Anticancer Res. 2003; 23: 3877-3881PubMed Google Scholar). Thus, CD98hc plays an important role in tumorigenesis; however, its mechanism of action has not been determined. The integrin family of cell-surface heterodimeric glycoproteins composed of α and β subunits function primarily as receptors for extracellular matrix ligands, which regulate many aspects of cell physiology, including morphology, adhesion, migration, proliferation, and differentiation (11Schwartz M.A. J. Cell Biol. 1997; 139: 575-578Crossref PubMed Scopus (305) Google Scholar). Many cancers show abnormalities of integrin function as a result of transformation by oncogenes (12Zou J.X. Liu Y. Pasquale E.B. Ruoslahti E. J. Biol. Chem. 2002; 277: 1824-1827Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). More importantly, the growth of several tumors depends on β1 integrin function (13Weaver V.M. Petersen O.W. Wang F. Larabell C.A. Briand P. Damsky C. amd Bissell M.J. J. Cell Biol. 1997; 137: 231-245Crossref PubMed Scopus (1205) Google Scholar). CD98hc constitutively and specifically associates with β1 integrins (14Fenczik C.A. Sethi T. Ramos J.W. Hughes P.E. Ginsberg M.H. Nature. 1997; 390: 81-85Crossref PubMed Scopus (260) Google Scholar, 15Zent R. Fenczik C.A. Calderwood D.A. Liu S. Dellos M. Ginsberg M.H. J. Biol. Chem. 2000; 275: 5059-5064Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar, 16Merlin D. Sitaraman S. Liu X. Eastburn K. Sun J. Kucharzik T. Lewis B. Madara J.L. J. Biol. Chem. 2001; 276: 39282-39289Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 17Miyamoto Y.J. Mitchell J.S. McIntyre B.W. Mol. Immunol. 2003; 390: 739-751Crossref Scopus (26) Google Scholar, 18Rintoul R.C. Buttery R.C. Mackinnon A.C. Wong W.S. Mosher D. Haslett C. Sethi T. Mol. Biol. Cell. 2002; 13: 2841-2852Crossref PubMed Scopus (79) Google Scholar), and accumulating evidence indicates that CD98hc plays a significant role in regulating integrin-mediated functions in cancer cells. Cross-linking CD98hc promotes activation of phosphatidylinositol 3-hydroxykinase (PI3K) 2The abbreviations used are: PI3K, phosphatidylinositol 3-hydroxykinase; CHO, Chinese hamster ovary; FAK, focal adhesion kinase; mAb, monoclonal antibody; DMEM, Dulbecco's modified Eagle's medium; FCS, fetal calf serum; FACS, fluorescence-activated cell sorting; PBS, phosphate-buffered saline; PIP3, phosphatidylinositol 3,4,5-trisphosphate; IP4, inositol 1,3,4,5-tetrakisphosphate; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; ANOVA, one-way analysis of variance; FRNK, focal adhesion kinase related non-kinase. (18Rintoul R.C. Buttery R.C. Mackinnon A.C. Wong W.S. Mosher D. Haslett C. Sethi T. Mol. Biol. Cell. 2002; 13: 2841-2852Crossref PubMed Scopus (79) Google Scholar) and Rap1 (19Suga K. Katagiri K. Kinashi T. Harazaki M. Iizuka T. Hattori M. Minato N. FEBS Lett. 2001; 489: 249-253Crossref PubMed Scopus (60) Google Scholar) and enhances β1 integrin-mediated cell adhesion in a number of cancer cells, including breast and small cell lung cancer (14Fenczik C.A. Sethi T. Ramos J.W. Hughes P.E. Ginsberg M.H. Nature. 1997; 390: 81-85Crossref PubMed Scopus (260) Google Scholar, 20Chandrasekaran S. Guo N.H. Rodrigues R.G. Kaiser J. Roberts D.D. J. Biol. Chem. 1999; 274: 11408-11416Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar), and clustering of α3β1 integrin on the surface of rhabdomyosarcoma cells (21Kolesnikova T.V. Mannion B.A. Berditchevski F. Hemler M.E. BMC Biochem. 2001; 2: 10-19Crossref PubMed Scopus (50) Google Scholar). The mechanism by which CD98hc associates with and regulates integrin function and what role this plays in transformation are unclear. The extracellular domain of CD98hc combines with at least six different light chains to form a series of disulfide-bonded heterodimers that are involved in l-amino acid transport. The role of the light chain in CD98hc interaction with or regulation of function of β1 integrins is controversial. Mutations of cysteine residues in CD98hc that disrupt covalent association with the light chain and that reduce amino acid transport also eliminate the transforming activity of CD98hc in BALB/3T3 cells (22Shishido T. Uno S. Kamohara M. Tsuneoka-Suzuki T. Hashimoto Y. Enomoto T. Masuko T. Int. J. Cancer. 2000; 87: 311-316Crossref PubMed Scopus (42) Google Scholar) and cause loss of β1 integrin association in low density light chain membrane fractions (21Kolesnikova T.V. Mannion B.A. Berditchevski F. Hemler M.E. BMC Biochem. 2001; 2: 10-19Crossref PubMed Scopus (50) Google Scholar). However, these mutants still bind to free β1A cytoplasmic tails (Tac-β1) in vitro and reverse Tac-β1-induced dominant integrin suppression in Chinese hamster ovary (CHO) cells (15Zent R. Fenczik C.A. Calderwood D.A. Liu S. Dellos M. Ginsberg M.H. J. Biol. Chem. 2000; 275: 5059-5064Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). However, titration of CD98hc by β1 tails is not the mechanism of Tac-β1 dominant suppression (15Zent R. Fenczik C.A. Calderwood D.A. Liu S. Dellos M. Ginsberg M.H. J. Biol. Chem. 2000; 275: 5059-5064Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). In contrast, other evidence suggests that the cytoplasmic/transmembrane domain of CD98hc is the critical region mediating CD98hc alteration of β1 integrin surface distribution and cytoskeletal architecture in Madin-Darby canine kidney cells and reversal of Tac-β1 dominant suppression in CHO cells (15Zent R. Fenczik C.A. Calderwood D.A. Liu S. Dellos M. Ginsberg M.H. J. Biol. Chem. 2000; 275: 5059-5064Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar, 16Merlin D. Sitaraman S. Liu X. Eastburn K. Sun J. Kucharzik T. Lewis B. Madara J.L. J. Biol. Chem. 2001; 276: 39282-39289Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). We have shown previously that cross-linking CD98hc stimulates PI3K activity in a β1 integrin-dependent manner (18Rintoul R.C. Buttery R.C. Mackinnon A.C. Wong W.S. Mosher D. Haslett C. Sethi T. Mol. Biol. Cell. 2002; 13: 2841-2852Crossref PubMed Scopus (79) Google Scholar). The aim of this study was to investigate the mechanism by which overexpression of CD98hc leads to cellular transformation, in particular assessing the relationship between transformation, PI3K activation, and β1 integrins. We found that cellular transformation by overexpression of CD98hc depends on activation of PI3K mediated by focal adhesion kinase (FAK). This PI3K activation depends on the interaction of β1 integrins with CD98hc and is associated with redistribution of the integrins. Finally, we found that the CD98hc transmembrane domain is necessary and sufficient for integrin association and PI3K activation and transformation by CD98hc. This protein plays an important role in the formation of certain tumors; this study defines the CD98hc interactions and resulting signaling events that lead to transformation. DNA Constructs—Human full-length CD69 was kindly provided by Dr. F Sanchez-Madrid (Universidad Autonoma de Madrid, Madrid, Spain). The CD98hc chimeras were made by overlap PCR or restriction digestion and religation. C98T69E98 contains amino acids 1–81 of CD98hc (Swiss-Prot accession number P08195), amino acids 121–183 of CD69 (Swiss-Prot accession number Q07108), and amino acids 105–529 of CD98hc. C69T98E98 contains amino acids 1–40 of CD69 and amino acids 82–529 of CD98hc. C98T98E69 contains amino acids 1–104 of CD98hc and amino acids 62–199 of CD69. C98T69E69 contains amino acids 1–81 of CD98hc and amino acids 41–199 of CD69. C69T98E69 contains amino acids 1–40 of CD69, amino acids 1–40 82–104 of CD98hc, and amino acids 1–40 62–199 of CD69. C69T69E98 contains amino acids 1–61 of CD69 and amino acids 105–529 of CD98hc. CD98hc(Δ2–77) has a deletion of amino acids 2–77, which removes the entire cytoplasmic domain of CD98hc, maintaining the initiator methionine as well as the presumptive stop transfer sequence Val-Arg-Thr-Arg. CD98hc(Δ1–86) (also previously termed D5; kindly provided by Drs. D. Merlin and J. L. Madara) (16Merlin D. Sitaraman S. Liu X. Eastburn K. Sun J. Kucharzik T. Lewis B. Madara J.L. J. Biol. Chem. 2001; 276: 39282-39289Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar) has a deletion of amino acids 1–86, which removes the entire cytoplasmic domain and the five proximal amino acids of the predicted transmembrane domain. The above cDNAs were subcloned into pcDNA3.1 (Invitrogen), which confers neomycin resistance. The subcloned plasmids were verified by sequencing. Plasmids were purified using the QIAGEN maxi plasmid kit. cDNA encoding human LAT1, a CD98 light chain, was a kind gift from Dr. F. Verrey (University of Zurich, Zurich, Switzerland). Antibodies—For Western blotting, the following antibodies were used: anti-protein kinase B and anti-phospho-Ser473 protein kinase B antibodies (New England Biolabs Inc., Beverly, MA), anti-FAK and anti-phospho-Tyr397 FAK antibodies (BIOSOURCE), anti-CD98 antibody (sc-7095, Santa Cruz Biotechnology), and anti-β1 integrin monoclonal antibody (mAb) (141720, Transduction Laboratories). For flow cytometry, protein A-purified 4F2 was used for identification of CD98hc and CD98hc chimeras containing the extracellular domain of CD98hc. Anti-human CD69 antibody (clone FN50, Dako Corp.) was used for identification of CD69 and chimeras containing the extracellular domain of CD69. For β1 integrin, rat clone 9EG7 was used (Pharmingen). For immunoprecipitation, anti-human β1 integrin antibody K20 (Dako Corp.) was used. Species-specific horseradish peroxidase-labeled IgG (Dako Corp.) was used for Western blotting, fluorescein isothiocyanate-labeled secondary antibodies (Dako Corp.) for flow cytometry, and Alexa Fluor 568 and Alexa Fluor 488 (Molecular Probes, Inc.) for confocal microscopy. Cell Culture and Transfection—CHO-K1 cells were obtained from the European Collection of Animal Cell Cultures and were grown in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal calf serum (FCS), 1% nonessential amino acids, 5 μg/ml glutamine, 50 units/ml penicillin, and 50 μg/ml streptomycin. The cell lines GD25β1 null, GD25β1A, and GD25β1A(Y783F/Y795F) have been described previously (23Fassler R. Pfaff M. Murphy J. Noegel A.A. Johansson S. Timpl R. Albrecht R. J. Cell Biol. 1995; 128: 979-988Crossref PubMed Scopus (215) Google Scholar, 24Sakai T. Zhang Q. Fassler R. Mosher D. J. Cell Biol. 1998; 141: 527-538Crossref PubMed Scopus (96) Google Scholar). GD25β1 null cells are fibroblasts derived from β1 null embryonic stem cells. The GD25β1A and GD25β1A(Y783F/Y795F) mutant cell lines were derived from GD25 cells upon stable transfection with cDNAs encoding the wild-type and mutant murine β1A integrin subunits, respectively (25Wennerberg K. Armulik A. Sakai T. Karlsson M. Fassler R. Schaefer E.M. Mosher D.F. Johansson S. Mol. Cell. Biol. 2000; 20: 5758-5765Crossref PubMed Scopus (79) Google Scholar). GD25β1 null cells were grown in DMEM containing 10% FCS, 5 μg/ml glutamine, 50 units/ml penicillin, and 50 μg/ml streptomycin; GD25β1A and GD25β1A(Y783F/Y795F) cells were grown in the same medium containing 10 μg/ml puromycin for selection. Transient transfection of cell lines with chimeric constructs was undertaken using Lipofectamine Plus (Invitrogen) following the manufacturer's instructions. Under optimal conditions, a transfection efficiency of at least 60% was achieved in each cell line. Control cells were transfected with control vector pcDNA 3.1. The hybridoma cell line 4F2 (C13) was purchased from American Type Culture Collection and cultured in DMEM containing 15% FCS, 50 units/ml penicillin, 50 μg/ml streptomycin, 2 mm l-glutamine, and OPI media supplement (Sigma). Secreted antibody was purified by protein G affinity chromatography. Construction of Stable Cell Lines—Subconfluent CHO-K1 cells were transfected using Lipofectamine following the manufacturer's instructions in serum-free medium for 5 h. Serum was added for the subsequent 48 h, and transfectants were selected in medium with 1.2 mg/ml G418 (Sigma). Clones selected from each construct were maintained in 0.8 mg/ml G418 and expanded. Clones showing equivalent wild-type, chimeric, or truncated human CD98hc expression by fluorescence-activated cell sorting (FACS) analysis were selected for this study. Flow Cytometry—Aliquots of 5 × 105 cells were washed and resuspended in 100 μl of phosphate-buffered saline (PBS) containing 1 μg of 4F2 (for CD98hc and chimeras containing the extracellular portion of CD98hc) or 1 μg of anti-CD69 antibody (for CD69 and chimeras containing the extracellular portion of CD69). Cells were incubated for 30 min at room temperature, followed by two washes with PBS. Samples were then incubated with species-specific fluorescein isothiocyanate-conjugated secondary antibody (1:50) for 30 min at 4 °C and again washed twice with PBS. Samples were finally resuspended in PBS and analyzed by flow cytometry using FACSCalibur™ (BD Biosciences). Control IgG2a and IgG1 antibodies for 4F2 and CD69, respectively, were also used. Clonogenic Assay—Cells (2 × 104/ml) were suspended in 0.3% (w/v) agarose in DMEM containing 1% FCS unless indicated otherwise. The cells were layered over a solid base of 0.5% (w/v) agarose in DMEM in 6-well culture dishes. Cultures were incubated in a humidified atmosphere of 5% CO2 and 95% air at 37 °C. After 6 days, colonies greater than four cells were counted under a light microscope. Cloning efficiency was calculated as a percentage of the initial number of seeded cells that formed colonies. PI3K Activity Assay—PI3K activity was measured as described previously (26Moore S.M. Rintoul R.C. Walker T.R. Chilvers E.R. Haslett C. Sethi T. Cancer Res. 1998; 58: 5239-5247PubMed Google Scholar). Briefly, cells were lysed in ice-cold buffer containing 50 mm HEPES (pH 7.4), 150 mm NaCl, 1.5 mm MgCl2, 1 mm EGTA, 10 mm sodium pyrophosphate, 100 mm sodium fluoride, 10% (v/v) glycerol, 1% (v/v) Triton X-100, 0.5 mm dithiothreitol, 1 mm sodium orthovanadate, and protease inhibitor mixture (Roche Applied Science). PI3K was immunoprecipitated from protein-equilibrated cell lysates using anti-PI3K p85α mAb (Upstate Biotechnology Inc., Lake Placid, NY) and assayed for activity using [γ-32P]ATP and phosphatidylinositol/phosphatidylserine as substrate. 3-Phosphorylated lipids were resolved by thin layer chromatography, identified by autoradiography, and quantified by liquid scintillation counting. Radioligand Displacement Assay for Mass Measurement of Phosphatidylinositol 3,4,5-Trisphosphate (PIP3)—PIP3 levels were measured as described previously (27Van der Kaay J. Cullen P.J. Downes C.P. Bird I.M. Phospholipid Signaling Protocols. Humana Press, Totowa, NJ1999: 109-125Google Scholar). In brief, CHO-K1 cells (5 × 106) were subjected to a standard Folch extraction, and lipid extracts containing PIP3 were then subjected to alkaline hydrolysis, resulting in the release of the polar head group inositol 1,3,4,5-tetrakisphosphate (IP4). The mass of IP4 was measured by [3H]IP4 (Amersham Biosciences) displacement from a recombinant IP4-glutathione S-transferase-binding protein using a calibration curve obtained with unlabeled IP4 standards. Immunoprecipitation and Western Blotting—Confluent cultures from 100-mm plates were quiesced overnight in 0.1% FCS and washed with PBS. Cells were lysed at 4 °C in lysis buffer containing 20 mm HEPES (pH 7.4), 1% CHAPS, 150 mm NaCl, 2 mm MgCl2, 1 mm MnCl2, 0.5 mm CaCl2, and EDTA-free protease inhibitor mixture (Roche Applied Science) and clarified by centrifugation for 10 min at 4 °C. Samples (20 μg of protein) were retained and solubilized in NuPAGE sample buffer (Invitrogen) for analysis of whole cell lysate by Western blotting. The remaining lysate was incubated overnight with 2 μg of immunoprecipitating antibody at 4 °C. Immune complexes were captured using 15 μl of protein G-agarose and washed three times with lysis buffer. Following elution with NuPAGE buffer, associated proteins were resolved on 4–12% gradient gels. The proteins were transferred to nitrocellulose membranes; blocked using 3% (w/v) albumin in 20 mm Tris-HCl (pH 7.4), 150 mm NaCl, and 0.02% (v/v) Tween 20 for 1 h at room temperature; and then incubated with primary antibody overnight at 4 °C. Species-specific horseradish peroxidase-conjugated antibodies were used for secondary labeling. Immunoreactive bands were identified by enhanced chemiluminescence (Amersham Biosciences) according to the manufacturer's instructions. Amino Acid Transport Assay—Cells (5 × 106) were washed twice and resuspended in amino acid-free and Na+-free uptake solution containing 100 mm choline chloride, 2 mm KCl, 1 mm MgCl2, 1 mm CaCl2, and 10 mm HEPES (pH 7.5). After equilibration at 37 °C for 30 min, 2 μCi of l-[4,5-3H]leucine (82 Ci/mmol) containing 2 mm unlabeled l-leucine was added to each tube, and incubation was continued for an additional 30 min at 37 °C. Cells were then placed on ice; pelleted; and washed three times with 1 ml of ice-cold wash buffer containing 80 μm choline chloride, 2 mm KCl, 1 mm MgCl2, 1 mm CaCl2, and 10 mm HEPES (pH 7.5). The washed cells were then digested with 200 μl of 0.2% SDS in 0.2 m NaOH for 1 h. Protein-equilibrated aliquots of 100 μl were added to scintillation fluid containing 100 μl of 0.2 m HCl, and activity was counted in a scintillation counter. Confocal Immunofluorescence—Cells were plated onto glass coverslips, fixed with 3% paraformaldehyde, and quenched in 50 mm NH4Cl. Nonspecific binding sites were blocked using 0.2% fish skin gelatin in PBS. Cells were then incubated sequentially with (i) 4F2 or fluorescein isothiocyanate-conjugated FN50, 9EG7, anti-phospho-FAK antibody, or IgG1 and IgG2A negative control antibodies and (ii) secondary Alexa Fluor antibodies. To assess the co-localization of CD98hc and β1 integrin, incubation with 9EG7 was carried out overnight at 4 °C prior to fixation. In these experiments, incubation with 4F2 or fluorescein isothiocyanate-conjugated FN50 was performed last of all, after secondary labeling of the β1 integrin. Confocal microscopy was performed with a Leica TCS NT confocal microscope system, and image analysis was performed using Leica TCS NT software. Statistical Analysis—Results are presented as means ± S.E. Significance of the differences between means was assessed using one-way analysis of variance (ANOVA) or two-tailed Student's t test. Values of p < 0.05 were considered significant. Unless stated otherwise, studies were performed on three to six independent occasions. CD98hc-induced Anchorage- and Serum-independent Growth Is Dependent on the Level of CD98hc Expression and PI3K Activation—To investigate the role that CD98hc plays in cancer, we examined the effect of overexpressing human CD98hc in CHO cells on anchorage-independent growth in soft agarose, a cardinal feature of malignant transformation that closely correlates with xenograft growth in nude mice, human tumor invasiveness, and clinical aggressiveness (28Bouck N. Di Mayorca G. Methods Enzymol. 1979; 58: 296-302Crossref PubMed Scopus (27) Google Scholar, 29Carney D.N. Gazdar A.F. Minna J.D. Cancer Res. 1980; 40: 1820-1823PubMed Google Scholar). Two clones stably expressing different cell-surface levels of CD98hc were selected. CHO cells stably expressing CD69 were used as controls. Like CD98hc, CD69 is a member of the type II transmembrane protein family. To exclude the possibility of clonal variation, three different stable clones were selected, and similar results were obtained. In addition, comparable results were obtained using a transient transfection system with transfection efficiencies >60%. Furthermore, chimeric expression did not affect β1 integrin expression as judged by flow cytometry (data not shown). The colony-forming efficiency of CHO cells stably expressing CD98hc was significantly higher than that of vector- or CD69-transfected cells. In addition, the efficiency of colony formation was greatest in the clone with the highest level of CD98hc cell-surface expression (CD98hc+) (Fig. 1A). Another feature of the transformed phenotype is the ability of cells to grow under serum-free conditions. Fig. 1B shows that overexpressing CD98hc supported anchorage-independent growth even under serum-free conditions. High saturation density is also regarded as an indicator of malignant transformation. In cell culture, the CD98hc clone exhibited higher saturation density compared with CD69-transfected cells after 10 days in culture, whereas the rate of growth was unaffected (Fig. 1C). PI3K plays a key role in integrin activation and cellular activation and transformation (30Carpenter C.L. Cantley L.C. Curr. Opin. Cell Biol. 1996; 8: 153-158Crossref PubMed Scopus (576) Google Scholar). We therefore examined the effect of overexpressing CD98hc on PI3K activity in CHO cells. Expression of CD98hc significantly increased PI3K activity compared with expression of CD69 by 2–2.5-fold and increased phosphorylation of protein kinase B (Fig. 2A). The PI3K inhibitor LY294002 (31Vlahos C.J. Matter W.F. Hui K.Y. Brown R.F. J. Biol. Chem. 1994; 269: 5241-5248Abstract Full Text PDF PubMed Google Scholar) caused a marked concentration-dependent inhibition of the colony-forming ability of CHO cells stably overexpressing CD98hc (IC50 = 2.1 μm) (Fig. 2B). Thus, increased expression of wild-type CD98hc acts like an oncogene, stimulating serum- and anchorage-independent clonal growth. These effects are dependent on the level of CD98hc cell-surface expression and are blocked by inhibiting PI3K activation. The Transmembrane Domain (Amino Acids 82–104) of CD98hc Is Necessary and Sufficient for PI3K Activation, Elevation of Intracellular PIP3, and Colony Formation—CD98hc/CD69 chimeras (in which the extracellular, transmembrane, and cytoplasmic domains of CD98hc were exchanged with those of the type II membrane protein CD69 as shown in Fig. 3) were transfected into CHO cells to investigate the structure/function relationship of CD98hc to PI3K activation and transformation. Stable CHO cell lines were generated expressing each chimera at comparable levels as judged by flow cytometry and Western blot analysis (Fig. 4). The membrane topography of CD98hc and each of the chimeras has been established previously (32Fenczik C.A. Zent R. Dellos M. Calderwood D.A. Satriano J. Kelly C. Ginsberg M.H. J. Biol. Chem. 2001; 276: 8746-8752Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar); the C terminus is extracellular, and the N terminus is cytoplasmic. Chimeric expression did not affect β1 integrin expression as judged by flow cytometry (data not shown).Fig. 4Expression of CD98hc/CD69 chimeras in CHO-K1 cells. CD98hc/CD69 chimeras were stably transfected into CHO-K1 cells. The results from FACS analysis of expression in the stable clones using either anti-CD98 antibody 4F2 or anti-CD69 antibody are shown (A). The results from Western blot analysis of expression of CD98hc/CD69 chimeras in stable clones are also shown (B). Cell lysates were probed with anti-CD98 antibody 4F2 or anti-CD69 antibody as indicated. Analysis of expression of chimeras containing the CD69 extracellular domain was performed under nonreducing conditions, as the anti-CD69 antibody recognizes only native antigen.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The effect of expressing CD98hc/CD69 chimeras on PI3K activity in CHO cells was examined both by in vitro kinase assay and by generation of the product PIP3 using a radioisotope dilution assay (27Van der Kaay J. Cullen P.J. Downes C.P. Bird I.M. Phospholipid Signaling Protocols. Humana Press, Totowa, NJ1999: 109-125Google Scholar). The transmembrane domain of CD98hc was necessary and sufficient to activate PI3K and to elevate intracellular PIP3 levels (Fig. 3, A and B). In particular, the C69T98E69 chimera (extracellular and intracellular CD69 and transmembrane CD98hc) and the truncation mutant CD98hc(Δ2–77) (in which the cytoplasmic domain (amino acids 2–77) is deleted) were both able to stimulate PI3K activation and elevation of intracellular PIP3 levels. In contrast, the chimera C98T69E98 (in which the transmembrane domain of CD98hc is substituted with the transmembrane domain of CD69) did not stimulate PI3K activity or elevate PIP3 levels. The effect of stable chimeric expression on colony formation in semisolid agarose and 1% FCS is shown in Fig. 3C. All CHO cells stably expressing the transmembrane domain of CD98hc showed cloning efficiencies comparable with to those of CHO cells overexpressing wild-type CD98hc. In contrast, chimeras containing the transmembrane domain of CD69 had cloning efficiencies
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