HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 279, Issue 52, 54731-54741, December 24, 2004

This Article Abstract Full Text (PDF) All Versions of this Article: 279/52/54731    most recent M408700200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Henderson, N. C. Articles by Sethi, T. Search for Related Content PubMed PubMed Citation Articles by Henderson, N. C. Articles by Sethi, T. Social Bookmarking                      What's this?

CD98hc (SLC3A2) Interaction with ₁ Integrins Is Required for Transformation^*

Neil C. Henderson, Elizabeth A. Collis, Alison C. Mackinnon, Kenneth J. Simpson, Christopher Haslett, Roy Zent||, Mark Ginsberg**, and Tariq Sethi

From the Lung Inflammation Group, Medical Research Council Centre for Inflammation Research, University of Edinburgh Medical School, Edinburgh EH8 9XD, Scotland, United Kingdom, the ^||Division of Nephrology, Veterans Affairs Medical Center, Vanderbilt University Medical Center, Nashville, Tennessee 37232, and the ^**Department of Medicine, University of California-San Diego, La Jolla, California 92093-0726

Received for publication, July 30, 2004 , and in revised form, October 4, 2004.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
CD98hc (SLC3A2) constitutively and specifically associates with ₁ 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 ₁ 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/₁ 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 ₁ integrins may provide a new target for cancer therapy.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
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 (1). 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 (2–4). 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 (5, 6). Deletion of CD98hc in embryonic stem cells blocks their ability to form teratomas in mice,¹ and overexpression of CD98hc in murine fibroblasts results in anchorage-independent growth (7). In addition, increased CD98hc expression correlates with the development, progression, and metastatic potential of tumors (8–10). 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 and subunits function primarily as receptors for extracellular matrix ligands, which regulate many aspects of cell physiology, including morphology, adhesion, migration, proliferation, and differentiation (11). Many cancers show abnormalities of integrin function as a result of transformation by oncogenes (12). More importantly, the growth of several tumors depends on ₁ integrin function (13). CD98hc constitutively and specifically associates with ₁ integrins (14–18), 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)² (18) and Rap1 (19) and enhances ₁ integrin-mediated cell adhesion in a number of cancer cells, including breast and small cell lung cancer (14, 20), and clustering of ₃₁ integrin on the surface of rhabdomyosarcoma cells (21). 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 ₁ 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 (22) and cause loss of ₁ integrin association in low density light chain membrane fractions (21). However, these mutants still bind to free _1A cytoplasmic tails (Tac-₁) in vitro and reverse Tac-₁-induced dominant integrin suppression in Chinese hamster ovary (CHO) cells (15). However, titration of CD98hc by ₁ tails is not the mechanism of Tac-₁ dominant suppression (15). In contrast, other evidence suggests that the cytoplasmic/transmembrane domain of CD98hc is the critical region mediating CD98hc alteration of ₁ integrin surface distribution and cytoskeletal architecture in Madin-Darby canine kidney cells and reversal of Tac-₁ dominant suppression in CHO cells (15, 16). We have shown previously that cross-linking CD98hc stimulates PI3K activity in a ₁ integrin-dependent manner (18). 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 ₁ 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 ₁ 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.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
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 [GenBank] ), amino acids 121–183 of CD69 (Swiss-Prot accession number Q07108 [GenBank] ), 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(2–77) 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(1–86) (also previously termed D5; kindly provided by Drs. D. Merlin and J. L. Madara) (16) 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-₁ 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 ₁ integrin, rat clone 9EG7 was used (Pharmingen). For immunoprecipitation, anti-human ₁ 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 GD25₁ null, GD25_1A, and GD25_1A(Y783F/Y795F) have been described previously (23, 24). GD25₁ null cells are fibroblasts derived from ₁ null embryonic stem cells. The GD25_1A and GD25_1A(Y783F/Y795F) mutant cell lines were derived from GD25 cells upon stable transfection with cDNAs encoding the wild-type and mutant murine _1A integrin subunits, respectively (25). GD25₁ null cells were grown in DMEM containing 10% FCS, 5 µg/ml glutamine, 50 units/ml penicillin, and 50 µg/ml streptomycin; GD25_1A and GD25_1A(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 x 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^TM (BD Biosciences). Control IgG_2a and IgG₁ antibodies for 4F2 and CD69, respectively, were also used.

Clonogenic Assay—Cells (2 x 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 (26). 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 p85 mAb (Upstate Biotechnology Inc., Lake Placid, NY) and assayed for activity using [-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 (27). In brief, CHO-K1 cells (5 x 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 x 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 IgG_2A negative control antibodies and (ii) secondary Alexa Fluor antibodies. To assess the co-localization of CD98hc and ₁ 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 ₁ 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.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
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 (28, 29). 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 ₁ 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).

View larger version (43K): [in this window] [in a new window]   FIG. 1. CD98hc increases clonal growth in CHO-K1 cells. A, stable transfection of CD69 or CD98hc into CHO-K1 cells. The results from FACS analysis of CD69 or CD98hc expression in the stable clones are shown (upper panels). Representative views from stained colonies expressing CD69 (left panel) and low (middle panel) and high (right panel) levels of CD98hc grown in semisolid agarose medium are shown (lower panels). B, clonal growth of CHO-K1 cells stably expressing CD98hc (closed bars) or CD69 (open bars) in semisolid agarose medium in the presence or absence of FCS. Cells (2 x 104/ml) were suspended in 0.3% agarose over a layer of 0.5% agarose in DMEM containing FCS as indicated. After 6 days, colonies greater than four cells were counted under a light microscope. Results are expressed as percent cloning efficiency and are the means ± S.E. of three independent experiments, *, significantly different from CD69-transfected cells (p < 0.05, Student's t test). C, CD98hc overexpression results in high saturation density. The growth of CD69-expressing () and CD98hc-expressing () cells in 10% FCS in standard tissue culture was measured. The results are the means ± S.E. of three independent experiments.

View larger version (29K): [in this window] [in a new window]   FIG. 2. CD98hc activates PI3K. A, 5 x 106 CHO-K1 cells stably expressing CD69 or CD98hc (low and high (+) expressing cells) were quiesced in 0.1% FCS overnight; lysates were equalized for protein; and PI3K activity was measured in p85 immunoprecipitates using an in vitro kinase assay as described under "Experimental Procedures." Aliquots of the lysate were subjected to SDS-PAGE and Western-blotted for the p85 subunit of PI3K to ensure equal immunoprecipitation (first panel). Radiolabeled PIP3 was resolved by thin layer chromatography, visualized by autoradiography, and quantified by liquid scintillation counting (second panel). Results are expressed as counts/min and represent the means ± S.E. of four independent experiments (third panel). An autoradiograph showing the 3-phosphorylated reaction product PIP3 is shown for a typical experiment. Aliquots of the lysate were Western-blotted with anti-phospho-Ser473 protein kinase B (P-PKB; fourth panel) or anti-protein kinase B (PKB; fifth panel) antibody. B, shown are the effects of LY294002 on CD98hc-stimulated clonal growth. The clonal growth of CD98hc+ cells in semisolid 0.3% agarose medium containing 1% FCS was determined at different concentrations of LY294002 as indicated. Results are expressed as percent cloning efficiency and are the means ± S.E. of three independent experiments.

View larger version (33K): [in this window] [in a new window]   FIG. 3. The transmembrane domain of CD98hc (amino acids 82–104) is necessary and sufficient for PI3K activation, elevation of intracellular PIP3, and colony formation. A, the effect of chimeras on PI3K activity. Quiesced CHO-K1 cells (5 x 106) stably expressing CD69, CD98hc, or CD98hc chimeras were lysed, and PI3K activity was measured in p85 immunoprecipitates by in vitro kinase assay as described under "Experimental Procedures." Aliquots of the lysate were subjected to SDS-PAGE and Western-blotted for the p85 subunit of PI3K to ensure equal immunoprecipitation. Radiolabeled PIP3 was resolved by thin layer chromatography, visualized by autoradiography, and quantified by liquid scintillation counting. An autoradiograph showing the 3-phosphorylated reaction product PIP3 is shown for a typical experiment. Untransfected cells stimulated with 10% FCS for 10 min or with 100 nM wortmannin for 30 min were included as controls. Results are expressed as percent activity of control untransfected cells and represent the means ± S.E. of four experiments. *, significantly different from control untransfected cells (p < 0.05, ANOVA). B, the effect of chimeras on intracellular PIP3 levels. PIP3 levels were measured in 5 x 106 quiesced CHO-K1 cells stably expressing CD69, CD98hc, or CD98hc chimeras. The levels of PIP3 were quantified by an isotope dilution assay as described under "Experimental Procedures." Results are expressed as picomoles/mg of protein and represent the means ± S.E. of four independent experiments. *, significantly different from CD69-transfected cells (p < 0.05, ANOVA). C, the effect of CD98hc chimeras on clonal growth. CHO-K1 cells stably expressing CD69, CD98hc, or CD98hc chimeras were grown in 0.3% agarose over a layer of 0.5% agarose in 1% FCS culture medium as described under "Experimental Procedures." After 6 days, colonies greater than four cells were counted by light microscopy. Results are expressed as percent cloning efficiency and are the means ± S.E. of four independent experiments. *, significantly different from CD69-transfected cells (p < 0.05, ANOVA).

View larger version (33K): [in this window] [in a new window]   FIG. 4. Expression 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.

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 that were not significantly different from those of vector- or CD69-transfected cells. In particular, a CD98hc mutant with the cytoplasmic domain deleted (CD98hc(2–77)) and the chimera C₆₉T₉₈E₆₉ (containing the transmembrane domain of CD98hc and the extracellular and cytoplasmic domains of CD69) both markedly enhanced colony formation when stably expressed in CHO cells (30.2 ± 6.2 and 33.2 ± 3.4, respectively) compared with CD69-expressing cells (2.2 ± 1.7, mean of four independent experiments done in triplicate, mean ± S.E., p < 0.05). However, the reciprocal chimera C₉₈T₆₉E₉₈ (in which the transmembrane domain of CD98hc is replaced with the CD69 transmembrane domain) did not enhance colony formation (12.1 ± 2.1, n = 4 in triplicate, mean ± S.E., p = not significant). Similar results were achieved using a transient transfection system with transfection efficiencies >60% and three different stable clones, eliminating the possibility of clonal selection. Therefore, the transmembrane domain of CD98hc is necessary and sufficient to stimulate PI3K activity, to elevate intracellular PIP₃ levels, and to promote anchorage-independent growth.

The Transmembrane Domain (Amino Acids 82–104) of CD98hc Is Required and Sufficient for CD98hc and ₁ Integrin Co-localization—Dual label confocal immunofluorescence microscopy was used to examine the physical relationship between native ₁ integrins and CD98hc domains in vivo. We have shown previously that CD98hc constitutively associates with ₁ integrins, regardless of activation state, using stimulating, inhibitory, and neutral anti-₁ integrin antibodies (18). As expected, CD98hc and ₁ integrin were co-localized in the plasma membrane (Fig. 5A). However, no co-localization was observed between CD69 (labeled by FN50) and ₁ integrins in CHO cells expressing CD69. To examine the biochemical basis for the interaction between CD98hc and ₁ integrins, the following chimeras were used: C₆₉T₉₈E₆₉ (which contains the transmembrane domain of CD98hc and the extracellular and cytoplasmic domains of CD69) and CD98hc(2–77) (which has the cytoplasmic domain (amino acids 2–77) deleted) labeled by mAb FN50 and mAb 4F2, respectively. When stably expressed in CHO cells, both these mutants associated with ₁ integrins (Fig. 5A). Amaris software, which analyzes three-dimensional pixel volume co-localization, demonstrated that 70% of all ₁ integrins co-localized with CD98hc, C₆₉T₉₈E₆₉, or CD98hc(2–77) (Fig. 5B). Conversely, when stably expressed in CHO cells, C₉₈T₆₉E₉₈ (which contains the transmembrane domain of CD69 and the extracellular and cytoplasmic domains of CD98hc) showed less ₁ integrin association.

View larger version (24K): [in this window] [in a new window]   FIG. 5. The transmembrane domain (amino acids 82–104) of CD98hc is required and sufficient for CD98hc and 1 co-localization. A, CHO-K1 cells stably expressing CD69, CD98hc, CD98hc(2–77), C69T98E69, or C98T69E98 were plated onto coverslips, and the extracellular portion was labeled with mAb FN50 (CD69 and C69T98E69) or mAb 4F2 (CD98hc, C98T69E98, and CD98hc(2–77)). 1 integrin was labeled with 9EG7, and immunolocalization visualized by fluorescence confocal microscopy with appropriate species-specific Alexa red (1 integrin) and Alexa green (CD98hc/CD69) secondary antibodies. In the merged panels, areas of co-localization appear yellow. Representative cells are shown. Scale bars = 2.5 µm. B, the percentage co-localization of 1 integrin with CD69, CD98hc, CD98hc(2–77), C69T98E69, or C98T69E98 was calculated using Leica TCS NT software. Results are expressed as percent co-localization and are the means ± S.E. of five independent experiments.

View larger version (46K): [in this window] [in a new window]   FIG. 6. The transmembrane domain (amino acids 82–104) of CD98hc is required and sufficient for CD98hc and 1 integrin co-immunoprecipitation. A, CHO-K1 cells stably expressing CD98hc (low and high (+) expressing cells), CD98hc(2–77), or C98T69E98 were lysed in 1% CHAPS immunoprecipitation buffer and adjusted to 1 mg/ml. An aliquot of the total cell lysate was resolved by SDS-PAGE and blotted with goat anti-CD98hc polyclonal antibody (upper panel). 1 integrins were immunoprecipitated (IP) from total cell lysates with 2 µg of antibody K20, and associated proteins were resolved by SDS-PAGE and blotted with anti-1 integrin antibody mAb (middle panel) or goat anti-CD98hc polyclonal antibody (lower panel). B, cell lysates from stable chimeric clones were immunoprecipitated with 2 µg of anti-CD69 mAb FN50 (CD69, C98T69E69, and C69T98E69) or mAb 4F2 (CD98hc, CD98hc(2–77), and C98T69E98). Associated proteins were resolved by SDS-PAGE, blotted with anti-1 integrin mAb, and visualized by horse-radish peroxidase-labeled anti-mouse IgG as described under "Experimental Procedures." The blots shown are representative of three independent experiments.

CD98hc Alters ₁ Integrin Surface Distribution and Promotes Extensive Focal Adhesion Complex Formation—

View larger version (40K): [in this window] [in a new window]   FIG. 7. CD98hc alters 1 integrin surface distribution and promotes extensive focal adhesion complex formation. CHO-K1 cells expressing CD69, CD98hc, or C69T98E69 were plated onto coverslips and fixed in paraformaldehyde. A, shown is the isosurface rendering of 1-labeled cells expressing CD69, CD98hc, or C69T98E69. 1 integrin localization is shown in red on a green background. Representative cells of at least five independent experiments are shown. B, cells were stained for phospho-Tyr397 FAK (P-FAK) and actin (rhodamine phalloidin) and analyzed by fluorescence confocal microscopy (upper panels). Scale bars = 2 µm. Quantitation of 20 random fields/clone was performed using Openlab image analysis software (lower panel). Representative cells are shown from four independent experiments. *, significantly different from CD69-transfected cells (p < 0.05, ANOVA).

CD98hc Signaling Is ₁ Integrin- and FAK-dependent—

View larger version (36K): [in this window] [in a new window]   FIG. 8. Signaling by the transmembrane domain (amino acids 82–104) of CD98hc is 1 integrin- and FAK-dependent. A, CHO-K1 cells stably expressing CD69, CD98hc, or C69T98E69 were lysed and resolved by SDS-PAGE. Blots were probed with anti-phospho-Thr397 FAK polyclonal antibody (P-FAK; upper panel) or anti-FAK mAb (lower panel). Bands were visualized by horseradish peroxidase-labeled secondary antibodies as described under "Experimental Procedures." Representative blots of three independent experiments are shown. B, GD25 cells expressing mouse wild-type 1 integrin, GD251 null cells, or GD251A(Y783F/Y795F) cells were transiently transfected with CD69 (open bars), CD98hc (closed bars), or C69T98E69 (hatched bars) and lysed, and PI3K activity was measured as described under "Experimental Procedures." Aliquots of the lysate were subjected to SDS-PAGE and blotted for the p85 subunit of PI3K to ensure equal immunoprecipitation (upper panel). PIP3 was resolved by thin layer chromatography, visualized by autoradiography, and quantified by liquid scintillation counting (middle panel). An autoradiograph showing the 3-phosphorylated reaction product is shown for a typical experiment. Results are expressed as PI3K activity (counts/min x 103) and represent the means ± S.E. of four independent experiments (lower panel). C, GD25 cells expressing mouse wild-type 1 integrin, GD251 null cells, or GD251A(Y783F/Y795F) cells were transiently transfected with CD69 (open bars), CD98hc (closed bars), or C69T98E69 (hatched bars). 24 h after transfection, 1 x 105 live cells were grown in 0.3% agarose over a layer of 0.5% agarose in 10% FCS culture medium as described under "Experimental Procedures." After 4 days, colonies greater than four cells were counted by light microscopy. Results are expressed as percent cloning efficiency and are the means ± S.E. of three independent experiments.

To further assess the dependence of PI3K activation on FAK, CHO cells stably expressing CD69, CD98hc, or CD98hc chimeras were transiently transfected with the dominant-negative FAK-related non-kinase (FRNK) (33). The FRNK construct completely abolished the increase in PI3K activity induced by stable expression of CD98hc or the chimera containing the transmembrane domain of CD98hc (Fig. 9), demonstrating that FAK phosphorylation is required for PI3K activation.

View larger version (37K): [in this window] [in a new window]   FIG. 9. Inhibition of PI3K activity by FRNK. Quiesced CHO-K1 cells stably expressing CD69, CD98hc, or CD98hc chimeras were transiently transfected with vector or 5 µg of FRNK. Cells were lysed; PI3K was immunoprecipitated; and activity was measured by an in vitro kinase assay as described under "Experimental Procedures." Aliquots of the lysate were subjected to SDS-PAGE and Western-blotted with anti-FAK mAb. The blots show expression of FRNK as a 55-kDa fragment in FRNK-transfected cells. PIP3 was resolved by thin layer chromatography and quantified by liquid scintillation counting. Results are expressed as counts/min and represent the means ± S.E. of four independent experiments.

View larger version (30K): [in this window] [in a new window]   FIG. 10. Amino acids 82–86 of CD98hc are required for integrin association, PI3K activation, and anchorage-independent growth but not amino acid transport. A, CD69, CD98hc, C69T98E69, or CD98hc(1–86) was stably transfected into CHO-K1 cells. Expression of CD98hc(1–86) was determined by flow cytometric analysis with mAb 4F2. B, CHO-K1 cells stably expressing CD98hc(1–86) were plated onto coverslips. CD98hc(1–86) was labeled with 4F2 (left panel), and 1 integrin was labeled with 9EG7 (middle panel). The merged image is shown in the right panel. Scale bars = 4.2 µm. C, the effect of stable CD98hc(1–86) expression was compared with those of C69T98E69 and CD69 expression on PI3K activity (left panel), colony formation (middle panel), and isoleucine (IsoLeu) transport (right panel). The data represent the means ± S.E. of four independent experiments. *, significantly different from CD69-transfected cells (p < 0.05, ANOVA).

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

CD98hc plays an important role in tumorigenesis. Deletion of CD98hc impairs the development of teratocarcinomas from embryonic stem cells,¹ and overexpression of CD98hc in NIH3T3 cells leads to anchorage-independent growth and tumor development in athymic mice (7). Furthermore, cross-linking CD98hc promotes anchorage-independent growth in small cell lung cancer cells (18). The ability of cells to grow in soft agar is a feature of anchorage independence and pathognomonic of the transformed phenotype, correlating with tumorigenicity and invasiveness of human tumors (29). Full oncogenic transformation is believed to require both serum- and anchorage-independent growth (11). We have shown here that CD98hc-transfected CHO cells are capable of both anchorage- and serum-independent growth, suggesting that overexpression of CD98hc mediates oncogenic transformation by providing signals that normally emanate from both integrins and growth factor receptors.

₁ integrins play a central role in cancer (13, 34). CD98hc physically associates with ₁ integrins (15, 16, 17), and we have shown previously that cross-linking CD98hc results in "integrin-like" signaling (18). However, other groups have been unable to chemically cross-link CD98hc directly to ₁ integrins; hence, the interaction may not be direct (21). Nevertheless, an abundance of evidence points to the functional relevance of the CD98hc-₁ integrin complex. In vitro binding data show that the cytoplasmic and transmembrane domains of CD98hc are both necessary and sufficient for binding to free _1A cytoplasmic tails and for the reversal of dominant suppression (32). The results presented here show that only those chimeras that bind to ₁ integrins promote integrin signaling (as evidenced by FAK phosphorylation, PI3K activation, and increased intracellular levels of PIP₃) and transformation. Chimeras in which the putative transmembrane domain of CD98hc is replaced with that of CD69 lost the capacity to associate and co-localize with ₁ integrins, to stimulate PI3K, and to promote anchorage-independent growth. Results obtained with truncation mutants of CD98hc suggest that amino acids 82–86 of the putative CD98hc transmembrane/membrane proximal region of the cytoplasmic domain are required for this activity. Although amino acids 82–86 may mediate CD98/₁ integrin interactions, it is also possible that loss of these amino acids may result in a conformational change in the truncation mutant, disrupting CD98hc/₁ integrin association. Nonetheless, results obtained with truncation mutants CD98hc(2–77) and CD98hc(1–86) suggest the importance of the CD98hc transmembrane/membrane proximal region of the cytoplasmic domain in regulating ₁ integrin function and transformation. Other integrin-binding proteins such as cytohesin-1, Rack-1, and skelemin also bind the membrane proximal region (35) and might contribute to the CD98hc/_1A tail interactions. CD98hc may also act as a "molecular facilitator" in the plasma membrane, regulating the association of integrins in the plane of the membrane with transmembrane domains from the superfamily members CD81, CD82, CD63, and CD53, modifying the positive or negative regulatory effects of these proteins on integrin activity.

CD98hc expression is high on the surface of human tumor cells (5, 6). However, in untransformed cells, the cyclical endogenous expression of CD98hc does not result in the display of malignant phenotypes. Hara et al. (7) have previously shown that increasing levels of CD98hc in NIH3T3 cells result in increased tumorigenicity. In this study, we have demonstrated that increasing levels of CD98hc cause increased levels of CD98hc-₁ integrin complexes with subsequent FAK phosphorylation, PI3K activation, and transformation. FAK is up-regulated in a wide variety of human epithelial cancers, with expression closely correlated to invasive potential. Furthermore, recent evidence demonstrated a direct link between FAK expression and tumor development in vivo and has therefore stimulated interest in strategies to block FAK function as a therapeutic intervention in cancer (36, 37). Similarly, constitutively active PI3K can transform chick embryonic fibroblasts (38), and a mutant p85 regulatory subunit of PI3K can transform fibroblasts in vitro (39). In addition, PI3K acting through protein kinase B has been shown to promote anchorage-independent growth (26, 40). Therefore, our results suggest that a major mechanism by which CD98hc overexpression drives transformation in human cancers is via an increase in CD98hc-₁ integrin complexes with subsequent ₁ integrin-dependent FAK phosphorylation and PI3K activation. The precise role of FAK in CD98hc-mediated transformation remains to be fully elucidated. FAK null cells and cells expressing dominant-negative FAK constructs have inefficient spreading or focal adhesion formation. Furthermore, confocal microscopy experiments in our laboratory (data not shown) confirm published results (24) that, although all three GD25 cell lines spread well on fibronectin and display focal adhesion contacts and F actin, qualitative differences in F actin and focal adhesion contacts are observed. GD25_1A cells have coarser and more heterogeneous focal contacts than GD25₁ null cells, whereas GD25_1A(Y783F/Y795F) cells have finer and more uniform focal contacts. Thus, although our results suggest that FAK phosphorylation plays a direct role in CD98hc/₁ integrin-mediated PI3K activation and transformation, it remains possible that blocking integrin-mediated FAK activation blocks PI3K activation and transformation indirectly as a result of qualitative differences in spreading or focal adhesion formation.

The physical interaction of CD98hc with amino acid transporter light chains has been proposed to regulate integrins (21). CD98hc also regulates y⁺L- and L-type amino acid transport (41, 42). This regulation appears to be due to disulfide-bonded heterodimer formation with a variety of light chains (41, 43). In addition to covalent association, there is also a noncovalent interaction between the heavy and light chains of CD98 (44). We used CD98hc chimeras lacking the CD98hc extracellular domain to completely eliminate the possibility of interactions with the light chain. Our results show that integrin association, PI3K activation, and stimulation of anchorage-independent growth by CD98hc are functions distinct and separable from the regulation of amino acid transport. Chimeras in which the transmembrane domain of CD98hc is replaced with that of CD69 lost the capacity to associate and co-localize with ₁ integrins, to stimulate PI3K, and to promote anchorage-independent growth. In contrast, these replacements had no significant effect on the amino acid transport function of CD98 (data not shown) (15). Exchange of the extracellular domain of CD98hc with that of CD69 resulted in a protein that was still capable of affecting integrin function but did not stimulate isoleucine transport. These results suggest that the association of CD98hc with a light chain is not required for its interaction with integrins or for the functional regulation of integrins. The C109S CD98hc mutant, which blocks disulfide linkage to the light chain, has been reported to decrease integrin association (21). However, as shown here, replacement of the entire extracellular domain of CD98hc leaves this function intact. These data suggest that the effect of the C109S mutant on these functions cannot be ascribed simply to loss of light chain interaction and may result from a change in the conformation or glycosylation of CD98hc. This hypothesis is supported by the marked antigenic change in CD98hc caused by the C103S mutant (22).

CD98hc is highly expressed on the surface of tumor cells irrespective of the tissue of origin (5, 6). Many cancer cells show abnormalities of integrin function as a result of transformation by oncogenes (12). CD98hc modulates integrin function in cancer cells. Cross-linking CD98hc stimulates integrin ₃₁-dependent adhesion in small cell lung cancer cells and certain breast cancer cell lines (14, 20). Our results suggest that the physical interaction between the transmembrane/membrane proximal cytoplasmic domain of CD98hc and ₁ integrins leads directly to activation of the full program of integrin stimulation. This induces receptor clustering stimulating the phosphorylation of FAK, resulting in PI3K activation and transformation. No tumor-associated point mutations have been reported in CD98hc to date; however, mutations that promote its interaction with integrins would be anticipated to promote tumorigenesis. Furthermore, increased expression of CD98hc promotes oncogenesis. Similarly, overexpression of wild-type epidermal growth factor receptor family members by gene amplification or increased transcription (45) has been implicated in a wide variety of human tumors. Receptor oncogenes such as members of the ERBb/epidermal growth factor receptor family are overexpressed in some tumor types. In contrast, there is increased expression of CD98hc on almost all tumor cells (5, 6). Therefore, the analysis of CD98hc-mediated transformation may reveal general mechanisms involved in the oncogenic process and may provide a novel target for cancer therapy.

	FOOTNOTES

 
^* This work was supported by the Wellcome Trust, United Kingdom (Clinical Training Fellowship to N. C. H. and Senior Research Leave Fellowship to T. S.), University of Edinburgh Faculty Studentship to E. A. C., the Association of International Cancer Research, and the British Lung Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Lung Inflammation Group, MRC Centre for Inflammation Research, Hugh Robson Bldg., Rm. 219, University of Edinburgh Medical School, George Square, Edinburgh EH8 9XD, Scotland, UK. Tel.: 44-131-651-1791; Fax: 44-131-651-1791; E-mail: t.sethi{at}ed.ac.uk.

¹ C. Feral and M. H. Ginsberg, submitted for publication.

² The 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.

	ACKNOWLEDGMENTS

 
We gratefully acknowledge Linda Wilson (University of Edinburgh Medical School Confocal Facility) and Kirsten Atkinson for expert technical assistance.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 

1. Haynes, B. F., Hemler, M. E., Mann, D., Eisenbarth, G., Shelhamer, J., Mostowski, H. S., Thomas, C. A., Strominger, J. L., and Fauci, A. (1981) J. Immunol. 126, 1409-1414[Medline] [Order article via Infotrieve]
2. Azzarone, B., Malpiece, Y., Zaech, P., Moretta, L., Fauci, A., and Suarez, H. (1985) Exp. Cell Res. 159, 451-462[CrossRef][Medline] [Order article via Infotrieve]
3. Suomalainen, H. A. (1986) J. Immunol. 137, 422-427[Abstract]
4. Parmacek, M. S., Karpinski, B. A., Gottesdiener, K. M., Thompson, C. B., and Leiden, J. M. (1989) Nucleic Acids Res. 17, 1915-1931[Abstract/Free Full Text]
5. Bellone, G., Alloatti, G., Levi, R., Geuna, M., Tetta, C., Peruzzi, L., Letarte, M., and Malavasi, F. (1989) Eur. J. Immunol. 19, 1-8[Medline] [Order article via Infotrieve]
6. Dixon. W. T., Sikora, L. K., Demetrick, D. J., and Jerry. L. M. (1990) Int. J. Cancer 45, 59-68[Medline] [Order article via Infotrieve]
7. Hara, K., Kudoh, H., Enomoto, T., Hashimoto, Y., and Masuko, T. (1999) Biochem. Biophys. Res. Commun. 262, 720-725[CrossRef][Medline] [Order article via Infotrieve]
8. Esteban, F., Ruiz-Cabello, F., Concha, A., Perez Ayala, M., Delgado, M., and Garrido, F. (1990) Cancer 66, 1493-1498[CrossRef][Medline]